CN114349816B - Aminopeptidase N/CD 13-based small molecule coupling molecule and preparation method and application thereof - Google Patents

Abstract

The present invention discloses a novel small molecule coupling compound based on aminopeptidase N/CD13 and a preparation method and application thereof. The compound has a structure shown in Formula I: The present invention uses the CD13 inhibitor Ubenimex as the targeting ligand of the small molecule conjugated drug, which can not only mediate the tumor targeting of the molecule, but also the cytotoxic substances and Ubenimex produced by degradation after entering the cell may produce a synergistic effect, providing a new strategy for the treatment of tumors.

Description

A small molecule coupling molecule based on aminopeptidase N/CD13 and its preparation method and application

Technical Field

The present invention relates to the technical field of CD13 inhibitors, and in particular to a novel small molecule coupling molecule based on aminopeptidase N/CD13, and a preparation method and application thereof.

Background Art

Since the 21st century, drug treatment for malignant tumors has entered an era of precision and personalized treatment (Nat Rev Genet. 2016, 17, 507-522). Traditional cytotoxic drugs such as 5-fluorouracil and gemcitabine have always been the first-line drugs for tumor treatment. However, due to the lack of selectivity, these drugs show strong toxic side effects in clinical use. How to accurately deliver these anti-tumor drugs to tumor tissues without affecting normal tissues and organs is a hot topic in the current development of precision tumor treatment drugs. Targeted drug conjugate technology is an emerging technology that chemically couples antibodies, peptides, and small molecule non-peptide ligands that target tumor tissues on the basis of the structure of cytotoxic drugs with high anti-tumor effects, so as to precisely deliver cytotoxic drugs to tumor tissues, thereby exerting a toxicity-reducing and synergistic effect (Lancet. 2019, 394, 793-804; Chem Soc Rev. 2021, 50, 1480-1494; Chem Rev. 2017, 117, 12133-12164).

Targeted drug conjugates mainly include antibody drug conjugates (ADC), peptide drug conjugates (PDC) and small-molecule drug conjugates (SMDC). Their general structural formulas are similar, including three parts: specific targeting ligands, connectors and cytotoxic substances. The difference between the three drug conjugates lies in the difference in specific targeting ligands: macromolecular antibodies, peptides and small molecules. Among them, antibody drug conjugates are the most mature type of research at present, with a total of 11 drugs on the market. They are mainly used in the treatment of hematological tumors in clinical practice, and solid tumors are less (Molecules.2020,25,4764-4797). Compared with ADCs with antibodies as ligands, SMDC has the advantages of no immune response, easier synthesis control, easy structure modification, small molecular weight, easier entry into solid tumor tissues, low preparation cost, and good stability in vivo and in vitro (Nat Rev Drug Discov.2015,14,203-219). SMDC is considered to have more advantages in the treatment of solid tumors and can complement ADC drugs in terms of indications. Therefore, small molecule conjugate drugs have received extensive attention and are in a rapid development stage. Currently, many candidate drugs have entered clinical research (Nat Rev Drug Discov. 2015, 14, 203-219).

Cytotoxic substances are the core part of small molecule conjugate drugs that kill tumors and are called "nuclear warheads" that attack tumors. Gemcitabine and pentafluorouracil are commonly used cytotoxic substances (Chem Soc Rev. 2021, 50, 1480-1494; J Med Chem. 2021, 64, 4450-4461). Gemcitabine is clinically used to treat pancreatic cancer, liver cancer, etc., but it has developed serious drug resistance during long-term use. For this reason, NUC-1031 (Figure 3) designed and synthesized using Pro tide prodrug technology overcomes gemcitabine resistance and is a new gemcitabine prodrug. It is currently in phase III clinical trials (J Med Chem. 2014, 57, 1531-1542). The Pro tide prodrug NUC-3373 for pentafluorouracil is currently in phase I clinical trials. The main resistance mechanism of NUC-1031 is that it is not dependent on the transport of the non-Na ⁺ -dependent equilibrium nucleoside transporter (hENT1) required for gemcitabine to cross the membrane, avoiding the rate-limiting process of gemcitabine-dependent deoxycytidine kinase (dCK) to generate a phosphate dFdCMP, and is insensitive to the main enzyme inactivating gemcitabine (cytidine deaminase (CDA)). Therefore, NUC-1031 has significantly better anti-tumor activity in vivo and in vitro than gemcitabine, and still has a certain inhibitory activity against gemcitabine-resistant tumors, making it a more ideal cytotoxic agent.

