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CN109280075B - Peptide amide compound, preparation method and medical application thereof - Google Patents

Peptide amide compound, preparation method and medical application thereof Download PDF

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CN109280075B
CN109280075B CN201810793881.5A CN201810793881A CN109280075B CN 109280075 B CN109280075 B CN 109280075B CN 201810793881 A CN201810793881 A CN 201810793881A CN 109280075 B CN109280075 B CN 109280075B
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amino
phenyl
tert
butoxycarbonylamino
hexanoyl
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CN109280075A (en
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张晨
黄安邦
黄正刚
严庞科
郑伟
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Tibet Haisike Pharmaceutical Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The invention relates to a compound shown in a general formula (I) or a stereoisomer, a solvate, a pharmaceutically acceptable salt or a eutectic crystal thereof, a composition, a preparation method and a medical application thereof, wherein the general formula (I) is shown as follows:

Description

Peptide amide compound, preparation method and medical application thereof
Technical Field
The invention relates to a peptide amide compound with analgesic effect, a preparation method thereof and application thereof in medicine.
Background
Opioids have been used for thousands of years in the treatment of pain, which exert physiological effects primarily through binding to the three known classical opioid receptors, μ, δ and κ. These three receptors are members of the G protein-coupled receptor family, are distributed primarily in the central nervous system, and are also present in many peripheral tissues. The most classical of these drugs is morphine, which exerts its analgesic effect primarily through the action of the mu opioid receptor.
In addition, the commonly used clinical analgesic drugs also comprise other mu opioid receptor drugs, such as traditional opioid drugs represented by hydromorphone and fentanyl.
However, mu opioid receptor drugs can cause a variety of side effects after long-term use, such as tolerance, dependence, and respiratory depression, as well as gastrointestinal motility, which not only increases the cost of treatment, but also affects the patient's recovery cycle. Some non-opioid injections, such as acetaminophen and NSAIDs (non-steroidal anti-inflammatory drugs), have limited application range and dosage due to their poor analgesic effect; in addition, there are certain side effects, such as acetaminophen increases liver toxicity, and NSAIDs (non-steroidal anti-inflammatory drugs) cause various gastrointestinal diseases.
With the continuous increase of the working pressure of modern society and the arrival of the elderly society and the vital function of opioid receptors on treating different types of pain, the search for novel opioid drugs with high analgesic activity and low toxic and side effects has important scientific and social significance.
Studies have found that by using kappa opioid receptor agonists, kappa opioid receptors can be targeted for intervention to treat pain and prevent a wide variety of diseases and conditions. The use of kappa opioid receptor agonists for the treatment of pain, including hyperalgesia, was described by Woold et al, Anesthesia and Analgesia (1993, 77, 362-379); wu et al 1999 Circulation Res (1999,84,1388-1395) proposed kappa opioid agonists as targets for the prevention and treatment of cardiovascular disease; the neuroprotective effects of kappa opioid receptor agonists were described by Kaushik et al in j.postgradate Medicine (2003, 49(1),90-95) in 2003; potter et al, Pharmacol. exp. Ther (2004, 209,548-553) described the use of kappa opioid agonists in ocular disorders and pain; in 2005 Wikstrom et al, j.am.soc.nephrol (2005,16, 3742-; Bileviciute-Ljungar et al in 2006 assessed the properties of kappa opioid receptor agonists for inflammatory diseases such as osteoarthritis, rheumatoid arthritis, etc., in Rheumatology (2006,45, 295-302); lembo evaluated the use of kappa opioid receptor agonists in gastrointestinal tract disease in diges.dis. (2006,24,91-98) in 2006; in 2006 Jolivalt et al in Diabetologia (2006,49(11), 2775-; schteingart, Claudio, D et al, 2008 Calla therapeutics, Inc. in WO2008057608A2 evaluated the effects of kappa opioid agonists on visceral pain, pain associated with activation of pH sensitive nociceptors, and capsaicin-induced ocular pain.
Disclosure of Invention
The invention aims to provide a kappa opioid receptor agonist with novel structure, better biological activity and better analgesic effect, a preparation method thereof and application thereof in medicine.
The invention provides a compound shown in a general formula (I) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt or a eutectic crystal thereof:
Figure BDA0001735584020000021
wherein,
R1independently selected from H, OH, C1-6Alkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl or 3-to 8-membered heterocyclyl, said alkyl, alkoxy, alkenyl, alkynyl, carbocyclyl or heterocyclyl being optionally further substituted by 0-5 substituents selected from F, Cl, Br, I, CF3Nitro, cyano, C1-6Alkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl or 3-to 8-membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from any of N, O or S;
R2、R3each independently selected from H, C1-6Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH)2)q-C3-8Carbocyclyl or-C (═ O) O- (CH2)q-3 to 8 membered heterocyclyl, said alkyl, carbocyclyl or heterocyclyl being optionally further substituted by 0-5 substituents selected from F, Cl, Br, I, OH, CF3Nitro, cyano, C1-6Alkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl or 3-to 8-membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from any of N, O or S;
R4each independently selected from F, OH, CF3、C1-6Alkyl or C1-6An alkoxy group;
R7is independently selected from C1-6Alkyl, said alkyl optionally further substituted with 1-5R7aSubstituted;
R7aindependently selected from F, OH, CF3Cyano, cyclopropyl, C2-6Alkenyl radical, C2-6Alkynyl or-C (═ O) NR7bR7cSaid cyclopropyl, alkenyl or alkynyl is optionally further substituted with 0-5 of F, Cl, Br, I, OH, CF3Nitro, cyano, C1-6Alkyl radical, C3-8Substituted with a carbocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from N, O or S;
R7b、R7ceach independently selected from H, C1-6Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH2)q-C3-8Carbocyclyl or-C (═ O) O- (CH2)q-3 to 8 membered heterocyclyl, said alkyl, alkoxy, carbocyclyl or heterocyclyl being optionally further substituted by 0-5 substituents selected from F, Cl, Br, I, OH, CF3Cyano, nitro, C1-6Alkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl or 3-to 8-membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from N, O or S;
R5、R6、R11、R12each independently selected from H, C1-6Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH)2)q-C3-8Carbocyclyl or-C (═ O) O- (CH2)q-3 to 8 membered heterocyclyl, said alkyl, carbocyclyl and heterocyclyl being optionally further substituted by 0-5 substituents selected from F, Cl, Br, I, OH, CF3Nitro, cyano, C1-6Alkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl or 3-to 8-membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from any of N, O or S;
q is selected from 1,2,3 or 4;
a is selected from 0, 1,2,3 or 4;
b is selected from 0, 1,2 or 3;
c is selected from 0, 1,2,3,4 or 5;
d is selected from 0, 1,2,3,4 or 5;
R9、R10each independently selected from F, Cl, Br, I, CF3Cyano, nitro, C1-4Alkyl, -OR9a、-C(O)OR9b、-SR9c、-S(O)R9d、-S(O)2R9eor-NR9fR9g；
R9a、R9b、R9c、R9d、R9e、R9fAnd R9gEach independently selected from H or C1-4An alkyl group;
alternatively, R9f、R9gThe nitrogen atom to which it is attached forms a 5-to 6-membered heterocyclic ring containing 1 to 3 heteroatoms selected from N, O or S.
In a preferred embodiment of the present invention, a compound represented by the general formula (I) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt or a co-crystal thereof, wherein:
R1independently selected from H, OH, C1-4Alkyl radical, C1-4Alkoxy radical, C2-4Alkenyl radical, C2-4Alkynyl, C3-6Carbocyclyl or 3-to 6-membered heterocyclyl, preferably OH, C1-4Alkyl radical, C1-4Alkoxy, said alkyl, alkoxy, alkenyl, alkynyl, carbocyclyl or heterocyclyl is optionally further substituted with 0-3 substituents selected from F, Cl, Br, I, CF3Nitro, cyano, C1-4Alkyl radical, C1-4Alkoxy radical, C2-4Alkenyl radical, C2-4Alkynyl, C3-6Substituted by carbocyclic groups or 3-to 6-membered heterocyclic groupsAnd (b) said heterocyclyl contains 1 to 3 heteroatoms selected from N, O or S;
R2、R3each independently selected from H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH)2)q-C3-6Carbocyclyl or-C (═ O) O- (CH2)q-3 to 6 membered heterocyclyl, preferably H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl or-C (═ O) O- (CH)2)q-C3-6Carbocyclyl, said alkyl, carbocyclyl or heterocyclyl being optionally further substituted by 0 to 3 substituents selected from F, Cl, Br, I, OH, CF3Nitro, cyano, C1-4Alkyl radical, C1-4Alkoxy radical, C2-4Alkenyl radical, C2-4Alkynyl, C3-6Carbocyclyl or 3 to 6 membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from any of N, O or S;
R4each independently selected from F, OH, CF3、C1-4Alkyl or C1-4Alkoxy, preferably F, CF3Or C1-4An alkyl group;
R7is independently selected from C1-4Alkyl, said alkyl optionally further substituted with 1-3R7aSubstituted;
R7aindependently selected from F, OH, CF3Cyano, cyclopropyl, C2-4Alkenyl radical, C2-4Alkynyl or-C (═ O) NR7bR7cSaid cyclopropyl, alkenyl or alkynyl is optionally further substituted with 0-3 of F, Cl, Br, I, OH, CF3Nitro, cyano, C1-4Alkyl radical, C3-6Substituted with a carbocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from N, O or S;
R7b、R7ceach independently selected from H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH2)q-C3-6Carbocyclyl or-C (═ O) O- (CH2)q-3 to 6 membered heterocyclyl, preferably H or C1-4Alkyl, said alkyl, alkoxy, carbocyclyl or heterocyclyl being optionally further substituted by 0-5 substituents selected from F, Cl, Br, I, OH, CF3Cyano radicalNitro, C1-4Alkyl radical, C1-4Alkoxy radical, C2-4Alkenyl radical, C2-4Alkynyl, C3-6Carbocyclyl or 3-to 6-membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from N, O or S;
R5、R6、R11、R12each independently selected from H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH)2)q-C3-6Carbocyclyl or-C (═ O) O- (CH2)q-3 to 6 membered heterocyclyl, preferably H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl, -C (═ O) O- (CH)2)q-C3-6Carbocyclyl, said alkyl, carbocyclyl and heterocyclyl being optionally further substituted by 0 to 3 substituents selected from F, Cl, Br, I, OH, CF3Nitro, cyano, C1-4Alkyl radical, C1-4Alkoxy radical, C2-4Alkenyl radical, C2-4Alkynyl, C3-6Carbocyclyl or 3 to 6 membered heterocyclyl, said heterocyclyl containing 1 to 3 heteroatoms selected from any of N, O or S;
q is selected from 1,2,3 or 4, preferably 1;
a is selected from 0, 1,2,3 or 4, preferably 0 or 1;
b is selected from 0, 1,2 or 3, preferably 3;
c is selected from 0, 1,2,3,4 or 5, preferably 0 or 1;
d is selected from 0, 1,2,3,4 or 5, preferably 0 or 1;
R9、R10each independently selected from F, Cl, Br, I, CF3Cyano, nitro, C1-4Alkyl or-NR9fR9gPreferably F, CF3Or C1-4An alkyl group;
R9fand R9gEach independently selected from H or C1-4An alkyl group.
In a preferred embodiment of the present invention, the present invention provides a compound represented by general formula (I), wherein the compound is selected from compounds represented by general formula (II) or stereoisomers, hydrates, metabolites, solvates, pharmaceutically acceptable salts or co-crystals thereof, wherein:
Figure BDA0001735584020000041
R1independently selected from OH or methoxy;
R2、R3each independently selected from H, C1-4Alkyl, -C (═ O) O-tert-butyl or-C (═ O) O-benzyl;
R7is independently selected from C1-6Alkyl optionally further substituted with 1-3R7aSubstituted;
R7aindependently selected from F, OH, CF3Cyano, C2-4Alkenyl radical, C2-4Alkynyl, -C (═ O) NH2Or cyclopropyl, said alkenyl, alkynyl or cyclopropyl being optionally further substituted by 0-3 of F, OH, CF3Methyl or phenyl;
R5、R6、R11、R12each independently selected from H, C1-4Alkyl, -C (═ O) O-C1-4Alkyl or-C (═ O) O-benzyl.
In a preferred embodiment of the present invention, a compound represented by the general formula (II) or a stereoisomer, a solvate, a pharmaceutically acceptable salt or a co-crystal thereof, wherein:
R1independently selected from OH or methoxy;
R2、R3each independently selected from H, methyl or-C (═ O) O-tert-butyl;
R7independently selected from methyl, ethyl, propyl, butyl, pentyl, isopropyl or
Figure BDA0001735584020000051
Said methyl, ethyl, propyl, butyl, pentyl, isopropyl or
Figure BDA0001735584020000052
Optionally further optionally substituted by 1-3R7aSubstituted;
R7aindependently selected from F, OH, CF3Cyano, and,
Figure BDA0001735584020000053
-C(＝O)NH2Or cyclopropyl;
R5、R6、R11、R12each independently selected from H, methyl, -C (═ O) O-tert-butyl.
In a preferred embodiment of the present invention, the present invention provides a compound represented by general formula (I) or (II), or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt or a co-crystal thereof, wherein the compound includes but is not limited to one of the compounds represented by the following structural formula:
Figure BDA0001735584020000061
Figure BDA0001735584020000071
。
the invention provides a pharmaceutical composition, which comprises a compound shown in a general formula (I) or (II) or a stereoisomer, a hydrate, a metabolite, a solvate, a pharmaceutically acceptable salt or a eutectic crystal thereof, and one or more pharmaceutically acceptable carriers and/or excipients.
Use of a compound of formula (I) or (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt or co-crystal thereof or a pharmaceutical composition comprising a compound of formula (I) or (II) or a stereoisomer, hydrate, metabolite, solvate, pharmaceutically acceptable salt or co-crystal thereof, for the manufacture of a medicament for the treatment or prevention of a kappa opioid receptor associated disease or condition in a mammal.
A preferred embodiment of the invention, wherein said kappa opioid receptor associated condition is selected from the group consisting of: pain, inflammation, pruritus, edema, hyponatremia, hypokalemia, ileus, cough and glaucoma.
A preferred embodiment of the present invention, wherein said pain is selected from the group consisting of: neuropathic pain, somatic pain, visceral pain, and skin pain.
A preferred embodiment of the present invention, wherein the pain is selected from the group consisting of: arthritic pain, kidney stone pain, uterine cramps, dysmenorrhea, endometriosis, dyspepsia, post-surgical pain, post-medical treatment pain, ocular pain, otitis pain, breakthrough cancer pain and pain associated with GI disorders.
Unless stated to the contrary, the terms used in the specification and claims have the following meanings.
Carbon, hydrogen, oxygen, sulfur, nitrogen or halogen referred to in the groups and compounds of the invention all include isotopes thereof, and carbon, hydrogen, oxygen, sulfur, nitrogen or halogen referred to in the groups and compounds of the invention are optionally further replaced by one or more of their corresponding isotopes, wherein isotopes of carbon include isotopes of carbon12C、13C and14c, isotopes of hydrogen including protium (H), deuterium (D, also known as deuterium), tritium (T, also known as deuterium), and isotopes of oxygen including16O、17O and18isotopes of O, sulfur including32S、33S、34S and36isotopes of S, nitrogen include14N and15isotopes of N, F19Isotopes of F, chlorine including35Cl and37cl, isotopes of bromine including79Br and81Br。
"alkyl" means a straight and branched chain monovalent saturated hydrocarbon group, the backbone comprising 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, further preferably 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, straight and branched chain groups, most preferably 1 to 2 carbon atoms, examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like; said alkyl group may optionally be further substituted by 0, 1,2,3,4 or 5 groups selected from F, Cl, Br, I, ═ O, hydroxy、-SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aWherein R is substituted by a substituent of (1)19And R19aEach independently selected from H, hydroxy, amino, carboxyl and C1-8Alkyl radical, C1-8Alkoxy radical, C2-8Alkenyl radical, C2-8Alkynyl, 3-to 10-membered carbocyclyl, 4-to 10-membered heterocyclyl, 3-to 10-membered carbocyclyloxy or 4-to 10-membered heterocyclyloxy, k is selected from 0, 1,2,3,4 or 5, and j is selected from 0, 1 or 2. Alkyl, k, j, R as appearing herein19And R19aAs defined above.
"alkylene" refers to a straight and branched chain divalent saturated hydrocarbon radical, including- (CH)2)v- (v is an integer of 1 to 10), examples of alkylene include, but are not limited to, methylene, ethylene, propylene, butylene, and the like; said alkylene may optionally be further substituted by 0, 1,2,3,4 or 5 groups selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aWhen the number of the substituents in the alkylene group is 2 or more, the substituents may be fused together to form a cyclic structure. Alkylene, as used herein, is defined as above.
"alkoxy" refers to a monovalent radical of an O-alkyl group, where alkyl is as defined herein, and examples of alkoxy include, but are not limited to, methoxy, ethoxy, 1-propoxy, 2-propoxy, 1-butoxy, 2-methyl-1-propoxy, 2-butoxy, 2-methyl-2-propoxy, 1-pentyloxy, 2-pentyloxy, 3-pentyloxy, 2-methyl-2-butoxy, 3-methyl-1-butoxy, 2-methyl-1-butoxy, and the like.
"alkenyl" means a straight and branched chain monovalent unsaturated hydrocarbon group having at least 1, and usually 1,2 or 3 carbon double bonds, and the main chain includes 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms in the main chain, examples of alkenyl include, but are not limited to, vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-3-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 2-hexenyl, and the like, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 1-octenyl, 3-octenyl, 1-nonenyl, 3-nonenyl, 1-decenyl, 4-decenyl, 1, 3-butadiene, 1, 3-pentadiene, 1, 4-hexadiene, and the like; said alkenyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aSubstituted with the substituent(s). Alkenyl as used herein, is defined as above.
"alkynyl" refers to straight and branched chain monovalent unsaturationsAnd a hydrocarbyl group having at least 1, and typically 1,2, or 3 carbon-carbon triple bonds, the backbone comprising 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms, and even more preferably 2 to 4 carbon atoms in the backbone, examples of alkynyl groups including, but not limited to, ethynyl, 1-propynyl, 2-propynyl, butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 4-pentynyl, 3-pentynyl, 1-methyl-2-butynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, and the like; said alkynyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aSubstituted with the substituent(s). Alkynyl, as found herein, is defined as above.
"cycloalkyl" refers to a monovalent saturated carbocyclic hydrocarbon group, typically of 3 to 10 carbon atoms, non-limiting examples including cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, and the like. Said cycloalkyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aBy a substituent of. Cycloalkyl as found herein, is as defined above.
"carbocyclic ring" means a saturated or unsaturated aromatic or non-aromatic ring which may be a 3 to 10 membered monocyclic, 4 to 12 membered bicyclic or 10 to 15 membered tricyclic ring system to which the carbocyclic group may be attached an endocyclic or spiro ring, non-limiting examples of which include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-alkenyl, 1-cyclopentyl-2-alkenyl, 1-cyclopentyl-3-alkenyl, cyclohexyl, 1-cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, phenyl or naphthyl. Said carbocyclyl may optionally be further substituted with 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aSubstituted with the substituent(s). Carbocycle as present herein is defined as above.