CD13 is a type of Zn2 ⁺ -dependent membrane protein that is highly expressed on the surface of tumor cells such as liver cancer and pancreatic cancer, as well as in blood vessels and matrix, and is lowly expressed in most normal tissue cells, with tumor tissue specificity (J Med Chem. 2018, 61, 6468-6490; Proc Natl Acad Sci US A. 2012, 109, 1637-1642). The protein structure of CD13 includes an intracellular region, a transmembrane region, and an extracellular region containing a catalytic active center. Studies have shown that monoclonal antibodies, peptides, and small molecule inhibitors of CD13 can bind to CD13 on the surface of tumor cells to mediate endocytosis (Trends Mol Med. 2008, 14, 361-371; J Hematol Oncol. 2020, 13, 32-47). CD13 has been used as a ligand target in antibody-drug conjugates and peptide-drug conjugates: (1) In 2020, Zapata et al. reported the first antibody-drug conjugate MI130110 with a CD13 monoclonal antibody as a ligand. By binding to CD13, the molecule is internalized into the cell, and the anti-tumor effect shows tumor tissue specificity (J Hematol Oncol. 2020, 13, 32-47); (2) The peptide conjugates NGR-hTNFα and tTF-NGR, which use the CD13-specific peptide NGR (Asn-Gly-Arg) as a ligand, are in Phase III and Phase I clinical trials, respectively (Med Res Rev. 2012, 32, 1078-1091). Therefore, CD13 is considered to be an ideal target for drug targeted delivery and an ideal target for the design of small molecule conjugates.

Compared with other major ligand targets for the development of small molecule conjugate drugs, the potential characteristics and advantages of CD13 are that it not only has tumor tissue specificity and mediates endocytosis, but is also an important target for the treatment of multiple tumors: CD13 is a surface marker of liver cancer stem cells and is closely related to the occurrence, drug resistance and recurrence of liver cancer (J Clin Invest. 2010, 120, 3326-3339); CD13 small molecule inhibitor Ubenimex plays multiple anti-tumor effects in clinical practice, including tumor immunoregulation, inhibition of tumor angiogenesis and reversal of drug resistance (Proc Natl Acad Sci U S A. 2012, 109, 1637-1642; J Clin Invest. 2010, 120, 3326-3339). Therefore, a targeting ligand that combines Ubenimex with cytotoxic agents is needed to further improve the inhibitory effect.

Summary of the invention

In view of the above-mentioned prior art, the purpose of the present invention is to provide a small molecule coupling molecule based on aminopeptidase N/CD13 and its preparation method and application. The present invention uses the CD13 inhibitor Ubenimex as a targeting ligand of a small molecule coupling drug, which can not only mediate the tumor targeting of the molecule, but also the cytotoxic substances produced by the degradation after entering the cell and Ubenimex may also produce a synergistic effect, providing a new strategy for the treatment of tumors.

To achieve the above object, the present invention adopts the following technical solution:

In a first aspect of the present invention, there is provided a compound of formula I, or an optical isomer, diastereomer, racemate or a mixture thereof, or a pharmaceutically acceptable salt, solvate or deuterated compound thereof;

Wherein: A is selected from NH or O;

B is CH ₂ , (CH ₂ ) _n , CHCH ₃ , OCH ₂ , O(CH ₂ ) _n , OCH(CH ₃ )CH ₂ , OC(CH ₃ ) ₂ CH ₂ , NHCH ₂ , NH(CH ₂ ) _n ,NHCHCH ₃ ,NHCH(CH ₃ )CH ₂ ,NHC(CH ₃ ) ₂ CH ₂ ,

Where n is 2-10; represents the bond to the carbonyl group; represents the bond connected to A;

C is wherein R ₁ is selected from -CH ₃ , -(CH ₂ ) _n CH ₃ , -CH(CH ₃ ) ₂ , C ₆ H ₅ CH ₂ -, 4-FC ₆ H ₅ CH ₂ -,

-CH ₂ CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -CHCH ₃ CH ₂ CH ₃ or C ₆ H ₅ -;

R ₂ is selected from

R ₃ is selected from Where n is 1-6;

R ₄ and R ₅ are selected from H, halogen, (C _1-2 ) alkyl, halomethyl, OH, OCH ₃ , O(CH ₂ ) _n CH ₃ , OC(CH ₃ ) ₃ , OCH(CH ₃ ) ₂ , cyclopropyloxy, 5-6 membered alkoxy, NH ₂ , N(CH ₃ ) ₂ , NH(CH ₂ ) _n CH ₃ , CN or N ₃ , wherein n is 0-9;

R ₆ is selected from -CH ₃ , -(CH ₂ ) _n CH ₃ , -CH(CH ₃ ) ₂ , C ₆ H ₅ CH ₂ -, 4-FC ₆ H ₅ CH ₂ -, -CH ₂ CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -CHCH ₃ CH ₂ CH ₃ or C ₆ H ₅ -;

R ₇ is selected from H, -CH ₃ or -CH ₂ CH ₃ ;

X is selected from O or NH;

D is H or F.

Preferably, the compound represented by formula I has a structure represented by formula II, formula III, formula IV or formula V;

Wherein: A is selected from NH or O;

B is CH ₂ , (CH ₂ ) _n , CHCH ₃ , OCH ₂ , O(CH ₂ ) _n , OCH(CH ₃ )CH ₂ , OC(CH ₃ ) ₂ CH ₂ , NHCH ₂ , NH(CH ₂ ) _n ,NHCHCH ₃ ,NHCH(CH ₃ )CH ₂ ,NHC(CH ₃ ) ₂ CH ₂ ,

Where n is 2-10; represents the bond to the carbonyl group; represents the bond connected to A;

R ₁ is selected from -CH ₃ , -(CH ₂ ) _n CH ₃ , -CH(CH ₃ ) ₂ , C ₆ H ₅ CH ₂ -, 4-FC ₆ H ₅ CH ₂ -,

-CH ₂ CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -CHCH ₃ CH ₂ CH ₃ or C ₆ H ₅ -;

R ₂ is selected from

R ₃ is selected from Where n is 1-6;

R ₄ and R ₅ are selected from H, halogen, (C _1-2 ) alkyl, halomethyl, OH, OCH ₃ , O(CH ₂ ) _n CH ₃ , OC(CH ₃ ) ₃ , OCH(CH ₃ ) ₂ , cyclopropyloxy, 5-6 membered alkoxy, NH ₂ , N(CH ₃ ) ₂ , NH(CH ₂ ) _n CH ₃ , CN or N ₃ , wherein n is 0-9;

R ₆ is selected from -CH ₃ , -(CH ₂ ) _n CH ₃ , -CH(CH ₃ ) ₂ , C ₆ H ₅ CH ₂ -, 4-FC ₆ H ₅ CH ₂ -,

-CH ₂ CH(CH ₃ ) ₂ , -C(CH ₃ ) ₃ , -CHCH ₃ CH ₂ CH ₃ or C ₆ H ₅ -;

R ₇ is selected from H, -CH ₃ or -CH ₂ CH ₃ ;

X is selected from O or NH.