"heterocyclic" means a saturated or unsaturated aromatic or non-aromatic ring which may be a 3-to 10-membered monocyclic, 4-to 12-membered bicyclic, or 10-to 15-membered tricyclic ring system and contains 1 to 4 heteroatoms selected from N, O or S, preferably a 3-to 8-membered heterocyclic group, the optionally substituted N, S ring of which may be oxidized to various oxidation states. The heterocyclic group may be attached at a heteroatom or carbon atom, the heterocyclic group may be attached to a bridged or spiro ring, non-limiting examples of which include epoxyethyl, epoxypropyl, aziridinyl, oxetanyl, azetidinyl, thietanyl, 1, 3-dioxolanyl, 1, 4-dioxolanyl, 1, 3-dioxanyl, azepinyl, oxepinyl, thiepinyl, via, or via, a,Azolozetyl, diazepinyl, thiazetyl, pyridyl, piperidyl, homopiperidinyl, furyl, thienyl, pyranyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, piperazinyl, homopiperazinyl, imidazolyl, piperidyl, perinyl, morpholinyl, thiomorpholinyl, thiaxanyl, 1, 3-dithianyl, dihydrofuranyl, dihydropyranyl, dithiapresented inyl, tetrahydrofuryl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydropyrazolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, benzimidazolyl, benzopyridyl, chromanyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolyl, 2H-pyranyl, 4H-pyranyl, dioxacyclohexyl, 1, 3-dioxopentyl, 1, 3-dihydropyranyl, Pyrazolinyl, dithianyl, dithienoalkyl, dihydrothienyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 1,2,3, 4-tetrahydroisoquinolinyl, 3-azabicyclo [3.1.0 ]]Hexyl, 3-azabicyclo [4.1.0]Heptyl, azabicyclo [2.2.2]Hexyl, 3H-indolyquinazine, N-pyridylurea, 1-dioxothiomorpholinyl, azabicyclo [3.2.1]Octyl, azabicyclo [5.2.0 ] groups]Nonyl, oxatricyclo [5.3.1.1 ]]Dodecyl, azaadamantyl and oxaspiro [3.3 ]]A heptalkyl group. Said heterocyclyl may optionally be further substituted by 0, 1,2,3,4 or 5 substituents selected from F, Cl, Br, I, ═ O, hydroxy, -SR19Nitro, cyano, C1-6Alkyl radical, C1-6Hydroxyalkyl radical, C1-6Alkoxy radical, C2-6Alkenyl radical, C2-6Alkynyl, C3-8Carbocyclyl, 3-to 8-membered heterocyclyl, - (CH)2)a-C(＝O)-R19、-(CH2)k-C(＝O)-O-R19、-(CH2)k-C(＝O)-NR19R19a、-(CH2)k-S(＝O)j-R19、-O-C(＝O)-O-R19or-NR19R19aSubstituted with the substituent(s). Heterocyclic rings, as found herein, are defined as above.
"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. Such as: "alkyl optionally substituted with F" means that the alkyl group may, but need not, be substituted with F, and the description includes the case where the alkyl group is substituted with F and the case where the alkyl group is not substituted with F.
"pharmaceutically acceptable salt" or "pharmaceutically acceptable salt thereof" means that the biological effectiveness and properties of the free acid or free base are maintained and the free acid is obtained by reaction with a non-toxic inorganic or organic base or a salt of the free acid obtained by reaction with a non-toxic inorganic or organic acid, including alkali metal salts such as sodium, potassium, lithium, and the like; alkaline earth metal salts such as calcium salts, magnesium salts, and the like; other metal salts such as iron salts, copper salts, cobalt salts, etc.; organic base salts such as ammonium salts, triethylamine salts, pyridine salts, picoline salts, 2, 6-lutidine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, cyclohexylamine salts, ethylenediamine salts, guanidine salts, isopropylamine salts, trimethylamine salts, tripropylamine salts, triethanolamine salts, diethanolamine salts, ethanolamine salts, dimethylethanolamine salts, dicyclohexylamine salts, caffeine salts, procaine salts, choline salts, betaine salts, benzamidine penicillin salts, glucamine salts, N-methylglucamine salts, theobromine salts, tromethamine salts, purine salts, piperazine salts, morpholine salts, piperidine salts, N-ethylpiperidine salts, tetramethylamine salts, dibenzylamine salts, phenylglycine alkyl ester salts and the like; hydrohalic acid salts such as hydrofluoride, hydrochloride, hydroiodide, hydrobromide and the like; inorganic acid salts such as hydrochloride, nitrate, sulfate, perchlorate, phosphate and the like; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, ethanesulfonate and the like; arylsulfonates such as benzenesulfonate, p-toluenesulfonate and the like; organic acid salts such as acetate, benzoate, fumarate, formate, trifluoroacetate, furoate, gluconate, glutamate, glycolate, isethionate, lactate, maleate, malate, mandelate, mucate, pamoate, pantothenate, stearate, succinate, sulfonamide, tartrate, malonate, 2-hydroxypropionate, citrate, salicylate, oxalate, glycolate, glucuronate, galacturonate, citrate, lysine, arginine, aspartate, cinnamate and the like.
"pharmaceutical composition" means a mixture of one or more compounds described herein or a physiologically/pharmaceutically acceptable salt thereof with other ingredients, wherein the other ingredients comprise physiologically/pharmaceutically acceptable carriers and excipients.
"carrier" refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
"excipient" refers to an inert substance added to a pharmaceutical composition to further depend on the administration of the compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and different types of starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oils, polyethylene glycols, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like.
"prodrug" refers to a compound that can be converted under physiological conditions or by solvolysis to a compound of the invention that is biologically active. Prodrugs of the invention are prepared by modifying functional groups in compounds of the invention, which modifications may be removed by routine manipulation or in vivo, to yield the parent compound.
"cocrystals" or "cocrystals" refers to crystals of an Active Pharmaceutical Ingredient (API) and a cocrystal former (CCF) that are bound together by hydrogen bonding or other non-covalent bonds, wherein the API and CCF are both solid in pure form at room temperature and a fixed stoichiometric ratio exists between the components. A co-crystal is a multi-component crystal that contains both a binary co-crystal formed between two neutral solids and a multicomponent co-crystal formed between a neutral solid and a salt or solvate. The "co-crystal former" includes, but is not limited to, various pharmaceutically acceptable acids, bases, non-ionic compounds, water, amino acids, alcohols or other solvents, non-limiting examples of which include alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met), glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (gin), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), pyroglutamic acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, hydrochloric acid, formic acid, acetic acid, propionic acid, benzenesulfonic acid, benzoic acid, phenylacetic acid, salicylic acid, alginic acid, anthranilic acid, benzoic acid, propionic acid, benzenesulfonic acid, benzoic acid, salicylic acid, and other solvents, Camphoric acid, citric acid, ethenesulfonic acid, formic acid, fumaric acid, furoic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, mucic acid, pamoic acid, pantothenic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, malonic acid, 2-hydroxypropionic acid, oxalic acid, glycolic acid, glucuronic acid, galacturonic acid, citric acid, lysine, arginine, aspartic acid, cinnamic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or trifluoromethanesulfonic acid, ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, caffeine, procaine, choline, and mixtures thereof, Betaine, phentermine, ethylenediamine, glucosamine, methylglucamine, theobromine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, methanol, ethanol, butynediol, 1, 2-propanediol, (R)1, 2-propanediol, (S)1, 2-propanediol or 1-methyl-1, 2-ethanediol.
"stereoisomers" refers to isomers resulting from the different arrangement of atoms in a molecule, including cis, trans isomers, enantiomers and conformational isomers.
An "effective dose" refers to an amount of a compound that causes physiological or medical translation in a tissue, system, or subject that is sought, including an amount of the compound that is sufficient to prevent, or alleviate to some extent, one or more symptoms of the condition or disorder being treated when administered to a subject.
"solvates" refers to compounds of the invention or salts thereof, which also include stoichiometric or non-stoichiometric solvents bound by non-covalent intermolecular forces. When the solvent is water, it is a hydrate.
Detailed Description
The following detailed description is provided for the purpose of illustrating the embodiments and the advantageous effects thereof, and is not intended to limit the scope of the present disclosure.
The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or (and) Mass Spectrometry (MS). NMR shift (. delta.) at 10-6The units in (ppm) are given. NMR was measured using (Bruker Avance III 400 and Bruker Avance 300) nuclear magnetic spectrometers in deuterated dimethyl sulfoxide (DMSO-d)6) Deuterated chloroform (CDCl)3) Deuterated methanol (CD)3OD), internal standard Tetramethylsilane (TMS).
MS was measured by Agilent 6120B (ESI) and Agilent 6120B (APCI).
HPLC was carried out using an Agilent 1260DAD high pressure liquid chromatograph (Zorbax SB-C18100X 4.6 mm).
The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.20 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.
The column chromatography generally uses 200-300 mesh silica gel of the Tibet Huanghai silica gel as a carrier.
Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as Tatan technology, Annaiji chemistry, Shanghai Demer, Chengdong chemical, Shaoshan far chemical technology, and Bailingwei technology.
The nitrogen atmosphere means that the reaction flask is connected with a nitrogen balloon with a volume of about 1L.
The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.
The hydrogenation reaction is usually carried out by vacuum pumping, hydrogen filling and repeated operation for 3 times.
In the examples, the reaction was carried out under a nitrogen atmosphere without specific mention.
In the examples, the solution means an aqueous solution unless otherwise specified.
In the examples, the reaction temperature is room temperature, unless otherwise specified.
The room temperature is the optimum reaction temperature and is 20-30 ℃.
Boc means t-butyloxycarbonyl.
Tf means trifluoromethanesulfonyl.
M represents mol/L.
Intermediate 1:
(2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1)
(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoic acid
Figure BDA0001735584020000141
The first step is as follows: (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid methyl ester (1b)
methyl(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoate
Figure BDA0001735584020000142
To a 50mL round bottom flask were added (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionic acid (1a) (3g, 11.0mmol), (2R) -2-amino-3-phenyl-propionic acid methyl ester (2.02g, 11mmol), 1-hydroxybenzotriazole (1.83g, 13.5mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (2.6g, 14mmol), and ethyl acetate (60mL) in that order, and after addition, the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude product of (2R) -methyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionate (1b) as a white solid (4.8g, yield 100%) and was directly charged to the next step.
MS m/z＝449.1[M+Na]+
1HNMR(400MHz,CDCl3)δ7.28-7.16(m,8H),7.01-6.95(m,2H),6.26(d,1H),4.92(br,1H),4.79-4.77(m,1H),4.38-4.27(m,1H),3.67(s,3H),3.18-2.92(m,4H),1.41(s,9H).
The second step is that: (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1)
(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoic acid
Figure BDA0001735584020000151
In a 50mL round-bottom flask, crude (2R) -methyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionate (1b) (2.7g, 6.3mmol) and methanol (50mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (10mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (40mL × 3), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 60:1) to give (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) as a white solid (1.85g, two-step yield 71%).
MS m/z＝435.2[M+Na]+
Intermediate 2:
1- [ (2R) -2-amino-6- (tert-Butoxycarbonylamino) hexanoyl ] -4- (tert-Butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2)
methyl1-[(2R)-2-amino-6-(tert-butoxycarbonylamino)hexanoyl]-4-(tert-butoxycarbonylamino)piperidine-4-carboxylate
Figure BDA0001735584020000152
The first step is as follows: 1- [ (2R) -2- (benzyloxycarbonylamino) -6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (2c)
methyl1-[(2R)-2-(benzyloxycarbonylamino)-6-(tert-butoxycarbonylamino)hexanoyl]-4-(tert-butoxycarbonylamino)piperidine-4-carboxylate
Figure BDA0001735584020000161
In a 50mL round-bottom flask, methyl 4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (2a) (4.61g, 17.85mmol), (2R) -2- (benzyloxycarbonylamino) -6- (tert-butoxycarbonylamino) hexanoic acid (2b) (8.0g, 21.03mmol), 1-hydroxybenzotriazole (3.24g, 24mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (5.74g, 29.94mmol) and ethyl acetate (91mL) were added in this order, and the reaction was stirred at room temperature for 24 h. The reaction mixture was washed once with 3mol/L aqueous hydrochloric acid (200mL), saturated aqueous sodium bicarbonate (200mL) and aqueous sodium chloride (200mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude methyl 1- [ (2R) -2- (benzyloxycarbonylamino) -6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (2c) as a white solid (8.9g, yield 78%), which was directly charged to the next reaction.
The second step is that: 1- [ (2R) -2-amino-6- (tert-Butoxycarbonylamino) hexanoyl ] -4- (tert-Butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2)
methyl1-[(2R)-2-amino-6-(tert-butoxycarbonylamino)hexanoyl]-4-(tert-butoxycarbonylamino)piperidine-4-carboxylate
Figure BDA0001735584020000162
In a 100mL round-bottom flask, crude methyl 1- [ (2R) -2- (benzyloxycarbonylamino) -6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (2c), (8.9g, 14mmol), palladium on carbon (1.78g, 20 wt%) and ethyl acetate (100mL) were added, and the reaction was stirred at room temperature under hydrogen (balloon) atmosphere for 24 h. The reaction mixture was then filtered through Celite, and the filtrate was concentrated under reduced pressure to give crude methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) as a white solid (6.8g, 100% yield).
MS m/z＝487.3[M+H]+
Example 1
4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 1-1)4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propanoyl ] amino ] -amino ] hex-4-ynoylamino ] hexamino ] piperidine-4-carboxylic acid.
Figure BDA0001735584020000171
The first step is as follows: 4-Methylbenzenesulfonic acid but-2-ynyl ester (1B)
but-2-ynyl 4-methylbenzenesulfonate
Figure BDA0001735584020000181
In a 250mL round bottom flask, 2-butyn-1-ol (1A) (2.09g, 29.8mmol), p-toluenesulfonic acid (7.5g, 39mmol) and tetrahydrofuran (100mL) were added and dissolved with stirring at room temperature. Then cooling to-40 ℃, adding potassium hydroxide powder (14.22g, 253mmol) into the reaction solution in batches for multiple times, keeping the temperature at-40 ℃ after the addition is finished, and continuing the reaction for 3 hours. Then, the reaction mixture was warmed to room temperature, water (100mL) was added to the reaction mixture, followed by extraction with ethyl acetate (100mL × 2), liquid separation, organic phases were combined and dried over anhydrous sodium sulfate, filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 50:1) to give but-2-ynyl 4-methylbenzenesulfonate (1B) as a yellow oil (6.42g, 96% yield).
MS m/z＝225.0[M+H]+
1HNMR(400MHz,CDCl3)δ7.81(d,2H),7.35(d,2H),4.67(q,2H),2.45(s,3H),1.72(t,3H).
The second step is that: 2- (Diphenylmethyleneamino) hex-4-ynoic acid ethyl ester (1C)
Ethyl 2-(benzhydrylideneamino)hex-4-ynoate
Figure BDA0001735584020000182
In a 250mL round-bottom flask, 4-methylbenzenesulfonic acid but-2-ynyl ester (1B) (8.07g, 36.0mmol), diphenylmethyleneglycine ethyl ester (7.61g, 28.5mmol), cesium carbonate (29.3g, 89.9mmol), tetrabutylammonium bromide (1.06g, 3.98mmol) and tert-butyl methyl ether (150mL) were added in this order, followed by heating and refluxing for 7 hours. Filtration was performed, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 500:1) to give ethyl 2- (diphenylmethyleneamino) hex-4-ynoate (1C) as a yellow oil (7.2g, yield 79%).
MS m/z＝320.1[M+H]+
Ethyl 2- (diphenylmethyleneamino) hex-4-ynoate (1C) (7.0g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: THar 200 preparatory SFC (SFC-7); column: pheno Lux Cellulose-2,300 × 50mm I.D.,10 μm; mobile phase: a for CO2and B for methane; gradient: b15 percent; flow rate: 180 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 5 minutes; sample preparation: ethyl 2- (diphenylmethyleneamino) hex-4-ynoate (1c) (7.0g) was dissolved in 400mL of methanol; and (3) injection: 3 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ via a rotary evaporator to obtain two optical isomers, compound 1C-1(2.66g, e.e. (optical purity enantiomer excess percentage) 99.5%), compound 1C-2(2.71g, e.e.: 97.9%).
Compound 1C-1:
1H NMR(400MHz,CDCl3)δ7.68-7.62(m,2H),7.49-7.42(m,3H),7.42-7.30(m,3H),7.27-7.21(m,2H),4.29-4.10(m,3H),2.95-2.75(m,1H),2.74-2.60(m,1H),1.72(t,3H),1.26(t,3H).
MS m/z＝320.2[M+H]+
chiral HPLC retention time: 9.610min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 1C-2:
1H NMR(400MHz,CDCl3)δ7.68-7.62(m,2H),7.49-7.42(m,3H),7.42-7.30(m,3H),7.27-7.21(m,2H),4.29-4.10(m,3H),2.95-2.75(m,1H),2.74-2.60(m,1H),1.72(t,3H),1.26(t,3H).
MS m/z＝320.1[M+H]+
chiral HPLC retention time: 12.790min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the third step: 2-Aminohexane-4-alkynoic acid ethyl ester (1D-1)
ethyl 2-aminohex-4-ynoate
Figure BDA0001735584020000191
In a 50mL round-bottom flask, ethyl 2- (diphenylmethyleneamino) hex-4-ynoate (1C-1) (1.94g, 6.07mmol) was dissolved in t-butyl methyl ether (10mL), and 3mol/L aqueous hydrochloric acid (50mL) was added at room temperature, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100mL × 2), liquid separation, combination of organic phases and drying over anhydrous sodium sulfate, filtration, concentration of the filtrate under reduced pressure, and separation and purification of the residue by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 150:1 elution) to give ethyl 2-aminohex-4-ynoate (1D-1) (0.65g, yield 69%) as a yellow oily product.
MS m/z＝156.2[M+H]+
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoic acid ethyl ester (1E-1)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]hex-4-ynoate
Figure BDA0001735584020000192
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (1.65g, 4.0mmol), ethyl 2-aminohex-4-ynoate (1D-1) (0.6g, 4.0mmol), 1-hydroxybenzotriazole (0.65g, 4.8mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.92g, 5.9mmol) and dichloromethane (25mL) were added in that order and, after addition, stirred at room temperature overnight. Then, the reaction mixture was washed with 1mol/L aqueous hydrochloric acid (40mL) and separated, and then saturated aqueous sodium bicarbonate (40mL) and triethylamine (1.9mL) were added to the organic phase, and the mixture was stirred at room temperature for 30 minutes and separated. Then, the organic phase was washed once with 1mol/L aqueous hydrochloric acid (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (5mL) in this order, and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoate (1E-1) as a white solid (1.8g, yield 82%) and was directly subjected to the next reaction.
MS m/z＝572.2[M+Na]+
The fifth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoic acid (1F-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]hex-4-ynoic acid
Figure BDA0001735584020000201
In a 50mL reaction flask, crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoate (1E-1) (1.9g, 3.5mmol) and methanol (30mL) were added and dissolved with stirring at room temperature. Then, a 1mol/L aqueous solution of sodium hydroxide (5mL) was added dropwise thereto, and the mixture was stirred at room temperature overnight after the addition. The reaction solution was concentrated under reduced pressure to remove methanol, then 3mol/L aqueous hydrochloric acid (30mL) was added, and extracted with ethyl acetate (60 mL. times.2), separated, the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoic acid (1F-1) as a white solid (1.8g, yield 98%), and directly charged to the next reaction.
And a sixth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [2- [ [ (2R) -2- [ [ (2R) - (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (1G-1)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]hex-4-ynoylamino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000202
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynoic acid (1F-1) (0.63g, 1.2mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.58g, 1.2mmol), 1-hydroxybenzotriazole (0.20g, 1.44mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.28g, 1.78mmol) and dichloromethane (25mL) were added in that order, then stirred at room temperature overnight. The reaction mixture was washed once with 3mol/L aqueous hydrochloric acid (60mL), saturated aqueous sodium bicarbonate (60mL) and saturated aqueous sodium chloride (40mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [2- [ [ (2R) - (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylate (1G-1) as a white solid (1.08G, 91% yield).