Preferably, the compound represented by formula I is selected from:

Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (13a);

Benzyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (13b);

Methyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (13c);

Cyclohexyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (13d);

Benzyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphono)-L-alaninate (13e);

Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphono)-L-alaninate (13f);

Benzyl ((((2R,3R,5R)-5-(4-(((3-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)propoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (22a);

Benzyl ((((2R,3R,5R)-5-(4-(((2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)ethoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (22b);

Benzyl ((((2R,3R,5R)-5-(4-(((4-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)butoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (22c);

Methyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetamido)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofurano[3,2-d][1,3,2]diphosphinilidene-2-yl)oxy)acetate (34a);

Ethyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetamido)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofurano[3,2-d][1,3,2]diphosphinilidene-2-yl)oxy)acetate (34b);

Phenyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofurano[3,2-d][1,3,2]diphosphinilidene-2-yl)oxy)acetate (34c);

3-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)propyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40a);

2-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)ethyl (1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40b);

4-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)butyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40c);

5-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)pentyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40d).

Unless otherwise defined, the compounds and salts provided herein may also contain all isotopes of atoms present in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers.

As used herein, "halogen" refers to F, Cl, Br, or I. In some embodiments, halogen is F, Cl, or Br. In some embodiments, halogen is F. In some embodiments, halogen is Cl. In some embodiments, halogen is Br. In some embodiments, halogen is I.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Wherein pharmaceutically acceptable salt refers to the derivative of the disclosed compound, wherein the parent compound is modified by converting the existing acid or base moiety into its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; and alkali metal or organic salts of acidic residues such as carboxylic acids, etc. The pharmaceutically acceptable salts of the present application include, for example, conventional non-toxic salts of the parent compound formed by non-toxic inorganic or organic acids, mainly including inorganic acid salts such as sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, hydrochloric acid, boric acid, sulfamic acid, etc.; or organic acids such as acetic acid, propionic acid, butyric acid, valproic acid, camphoric acid, capric acid, caproic acid, caprylic acid, suberic acid, carbonic acid, cinnamic acid, glycolic acid, trifluoroacetic acid, adipic acid, pyruvic acid, salicylic acid, methanesulfonic acid, alginic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, stearic acid, lactic acid, citric acid, oxalic acid, malonic acid, succinic acid, pyroglutamic acid, ascorbic acid, aspartic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, hydroxymaleic acid, palmitic acid, cinnamic acid, isobutylene glycol, Acid, lauric acid, mandelic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-aminobenzenesulfonic acid, 2-acetoxy-benzoic acid, 2-hydroxy-1,2,3-propanetriol, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, formic acid, fumaric acid, mucic acid, gentisic acid, ethylsulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfinic acid, isethionic acid, ethanedisulfonic acid, 4-(鬏 methoxycarbonylamino)butyric acid, dichloroacetic acid, 1,2-ethanedisulfonic acid, camphor-10-sulfonic acid, 2,4-dihydroxybenzoic acid, α-ketoglutaric acid, 1-hydroxy-2-naphthoic acid, p-acetylaminobenzoic acid, 2-hydroxybenzoic acid, 4-amino-2-hydroxybenzoic acid, all-trans retinoic acid. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing a basic or acidic part by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, alcohols (e.g., methanol, ethanol, isopropanol or butanol), or acetonitrile (MeCN) are preferred.

In a second aspect of the present invention, there is provided a method for preparing a compound of formula I, or an optical isomer, diastereomer, racemate or a mixture thereof, or a pharmaceutically acceptable salt, solvate or deuterated product thereof, comprising the following steps:

(1) Using the compound Boc-AHPA as a raw material, the intermediate Boc-Bestatin is obtained by condensation and hydrolysis demethylation reaction;

(2) using gemcitabine or 5'-deoxy-5-fluorocytidine as a cytotoxic agent, modifying and protecting the hydroxyl groups on the cytotoxic agent to obtain a modified cytotoxic agent;

(3) The target product is obtained by condensing the amino terminus of the modified cytotoxic agent with the carboxyl group of Boc-Bestatin and removing the protecting group.

The modification and protection of the hydroxyl groups on the cytotoxic agent are as follows: the hydroxyl group at the 3' position of the cytotoxic agent gemcitabine is Boc protected, the hydroxymethyl group at the 5' position is modified by phosphorylated protide prodrug, and the two hydroxyl groups of 5'-deoxy-5-fluorocytosine nucleoside are acetylated and protected.

The preparation method of some compounds having the structure of formula II and V is prepared by the following reaction:

Reagents and conditions: (a) POCl ₃ , Et ₃ N, dry Et ₂ O, -78℃; (b) Et ₃ N, dry DCM; (c) (Boc) ₂ O, Na ₂ CO ₃ ,1,4 -dioxane/H ₂ O; (d) tert-BuMgCl, THF, rt; (e) EDCI, HOBt, dry DCM; (f) TFA, dry DCM; (g) EDCI, HOBt, dry DCM; (h) 1.5N NaOH, MeOH; (i) EDCI, HOBt, dry DCM; (j) dry HCl inEtOAc; 1N NaHCO ₃ .

Compound 1 reacts with trichlorophosphine to obtain compound 2, which is further condensed to obtain intermediate 4. Gemcitabine is monoprotected with (Boc) ₂ O and reacted with intermediate 4 to obtain 6, which is further condensed with Boc-L-glycine to generate compound 7, and 7 is deprotected to obtain amine compound 8. Boc-AHPA (9) is condensed and demethylated to obtain compound 11, which is further condensed with 8 to obtain intermediate 12. 12 is then deamino protected and the target product 13 is obtained by adjusting the base.

The specific conditions of each reaction in the above reaction scheme can be conventional reaction conditions in the art.

The preparation method of some compounds having the structure of formula II and V can also be prepared by the following reaction:

Reagents and conditions: (a) EDCI, HOBt, dry DCM; (b) Et ₃ N, dry DCM; (c) Et ₃ N, dry DCE, DMAP, 60℃; (d) TFA, dry DCM; (e) EDCI, HOBt, dry DCM, Et ₃ N; (f) dry HCl in EtOAc.

Compound 14 was condensed with 15 to give intermediate 16, which was further reacted with 17 to give 18. Intermediate 6 was reacted with 18 to give 19, which was deprotected to give amine compound 20. Boc-AHPA (9) was condensed with 19 to give 21, which was further deprotected to give target product 22.

The specific conditions of each reaction in the above reaction scheme can be conventional reaction conditions in the art.

The preparation method of some compounds having the structure of formula III is prepared by the following reaction:

Reagents and conditions: (a) TMSCl, pyridine, -5℃; (b) DMTrCl, DMAP, 55℃; (c) NH ₄ F, MeOH, 60℃; (d) POCl ₃ , Et ₃ N, -15℃ ; (e) Et ₃ N, NMI, dry DCM; (f) Et ₃ SiH, TFA, dry DCM; (g) EDCI, HOBt, dry DCM; (h) TFA, DCM; (i) EDCI, HOBt, dry DCM; (j) TFA, dry DCM; 1N NaHCO ₃ .

After gemcitabine was protected by TMS, it was further reacted with DMTrCl to obtain compound 24. After removing TMS, compound 24 was reacted with intermediate 27 to obtain 28, and further deamino protecting group was obtained to obtain intermediate 29. Boc-glycine was condensed with 29 to obtain 31, and the Boc protecting group was removed to obtain 32, which was further condensed with compound 11 to obtain 33. After removing the amino protecting group of compound 33, the target compound 34 was obtained by adjusting the base.

The specific conditions of each reaction in the above reaction scheme can be conventional reaction conditions in the art.

The preparation method of some compounds having the structure of formula IV is prepared by the following reaction:

Reagents and conditions: (a) Et ₃ N, DCE, 60°C; (b) TFA, DCM; (c) EDCI, HOBt, dry DCM; (d) K ₂ CO ₃ , MeOH; (e) dry HCl in EtOAc.

Compound 18 reacts with 35 to obtain compound 36, which is then further condensed with Boc-AHPA to obtain intermediate 38 after removing the Boc protecting group. Compound 38 is then deacetylated and deacetylated to obtain target compound 40.