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (1H-1)
methyl 4-amino-1-[(2R)-6-amino-2-[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]hex-4-ynoylamino]hexanoyl]piperidine-4-carboxylate；2，2，2-trifluoroacetic acid
Figure BDA0001735584020000211
In a 50mL round-bottom flask, methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [2- [ [ (2R) -2- [ [ (2R) -3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylate (1G-1) (0.92G, 0.93mmol) and dichloromethane (9mL) were added and dissolved with stirring at room temperature. Trifluoroacetic acid (3.5mL) was then added dropwise and the reaction stirred at room temperature for 2h after the addition was complete. The reaction mixture was directly concentrated under reduced pressure to give a crude product of methyl 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate salt (1H-1) as a pale yellow oil (0.64g, 95% yield), which was directly subjected to the next reaction.
MS m/z＝345.8[M+2H]+/2。
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynoylamino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 1-1)
4-amino-1-[(2R)-6-amino-2-[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]hex-4-ynoylamino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000212
In a 50mL round bottom flask, crude methyl 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylate (1H-1) (0.64g, 0.93mmol) and methanol (6mL) were added and dissolved with stirring at room temperature. Then, 1mol/L aqueous sodium hydroxide solution (5mL) was added, and the reaction was stirred at room temperature overnight. The reaction mixture was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by a preparative liquid phase (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynoylamino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (1I-1) as a white powder (0.41g, yield 43%).
MS m/z＝676.4[M+H]+
Tritrifluoroacetate salt of 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynoylamino ] hexanoyl ] piperidine-4-carboxylic acid (1I-1) obtained in the above step is ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, and eluted with aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL), and the eluted solution is concentrated under reduced pressure (water temperature 60 ℃ C., reduced pressure to 25mL), and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [2- [ [ (2R) -2-, [2 ] [ (2R) -2-amino-3-phenyl-propionyl ] amino ] hex-4-ynylamino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 1-1) as a white solid (0.13g, 47.5% yield).
MS m/z＝676.4[M+H]+
1HNMR(400MHz,D2O)δ7.41-7.28(m,6H),7.23(d,2H),7.16(d,2H),4.83(dd,1H),4.63(dd,1H),4.38-4.30(m,1H),3.80-3.51(m,5H),3.20-3.03(m,1H),3.02-2.90(m,3H),2.85(d,2H),2.59(dd,2H),2.18-2.07(m,1H),2.07-1.96(m,1H),1.75(d,4H),1.73-1.52(m,5H),1.50-1.29(m,2H).
Example 2:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 2-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000231
The first step is as follows: 2- (Diphenylmethyleneamino) -3-cyclopropyl-propionic acid ethyl ester (2B)
ethyl 2-(benzhydrylideneamino)-3-cyclopropyl-propanoate
Figure BDA0001735584020000232
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (7.61g, 28.5mmol) and tetrahydrofuran (150mL) were added and the solution stirred at room temperature, then cooled to-78 deg.C and potassium tert-butoxide solid (3.36g, 29.9mmol) was added in portions and stirred for 1h at 0 deg.C. Bromomethylcyclopropane (2A) (5g, 3.6mL, 37mmol) was then added dropwise, and after addition was complete, the reaction was allowed to warm to room temperature and stirred for 8 h. Then, water (100mL) was added to the reaction solution, and extraction was performed with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1 elution) to give ethyl 2- (diphenylmethyleneamino) -3-cyclopropyl-propionate (2B) as a yellow oil (5.1g, yield 57%).
MS m/z＝322.2[M+H]+
2- (diphenylmethyleneamino) -3-cyclopropyl-propionic acid ethyl ester (2B) (5.1g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: THar 80 preparatory SFC (SFC-16); column: whelk O1(S, S),250 × 30mm i.d.,5 μm; mobile phase: a for CO2and B for iso-propanol; gradient: b, 40 percent; flow rate: 70 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 5 minutes; sample preparation: 2- (Diphenylmethyleneamino) -3-cyclopropyl-propionic acid ethyl ester (2b) (5.1g) was dissolved in 100mL of methanol; and (3) injection: 2 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 2B-1(1.84g, e.e.: 100%), compound 2B-2(1.68g, e.e.: 99.3%).
Compound 2B-1:
MS m/z＝322.2[M+H]+
chiral HPLC retention time: 8.117min
The analysis conditions are as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 2B-2:
MS m/z＝322.1[M+H]+
chiral HPLC retention time: 9.229min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the second step: 2-amino-3-cyclopropyl-propionic acid ethyl ester (2C-1)
ethyl 2-amino-3-cyclopropyl-propanoate
Figure BDA0001735584020000241
In a 50mL round-bottom flask, 2- (diphenylmethyleneamino) -3-cyclopropyl-propionic acid ethyl ester (Compound 2B-1) (1.94g, 6.07mmol) and tert-butyl methyl ether (10mL) were added, followed by 3mol/L aqueous hydrochloric acid (50mL) and stirred at room temperature overnight. The pH of the reaction solution was adjusted to about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 150:1 elution) to give ethyl 2-amino-3-cyclopropyl-propionate (2C-1) as a yellow oil (0.28g, yield 38%).
MS m/z＝156.2[M-H]-
The third step: 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid ethyl ester (2D-1)
Ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoate
Figure BDA0001735584020000251
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (1.21g, 2.93mmol), ethyl 2-amino-3-cyclopropyl-propionate (2C-1) (0.28g, 1.8mmol), 1-hydroxybenzotriazole (0.40g, 2.93mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.56g, 2.9mmol) and dichloromethane (50mL) were added in that order and stirred at room temperature overnight. A1 mol/L aqueous hydrochloric acid solution (40mL) was added to the reaction mixture, followed by liquid separation. To the organic phase were added a saturated aqueous sodium bicarbonate solution (40mL) and triethylamine (1.38g, 13.6mmol), and the mixture was stirred for 30 minutes and then separated. The organic phase was washed once with 1mol/L aqueous hydrochloric acid (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated aqueous sodium chloride (40mL) in this order, and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid ethyl ester (2D-1) as a white solid (0.81g, 92% yield) which was directly subjected to the next reaction.
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid (2E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoic acid
Figure BDA0001735584020000252
In a 50mL reaction flask, crude 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid ethyl ester (2D-1) (1.9g, 3.5mmol) and methanol (30mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (4mL) and stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid (2E-1) as a white solid (1.8g, yield 98%), which was directly subjected to the next reaction.
MS m/z＝522.3[M-H]-
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (2F-1)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000261
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionic acid (2E-1) (0.40g, 0.8mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.428g, 0.88mmol), 1-hydroxybenzotriazole (0.20g, 1.44mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.28g, 1.78mmol) and dichloromethane (25mL), then stirred at room temperature overnight. The reaction mixture was washed successively with 3mol/L aqueous hydrochloric acid (60mL), saturated aqueous sodium bicarbonate (60mL), and saturated aqueous sodium chloride (40mL), and the organic phases were combined and dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] hexanoyl ] piperidine-4-carboxylate (2F-1) as a white solid (0.74g, 93% yield).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (2G-1)
methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000262
In a 50mL round bottom flask, methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylate (2F-1) (0.60g, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3.5mL) was added dropwise at room temperature, and the reaction mixture was stirred at room temperature for 2h after completion of addition. Then, the reaction mixture was directly concentrated under reduced pressure to obtain a crude product of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (2G-1) as a pale yellow oil (0.4G, yield 98%), which was directly subjected to the next reaction.
MS m/z＝346.8[M+2H]/2
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 2-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-3-cyclopropyl-propanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000271
In a 50mL round bottom flask was added crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoracetic acid (2G-1) (0.4G, 0.6mmol), methanol (2mL) and water (10 mL). The reaction solution was adjusted to pH 7 with 1mol/L NaOH solution, and then 1mol/L NaOH solution (1.0mL) was added thereto, followed by stirring at room temperature overnight. . The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was purified by preparative liquid phase separation (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropyl-propionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (2I-1) as a white powder (0.24g, 59% yield).
MS m/z＝339.8[M+2H]+/2
The 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropylpropionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (2I-1) obtained in the above step was ion-exchanged with an ion-exchange resin (25mL of ion-exchange resin, and eluted with aqueous ammonia/distilled water (v/v) ═ 1:8(800mL), and the eluted solution was concentrated under reduced pressure (at 60 ℃ C. under reduced pressure to 25mL), followed by lyophilization to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -3-cyclopropylpropionyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 2-1) as a white solid (0.08g, 67% yield).
MS m/z＝339.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.40-7.27(m,6H),7.22(d,2H),7.16(d,2H),4.83(q,1H),4.61(t,1H),4.30(t,1H),3.84-3.46(m,5H),3.13-2.90(m,4H),2.86(d,2H),2.25-1.97(m,2H),1.86-1.58(m,7H),1.58-1.49(m,1H),1.49-1.31(m,2H),0.75-0.60(m,1H),0.55-0.35(m,2H),0.14-0.03(m,2H).
Example 3:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 3-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000281
The first step is as follows: 2-fluoro-2-methyl-propan-1-ol (3B)
2-fluoro-2-methyl-propan-1-ol
Figure BDA0001735584020000282
2-Methylpropylene oxide (3A) (100g, 1.387mol) and methyl tert-butyl ether (800mL) were added to a 1000mL round bottom flask, the reaction was cooled to-78 deg.C, and then hydropyridine fluoride (275g, 2.775mol) was added slowly dropwise over half an hour, after which it was allowed to warm to room temperature and stirred overnight. Water (200mL) was added to the reaction mixture, and the organic phase was washed with saturated brine (300mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-fluoro-2-methyl-propan-1-ol (3B) as a yellow oil (30.7g, 24% yield).
1HNMR(400MHz,DMSO-d6)δ4.91(t,1H),3.35(dd,2H),1.26(d,6H).
The second step is that: (2-fluoro-2-methyl-propyl) trifluoromethanesulfonate (3C)
(2-fluoro-2-methyl-propyl)trifluoromethanesulfonate
Figure BDA0001735584020000291
2, 6-lutidine (55.4mL, 480mmol) and dichloromethane (100mL) were added to a 250mL reaction flask, the reaction was cooled to-25 deg.C, trifluoroacetic anhydride (74.6g, 443mmol) was added dropwise, after which time reaction was continued at-25 deg.C for 10min, 2-fluoro-2-methyl-propan-1-ol (3B) (27.1g, 294mmol) was slowly added dropwise, and after addition was complete reaction was allowed to warm to room temperature and stirred overnight. The reaction was concentrated under reduced pressure to give crude (2-fluoro-2-methyl-propyl) trifluoromethanesulfonate (3C) as a yellow oily product (6.7g, yield 100%).
The third step: 2- (Diphenylmethyleneamino) -4-fluoro-4-methyl-pentanoic acid ethyl ester (3D)
ethyl 2-(benzhydrylideneamino)-4-fluoro-4-methyl-pentanoate
Figure BDA0001735584020000292
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (32.04g, 120mmol) and N, N-dimethylformamide (150mL) were added, then the reaction solution was cooled to 0 ℃, potassium tert-butoxide (14.82g, 132mmol) was added in portions, after the addition, the reaction was stirred at 0 ℃ for 1h, then crude (2-fluoro-2-methyl-propyl) trifluoromethanesulfonate (3C) (29.6g, 132mmol) was added dropwise to the reaction solution, and after the addition, the reaction solution was warmed to room temperature and stirred for 8 h. The reaction solution was added with water (150mL), followed by extraction with ethyl acetate (200mL × 2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (diphenylmethyleneamino) -4-fluoro-4-methyl-pentanoate (3D) as a yellow oily product (15g, yield 29%).
MS m/z＝342.1[M+H]+
Ethyl 2- (diphenylmethyleneamino) -4-fluoro-4-methyl-pentanoate (3D) (10g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: THar 80 preparatory SFC (SFC-17); column: phenomenex Lux Cellulose-2,250 × 30mm i.d.,5 μm.; mobile phase: a for CO2 and B for EtOH; gradient: b15 percent; flow rate: 50 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 3 minutes; sample preparation: 2- (diphenylmethyleneamino) -4-fluoro-4-methyl-pentanoic acid ethyl ester (3D) (10g) dissolved in 400mL of methanol; and (3) injection: 2.5 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 3D-1(2.51g, e.e.: 100%), compound 3D-2(3.65g, e.e.: 98.1%).
Compound 3D-1:
chiral HPLC retention time: 23.612min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 3D-2:
chiral HPLC retention time: 25.688min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the fourth step: 2-amino-4-fluoro-4-methyl-pentanoic acid ethyl ester (3E-1)
ethyl 2-amino-4-fluoro-4-methyl-pentanoate
Figure BDA0001735584020000301
In a 50mL round-bottom flask, 2- (diphenylmethyleneamino) -4-fluoro-4-methyl-pentanoic acid ethyl ester (3D-1) (1.03g, 2.95mmol) was dissolved in t-butyl methyl ether (10mL), and 3mol/L aqueous hydrochloric acid solution (30mL) was added at room temperature, followed by stirring at room temperature overnight. The organic layer was separated and discarded, the aqueous phase was adjusted to pH about 13 with aqueous ammonia, then extracted with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give ethyl 2-amino-4-fluoro-4-methyl-pentanoate (3E-1) as a yellow oily product (0.39g, yield 74%).
MS m/z＝178.2[M+H]+
The fifth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoic acid ethyl ester (3F-1)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoate
Figure BDA0001735584020000311
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.825g, 2.0mmol), ethyl 2-amino-4-fluoro-4-methyl-pentanoate (3E-1) (0.354g, 2.0mmol), 1-hydroxybenzotriazole (0.297g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.15g, 6mmol) and dichloromethane (45mL) were added in that order and stirred at room temperature overnight after the addition. The reaction mixture was washed with a 1mol/L aqueous hydrochloric acid solution (35mL), a saturated aqueous sodium bicarbonate solution (35mL) and a saturated aqueous sodium chloride solution (5mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 5:1) to give 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoic acid ethyl ester (3F-1) as a white solid (0.65g, yield 68%).
1HNMR(400MHz,CDCl3)δ7.33-7.09(m,10H),6.43(d,1H),6.34(br,1H),4.78(br,1H),4.60(dt,1H),4.57-4.49(m,1H),4.36-4.24(m,1H),4.21-4.12(m,2H),3.17-2.89(m,4H),2.16-2.01(m,1H),1.99-1.85(m,1H),1.40-1.20(m,18H).
And a sixth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoic acid (3G-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoic acid
Figure BDA0001735584020000312
In a 50mL reaction flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoate (3F-1) (0.65g, 1.1mmol) and methanol (30mL) were added and dissolved with stirring at room temperature. Then, a 1mol/L aqueous solution of sodium hydroxide (1.5mL) was added dropwise thereto, and the mixture was stirred at room temperature overnight after completion of the addition. The reaction solution was concentrated under reduced pressure to remove methanol, then 3mol/L aqueous hydrochloric acid (30mL) was added and extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoic acid (3G-1) as a white solid product (0.62G, yield 100%), and was directly put into the next reaction.
MS m/z＝566.3[M+H]+
The seventh step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (3H-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000321
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoic acid (3G-1) (0.62G, 1.1mmol), 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2) (0.487G, 1.0mmol), 1-hydroxybenzotriazole (0.155G, 1.15mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.506G), 2.64mmol) and dichloromethane (25mL), then stirred at room temperature overnight. The reaction mixture was washed once with 3mol/L aqueous hydrochloric acid (60mL), saturated aqueous sodium bicarbonate (60mL) and saturated aqueous sodium chloride (40mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (3H-1) as a white solid (0.87g, yield 86%).
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester trifluoroacetate (3I-1)
methyl4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000331
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (3H-1) (0.79g, 0.78mmol) and dichloromethane (9mL) were added and dissolved with stirring at room temperature. Trifluoroacetic acid (2.0mL) was then added dropwise, and after addition was complete the reaction was stirred at room temperature for 2 h. The reaction solution was directly concentrated to dryness to give a pale yellow oily substance, 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester, a crude product of trifluoro acetic acid (3I-1), and a pale yellow solid (0.82g, 100%) which was directly used in the next reaction.
MS m/z＝356.8[M+2H]2+/2
The ninth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 3-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-fluoro-4-methyl-pentanoyl]amino]hexanoyl]piperidine-4-carboxylicacid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000332
In a 50mL round bottom flask, 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester was added; crude Tritrifluoroacetic acid (3I-1) (0.82g, 0.78mmol) and methanol (6mL) were dissolved with stirring at room temperature. Then, 1mol/L aqueous sodium hydroxide solution (5mL) was added, and the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was purified by preparative liquid phase (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-fluoro-4-methyl-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetic acid (compound 3-1), white powder (100mg, yield 23%).
MS m/z＝349.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.44-7.27(m,6H),7.27-7.20(m,4H),4.69-4.63(m,2H),4.54-4.45(m,1H),4.26-4.19(m,1H),3.94-3.75(m,3H),3.73-3.50(m,2H),3.24-3.03(m,3H),3.03-2.91(m,3H),2.37-2.25(m,1H),2.24-2.07(m,2H),2.06-1.88(m,2H),1.88-1.57(m,6H),1.44(d,3H),1.38(d,3H).
Example 5:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000351
The first step is as follows: 2- (Diphenylmethyleneamino) -6-fluoro-hexanoic acid ethyl ester (4B)
ethyl 2-(benzhydrylideneamino)-6-fluoro-hexanoate
Figure BDA0001735584020000352
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (7.61g, 28.5mmol) and tetrahydrofuran (150mL) were added, the reaction cooled to-78 deg.C, then potassium tert-butoxide (3.36g, 29.9mmol) was added in portions, the mixture was stirred for 1h while heating to 0 deg.C, 1-bromo-4-fluorobutane (4A) (5.74g, 37mmol) was added dropwise, and after addition the reaction was stirred for 8h at room temperature. Water (100mL) was added to the reaction solution, followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (diphenylmethyleneamino) -6-fluoro-hexanoate (4B) as a yellow oil (8.0g, yield 63%).
MS m/z＝342.1[M+H]+
Ethyl 2- (diphenylmethyleneamino) -6-fluoro-hexanoate (4B) (8.0g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: MG II preparatory SFC (SFC-13); column: whelk O1(S, S),250 × 30mm i.d.,5 μm; mobile phase: a for CO2and B for iso-propanol; gradient: b, 40%; flow rate: 60 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 3.5 minutes; sample preparation: 2- (Diphenylmethyleneamino) -6-fluoro-hexanoic acid ethyl ester (4B) (8.0g) was dissolved in 150mL of methanol; and (3) injection: 1.2 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 4B-1(3.10g, e.e.: 100%), and compound 4B-2(3.04g, e.e.: 100%).
Compound 4B-1:
MS m/z＝342.1[M+H]+
chiral HPLC retention time: 10.328min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm.
Compound 4B-2:
MS m/z＝342.1[M+H]+
chiral HPLC retention time: 12.028min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: (ii) greater xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm.
The second step is that: 2-amino-6-fluoro-hexanoic acid ethyl ester (4C-1)
ethyl 2-amino-6-fluoro-hexanoate
Figure BDA0001735584020000361
In a 50mL round-bottom flask, ethyl 2- (diphenylmethyleneamino) -6-fluoro-hexanoate (4B-1) (205g, 60mmol) and tert-butyl methyl ether (10mL) were added followed by 3mol/L aqueous hydrochloric acid (50mL), and after the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 13 with aqueous ammonia, extracted with ethyl acetate (100mL × 2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 150:1) to give ethyl 2-amino-6-fluoro-hexanoate (4C-1) as a yellow oil (0.59g, 56% yield).
MS m/z＝178.1[M+H]+
1HNMR(400MHz,CDCl3)δ4.58-4.34(dt,2H),4.18(q,2H),3.44(dd,1H),1.82-1.72(m,4H)，1.72-1.66(m，1H)，1.66-1.58(m，1H)，1.58-1.45(m，2H)，1.28(t，3H).