The third aspect of the present invention provides the use of the compound shown in Formula I, or its optical isomers, diastereomers, racemates or mixtures thereof, or its pharmaceutically acceptable salts, solvates, deuterated substances in the preparation of targeting ligands.

The compound shown in Formula I can be used as a CD13 inhibitor, which can precisely deliver the coupled cytotoxic drug to the tumor tissue by targeting the tumor tissue surface receptor CD13, thereby exerting a toxicity-reducing and efficacy-enhancing effect.

In a fourth aspect of the present invention, there is provided the use of a compound of formula I, or its optical isomers, diastereomers, racemates or mixtures thereof, or its pharmaceutically acceptable salts, solvates, deuterated derivatives in the preparation of drugs for preventing or treating tumors.

Preferably, the tumor includes: multiple myeloma, lymphoma, leukemia and solid tumor.

The solid tumors are prostate cancer, pancreatic cancer, liver cancer, cervical cancer, breast cancer, melanoma, genital urinary system tumors, colorectal cancer, non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, brain tumors, gliomas, gastric cancer, laryngeal cancer, nasopharyngeal cancer, kidney cancer, skin cancer, epithelial cell cancer, bile duct cancer, ovarian cancer, nasopharyngeal cancer, bladder cancer, oral cancer, tongue cancer, and human fibrosarcoma.

In a fifth aspect of the present invention, a pharmaceutical composition is provided, which comprises a compound of Formula I, or its optical isomers, diastereomers, racemates or mixtures thereof, or its pharmaceutically acceptable salts, solvates or deuterated derivatives as an active ingredient.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients.

The sixth aspect of the present invention provides the use of the above-mentioned pharmaceutical composition in the preparation of a pharmaceutical preparation for treating tumor diseases.

As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent required to treat, improve a targeted disease or condition, or to exhibit a detectable therapeutic effect.

The active ingredient can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, it will be understood that the amount of compound actually administered will generally be determined by a physician based on relevant circumstances, including the condition to be treated, the route of administration selected, the actual compound administered, the age, weight and response of the individual subject, and the severity of the subject's symptoms, etc.

Beneficial effects of the present invention:

(1) The compound prepared by the present invention can be used as a CD13 inhibitor, and the coupled cytotoxic drug can be accurately delivered to the tumor tissue by targeting the tumor tissue surface receptor CD13, thereby playing a role in reducing toxicity and enhancing efficacy. In addition, using the CD13 inhibitor Ubenimex as a targeting ligand for small molecule coupled drugs can not only mediate the tumor targeting of the molecule, but also the cytotoxic substances produced by degradation after entering the cell and Ubenimex may also produce a synergistic effect, providing a new strategy for the treatment of tumors.

(2) The compounds of the general structural formula (I) provided by the present invention all exhibit significant CD13 inhibitory activity, and most of the compounds have better CD13 activity than the CD13 inhibitor ubenimex. In vitro experiments on the inhibition of tumor cell proliferation in multiple strains have shown that these compounds have significant tumor cell proliferation inhibitory activity, and the inhibitory activity ranges from several to several hundred nanomoles. In addition, in the in vivo anti-tumor activity study in mice, the representative compound 13b has significant inhibitory activity against mouse H22 liver cancer, and its growth inhibitory activity against liver cancer H22 reaches 75%.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Stability of 13b in human plasma.

Figure 2: Comparison of tumor weights in the mouse liver cancer cell H22 tumor-bearing model.

Figure 3: Tumor images of each group in the mouse liver cancer cell H22 tumor-bearing model.

DETAILED DESCRIPTION

It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

As introduced in the background technology section, the CD13 small molecule inhibitor ubenimex has multiple anti-tumor effects in clinical practice, including tumor immunomodulation, inhibition of tumor angiogenesis and reversal of drug resistance. Cytotoxic agents are the core part of small molecule conjugate drugs that kill tumors and are called "nuclear warheads" that attack tumors. Gemcitabine and pentafluorouracil are commonly used cytotoxic agents. Gemcitabine is clinically used to treat pancreatic cancer, liver cancer, etc., but serious drug resistance has been developed during long-term use.