The third step: 2- [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid ethyl ester (4D-1)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoate
Figure BDA0001735584020000371
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (1.65g, 4.0mmol), ethyl 2-amino-6-fluoro-hexanoate (4C-1) (0.59g, 3.3mmol), 1-hydroxybenzotriazole (0.65g, 4.8mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.92g, 4.8mmol) and dichloromethane (50mL) were added in that order, and the reaction was stirred at room temperature overnight. A1 mol/L aqueous hydrochloric acid solution (40mL) was added to the reaction mixture, and the mixture was separated. To the organic phase were added a saturated aqueous sodium bicarbonate solution (40mL) and triethylamine (1.38g, 13.6mmol), and the mixture was stirred for 30 minutes and then separated. Then, the organic phase was washed once with 1mol/L aqueous hydrochloric acid (40mL), saturated aqueous sodium bicarbonate (40mL) and saturated sodium chloride (40mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude ethyl 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoate (4D-1) as a white solid (1.38g, 72% yield) which was directly charged to the next reaction.
The fourth step: 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoic acid
Figure BDA0001735584020000372
In a 50mL reaction flask, crude 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid ethyl ester (4D-1) (1.377g, 2.4mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2.5mL) and reaction with stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-1) as a white solid (1.09g, yield 79%), and the next reaction was carried out as it is.
MS m/z＝542.2[M-H]-
The fifth step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoic acid methyl ester (4F-1)
methyl(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoate
Figure BDA0001735584020000381
In a 50mL round-bottom flask, crude [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-1) (1.09g, 2.0mmol), (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoic acid methyl ester () (0.59g, 2.28mmol), 1-hydroxybenzotriazole (0.32g, 2.4mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.7mmol) and dichloromethane (50mL) were added in this order, followed by stirring at room temperature overnight. The reaction mixture was washed successively with 3mol/L aqueous hydrochloric acid (60mL), saturated aqueous sodium bicarbonate (60mL), and saturated aqueous sodium chloride (40mL), and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoate (4F-1) as a white solid (1.2g, 76% yield) which was directly charged to the next reaction.
And a sixth step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoic acid (4G-1)
(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoic acid
Figure BDA0001735584020000382
In a 50mL round-bottom flask, crude methyl (4F-1) 6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoate (4F-1) (1.2g, 1.5mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2mL) and reaction with stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoic acid (4G-1) as a white solid (1.10G, yield 93%), and was directly subjected to the next reaction.
The seventh step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2-tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (4H-1)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000391
In a 50mL round bottom flask were added crude (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoic acid (4G-1) (0.55G, 0.71mmol), methyl 4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (2a) (0.18G, 0.71mmol), 1-hydroxybenzotriazole (0.12G, 0.85mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.27G, 1.4mmol) and dichloromethane (50mL) in that order, and the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 3mol/L aqueous hydrochloric acid solution (60mL), a saturated aqueous sodium bicarbonate solution (60mL), and a saturated aqueous sodium chloride solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave a crude product of methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2-tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (4H-1) as a white solid (0.65g, yield 90%) which was directly charged to the next reaction.
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (4I-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000392
In a 50mL round bottom flask was added crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2-tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (4H-1) (0.60g, 0.60mmol) and dichloromethane (9mL), the solution was stirred at room temperature, then trifluoroacetic acid (3.5mL) was added dropwise, and after completion of addition the reaction was stirred at room temperature for 2H. The reaction mixture was directly concentrated to dryness to give a crude product of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (4I-1) as a pale yellow oil (0.632g, 100%) and directly subjected to the next reaction.
The ninth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000401
In a 50mL round bottom flask was added crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2-tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (4H-1) (0.63g, 0.60mmol), methanol (2mL) and water (10mL) were added to the reaction mixture, the pH of the reaction mixture was adjusted to about 7 with 1mol/L aqueous sodium hydroxide solution, and 1mol/L aqueous sodium hydroxide solution (1.5mL) was added to the reaction mixture, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (liquid phase preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution B: A25%, elution time 20 minutes), tritrifluoroacetate salt of 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (4J-1) was obtained as a white powder (0.30g, 45% yield).
MS m/z＝696.3[M-H]-
The 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 4J-1) obtained in the above step is ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, elution with aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL), and the resulting eluate is concentrated (water temperature 60 ℃ C., reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R)) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-1) as a white solid (0.1g, 74.5% yield).
MS m/z＝698.3[M+H]+
1HNMR(400MHz,D2O)δ7.40-7.28(m,6H),7.22(d,2H),7.17(d,2H),4.82-4.78(m,1H),4.65-4.55(m,2H),4.50-4.42(m,1H),4.30-4.17(m,1H),3.85-3.74(m,2H),3.73-3.67(m,2H),3.67-3.50(m,1H),3.10-2.92(m,4H),2.88(d,2H),2.26-2.15(m,1H),2.15-2.05(m,1H),1.88-1.57(m,10H),1.55-1.31(m,4H).
Example 6:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000411
The first step is as follows: 2-amino-6-fluoro-hexanoic acid ethyl ester (4C-2)
ethyl 2-amino-6-fluoro-hexanoate
Figure BDA0001735584020000421
In a 50mL round-bottom flask, the isomer 2- (diphenylmethyleneamino) -6-fluoro-hexanoic acid ethyl ester (4B-2) (2.05g, 6.0mmol) resolved by the first step of example 5 and ethyl acetate (20mL) were added, followed by 1mol/L aqueous hydrochloric acid (10mL), and the reaction was stirred at room temperature for 3 h. The organic phase was separated and discarded, the aqueous phase was adjusted to pH 13 with aqueous ammonia, then extracted with dichloromethane (25 mL. times.2), and the combined organic phases were dried over anhydrous sodium sulfate to give a solution of ethyl 2-amino-6-fluoro-hexanoate (4C-2) which was directly subjected to the next reaction.
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid ethyl ester (4D-2)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoate
Figure BDA0001735584020000422
In a 100mL round bottom flask were added sequentially (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the ethyl 2-amino-6-fluoro-hexanoate solution obtained in the previous step (50mL) and the reaction was stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was washed once with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoate (4D-2) as a white solid (0.8g, two-step yield 70%).
The third step: 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoic acid
Figure BDA0001735584020000423
In a 50mL reaction flask, ethyl 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoate (4D-2) (0.8g, 1.4mmol) and methanol (10mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2mL) and reaction with stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and extracted with ethyl acetate (30 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-2) as a white solid (0.57g, yield 75%).
MS m/z＝542.2[M-H]-
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (4H-2)
methyl
4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000431
In a 50mL round bottom flask, crude 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6-fluoro-hexanoic acid (4E-2) (0.38g, 0.7mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21g, 1.05mmol) and dichloromethane (30mL), then the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with 1mol/L aqueous hydrochloric acid (10mL), saturated aqueous sodium bicarbonate (10mL), and saturated aqueous sodium chloride (10mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 2:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (4H-2) as a white solid (0.64g, yield 90%).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (4I-2)
methyl
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000432
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (4H-2) (0.60g, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3.0mL) was added dropwise at room temperature, and after completion of addition, stirring was carried out at room temperature for 3H. The reaction mixture was concentrated to give a crude product (0.632g, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (4I-2) as a pale yellow oily substance, which was directly subjected to the next reaction.
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6-fluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000441
To a 50mL round bottom flask was added the crude reaction product methyl 4-amino-1- ((2R) -6-amino-2- (2- ((R) -2- ((R) -2-amino-3-phenylpropionylamino) -6-fluorocaproamide) hexanoyl) piperidine-4-carboxylate tritrifluoroacetate (4G-2) crude (0.632G, 0.6mmol), methanol (2mL), and water (10mL), the reaction was adjusted to pH about 7 with 1mol/L aqueous sodium hydroxide, followed by addition of 1mol/L aqueous sodium hydroxide (1.5mL), and stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (conditions for preparing liquid phase: C18 reverse phase preparative column using deionized water (a) containing 0.05% TFA as mobile phase, acetonitrile (B), and isocratic elution of B: 25% of a, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate salt (4K-2) as a white solid (0.23g, 37% yield).
MS m/z＝698.3[M+H]+
White solid 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (4K-2) obtained in the above step was subjected to ion exchange with an ion exchange resin (35mL of ion exchange resin, ammonia/distilled water (v/v) ═ 1:8(1000mL) elution), and the received elution solution was concentrated (water temperature 60 ℃ C., reduced pressure concentration to 25mL), and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -6-amino-2- [ [ (2R-propionyl ] 2 [) ) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6-fluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 4-2) as a white solid (0.16g, 39% yield).
MS m/z＝698.4[M+H]+
1HNMR(400MHz,D2O)δ7.35-7.28(m,6H),7.23-7.21(m,2H),7.16-7.14(m,2H),4.7(m,1H),4.53-4.46(m,2H),4.37-4.34(m,1H),4.10-4.09(m,1H),3.65-3.43(m,5H),3.05-2.87(m,6H),2.05-1.74(m,2H),1.55-1.4(m,10H),1.28-1.26(m,2H),0.89(m,2H).
Example 7:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 5-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000451
The first step is as follows: 2- (Diphenylmethyleneamino) -6,6, 6-trifluorohexanoic acid ethyl ester (5B)
ethyl 2-(benzhydrylideneamino)-6,6,6-trifluoro-hexanoate
Figure BDA0001735584020000461
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (9.28g, 34.7mmol) and tetrahydrofuran (150mL) were added, then cooled to-78 deg.C, and potassium tert-butoxide (4.29g, 38.2mmol) was added in portions, which after addition rose to 0 deg.C and the reaction stirred for 1 h. 1-bromo-4, 4, 4-trifluorobutane (5A) (5.0g, 26.0mmol) was then added dropwise, after which time the reaction was allowed to warm to room temperature and stirred for 8 h. Water (100mL) was added to the reaction solution, followed by extraction with ethyl acetate (100mL × 2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5B) as a yellow oil (9.8g, yield 100%).
MS m/z＝378.2[M+H]+
Ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5B) (9.8g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: MG II preparatory SFC (SFC-1); column: pheno Lux Cellulose-2, 250X 30mm I.D.,5 μm; mobile phase: a for CO2and B for iso-propanol; gradient: b20 percent; flow rate: 60 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: 2 minutes; sample preparation: ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5B) (9.8g) was dissolved in 250mL of methanol; and (3) injection: 1 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 5B-1(2.27g, e.e.: 99.7%) and compound 5B-2(2.81g, e.e.: 100%).
Compound 5B-1:
chiral HPLC retention time: 7.086min
The analysis conditions are as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 5B-2:
chiral HPLC retention time: 8.895min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the second step is that: 2-amino-6, 6, 6-trifluorohexanoic acid ethyl ester (5C-1)
ethyl 2-amino-6,6,6-trifluoro-hexanoate
Figure BDA0001735584020000471
In a 50mL round-bottom flask, ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5B-1(0.9g, 2.0mmol) and tert-butyl methyl ether (10mL) were added, followed by 3mol/L aqueous hydrochloric acid (50mL), and the reaction was stirred at room temperature overnight, the pH of the reaction solution was adjusted to about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give ethyl 2-amino-6, 6, 6-trifluorohexanoate (5C-1) as a yellow oil (0.4g, yield 78%).
MS m/z＝214.1[M+H]+
1HNMR(400MHz,CDCl3)δ4.28-4.11(m,2H),3.45-3.39(m,1H),2.22-2.03(m,2H),1.90-1.55(m,6H),1.28(t,3H).
The third step: 2- [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoic acid ethyl ester (5D-1)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoate
Figure BDA0001735584020000472
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.825g, 2.0mmol), ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5C-1) (0.4g, 2.0mmol), 1-hydroxybenzotriazole (0.30g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and dichloromethane (50mL) were added in this order and the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give crude ethyl 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoate (5D-1) as a white solid (1.21g, 100% yield).
The fourth step: 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoic acid (5E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluorohexanoic acid
Figure BDA0001735584020000481
In a 50mL round-bottom flask, crude ethyl 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoate (5D-1) (1.21g, 2.0mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2.5mL) and reaction stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and then extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoic acid (5E-1) as a white solid (0.95g, yield 82%).
MS m/z＝578.2[M-H]-
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (5F-1)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000482
In a 50mL round bottom flask, crude 2- [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoic acid (5E-1) (0.51g, 0.88mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.428g, 0.88mmol), 1-hydroxybenzotriazole (0.20g, 1.44mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.28g, 1.78mmol) and dichloromethane (25mL), then the reaction was stirred at room temperature overnight. The reaction solution was washed successively with a saturated aqueous sodium hydrogencarbonate solution (60mL) and a 3mol/L aqueous hydrochloric acid solution (40mL), and then dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (5F-1) as a white solid (0.90g, 97% yield).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (5G-1)
methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000491
In a 50mL round bottom flask, crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (5F-1) (0.60g, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3.5mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 2 h. The reaction mixture was directly concentrated under reduced pressure to give a crude product of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (5G-1) as a pale yellow oil (0.4G, 96% yield).
MS m/z＝374.8[M+H]+。
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 5-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000492
In a 50mL round-bottomed flask, 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester crude Tritrifluoroacetate (5G-1) (0.4G, 0.6mmol), methanol (2mL) and water (10mL) were added, and the reaction solution was adjusted to pH about 7 with 1mol/L aqueous sodium hydroxide solution, followed by addition of 1mol/L aqueous sodium hydroxide solution (1.5mL) and stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (5H-1) as a white powder (0.10g, yield 15%).
MS m/z＝734.4[M+H]+。
The compound 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tri-trifluoroacetate (5H-1) (0.06g, 0.06mmol) obtained in the above step was ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, elution with aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL), and the eluate thus obtained was concentrated (water temperature 60 ℃ C. and reduced pressure to 25mL), and lyophilized to give 4-amino-1- [ (2R) -6-amino-2- [ ] 2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 5-1) as a white solid (0.03g, 67% yield).
MS m/z＝734.3[M+H]+
1HNMR(400MHz,D2O)δ7.42-7.30(m,6H),7.26-7.16(m,4H),4.65-4.56(m,1H),4.30-4.20(m,1H),3.93-3.55(m,5H),3.12-2.89(m,6H),2.36-2.10(m,4H),2.00-1.33(m,13H)。
Example 8:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 5-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000511
The first step is as follows: 2-amino-6, 6, 6-trifluoro-hexanoic acid ethyl ester (5C-2)
ethyl 2-amino-6,6,6-trifluoro-hexanoate
Figure BDA0001735584020000512
In a 50mL round-bottom flask, the isomer ethyl 2- (diphenylmethyleneamino) -6,6, 6-trifluorohexanoate (5b-2) (0.75g, 2.0mmol) resolved by the first step of example 7 and ethyl acetate (10mL) were added, followed by 1mol/L aqueous hydrochloric acid (10mL), and the reaction was stirred at room temperature for 3 h. Separating and discarding the organic phase. The aqueous phase was adjusted to pH 13 with aqueous ammonia, extracted with dichloromethane (30 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate to give a solution of ethyl 2-amino-6, 6, 6-trifluoro-hexanoate (5C-2) which was directly subjected to the next reaction.
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoic acid ethyl ester (5D-2)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoate
Figure BDA0001735584020000521
In a 100mL round bottom flask, a solution (50mL) of (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and ethyl 2-amino-6, 6, 6-trifluoro-hexanoate (5C-2) obtained in the previous step were added in this order, followed by stirring at room temperature for 3 h. After the reaction, the reaction mixture was washed successively with 1mol/L aqueous hydrochloric acid (20mL), saturated aqueous sodium bicarbonate (20mL), and saturated aqueous sodium chloride (20mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration, concentration of the filtrate under reduced pressure and separation and purification of the residue by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 2:1) gave crude ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoate (5D-2) as a white solid (1.01g, 83% yield over two steps).
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoic acid (5E-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoic acid
Figure BDA0001735584020000522
In a 100mL round bottom flask, crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluorohexanoate (5D-2) (0.97g, 1.6mmol) and methanol (12mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2.3mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and then extracted with ethyl acetate (20 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoic acid (5E-2) as a white solid (0.92g, yield 100%).
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (5F-2)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000531
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoic acid (5E-2) (0.40g, 0.7mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21 g), 1.05mmol) and dichloromethane (30mL), after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed once with 1mol/L aqueous hydrochloric acid solution (10mL), saturated aqueous sodium bicarbonate solution (10mL) and saturated aqueous sodium chloride solution (10mL) in this order, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (5F-2-methyl 5F-propionyl) 3) Crude, white solid (0.59g, 80% yield over two steps).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (5G-2)
methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000532
In a 50mL round-bottom flask, crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (5F-2) (0.60g, 0.60mmol) and dichloromethane (9mL) were added, and trifluoroacetic acid (3mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was directly concentrated to dryness to give methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate as tri-trifluoroacetate (5G-2) as a pale yellow oil (0.8G, 100%) which was directly subjected to the next reaction.
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 5-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-6,6,6-trifluoro-hexanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000541
In a 50mL round bottom flask, crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (5G-2) from the previous step was added crude trifluoro acetate (0.80G, 0.60mmol), methanol (2mL) and water (10mL) were added to the reaction mixture, the pH of the reaction mixture was adjusted to about 7 with 1mol/L aqueous sodium hydroxide solution, and then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (5H-2) as a white powdery solid.
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (5H-2) obtained in the above step was ion-exchanged with an ion-exchange resin (ion-exchange resin 35mL, aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL) and the received elution solution was concentrated (water temperature 60 ℃ C., reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) ) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -6,6, 6-trifluoro-hexanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 5-2) as a white solid (181mg, 40% yield).
MS m/z＝367.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.46-7.01(m,10H),4.81(dd,1H),4.61-4.50(m,1H),4.18-4.07(m,1H),3.97(dt,1H),3.88-3.46(m,4H),3.17-2.79(m,6H),2.27-1.97(m,4H),1.87-1.81(m,1H),1.72-1.53(m,6H),1.46-1.39(m,1H),1.32-1.30(m,2H),1.08-1.06(m,2H).
Example 9:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 6-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000551
The first step is as follows: 2- (Diphenylmethyleneamino) -7-fluoro-heptanoic acid ethyl ester (6B)
ethyl 2-(benzhydrylideneamino)-7-fluoro-heptanoate
Figure BDA0001735584020000552
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (13.9g, 52mmol) and tetrahydrofuran (150mL) were added, then cooled to-78 deg.C, and potassium tert-butoxide (7.59g, 67.6mmol) was added in portions, after which time it was raised to 0 deg.C and stirred for 1h, then 1-bromo-5-fluoropentane (6A) (9.67g, 57.2mmol) was added dropwise, after which time it was raised to room temperature and stirred for 8 h. Water (100mL) was added to the reaction solution, followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (diphenylmethyleneamino) -7-fluoro-heptanoate (6B) as a yellow oil (16.7g, yield 82%).
MS m/z＝356.3[M+H]+
Ethyl 2- (diphenylmethyleneamino) -7-fluoro-heptanoate (6B) (10g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises: THar 350 preparatory SFC (SFC-7); column: phenomenex Lux Cellulose-2,300 × 50mm i.d.,10 μm; mobile phase: a for CO2and B for iso-propanol; gradient: b20 percent; flow rate: 200 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; cycle: about 3.5 minutes; sample preparation: 2- (Diphenylmethyleneamino) -7-fluoro-heptanoic acid ethyl ester (6B) (10g) was dissolved in 500mL of methanol; and (3) injection: 3.0 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain compound 6B-1(3.61g, 99.5% e.e.) which is two optical isomers, and compound 6B-2(3.75g, 99.1% e.e.).
Compound 6B-1:
chiral HPLC retention time: 12.231min
The analysis conditions are as follows: the instrument comprises the following steps: HPLC agilent 1260; column: (ii) greater xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 6B-2:
chiral HPLC retention time: 15.539min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the second step is that: 2-amino-7-fluoro-heptanoic acid ethyl ester (6C-1)
ethyl 2-amino-7-fluoro-heptanoate
Figure BDA0001735584020000561
In a 50mL round-bottom flask, ethyl 2- (diphenylmethyleneamino) -7-fluoro-heptanoate (6B-1) (071g, 21mmol) and ethyl acetate (10mL) were added, followed by 1mol/L aqueous hydrochloric acid (10mL), and the reaction was stirred at room temperature for 3 h. The organic layer was separated and discarded, and the aqueous phase was adjusted to pH 13 with aqueous ammonia, extracted with dichloromethane (50 mL. times.2), and the organic phases were combined and dried over anhydrous sodium sulfate to give a solution of ethyl 2-amino-7-fluoro-heptanoate (6C-1) which was directly subjected to the next reaction.
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid ethyl ester (6D-1)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoate
Figure BDA0001735584020000571
To a 100mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the solution obtained in the previous step ethyl 2-amino-7-fluoro-heptanoate (6C-1) (50mL) in this order, and after completion of addition, the reaction was stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL), and a saturated aqueous sodium chloride solution (20mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoate (6D-1) as a white solid (0.88g, 75% yield over two steps).
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoic acid
Figure BDA0001735584020000572
In a 50mL round-bottom flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoate (6D-1) (0.88g, 1.5mmol) and methanol (12mL) were added, followed by dropwise addition of a 1mol/L aqueous solution of sodium hydroxide (2.2mL) and stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (20 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-1) as a white solid (0.4g, yield 100%).
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (6F-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000581
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-1) (0.40g, 0.7mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21g, 1.05mmol) and dichloromethane (30mL), the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with 1mol/L aqueous hydrochloric acid (10mL), saturated aqueous sodium bicarbonate (10mL), and saturated aqueous sodium chloride (10mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration, concentration of the filtrate under reduced pressure and isolation and purification of the residue by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) gave methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylate (6F-1) as a white solid (0.59g, 80% yield over two steps).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (6G-1)
methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000582
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (6F-1) (0.60g, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was directly concentrated to give a crude product of methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (6G-1) as a pale yellow oil (1.2G), which was then subjected to the next reaction.
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 6-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000591
To a 50mL round-bottomed flask was added the crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (6G-1) crude product (1.2G) of the above-mentioned reaction crude product, methanol (2mL) and water (10mL), the reaction solution was adjusted to pH about 7 with 1mol/L aqueous sodium hydroxide solution, and then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (conditions for preparing liquid phase: C18 reverse phase preparative column using deionized water (A) containing 0.05% TFA as mobile phase, acetonitrile (B), isocratic elution B: 25% A, elution time 20 minutes), and the resulting eluted solution was concentrated (water temperature 60 ℃ C. and reduced pressure concentration to 25mL) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate salt, white powdery solid.
The 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate obtained in the above step was ion-exchanged with an ion-exchange resin (ion-exchange resin 35mL, aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL) eluted), and lyophilized to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] Amino ] -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 6-1) as a white solid (148mg, 37% yield)
MS m/z＝356.6[M+2H]+/2
1HNMR(400MHz,D2O)δ7.32-7.26(m,6H),7.18(d,2H),7.13(d,2H),4.77-4.70(m,1H),4.55(dd,2H),4.43(t,1H),4.18(t,1H),3.68-3.47(m,5H),3.04-2.81(m,6H),2.12-2.20(m,2H),1.72-1.70(m,10H),1.41-1.28(m,6H).
Example 10:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 6-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000601
The first step is as follows: 2-amino-7-fluoro-heptanoic acid ethyl ester (6c-2)
ethyl 2-amino-7-fluoro-heptanoate
Figure BDA0001735584020000602
In a 50mL round-bottom flask, 2- (diphenylmethyleneamino) -7-fluoro-heptanoic acid ethyl ester (6B-2) (0.95g, 2.7mmol) and tert-butyl methyl ether (10mL) were added, followed by addition of 3mol/L aqueous hydrochloric acid (50mL), and the reaction was stirred at room temperature overnight. The pH of the reaction solution was adjusted to about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100 mL. times.2), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give ethyl 2-amino-7-fluoro-heptanoate (6C-2) as a yellow oil (0.39g, 76% yield).
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid ethyl ester (6D-2)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoate
Figure BDA0001735584020000611
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.536g, 1.3mmol), ethyl 2-amino-7-fluoro-heptanoate (6C-2) (0.25g, 1.3mmol), 1-hydroxybenzotriazole (0.193g, 1.43mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.38g, 2.0mmol) and dichloromethane (50mL) were added in that order and the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoate (6D-2) as a white solid (0.68g, 43% yield).
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoic acid
Figure BDA0001735584020000612
In a 50mL round-bottom flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoate (6D-2) (0.67g, 1.1mmol) and methanol (15mL) were added in this order, followed by dropwise addition of 1mo/L aqueous sodium hydroxide solution (5mL) and stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (60 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-2) as a white solid (0.72g, yield 100%).
MS m/z＝556.2[M-H]-
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (6F-2)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000621
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoro-heptanoic acid (6E-2) (0.54g, 0.97mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.428g, 0.88mmol), 1-hydroxybenzotriazole (0.16g, 1.15mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.506g, 2.64mmol) and dichloromethane (25mL), the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (60mL) and a 3mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylate (6F-2) as a white solid (0.78g, 79% yield).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (6G-2)
Methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000622
In a 50mL round bottom flask was charged crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -3-phenyl-propionyl ] amino ] -7-fluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylate (6F-2) (0.70g, 0.70mmol) and dichloromethane (9mL), followed by dropwise addition of trifluoroacetic acid (3.5mL) at room temperature and stirring of the reaction at room temperature for 2h after completion of the addition. The reaction mixture was directly concentrated to give a crude product (0.74G, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate salt (6G-2) as a pale yellow oil, which was directly subjected to the next reaction.
MS m/z＝363.9[M+2H]+/2。
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 6-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7-fluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000631
Crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate Tritrifluoroacetate (6G-2) (0.74G) obtained in the above step was adjusted to pH 7 with 1mol/L aqueous sodium hydroxide solution, and then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (conditions for preparation of liquid phase: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate salt (6H-2) as a white powder (0.10g, 14% yield).
MS m/z＝356.8[M+2H]+/2。
Tritrifluoroacetate 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (6G-2) obtained in the above step was ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, elution with aqueous ammonia/distilled water (v/v) ═ 1:8(1000 mL)), the resulting eluate was concentrated (water temperature 60 ℃ C., reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino group.
-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7-fluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 6-2) as a white solid (40mg, 75% yield)
MS m/z＝356.7[M+2H]+/2
1HNMR(400MHz,D2O)δ7.32-7.26(m,6H),7.18(d,2H),7.13(d,2H),4.77-4.70(m,1H),4.55(dd,2H),4.43(t,1H),4.18(t,1H),3.68-3.47(m,5H),3.04-2.81(m,6H),2.12-2.20(m,2H),1.72-1.70(m,10H),1.41-1.28(m,6H).
Example 11:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 7-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000641
The first step is as follows: 3,3, 3-Trifluoropropyltrifluoromethanesulfonate (7B)
3,3,3-trifluoropropyl trifluoromethanesulfonate
Figure BDA0001735584020000642
2, 6-lutidine (17g, 160mmol) and dichloromethane (120mL) were added to a 250mL single-neck flask, the reaction was cooled to-25 deg.C, trifluoroacetic anhydride (45.0g, 160mmol) was added dropwise, reaction was continued at-25 deg.C for 2h after addition was complete, trifluoroethanol (12.0g, 105mmol) was added dropwise slowly, and the reaction was stirred overnight at room temperature after addition was complete. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluting with methylene chloride) to give 3,3, 3-trifluoropropyltrifluoromethanesulfonate (7B) as a yellow oily product (6.6g, yield 26%).
1HNMR(400MHz,CDCl3)δ4.70(t,2H),2.68(m,2H).
The second step is that: 2- (Diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoic acid ethyl ester (7C)
ethyl 2-(benzhydrylideneamino)-5,5,5-trifluoro-pentanoate
Figure BDA0001735584020000651
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (13.9g, 52.0mmol) and tetrahydrofuran (150mL) were added, the reaction was cooled to-78 deg.C, potassium tert-butoxide (7.59g, 67.6mmol) was added in portions, after addition, the temperature was raised to 0 deg.C and the reaction was stirred for 1h, 1-bromo-5-fluoropentane (6A) (9.67g, 57.2mmol) was added dropwise to the reaction, and after addition, the reaction was allowed to warm to room temperature and stirred for 8 h. The reaction solution was added with water (100mL), followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give 2- (diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoic acid ethyl ester (7C) as a yellow oily product (16.7g, yield 82%).
MS m/z＝364.2[M+H]+
Ethyl 2- (diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoate (7C) (10g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: THar 200 preparatory SFC (SFC-10); column: whelk O1(S, S),300 × 50mm i.d.,10 μm; mobile phase: afor CO2and B for iso-propanol; gradient: b25 percent; flow rate: 200 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 4 minutes; sample preparation: 2- (Diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoic acid ethyl ester (7C) (10g) dissolved in 180mL of methanol; and (3) injection: 3.0 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 7C-1(1.87g, e.e.: 100%), compound 7C-2(1.79g, e.e.: 100%).
Compound 7C-1:
chiral HPLC retention time: 5.872min
Analysis conditions were as follows: the instrument comprises: HPLC agilent 1260; column: (ii) greater xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 7C-2:
chiral HPLC retention time: 6.584min
The analysis conditions are as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the third step: 2-amino-5, 5, 5-trifluoro-pentanoic acid ethyl ester (7D-1)
ethyl 2-amino-5,5,5-trifluoro-pentanoate
Figure BDA0001735584020000652
In a 50mL round-bottom flask, 2- (diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoic acid ethyl ester (7C-1) (0.76g, 2.1mmol) and methyl tert-butyl ether (10mL) were added, 1mol/L hydrochloric acid aqueous solution (10mL) was added, and the reaction was stirred at room temperature for 3 h. The organic layer was separated and discarded, and the aqueous phase was adjusted to pH 13 with aqueous ammonia, then extracted with dichloromethane (25 mL. times.2), and the organic phases were combined and dried over anhydrous sodium sulfate to obtain a solution (50mL) of ethyl 2-amino-5, 5, 5-trifluoro-valerate (7D-1) which was directly subjected to the next reaction. The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butyloxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoic acid ethyl ester (7E-1)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoate
Figure BDA0001735584020000661
In a 100mL round bottom flask were added sequentially (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the solution of ethyl 2-amino-5, 5, 5-trifluoro-pentanoate (7D-1) obtained in the previous step (50mL), followed by stirring at room temperature for 3 h. After completion of the reaction, the reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel separation (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoate (7E-1) as a white solid (0.83g, 70% yield over two steps).
The fifth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoic acid (7F-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoic acid
Figure BDA0001735584020000662
In a 100mL round-bottom flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoate (7E-1) (0.83g, 1.4mmol) and methanol (10mL) were added, followed by dropwise addition of a 1mol/L aqueous solution of sodium hydroxide (2mL) and reaction with stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (30 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -5,5, 5-trifluoro-pentanoic acid (7F-1) as a white solid (0.4g, 100%).
And a sixth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (7G-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000671
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoic acid (7F-1) (0.4g, 0.7mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21 g), 1.05mmol) and dichloromethane (30mL), the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (10mL), a saturated aqueous sodium bicarbonate solution (10mL) and a saturated aqueous sodium chloride solution (10mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (7G-1) as a white solid (0.66G, two-step yield 90%).
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (7H-1)
methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate；2，2，2-trifluoroacetic acid
Figure BDA0001735584020000672
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (7G-1) (0.40G, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was concentrated under reduced pressure to give a crude product (0.65g, yield 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (7H-1) as a pale yellow oil, which was directly subjected to the next reaction.
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 7-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000681
In a 50mL round-bottomed flask, 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester crude Tritrifluoroacetate (7H-1) (0.65g), methanol (2mL) and water (10mL) were added, and the reaction solution was adjusted to pH about 7 with 1mol/L aqueous sodium hydroxide solution, then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (conditions for preparing liquid phase: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: 25% of a, elution time 20 minutes), to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tris (trifluoroacetate) (7I-1) as a white powder.
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid obtained in the above step; the tris-trifluoroacetate salt was ion-exchanged with an ion exchange resin (ion exchange resin 35mL, aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL) elution), and the received eluted solution was concentrated (water temperature 60 ℃ c. and reduced pressure to 25mL), and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 7-1) as a white solid (0.19g, yield 40%).
MS m/z＝360.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.33-7.13(m,10H),4.71-4.70(m,1H),5.54(t,1H),4.72(t,1H),3.70-3.46(m,5H),2.99-2.83(m,6H),2.20-1.94(m,5H),1.88-1.82(m,1H),1.73-1.57(m,6H),1.44-1.36(m,2H).
Example 12:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 7-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000691
The first step is as follows: 2-amino-5, 5, 5-trifluoro-pentanoic acid ethyl ester (7D-2)
ethyl 2-amino-5,5,5-trifluoro-pentanoate
Figure BDA0001735584020000692
To a 50mL round-bottom flask, the isomer 2- (diphenylmethyleneamino) -5,5, 5-trifluoro-pentanoic acid ethyl ester resolved by the first step of example 11 (7C-2) (0.85g, 2.3mmol), tert-butyl methyl ether (10mL), and 3mol/L hydrochloric acid aqueous solution (50mL) were added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give ethyl 2-amino-5, 5, 5-trifluoro-valerate (7D-2) as a yellow oil (0.30g, 66% yield).
MS：m/z＝200.2[M+H]+
1HNMR(400MHz,CDCl3)δ4.24-4.16(m,2H),3.46(dd,1H),2.40-2.13(m,2H),2.07-1.94(m,1H),1.76(m,1H),1.67(br,2H),1.29(t,3H).
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoic acid ethyl ester (7E-2)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoate
Figure BDA0001735584020000701
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.536g, 1.3mmol), ethyl 2-amino-5, 5, 5-trifluoro-pentanoate (7D-2) (0.258g, 1.3mmol), 1-hydroxybenzotriazole (0.193g, 1.43mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.38g, 2.0mmol) and dichloromethane (50mL) were added in this order, followed by stirring at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoropentanoate (7E-2) as a white solid (0.72g, 93% yield).
MS m/z＝616.3[M+Na]+
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoropentanoic acid (7F-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoic acid
Figure BDA0001735584020000702
In a 50mL round-bottom flask, crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoropentanoate (7E-2) (0.67g, 1.1mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (1.5mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L hydrochloric acid (30mL) and then extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoropentanoic acid (7F-2) as a white solid (0.46g, 74% yield).
MS m/z＝588.8[M+Na]+
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (7G-2)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000711
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoropentanoic acid (7F-2) (0.46g, 0.81mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.394g, 0.81mmol), 1-hydroxybenzotriazole (0.155g, 1.15mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.506 g) were added in this order, 2.64mmol) and dichloromethane (25mL), after addition was complete the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (60mL) and a 3mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (7G-2) as a white solid (0.70G, 84% yield).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (7H-2)
Methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000712
In a 50mL round bottom flask, crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (7G-2) (0.70G, 0.70mmol) and dichloromethane (9mL) were added, followed by dropwise addition of trifluoroacetic acid (3.5mL) at room temperature, and after completion of addition, the reaction was stirred at room temperature for 2 h. The reaction mixture was directly concentrated to give a crude product (0.76g, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (7H-2) as a pale yellow oil, which was directly subjected to the next reaction.
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 7-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5,5,5-trifluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000721
Crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (7H-2) crude Tritrifluoroacetate (0.76g) obtained in the above step was adjusted to pH 7 with 1mol/L NaOH solution, and 1mol/L NaOH solution (1.5mL) was added and the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (liquid phase preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5,5, 5-trifluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (compound 7-2), white powder (0.06g, yield 8%).
MS m/z＝360.7[M+H]/2。
1HNMR(400MHz,D2O)δ7.63-6.98(m,10H),4.94-4.84(m,1H),4.62-4.58(m,1H),4.30-4.15(m,2H),3.89-3.47(m,4H),3.26-2.82(m,6H),2.34-1.23(m,14H).
Example 13:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 8-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000731
The first step is as follows: 2- (dibenzylideneamino) -7,7, 7-trifluoro-heptanoic acid ethyl ester (8B)
ethyl 2-(benzhydrylideneamino)-7,7,7-trifluoro-heptanoate
Figure BDA0001735584020000732
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (11.38g, 42.57mmol) and tetrahydrofuran (100mL) were added and stirred at room temperature to dissolve, then the reaction was cooled to 0 deg.C, potassium tert-butoxide (6.21g, 55.3mmol) was added in portions, after addition was maintained at 0 deg.C and stirred for 1h, 1-bromo-5-fluoropentane (8A) (9.60g, 46.8mmol) was added dropwise, after addition was raised to room temperature and stirred for 8 h. The reaction solution was added with water (100mL), followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (dibenzylideneamino) -7,7, 7-trifluoro-heptanoate (8B) as a yellow oil (14.0g, yield 84%).
MS m/z＝392.1[M+H]+
Ethyl 2- (dibenzylideneamino) -7,7, 7-trifluoro-heptanoate (8B) (9.2g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: MG Π preparatory SFC (SFC-1); column: phenomenex Lux Cellulose-2,250 × 30mm i.d.,5 μm; mobile phase: a for CO2and B for methane; gradient: b15 percent; flow rate: 60 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; cycle: about 2.2 minutes; sample preparation: ethyl 2- (dibenzylideneamino) -7,7, 7-trifluoro-heptanoate (8B) (9.2g) was dissolved in 50mL of methanol; and (3) injection: 0.5 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 8B-1(4.00g, e.e.: 99.5%) and compound 8B-2(4.00g, e.e.: 97.1%).
Compound 8B-1:
chiral HPLC retention time: 7.896min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 8B-2:
chiral HPLC retention time: 8.803min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the second step is that: 2-amino-7, 7, 7-trifluoroheptanoic acid ethyl ester (8C-1)
ethyl 2-amino-7,7,7-trifluoro-heptanoate
Figure BDA0001735584020000741
In a 50mL round-bottom flask, ethyl 2- (dibenzylideneamino) -7,7, 7-trifluoro-heptanoate (8B-1) (0.76g, 1.9mmol) and methyl t-butyl ether (10mL) were added, and the mixture was stirred at room temperature to dissolve, followed by addition of 1mol/L aqueous hydrochloric acid (10mL), followed by stirring at room temperature for 3 hours. The organic layer was separated and discarded, the aqueous phase was adjusted to pH 13 with aqueous ammonia, then extracted with dichloromethane (25 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the resulting ethyl 2-amino-7, 7, 7-trifluoroheptanoate (8C-1) solution was directly subjected to the next reaction.
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoic acid ethyl ester (8D-1)
Ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoate
Figure BDA0001735584020000742
To a 100mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.78g, 1.9mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the ethyl 2-amino-7, 7, 7-trifluoroheptanoate (8C-1) solution (50mL) in that order, and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration was carried out, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoate (8D-1) as a white solid (1.03g, 90% yield in two steps).
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoic acid (8E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoic acid
Figure BDA0001735584020000751
In a 50mL round-bottom flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoate (8D-1) (0.99g, 1.6mmol) and methanol (12mL) were added, and the clear solution was stirred at room temperature, followed by dropwise addition of a 1mol/L aqueous solution of sodium hydroxide (2.0mL) and stirring at room temperature overnight after the completion of the addition. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), extracted with ethyl acetate (20 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoic acid (8E-1) as a white solid (0.94g, 44% yield).
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (8F-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000752
In a 50mL round-bottom flask, 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoic acid (8E-1) (0.40g, 0.7mmol), 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21 g), 1.05mmol) and dichloromethane (30mL), after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (10mL), a saturated aqueous sodium bicarbonate solution (10mL) and a saturated aqueous sodium chloride solution (10mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (8F-1) as a white solid (0.65g, 90% yield in two steps).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (8G-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000761
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (8F-1) (0.50g, 0.60mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3 h. Directly concentrating and drying the reaction liquid to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-methyl formate; tritrifluoroacetate (8G-1) crude (0.66G, 100%) was taken as a pale yellow oil and reacted directly in the next step.
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 8-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000762
In a 50mL round bottom flask, 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester was added; tritrifluoroacetate (8G-1) crude product (0.66G), methanol (2mL) and water (10mL), the reaction mixture was adjusted to pH 7 with 1mol/L aqueous sodium hydroxide solution, 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was purified by preparative liquid phase separation (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes), to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate as a white powder.
The 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate obtained in the above step was ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, ammonia/distilled water (v/v) ═ 1:8(1000mL) and the resulting eluate was concentrated (water temperature 60 ℃ C.) under reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 8-1) as a white solid (153mg, 33% yield).
MS m/z＝374.7[M+2H]+/2
1HNMR(400MHz,D2O)δ7.34-7.12(m,10H),4.71-4.70(m,1H),4.53(t,1H),4.19(t,1H),3.69-3.48(m,5H),3.10-2.70(m,6H),2.22-2.08(m,3H),2.02-1.95(m,1H)，1.80-1.20(m,14H)
Example 14:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 8-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylicacid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000781
The first step is as follows: 2-amino-7, 7, 7-trifluoroheptanoic acid ethyl ester (8C-2)
ethyl 2-amino-7,7,7-trifluoro-heptanoate
Figure BDA0001735584020000782
To a 50mL round-bottom flask, the isomer 2- (diphenylmethyleneamino) -7-fluoro-heptanoic acid ethyl ester (8B-2) (1.55g, 3.96mmol) resolved by the first step of example 13, t-butyl methyl ether (10mL) and 3mol/L aqueous hydrochloric acid solution (50mL) were added, and after completion of the addition, the reaction was stirred at room temperature overnight. The pH was adjusted to about 13 with aqueous ammonia, followed by extraction with ethyl acetate (100 mL. times.2), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give ethyl 2-amino-7, 7, 7-trifluoroheptanoate (8C-2) as a yellow oil (0.60g, 67% yield).
MS：m/z＝228.1[M+H]+
1HNMR(400MHz,CDCl3)δ4.18(q,2H),3.48-3.39(m,1H),2.17-1.99(m,2H),1.82-1.39(m,8H),1.28(t,3H).
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoic acid ethyl ester (8D-2)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoate
Figure BDA0001735584020000791
In a 50mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.825g, 2.0mmol), ethyl 2-amino-7, 7, 7-trifluoroheptanoate (8C-2) (0.454g, 2.0mmol), 1-hydroxybenzotriazole (0.297g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.15g, 6.0mmol) and dichloromethane (20mL) in that order, and after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude product of ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoate (8D-2) as a white solid (1.25g, yield 100%).
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7,7 trifluoroheptanoic acid (8E-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoic acid
Figure BDA0001735584020000792
In a 50mL round bottom flask, crude product of ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoate (8D-2) (1.25g, 2.01mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2.5mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and then extracted with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7,7 trifluoroheptanoic acid (8E-2) as a white solid (1.1g, 92% yield).
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (8F-2)
4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000801
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoroheptanoic acid (8E-2) (0.59g, 1.0mmol), 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2) (0.487g, 1.0mmol), 1-hydroxybenzotriazole (0.16g, 1.15mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.506 g) were successively charged, 2.64mmol) and dichloromethane (25mL), after addition was complete the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (60mL) and a 3mol/L aqueous hydrochloric acid solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7, 7-trifluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylate (8F-2) as a white solid (0.56g, 52.7% yield).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (8G-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000802
In a 50mL round bottom flask was added crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -7,7,7 trifluoroheptyl ] amino ] hexanoyl ] piperidine-4-carboxylate (8F-2) (0.55g, 0.52mmol) and dichloromethane (9mL), trifluoroacetic acid (3.5mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 2 h. The reaction mixture was directly concentrated under reduced pressure to give a crude product (574mg, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (8G-2) as a pale yellow oil, which was directly subjected to the next reaction.
MS m/z＝381.8[M+2H]+/2。
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 8-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-7,7,7-trifluoro-heptanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000811
In a 50mL reaction flask, crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (8G-2) (0.55G) was added, then the pH was adjusted to about 7 with 1mol/L aqueous sodium hydroxide solution, 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (preparation conditions: C18 reverse phase preparative column, mobile phase was deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -7,7, 7-trifluoro-heptanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tris trifluoroacetate (compound 8-2) as a white powder (0.14g, yield 24.7%).
MS m/z＝374.8[M+H]+/2
1HNMR(400MHz,CD3OD)δ7.46-7.20(m,10H),4.63-4.45(m,2H),4.37-4.05(m,4H),4.04-3.69(m,4H),3.68-3.51(m,2H),3.25-2.82(m,8H),2.40-2.00(m,6H),1.90-1.57(m,9H),1.57-1.30(m,7H),1.15-0.90(m,3H).
Example 15:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 9-1)4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propanoyl ] amino ] -3-phenyl-propanoyl ] amino ] -4-cyclopropy l-butanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid.
Figure BDA0001735584020000821
The first step is as follows: 2-Cyclopropylethyl trifluoromethanesulfonate (9B)
2-cyclopropylethyl trifluoromethanesulfonate
Figure BDA0001735584020000822
2, 6-lutidine (5.1g, 48.0mmol) and dichloromethane (40mL) were added to a 250mL single neck flask, the reaction was cooled to-25 deg.C, trifluoroacetic anhydride (12.5g, 44.3mmol) was added dropwise, and the reaction was stirred at-25 deg.C for 2 h. Trifluoroethanol (2.71g, 31.5mmol) was then slowly added dropwise, and after addition was complete, the mixture was warmed to room temperature and stirred overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluting with methylene chloride) to give 2-cyclopropylethyl trifluoromethanesulfonate (9B) as a yellow oil (4.8g, yield 70%).
1HNMR(400MHz,CDCl3)δ4.60(t,2H),1.73(q,2H),0.86-0.71(m,1H),0.61-0.51(m,2H),0.17-0.10(m,2H).
The second step is that: 2- (Diphenylmethyleneamino) -4-cyclopropyl-butyric acid ethyl ester (9C)
ethyl 2-(benzhydrylideneamino)-4-cyclopropyl-butanoate
Figure BDA0001735584020000831
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (2.67g, 10mmol) and tetrahydrofuran (100mL) were added, the reaction cooled to-78 deg.C, followed by the addition of potassium tert-butoxide (1.46g, 13mmol) in portions, which increased to 0 deg.C after the addition, followed by stirring for 1h, 2-cyclopropylethyl triflate (9B) (2.84g, 13mmol) was added dropwise, which was allowed to warm to room temperature after the addition, and the reaction was stirred for 8 h. The reaction solution was added with water (100mL), followed by extraction with ethyl acetate (100mL × 2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by separation on silica gel (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give 2- (diphenylmethyleneamino) -4-cyclopropyl-butyric acid ethyl ester (9C) as a yellow oil (2.38g, yield 89%).
Ethyl 2- (diphenylmethyleneamino) -4-cyclopropyl-butyrate (9C) (9.9g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises: THar 80 preparatory SFC (SFC-16); column: phenomenex Lux Cellulose-2, 250X 30mm I.D.,5 μm; mobile phase: a for CO2and B for ethanol; gradient: b15 percent; flow rate: 60 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 4 minutes; sample preparation: 2- (Diphenylmethyleneamino) -4-cyclopropyl-butyric acid ethyl ester (9C) (9.9g) was dissolved in 100mL of methanol; and (3) injection: 1 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 9C-1(4.00g, e.e.: 99.5%) and compound 9C-2(4.00g, e.e.: 96.5%).
Compound 9C-1:
chiral HPLC retention time: 7.757min
The analysis conditions are as follows: the instrument comprises the following steps: HPLC agilent 1260; column: (ii) greater xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 9C-2:
chiral HPLC retention time: 8.518min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the third step: 2-amino-4-cyclopropyl-butyric acid ethyl ester (9D-1)
ethyl 2-amino-4-cyclopropyl-butanoate
Figure BDA0001735584020000832
To a 50mL round-bottom flask were added 2- (diphenylmethyleneamino) -4-cyclopropyl-butyric acid ethyl ester (9C-1) (0.8g, 2.0mmol), ethyl acetate (10mL) and 1mol/L hydrochloric acid aqueous solution (10mL), and after completion of the addition, the reaction was stirred at room temperature for 3 hours. The organic layer was discarded. The aqueous layer was adjusted to pH 13 with aqueous ammonia, then extracted with dichloromethane (25 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate to give a solution (50mL) of ethyl 2-amino-4-cyclopropyl-butyrate (9D-1) which was directly fed to the next reaction.
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutanoic acid ethyl ester (9E-1)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoate
Figure BDA0001735584020000841
To a 100mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the solution of ethyl 2-amino-4-cyclopropyl-butyrate (9D-1) obtained in the previous step (50mL) in that order, and after the addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL), and the organic phase was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure, the residue was purified by silica gel separation (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyrate (9E-1) as a white solid (0.79g, 70% yield over two steps).
The fifth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutanoic acid (9F-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoic acid
Figure BDA0001735584020000842
Ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyrate (9E-1) (0.79g, 1.4mmol) and methanol (12mL) were charged in a 50mL round-bottomed flask, and the solution was stirred at room temperature, followed by dropwise addition of a 1mol/L aqueous solution of sodium hydroxide (2mL) and stirring at room temperature overnight after completion of the addition. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (20 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyric acid (9F-1) as a white solid (0.54g, yield 71%).
And a sixth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (9G-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000851
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyric acid (9F-1) (0.40g, 0.7mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21g, 1.05mmol) and dichloromethane (30mL), after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with 1mol/L aqueous hydrochloric acid (10mL), saturated aqueous sodium bicarbonate (10mL) and saturated aqueous sodium chloride (10mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure, the residue was purified by silica gel separation (dichloromethane/methanol (v/v) ═ 100:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate (9G-1) as a white solid (0.58G, 80% yield).
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (9H-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000861
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (9G-1) (0.58G, 0.60mmol) and dichloromethane (9mL) were added, followed by dropwise addition of trifluoroacetic acid (3mL), and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was directly concentrated to give a crude product (628mg, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate (9H-1) as a pale yellow oil, which was then directly subjected to the next reaction.
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 9-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000862
To a 50mL round-bottomed flask, the crude product (0.63g) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (9H-1) in the above step, methanol (2mL) and water (10mL) were added, and the reaction mixture was adjusted to pH 7 with 1mol/L aqueous sodium hydroxide solution, then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, and the reaction was stirred at room temperature overnight after completion of the addition. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (conditions for preparation of liquid phase: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes), to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (9I-1) as a white powder.
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (9I-1) obtained in the above step was subjected to ion exchange with an ion exchange resin (ion exchange resin 35mL, ammonia/distilled water (v/v) ═ 1:8(1000mL) elution), and the resulting eluate was concentrated (water temperature 60 ℃ C. and reduced pressure to 25mL), followed by lyophilization to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 9-1) as a white solid (129mg, 33% yield).
MS m/z＝346.7[M+2H]+/2
1HNMR(400MHz,D2O)δ7.41-7.05(m,10H),4.78-4.71(m,1H),4.57(t,1H),4.33-4.21(m,1H),3.84-3.38(m,5H),3.14-2.78(m,6H),2.23-1.93(m,2H),1.91-1.50(m,8H),1.25(m,4H),0.67(s,1H),0.41(d,2H),0.00(s,2H).
Example 16:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 9-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020000881
The first step is as follows: 2-amino-4-cyclopropyl-butyric acid ethyl ester (9D-2)
ethyl 2-amino-4-cyclopropyl-butanoate
Figure BDA0001735584020000882
To a 50mL round-bottom flask, the isomer 2- (diphenylmethyleneamino) -4-cyclopropyl-butyric acid ethyl ester (9C-2) (0.8g, 2.0mmol) resolved by the first step of example 15, tert-butyl methyl ether (10mL) and 3mol/L hydrochloric acid aqueous solution (50mL) were added, and after completion of the addition, the reaction was stirred at room temperature overnight, followed by separation and discarding of the organic phase. The aqueous phase was adjusted to pH about 13 with aqueous ammonia, then extracted with ethyl acetate (100 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give ethyl 2-amino-4-cyclopropyl-butyrate (9D-2) as a yellow oil (0.35g, 84% yield).
MS：m/z＝172.1[M+H]+
The second step is that: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutanoic acid ethyl ester (9E-2)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoate
Figure BDA0001735584020000891
In a 50mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.536g, 1.3mmol), ethyl 2-amino-4-cyclopropyl-butanoate (9D-2) (0.223g, 1.3mmol), 1-hydroxybenzotriazole (0.193g, 1.43mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.38g, 2.0mmol) and dichloromethane (50mL) in that order, and after addition was complete the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium bicarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyrate (9E-2) as a white solid (0.5g, 68% yield).
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutanoic acid (9F-2)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoic acid
Figure BDA0001735584020000892
In a 50mL round-bottom flask, crude ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyrate (9E-2) (1.2g, 2.1mmol) and methanol (15mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2.5mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (60 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyric acid (9f-2) as a white solid (0.9g, yield 80%).
The fourth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (9G-2)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000901
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4-cyclopropylbutyric acid (9F-2) (0.54g, 1.0mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.487g, 1.0mmol), 1-hydroxybenzotriazole (0.155g, 1.15mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.506g, 2.64mmol) and dichloromethane (25mL), after addition was complete the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (60mL) and a 3mol/L aqueous hydrochloric acid solution (40mL), and the organic layer was dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate under reduced pressure gave crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate (9G-2) as a white solid (0.70G, 70% yield).
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (9H-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000902
In a 50mL round bottom flask was added crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate (9G-2) (0.70G, 0.70mmol) and dichloromethane (9mL), trifluoroacetic acid (3.5mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 2 h. The reaction mixture was concentrated to dryness to give a crude product (0.73g, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate (9H-2) as a pale yellow oil, which was then directly charged into the next step.
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 9-2)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4-cyclopropyl-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000911
In a 50mL reaction flask, the crude product (0.73g) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate trifluoro acetate (9H-2) obtained in the above step was added, followed by adjusting the pH to 7 with 1mol/L aqueous sodium hydroxide solution, addition of 1mol/L aqueous sodium hydroxide solution (1.5mL) and then stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4-cyclopropyl-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid; tritrifluoroacetate salt (Compound 9-2), white powder (0.12g, 16.6% yield).
MS m/z＝346.8[M+H]+/2。
1HNMR(400MHz,CD3OD)δ7.44-7.18(m,10H),4.51(dd,1H),4.29-3.95(m,3H),3.90-3.44(m,4H),3.30-3.16(m,1H),3.17-2.81(m,5H),2.37-2.09(m,2H),1.99-1.47(m,8H),1.46-1.30(m,2H),1.00-0.71(m,2H),0.61-0.46(m,1H),0.46-0.30(m,2H),-0.03--0.16(m,2H)。
Example 18:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate salt (Compound 11-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000921
The first step is as follows: 2- (Diphenylmethyleneamino) -5-fluoro-pentanoic acid ethyl ester (11b)
ethyl 2-(benzhydrylideneamino)-5-fluoro-pentanoate
Figure BDA0001735584020000922
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (7.61g, 28.5mmol) and tetrahydrofuran (150mL) were added, the reaction cooled to-78 deg.C, followed by the addition of potassium tert-butoxide (3.36g, 29.9mmol) in portions, which was then raised to 0 deg.C and stirred for 1h, followed by the addition of 1-bromo-3-fluoropropane (11A) (5.21g, 3.6mL, 37mmol) dropwise, which was then raised to room temperature and stirred for 8 h. Water (100mL) was added to the reaction solution, followed by extraction with ethyl acetate (100mL × 2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by separation on silica gel (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give 2- (diphenylmethyleneamino) -5-fluoro-pentanoic acid ethyl ester (11B) as a yellow oil (5.2g, yield 56%).
MS:m/z＝328.1[M+H]+
2- (Diphenylmethyleneamino) -5-fluoro-pentanoic acid ethyl ester (11B) (5.2g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: MG II preparatory SFC (SFC-11); column: whelk O1(S, S),250 × 30mm i.d.,5 μm; mobile phase: a for CO2and B for ethanol; gradient: b35 percent; flow rate: 60 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 4 minutes; sample preparation: 2- (Diphenylmethyleneamino) -5-fluoro-pentanoic acid ethyl ester (11B) (5.2g) dissolved in 100mL of methanol; and (3) injection: 2 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain two optical isomers, compound 11B-1(1.68g, e.e.: 100%), compound 11B-2(2.41g, e.e.: 99.5%).
Compound 11B-1:
chiral HPLC retention time: 9.846min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 11B-2:
chiral HPLC retention time: 12.375min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the second step is that: 2-amino-5-fluoro-pentanoic acid ethyl ester (11C-1)
ethyl 2-amino-5-fluoro-pentanoate
Figure BDA0001735584020000931
In a 50mL round-bottom flask, 2- (diphenylmethyleneamino) -5-fluoro-pentanoic acid ethyl ester (11b-1) (1.4g, 4.3mmol), tert-butyl methyl ether (10mL) and 3mol/L hydrochloric acid aqueous solution (50mL) were added, and after completion of addition, the reaction was stirred at room temperature overnight, followed by separation and removal of the organic phase. The aqueous phase was adjusted to pH about 13 with aqueous ammonia and then extracted with ethyl acetate (100 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give ethyl 2-amino-5-fluoro-valerate (11C-1) as a yellow oil (0.70g, 98% yield).
The third step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butyloxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid ethyl ester (11D-1)
ethyl 2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoate
Figure BDA0001735584020000932
In a 50mL round bottom flask, (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (1.8g, 4.4mmol), ethyl 2-amino-5-fluoro-pentanoate (11C-1) (0.7g, 4.3mmol), 1-hydroxybenzotriazole (0.62g, 4.6mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (1.2g, 6.3mmol) and dichloromethane (50mL) were added in this order, and after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a saturated aqueous sodium hydrogencarbonate solution (40mL) and a 1mol/L aqueous hydrochloric acid solution (40mL), and the organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give a crude product of 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid ethyl ester (11D-1) as a white solid (1.47g, yield 62%).
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid (11E-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoic acid
Figure BDA0001735584020000941
In a 50mL round-bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid ethyl ester (11D-1) (1.47g, 2.6mmol), methanol (15mL) and 1mol/L aqueous sodium hydroxide solution (3mL) were added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (60 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure after filtration to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid (11E-1) as a white solid (0.99g, yield 71%).
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- ((tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (11F-1)
methyl 4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000942
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoic acid (11E-1) (0.99g, 1.88mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.91g, 1.88mmol), 1-hydroxybenzotriazole (0.25g, 1.88mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.54g, 2.8mmol) and dichloromethane (50mL), after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 3mol/L aqueous hydrochloric acid solution (60mL), a saturated aqueous sodium bicarbonate solution (60mL) and a saturated aqueous sodium chloride solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- ((tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (11F-1) as a pale yellow solid (1.3g, yield 69%).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (11G-1)
Methyl 4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000951
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- ((tert-butoxycarbonylamino) -3-phenylpropionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (11F-1) (1.3g, 1.3mmol) and dichloromethane (9mL) were charged, trifluoroacetic acid (3.5mL) was added dropwise at room temperature, and after completion of the addition, the reaction mixture was reacted at room temperature for 2 hours to obtain 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2R ] ((2R) -2R) after direct concentration to dryness ) Methyl (2-amino-3-phenyl-propionyl) amino ] -3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate crude trifluoro acetate (11G-1) (1.35G, 100%) as a pale yellow oil, which was directly subjected to the next reaction.
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate salt (Compound 11-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-fluoro-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000952
To a 50mL round bottom flask was added crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate salt (11G-1) (1.35G), methanol (2mL), and water (10mL), the reaction solution was adjusted to pH 7 with 1mol/L aqueous sodium hydroxide solution, and 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (liquid phase preparation conditions: C18 reverse phase preparative column, mobile phase of deionized water (a) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: a ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-fluoro-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (compound 11-1), white powder (0.63g, yield 58%).
MS:m/z＝342.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.52–7.11(m,10H),4.68–4.55(m,2H),4.50-4.43(m,1H),4.35-4.20(m,2H),4.05–3.65(m,4H),3.60-3.45(m,1H),3.18(d,2H),3.06–2.92(m,4H),2.43–2.18(m,2H),2.07–1.61(m,10H),1.55-1.30(m,2H).
Example 19:
4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (Compound 12-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020000961
The first step is as follows: 2,2, 2-trifluoroethyl trifluoromethanesulfonate (12B)
2,2,2-trifluoroethyl trifluoromethanesulfonate
Figure BDA0001735584020000971
Triethylamine (5.54mL, 39.7mmol) and dichloromethane (40mL) were added to a 100mL reaction flask, the reaction was cooled to-25 deg.C, trifluoroacetic anhydride (12.5g, 44.3mmol) was added dropwise, reaction was continued at-25 deg.C for 2h after addition, trifluoroethanol (2.71g, 27.1mmol) was added dropwise slowly, and the reaction was allowed to warm to room temperature and stirred overnight after addition. The reaction was concentrated under reduced pressure to give crude 3,3, 3-trifluoropropyltrifluoromethanesulfonate (7b) as a yellow oily product (6.7g, yield 100%).
The second step is that: 2- (Diphenylmethyleneamino) -4,4, 4-trifluorobutanoic acid ethyl ester (12C)
ethyl 2-(benzhydrylideneamino)-4,4,4-trifluoro-butanoate
Figure BDA0001735584020000972
In a 250mL round bottom flask, diphenylmethylene glycine ethyl ester (5.47g, 20.85mmol) and tetrahydrofuran (80mL) are added, then the reaction liquid is cooled to-78 ℃, potassium tert-butoxide (2.57g, 22.94mmol) is added in batches, after the addition, the temperature is raised to 0 ℃ for stirring reaction for 1h, then crude 3,3, 3-trifluoropropyl trifluoromethanesulfonate (7B) (6.7g, 27.1mmol) is added dropwise to the reaction liquid, and after the addition, the reaction liquid is raised to room temperature for stirring reaction for 8 h. The reaction solution was added with water (100mL), followed by extraction with ethyl acetate (100mL × 2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 100:1) to give ethyl 2- (diphenylmethyleneamino) -4,4, 4-trifluorobutyrate (12c) as a yellow oily product (2.5g, yield 26%).
MS m/z＝350.1[M+H]+
Ethyl 2- (diphenylmethyleneamino) -4,4, 4-trifluorobutyrate (12C) (2.5g) was taken for chiral resolution.
The preparation conditions are as follows: the instrument comprises the following steps: shimadzu LC-20AP Prep HPLC (PrepL-GA); column: phenomenex Lux Cellulose-2,300 × 50mm i.d.,10 μm; mobile phase: a for CO2and B for iso-propanol; gradient: b5 percent; flow rate: 80 mL/min; back pressure: 100 bar; column temperature: 38 ℃; wavelength: 220 nm; and (3) period: about 5 minutes; sample preparation: ethyl 2- (diphenylmethyleneamino) -4,4, 4-trifluorobutanoate (12C) (2.5g) was dissolved in 40mL of methanol; and (3) injection: 2.0 mL/needle.
After separation, the fractions were concentrated and dried at 40 ℃ by a rotary evaporator to obtain 12C-1(0.93g, e.e.: 99.2%) which is two optical isomers, and 12C-2(1.06g, e.e.: 98.3%).
Compound 12C-1:
chiral HPLC retention time: 5.631min
Analysis conditions were as follows: the instrument comprises the following steps: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
compound 12C-2:
chiral HPLC retention time: 6.658min
Analysis conditions were as follows: the instrument comprises: HPLC agilent 1260; column: xylonite CHIRALPAK, AD-H4.6 x 250mm, 5 μm; mobile phase: n-hexane/isopropanol 99: 1; gradient: isocratic elution for 30 minutes; flow rate: 1.0 mL/min; back pressure: 35.28 bar; column temperature: 35 ℃; wavelength: 210 nm;
the third step: 2-amino-4, 4, 4-trifluorobutanoic acid ethyl ester (12D-1)
ethyl 2-amino-4,4,4-trifluoro-butanoate
Figure BDA0001735584020000981
In a 50mL round-bottom flask, ethyl 2- (diphenylmethyleneamino) -4,4, 4-trifluorobutanoate (12C-1) (0.73g, 2.1mmol), methyl tert-butyl ether (10mL), and 1mol/L aqueous hydrochloric acid (10mL) were added, and the reaction was stirred at room temperature for 3 h. The organic layer was separated and discarded, and the aqueous phase was adjusted to pH 13 with aqueous ammonia, extracted with dichloromethane (25 mL. times.2), and the organic phases were combined and dried over anhydrous sodium sulfate to give a solution (50mL) of ethyl 2-amino-4, 4, 4-trifluorobutyrate (12D-1) which was directly subjected to the next reaction.
The fourth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoic acid ethyl ester (12E-1)
ethyl2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoate
Figure BDA0001735584020000982
To a 100mL round bottom flask were added (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1) (0.83g, 2.0mmol), 1-hydroxybenzotriazole (0.3g, 2.2mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.58g, 3.0mmol) and the ethyl 2-amino-4, 4, 4-trifluorobutyrate (12D-1) solution (50mL) in that order, and after the addition was complete, the reaction was stirred at room temperature for 3 h. After completion of the reaction, the reaction mixture was washed once with a 1mol/L aqueous hydrochloric acid solution (20mL), a saturated aqueous sodium bicarbonate solution (20mL) and a saturated aqueous sodium chloride solution (20mL), respectively, and the organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel separation (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give ethyl 2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutyrate (12E-1) as a white solid (0.81g, 70% yield in two steps).
MS m/z＝602.3[M+Na]+
The fifth step: 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -4,4, 4-trifluorobutanoic acid (12F-1)
2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoic acid
Figure BDA0001735584020000991
In a 100mL round-bottomed flask, ethyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoate (12E-1) (0.81g, 1.4mmol) and methanol (10mL) were added, followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (2mL) and reaction with stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified (30mL) with 3mol/L aqueous hydrochloric acid solution, followed by extraction with ethyl acetate (30 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give a crude product of 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -4,4, 4-trifluorobutanoic acid (12F-1) as a white solid (0.77g, yield 100%).
MS m/z＝574.3[M+Na]+
And a sixth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (12G-1)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020000992
In a 50mL round bottom flask, crude 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoic acid (12F-1) (0.39g, 0.7mmol), 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2) (0.34g, 0.7mmol), 1-hydroxybenzotriazole (0.14g, 1.05mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.21 g), 1.05mmol) and dichloromethane (30mL), after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (10mL), a saturated aqueous sodium bicarbonate solution (10mL) and a saturated aqueous sodium chloride solution (10mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate (12G-1) as a white solid (0.38G, 52% yield in two steps).
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (12H-1)
methyl4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoyl]amino]hexanoyl]piperidine-4-carboxylate；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001001
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (12G-1) (0.38G, 0.38mmol) and dichloromethane (9mL) were added, trifluoroacetic acid (3mL) was added dropwise at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was directly concentrated to give a crude product (403mg, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (12H-1) as a pale yellow oil, which was then directly subjected to the next reaction.
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid; tritrifluoroacetate salt (Compound 12-1)
4-amino-1-[(2R)-6-amino-2-[[2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-4,4,4-trifluoro-butanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001002
To a 50mL round-bottomed flask, the crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluoro-butyryl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (12H-1) product (0.4g), methanol (2mL) and water (10mL) obtained in the above step were added, and the reaction solution was adjusted to pH about 7 with 1mol/L aqueous sodium hydroxide solution, then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added, and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (conditions for preparation of liquid phase: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -4,4, 4-trifluorobutanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate salt, white powder (compound 12-1) (0.12g, yield 30.7%).
MS m/z＝353.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.42-7.02(m,10H),4.89-4.81(m,1H),4.66-4.64(m,2H),4.19(t,1H),3.86-3.31(m,4H),3.19-2.85(m,6H),2.68-1.54(m,10H),1.46-1.24(m,2H).
Example 20:
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 13)
4-amino-1-[(2R)-6-amino-2-[[(2R)-5-amino-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020001021
The first step is as follows: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- (9H-fluoren-9-ylmethoxy) -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoic acid methyl ester (13B)
methyl(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-5-(tritylamino)pentanoyl]amino]hexanoate
Figure BDA0001735584020001022
To a 50mL round-bottom flask were added methyl (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoate (13A) (11.73g, 19.2mmol), (2R) -2- (9H-fluoren-9-ylmethoxycarbonylamino) -5-oxo-5- (tritylamino) pentanoic acid (5g, 19.2mmol), 1-hydroxybenzotriazole (3.1g, 23mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.4g, 23mmol) and dichloromethane (200mL) in this order, and after completion of addition, the reaction was stirred at room temperature for 3H. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (50mL), a saturated aqueous sodium bicarbonate solution (50mL) and a saturated aqueous sodium chloride solution (50mL), and the organic layer was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude product of methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- (9H-fluoren-9-ylmethoxy) -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoate (13B) as a white solid (16.53g, yield 100%).
The second step is that: (2R) -methyl 2- [ [ (2R) -2-amino-5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) hexanoate (13c)
methyl(2R)-2-[[(2R)-2-amino-5-oxo-5-(tritylamino)pentanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate
Figure BDA0001735584020001031
In a 50mL round bottom flask was added crude methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- (9H-fluoren-9-ylmethoxy) -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoate (13B) (10g, 11.7mmol), piperidine (4mL) and N, N-dimethylformamide (100mL), and after the addition was complete, it was stirred at room temperature for 6 minutes. Ethyl acetate (500mL) was added to the reaction mixture, which was then washed with saturated brine (100 mL. times.2), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude product of methyl (2R) -2- [ [ (2R) -2-amino-5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) hexanoate (13C) as a white solid (7.3g, yield 100%) and charged directly to the next reaction.
MS m/z＝631.4[M+H]+
The third step: (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (benzyloxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ]6- (tert-butoxycarbonylamino) hexanoic acid methyl ester (13D)
methyl(2R)-2-[[(2R)-2-[[(2R)-2-(benzyloxycarbonylamino)-3-phenyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate
Figure BDA0001735584020001041
In a 100mL round bottom flask were added (2R) -2- (benzyloxycarbonylamino) -3-phenyl-propionic acid (0.66g, 2.2mmol), (2R) -2- [ [ (2R) -2-amino-5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) methyl hexanoate (13C) crude (1.4g, 2.2mmol), 1-hydroxybenzotriazole (0.36g, 2.7mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.51g, 2.7mmol) and dichloromethane (20mL) and after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed with a 1mol/L aqueous hydrochloric acid solution (5mL), a saturated aqueous sodium bicarbonate solution (5mL) and a saturated aqueous sodium chloride solution (5mL) in this order, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 3:1) to give methyl (2R) -2- [ [ (2R) -2- (benzyloxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ]6- (tert-butoxycarbonylamino) hexanoate (13D) as a white solid (0.98g, 48% yield in three steps).
The fourth step: (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) methyl hexanoate (13E)
methyl(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]-6-(tert-butoxycarbonylamino)hexanoate
Figure BDA0001735584020001042
In a 100mL round-bottom flask, (2R) -methyl 2- [ [ (2R) -2- [ [ (2R) -2- (benzyloxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ]6- (tert-butoxycarbonylamino) hexanoate (13D) (0.8g,7mmol), palladium on carbon (0.16g, 20 wt%) and methanol (6mL) were added, and the reaction was stirred at room temperature for 4h under a hydrogen (balloon) atmosphere. The reaction mixture was filtered through celite, and the filtrate was concentrated under reduced pressure to give crude methyl (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) hexanoate (13E) as a white solid (0.7g, yield 100%), which was directly subjected to the next reaction.
The fifth step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionamido ] amino ] -3-phenylpropionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoic acid methyl ester (13F)
methyl(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]hexanoate
Figure BDA0001735584020001051
In a 50mL round bottom flask, crude (0.35g, 0.45mmol) of (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionic acid (1a) (0.11g, 0.42mmol), (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] -6- (tert-butoxycarbonylamino) hexanoic acid methyl ester (13E) was added in this order, 1-hydroxybenzotriazole (0.09g, 0.67mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.1g, 0.54mmol) and dichloromethane (10mL) were added and stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (3mL), a saturated aqueous sodium bicarbonate solution (3mL) and a saturated aqueous sodium chloride solution (3mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure after filtration to give a crude product of methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionamido ] amino ] -3-phenylpropionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoate (13F) as a white solid (0.3g, 65% yield in two steps).
And a sixth step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoic acid (13G)
(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]hexanoic acid
Figure BDA0001735584020001052
In a 50mL round-bottom flask, crude methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionamido ] amino ] -3-phenylpropionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoate (13F) (3.4g, 3.3mmol) was dissolved in methanol (30mL), followed by dropwise addition of 1mol/L aqueous sodium hydroxide solution (3.5mL), and after completion of addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and then extracted with ethyl acetate (50 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure after filtration to give a crude product of (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoic acid (13G) as a white solid (2.5G, yield 75%)
The seventh step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (13H)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-5-(tritylamino)pentanoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020001061
In a 50mL round bottom flask, 4- ((tert-butoxycarbonylamino) amino) piperidine-4-carboxylic acid methyl ester (0.21g, 0.82mmol), (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl group) was added in this order]Amino group]-3-phenyl-propanoyl group]Amino group]-5-oxo-5- (tritylamino) pentanoyl]Amino group]Crude hexanoic acid (13G) (0.83G, 0.82mmol), 1-hydroxybenzotriazole (0.13G, 0.98mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.15G, 0.98mmol) and dichloromethane (10mL) were stirred at room temperature after addition was complete overnight. The reaction solution was sequentially treated with 1mol/L HCl solution (3mL) and saturated NaHCO3Washed (3mL) with saturated NaCl (3mL), and the organic layer was dried over anhydrous sodium sulfate. Filtering, concentrating the filtrate under reduced pressure to obtain white solid 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl]Amino group]-3-phenyl-propionyl group]Amino group]-5-oxo-5- (tritylamino) pentanoyl]Amino group]Hexanoyl radical]Crude piperidine-4-carboxylic acid methyl ester (13H) (0.8g, 81% yield).
Eighth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (13I)
Methyl 4-amino-1-[(2R)-6-amino-2-[[(2R)-5-amino-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001071
In a 50mL round bottom flask, crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-oxo-5- (tritylamino) pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (13H) (0.80g, 0.60mmol) and dichloromethane (9mL) were added, followed by dropwise addition of trifluoroacetic acid (3mL) at room temperature, and after completion of addition, the reaction was stirred at room temperature for 3H. The reaction mixture was directly concentrated to dryness to give a crude product (630mg, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tri-trifluoroacetate (13I) as a pale yellow oil, which was then directly subjected to the next reaction.
The ninth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 13)
4-amino-1-[(2R)-6-amino-2-[[(2R)-5-amino-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-oxo-pentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020001072
To a 50mL round bottom flask was added crude methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (13I) trifluoro acetate obtained in the above step (0.63g), methanol (2mL) and water (10mL) were added to the reaction mixture, the pH of the reaction mixture was adjusted to about 7 with 1mol/L aqueous sodium hydroxide solution, and then 1mol/L aqueous sodium hydroxide solution (1.5mL) was added thereto, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (liquid phase preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution B: A25%, elution time 20 minutes), this gave 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tri-trifluoroacetate as a white powder.
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid obtained in the above step; the tris-trifluoroacetate salt was ion-exchanged with an ion exchange resin (ion exchange resin 35mL, aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL) elution), and the received eluted solution was concentrated (water temperature 60 ℃ c. and reduced pressure to 25mL), and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -5-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-oxo-pentanoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 13), a white solid (0.12g, yield 36%)
MS m/z＝695.3[M+H]+
1HNMR(400MHz,D2O)δ7.32-7.26(m,6H),7.18-7.12(m,4H),4.7(m,1H),4.56-4.52(m,1H),4.22-4.15(m,1H),3.70-3.63(m,5H),2.97-2.93(m,4H),2.83-2.81(m,2H),2.23-1.96(m,6H),1.72-1.64(m,6H),1.40-1.38(m,2H).
Example 21:
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-alkynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 14)4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propanoyl ] amino ] -3-phenyl-propanoyl ] amino ] penta ino ] -4-carboxylic acid.
Figure BDA0001735584020001091
The first step is as follows: (2R) -2-Aminopent-4-enoic acid methyl ester (14B)
methyl(2R)-2-aminopent-4-ynoate
Figure BDA0001735584020001092
In a 50mL round bottom flask, (2R) -2-aminopentane-4-ynoic acid (14A) (3g, 26mmol) and methanol (54mL) were added, the reaction was cooled to 0 deg.C, then thionyl chloride (3mL) was slowly added dropwise and stirred overnight at room temperature after dropping. The reaction mixture was concentrated under reduced pressure to give crude (2R) -methyl 2-aminopent-4-enoate (14B) as a pale yellow oil (3.2g, 25mmol, 95%).
Second step methyl (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoate (14C)
methyl(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-ynoate YH2Z00000763
Figure BDA0001735584020001101
In a 50mL round bottom flask, crude (7.26g, 17.6mmol) of (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenyl-propionic acid (intermediate 1), crude (2.88g, 17.6mmol) of methyl 2(R) -2-aminopentan-4-ynoate (14B), crude (2.85g, 17.6mmol), 1-hydroxybenzotriazole (2.85g, 21.1mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (3.28g, 21.1mmol) and dichloromethane (180mL) were added in this order, and the reaction was stirred at room temperature overnight after the addition. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (40mL), a saturated aqueous sodium bicarbonate solution (40mL) and a saturated aqueous sodium chloride solution (40mL), and the organic phase was dried over anhydrous sodium sulfate. The filtrate was concentrated under reduced pressure after filtration to give crude methyl (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoate (14C) as a white solid (8.1g, 88% yield).
MS m/z＝544.2[M+Na]+
The third step: (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoic acid (14D)
(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-ynoic acid
Figure BDA0001735584020001102
In a 50mL round bottom flask was added crude methyl (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoate (14C) (4g, 7.7mmol) and methanol (77mL), followed by dropwise addition of NaOH solution (1mol/L, 8mL) and stirring at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL) and then extracted with ethyl acetate (50 mL. times.2), the organic layers were combined and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product of (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoic acid (14D) as a white solid (3.3g, yield 85%).
The fourth step methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (14E)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020001111
In a 50mL round bottom flask was added crude (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoic acid (14D) (0.51g, 1.0mmol), methyl 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylate (intermediate 2) (0.49g, 1.0mmol), 1-hydroxybenzotriazole (0.14g, 1.03mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.17 g), 1.1mmol) and dichloromethane (20mL), after addition was complete the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 3mol/L aqueous hydrochloric acid solution (5mL), a saturated aqueous sodium bicarbonate solution (5mL) and a saturated aqueous sodium chloride solution (5mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol (v/v) ═ 50:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (14E) (0.75g, yield 77%) as a white solid.
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (14F)
4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001121
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (14E) (0.70g, 0.70mmol), dichloromethane (9mL) and trifluoroacetic acid (3mL) were added and stirred at room temperature for 3h after the addition was complete. The reaction mixture was directly concentrated to dryness to give a crude product (712mg, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate (14F) as a pale yellow oil, which was then directly subjected to the next reaction.
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 14)
4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020001122
In a 50mL round bottom flask, 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl group obtained in the above reaction was added]Amino group]-3-phenyl-propanoyl group]Amino group]Pent-4-ynoyl]Amino group]Hexanoyl radical]Piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (14F) crude (0.71g), methanol (2mL) and H2O (10mL), which was dissolved by stirring at room temperature, was adjusted to pH 7 with 1mol/L NaOH solution, and 1mol/L NaOH solution (1.5mL) was added thereto, followed by stirring at room temperature overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase (conditions for preparation of liquid phase: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl ] as a white powder-propionyl group]Amino group]-3-phenyl-propionyl group]Amino group]Pent-4-ynoyl]Amino group]Hexanoyl radical]Piperidine-4-carboxylic acid; tritrifluoroacetate salt.
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid obtained in the above step; the tris-trifluoroacetate salt was ion-exchanged with an ion exchange resin (ion exchange resin 35mL, aqueous ammonia/distilled water (v/v) ═ 1:8(1000mL) and the resulting eluate was concentrated (water temperature 60 ℃ C., reduced pressure concentration to 25mL), and further lyophilized to give 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (compound 14) (0.12g, 26% yield) as a white solid
MS m/z＝331.7[M+2H]+/2
1HNMR(400MHz,D2O)δ7.33-7.26(m,6H),7.20-7.18(m,2H),7.13-7.11(m,2H),4.59(t,1H),4.59(t,1H),4.38(t,1H),3.7-3.6(m,5H),3.05(dd,1H),2.95-2.89(m,3H),2.81(d,2H),2.63-2.62(m,2H),2.44-2.43(m,1H),2.18-2.03(m,2H),1.76-1.61(m,6H),1.41-1.34(m,2H)
Example 22:
4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-alkynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 15)4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propanoyl ] amino ] -3-phenyl-propanoyl ] amino ] -5-phenyl-pentanyl-pent-4-ynoyl ] amino ] hexano ] piperidine -4-carboxylic acid
Figure BDA0001735584020001141
The first step is as follows: (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoic acid methyl ester (15A)
Methyl(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoate
Figure BDA0001735584020001142
In a 50mL round bottom flask were added crude (2R) -methyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoate (14C) (0.52g, 1.0mmol), acetonitrile (3mL) and iodobenzene (0.21g, 1.0mmol) in that order, the mixture was stirred at room temperature, and then palladium dichloride (0.015g, 0.02mmol), cuprous iodide (0.0026g, 0.014mmol) and triethylamine (0.45mL) were added in that order, and after completion of the addition, the reaction was stirred at room temperature for 1.5 h. The reaction solution was quenched by adding saturated aqueous ammonium chloride (10mL), followed by extraction with ethyl acetate (6mL × 3), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 2:1) to give (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoic acid methyl ester (15A) as a white solid (0.5g, yield 90%).
MS m/z＝620.3[M+Na]+
The second step: (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenylpentan-4-ynoic acid (15B)
(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoic acid
Figure BDA0001735584020001151
In a 50mL round bottom flask, (2R) -methyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoate (15A) (0.5g, 0.8mmol) and methanol (8mL) were added, followed by dropwise addition of a 1mol/L aqueous solution of sodium hydroxide (1.1mL) and stirring at room temperature overnight after completion of the addition. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (20 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure after filtration to give a crude product of (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenylpentan-4-ynoic acid (15B) as a white solid (0.42g, yield 86%).
The third step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-Butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoic acid methyl ester (15C)
methyl(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoyl]amino]hexanoate
Figure BDA0001735584020001161
In a 50mL round bottom flask were added crude (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenylpentan-4-ynoic acid (15B) (0.2g, 0.7mmol), 1-hydroxybenzotriazole (0.11g, 0.8mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.15g, 0.8mmol) and dichloromethane (20mL) in that order, and after addition the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (5mL), a saturated aqueous sodium bicarbonate solution (5mL) and a saturated aqueous sodium chloride solution (5mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 1:1) to give methyl (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoate (15C) as a white solid (0.5g, yield 90%).
The fourth step: (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoic acid (15D)
(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoyl]amino]hexanoic acid
Figure BDA0001735584020001162
In a 50mL round bottom flask were added (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoic acid methyl ester (15C) (0.5g, 0.6mmol), methanol (6mL) and 1mol/L aqueous sodium hydroxide solution (0.8mL), and the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (20 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure after filtration to give a crude product of (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoic acid (15D) as a white solid (0.45g, 92% yield).
The fifth step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (15E)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020001171
In a 50mL round bottom flask, 4- ((tert-butoxycarbonylamino) amino) piperidine-4-carboxylic acid methyl ester (0.2g, 0.7mmol), (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoic acid (15D) crude product (0.4g, 0.7mmol), 1-hydroxybenzotriazole (0.11g, 0.8mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.15 g), 0.8mmol) and dichloromethane (15mL) were added and the reaction stirred at room temperature overnight. The reaction mixture was washed successively with a 1mol/L aqueous hydrochloric acid solution (3mL), a saturated aqueous sodium bicarbonate solution (3mL) and a saturated aqueous sodium chloride solution (3mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give a crude product of methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (15E) as a white solid (0.68g, 90% yield).
And a sixth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid Tritrifluoroacetate (15F)
methyl4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001181
In a 50mL round bottom flask was added crude methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (15E) (0.48g, 0.46mmol) and dichloromethane (5mL), followed by dropwise addition of trifluoroacetic acid (1.5mL) at room temperature and stirring at room temperature for 3h after completion of addition. The reaction mixture was directly concentrated to dryness to give a crude product (503mg, 100%) of 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate (15F) as a pale yellow oil, which was then directly subjected to the next reaction.
The seventh step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 15)
4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]-5-phenyl-pent-4-ynoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020001182
In a 50mL round-bottomed flask, the crude Tritrifluoroacetate salt (15F) of 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid obtained in the above step was added (0.5g), methanol (2mL) and water (5mL) were added to the reaction mixture, the pH of the reaction mixture was adjusted to about 7 with 1mol/L aqueous sodium hydroxide solution, and 1mol/L aqueous sodium hydroxide solution (0.8mL) was added thereto, followed by stirring at room temperature for overnight reaction. The reaction mixture was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (conditions for preparation of liquid phase: C18 reverse phase preparative column, mobile phase: deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution B: A ═ 25%, elution time 20 minutes), to give 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate as a white powder.
The 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate obtained in the above step was ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, and eluted with aqueous ammonia/distilled water (v/v) ═ 1:8(1000 mL)), and the thus-received eluted solution was concentrated (at a water temperature of 60 ℃ C. under reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] -5-phenyl-pent-4-ynoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid as a white solid (0.09g, 26% yield)
MS m/z＝369.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.43-7.26(m,11H),7.21-7.20(m,2H),7.09-7.07(m,2H),4.81-4.78(m,1H),4.64-4.60(m,1H),4.49-4.46(m,1H),3.62-3.46(m,5H),3.08-2.72(m,8H),2.09-1.97(m,2H),1.72-1.34(m,8H).
Example 23:
4-amino-1- (R) -6-amino-2- ((R) -2- ((R) -2- ((R) -2-amino-3-phenylpropionylamino) pentanoic acid-4-amido) hexanoyl) piperidine-4-carboxylic acid (Compound 16)
4-amino-1-((R)-6-amino-2-((R)-2-((R)-2-((R)-2-amino-3-phenylpropanamido)-3-phenylpropanamido)pent-4-enamido)hexanoyl)piperidine-4-carboxylic acid
Figure BDA0001735584020001191
The first step is as follows: (2R) -methyl 2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] enoate (16A)
methyl(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-enoate
Figure BDA0001735584020001201
In a 50mL round bottom flask, palladium/calcium carbonate (34mg), ethyl acetate (10mL) and quinoline (58. mu.L) were added, followed by dropwise addition of a solution of crude (0.65g,1.2mmol) of methyl (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-ynoate (14C) in ethyl acetate (5mL), and after completion of the addition, the reaction was stirred at room temperature for 3 h. The reaction mixture was filtered through Celite, the filtrate was washed with a 1mol/L aqueous hydrochloric acid solution (10mL), a saturated aqueous sodium bicarbonate solution (10mL) and a saturated aqueous sodium chloride solution (10mL), respectively, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 2:1) to give methyl (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] enoate (16A) as a white solid (0.58g, yield 90%).
MS m/z＝546.3[M+Na]+
The second step is that: (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] pent-4-enoic acid (16B)
(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-enoic acid
Figure BDA0001735584020001202
To a 50mL round bottom flask were added methyl (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] enoate (16A) (0.6g, 1.0mmol), methanol (10mL) and 1mol/L aqueous sodium hydroxide solution (1.1mL), and after completion of the addition, the reaction was stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to remove methanol, the aqueous phase was acidified with 3mol/L aqueous hydrochloric acid (30mL), followed by extraction with ethyl acetate (30 mL. times.2), the organic phases were combined and dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure after filtration to give a crude product of (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] pent-4-enoic acid (16B) as a white solid (0.54g, 93% yield).
The third step: 4- (tert-Butoxycarbonylamino) -1- [ (2R) -6- (tert-Butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (16C)
methyl4-(tert-butoxycarbonylamino)-1-[(2R)-6-(tert-butoxycarbonylamino)-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-(tert-butoxycarbonylamino)-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-enoyl]amino]hexanoyl]piperidine-4-carboxylate
Figure BDA0001735584020001211
In a 50mL round bottom flask, crude (2R) -2- [ [ (2R) -2- [ [ (2R) -2- (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] -3-phenylpropionyl ] amino ] pent-4-enoic acid (16B) (0.51g, 1mmol), 1- [ (2R) -2-amino-6- (tert-butoxycarbonylamino) hexanoyl ] -4- (tert-butoxycarbonylamino) piperidine-4-carboxylic acid methyl ester (intermediate 2) (0.49g, 1mmol), 1-hydroxybenzotriazole (0.14g, 1.03mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.17g, 1.1mmol), and dichloromethane (20mL) were added in this order, after the addition was complete, the reaction was stirred at room temperature overnight. The reaction mixture was washed successively with a 3mol/L aqueous hydrochloric acid solution (5mL), a saturated aqueous sodium bicarbonate solution (5mL) and a saturated aqueous sodium chloride solution (5mL), and the organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate (v/v) ═ 1:1) to give methyl 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylate (16C) as a white solid (0.75g, 77% yield).
The fourth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester Tritrifluoroacetate (16D)
methyl4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-enoyl]amino]hexanoyl]piperidine-4-carboxylic acid；2,2,2-trifluoroacetic acid
Figure BDA0001735584020001221
In a 50mL round bottom flask, 4- (tert-butoxycarbonylamino) -1- [ (2R) -6- (tert-butoxycarbonylamino) -2- [ [ (2R) -2- [ [ (2R) -2- [ [ (tert-butoxycarbonylamino) -3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid methyl ester (16C) (0.75g, 0.77mmol) and dichloromethane (9mL) were added, followed by dropwise addition of trifluoroacetic acid (3.0mL) at room temperature, followed by stirring at room temperature for 3 h. The reaction mixture was directly concentrated to dryness to give a crude product (0.785g, 100%) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate (16D) as a pale yellow oil, which was then directly subjected to the next reaction.
The fifth step: 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid (Compound 16)
4-amino-1-[(2R)-6-amino-2-[[(2R)-2-[[(2R)-2-[[(2R)-2-amino-3-phenyl-propanoyl]amino]-3-phenyl-propanoyl]amino]pent-4-enoyl]amino]hexanoyl]piperidine-4-carboxylic acid
Figure BDA0001735584020001222
To a 50mL round-bottomed flask, the crude product (0.785g) of methyl 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylate tritrifluoroacetate (16D) obtained in the above reaction was charged, followed by adjusting the pH to about 7 with 1mol/L aqueous sodium hydroxide solution, adding 1mol/L aqueous sodium hydroxide solution (1.5mL) and stirring at room temperature for reaction overnight. The reaction solution was adjusted to pH 5 with trifluoroacetic acid, and then concentrated under reduced pressure, and the residue was separated and purified by preparative liquid phase separation (preparative conditions: C18 reverse phase preparative column, mobile phase of deionized water (A) containing 0.05% TFA, acetonitrile (B), isocratic elution of B: A ═ 25%, elution time 20 minutes) to give 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid trifluoroacetate salt as a white powder.
The 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid tritrifluoroacetate obtained in the above step was ion-exchanged with an ion-exchange resin (35mL of ion-exchange resin, ammonia/distilled water (v/v) ═ 1:8(1000mL) and the resulting eluate was concentrated (water temperature 60 ℃ C.) under reduced pressure to 25mL) and lyophilized to obtain 4-amino-1- [ (2R) -6-amino-2- [ [ (2R) -2- [ [ (2R) -2- [ [ (2R) -2-amino-3-phenyl-propionyl ] amino ] pent-4-enoyl ] amino ] hexanoyl ] piperidine-4-carboxylic acid as a white solid (0.16g, 31% yield).
MS m/z＝332.8[M+2H]+/2
1HNMR(400MHz,D2O)δ7.35-7.26(m,6H),7.20-7.18(m,2H),7.14-7.12(m,2H),5.74-5.60(m,1H),5.15-5.1(m,2H),4.79-4.74(m,1H),4.57(t,1H),4.27(t,1H),3.74-3.49(m,5H),3.06-2.81(m,6H),2.50-2.36(m,2H),2.18–1.98(m,2H),1.75-1.59(m,6H),1.41-1.38(m,2H)
Biological test example
Test 1: agonistic activity at human kappa-opioid receptors
Forskolin stimulates the release of cAMP from OPRK1 cell (DiscoveRx) which is a human kappa-opioid receptor high-expression cell line, and kappa-opioid receptor agonists inhibits Forskolin-stimulated cAMP release. Inhibition of forskolin stimulated cAMP release by test compoundsIn use, the agonistic activity of a compound at the human kappa opioid receptor can be determined. First, the forskolin with a certain concentration and the test compound with different concentrations are incubated with the human kappa-opioid receptor high-expression cell strain. cAMP immunoassay using time-resolved fluorescence resonance energy transfer (TR-FRET) ((
Figure BDA0001735584020001231
PerkinElmer) to determine cAMP levels in stimulated OPRK1 cells. The specific method comprises the following steps:
OPRK1 cells (DiscoverX) highly expressing human kappa-opioid receptors were cultured in McCoy's 5A (Gibco 16600-082) medium containing 10% FBS (Gibco 10099-141). On the day of the experiment, cells in exponential growth phase were washed with PBS/5mM EDTA for isolation, harvested by centrifugation, and resuspended and counted in a Stimulation Buffer, adjusting the cell concentration to 3 x 105cells/ml. Forskolin and test compounds were dissolved in DMSO respectively to give a stock solution of 10mM, then the stabilization Buffer was used to dilute Forskolin to 4. mu.M, test compounds of different concentrations (concentrations of 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, 0.001024, 0. mu.M in this order) were added to the solution, and 5. mu.l/well of the test compounds were added to a 384-well plate. Mu.l of cell suspension was added to each well and incubated at room temperature for 30 min. Subsequently, 5. mu.l of 4 XTUR-cAMP tracer working solution (Eu-cAMP stock solution diluted 50 times with cAMP Detection Buffer) and 5. mu.l of 4 XTUR-anti-cAMP working solution (ULight-anti-cAMP stock solution diluted 150 times with cAMP Detection Buffer) were added to each well, respectively, and incubated at room temperature for 1 hour. The 384 well plates were assayed for cAMP levels using a microplate reader (Perkin Elmer, Envision) TR-FRET method. The resulting data were processed and fitted with origin7.5 software EC 50. The agonistic activity of the human kappa-opioid receptors of the compounds of the present invention was determined by the above experiments and the EC50 values were determined as shown in table 1.
The simulation Buffer preparation method comprises the following steps: 14mL of 1 × HBSS (Invitrogen, cat. # 14025-.
TABLE 1 agonistic activity of test compounds at human kappa opioid receptors
Compound numbering EC50(nM)
Compound 2-1 0.53
Compound 4-1 0.91
Compound 5-1 0.90
Compound 6-1 0.44
Compound 7-1 0.33
Compound 9-1 0.31
Compound 11-1 1.21
Compound 16 5.30
And (4) conclusion: the compound has obvious agonism on human kappa-opioid receptors.
And (3) testing 2: mouse writhing experiment
The mice can be caused to wriggle after being injected with acetic acid in the abdominal cavity. Writhing response refers to a behavioral response in which the mouse exhibits the characteristic abdominal muscle contraction or extension. The analgesic activity of the compound can be reflected by detecting the inhibition effect of the compound on the writhing behavior of mice caused by acetic acid. The specific method comprises the following steps:
ICR mice (purchased from Soken-Doucho Biotech, Inc., license number: SCXK (Chuan) 2008-24 (NO: 51203500002150)) at 8 weeks of age. Randomly grouping, wherein each group comprises 10 animals, and each animal is male and female; no water is forbidden 12h before experiment. On the day of the experiment, 1.0mg/kg of test compound was administered intravenously and the control group was administered with the blank reagent. 0.6% (v/v) acetic acid solution was injected intraperitoneally at a dose of 0.4 mL/body for 15min after administration. The number of writhing of mice within 15min after acetic acid injection was recorded, and the percentage of inhibition of the tested compound on writhing behavior of mice caused by acetic acid was calculated, respectively, and the analysis results are shown in table 2.
Percent inhibition is ═ number of twists in control group-number of twists in administration group)/number of twists in control group.
TABLE 2 percent inhibition of acetic acid induced writhing behavior in mice by test compounds
Figure BDA0001735584020001251
And (4) conclusion: the compound of the invention has obvious analgesic effect.

Claims (8)

1. A compound of the formula or a pharmaceutically acceptable salt thereof,
Figure FDA0003537630770000011
wherein,
R1independently selected from OH or methoxy;
R2、R3each independently selected from H, -C (═ O) O-tert-butyl or-C (═ O) O-benzyl;
R7is independently selected from C1-6Alkyl, said alkyl being further substituted by 1-3R7aSubstituted;
R7aindependently selected from F, OH, CF3Cyclopropyl, said cyclopropyl being further substituted with 0-3 of F, OH, CF3Methyl, substituted by a substituent;
R5、R6、R11、R12each independently selected from H, -C (═ O) O-tert-butyl or-C (═ O) O-benzyl.
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof,
wherein,
R1independently selected from OH or methoxy;
R2、R3each independently selected from H or-C (═ O) O-tert-butyl;
R7independently selected from methyl, ethyl, propyl, butyl, pentyl, isopropyl or
Figure FDA0003537630770000012
Said methyl, ethyl, propyl, butyl, pentyl, isopropyl or
Figure FDA0003537630770000013
Further substituted by 1-3R7aSubstituted;
R7aindependently selected from F, OH, CF3A cyclopropyl group;
R5、R6、R11、R12each independently selected from H or-C (═ O) O-tert-butyl.
3. A compound according to any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, selected from one of the following structures:
Figure FDA0003537630770000021
Figure FDA0003537630770000031
。
4. a pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers therefor.
5. Use of a compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 4, for the manufacture of a medicament for treating or preventing a kappa opioid receptor-associated disease or condition in a mammal.
6. The use of claim 5, wherein the kappa opioid receptor associated condition is selected from the group consisting of: pain, inflammation, itching, edema, hyponatremia, hypokalemia, ileus, cough and glaucoma.
7. The use of claim 6, wherein the pain is selected from the group consisting of: neuropathic pain, somatic pain, visceral pain, and skin pain.
8. The use of claim 6, wherein the pain is selected from the group consisting of: arthritic pain, kidney stone pain, uterine cramps, dysmenorrhea, endometriosis, dyspepsia, post-surgical pain, post-medical treatment pain, ocular pain, otitis pain, breakthrough cancer pain and pain associated with GI disorders.
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