Based on this, the present invention provides a small molecule coupling molecule based on aminopeptidase N/CD13 and its preparation method and application. Modifying gemcitabine using ProTide technology can avoid the rate-limiting process of gemcitabine generating a phosphate, effectively overcome gemcitabine's own drug resistance, and have significant liver targeting. Therefore, the present invention selects the ProTide prodrug of gemcitabine as a more ideal cytotoxic. The present invention is based on the design mode of small molecule coupling drugs, preferably gemcitabine (pentafluorouracil, etc.) Protide prodrug and its analogs as cytotoxics, ubenimex as a small molecule ligand targeting CD13, and synthesizes a small molecule coupling compound targeting CD13 by designing a cleavage-type connecting chain. Using CD13 inhibitors as the targeting ligand of small molecule coupling drugs can not only mediate the tumor targeting of molecules, but also the cytotoxic substances and ubenimex produced by degradation after entering the cells may also produce a synergistic effect.

The compounds of the present invention may form hydrates or solvates. Methods for forming hydrates when a compound is lyophilized with water or solvates when concentrated with a suitable organic solvent in solution are known to those skilled in the art.

The present invention comprises a pharmaceutical composition containing a therapeutic amount of a compound of the present invention, and one or more pharmaceutically acceptable carriers and/or excipients. Carriers include, for example, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof, as discussed in more detail below. If desired, the composition may also contain a smaller amount of a wetting agent or emulsifier, or a pH buffer. The composition may be a liquid, a suspension, an emulsion, a tablet, a pill, a capsule, a sustained release formulation, or a powder. The composition may be formulated into a suppository with conventional adhesives and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate, and the like. Depending on the desired formulation, the formulation may be designed to mix, granulate, and compress or dissolve components. In another approach, the composition may be formulated into nanoparticles and enteric pellets.

The pharmaceutical carrier used may be either solid or liquid.

Typical solid carriers include lactose, gypsum, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid and the like. A solid carrier may include one or more substances that may simultaneously act as a flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder or tablet-disintegrant; it may also be an encapsulating material. In a powder, the carrier is a finely pulverized solid, which is mixed with a finely pulverized active ingredient. In a tablet, the active ingredient is mixed with a carrier having the necessary compression properties in a suitable ratio and compressed in the desired shape and size. Powders and tablets preferably contain up to 99% active ingredients. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, low melting wax and ion exchange resins.

Typical liquid carriers include syrups, peanut oil, olive oil, water, etc. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, tinctures and sealed compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of the two or a pharmaceutically acceptable oil or fat. The liquid carrier can include other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, pigments, viscosity modifiers, stabilizers or osmotic pressure-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially including additives such as those described above, such as cellulose derivatives, preferably carboxymethyl cellulose sodium salt solutions), alcohols (including monohydric alcohols and polyhydric alcohols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). Carriers for parenteral administration can also be fats and oils such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are used for sterile liquid compositions for parenteral administration. The liquid carrier for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. Sterile solutions or suspensions of liquid pharmaceutical compositions can be used, for example, for intravenous, intramuscular, intraperitoneal or subcutaneous injection. The injection can be a single push or gradually injected, such as a 30-minute intravenous infusion. The compound can also be administered orally in the form of a liquid or solid composition.

The carrier or excipient may include time-delay materials known in the art, such as glyceryl monostearate or glyceryl distearate, and may also include wax, ethylcellulose, hydroxypropylmethylcellulose, methyl methacrylate, etc. When the formulation is for oral administration, it is recognized that 0.01% Tween 80 in PHOSALPG-50 (phospholipids and 1,2-propylene glycol concentrate, A. Nattermann & Cie. GmbH) is used for the preparation of acceptable oral formulations of other compounds and can be adapted to the preparation of various compounds of the present invention.

A wide variety of pharmaceutical forms can be used when administering the compounds of the invention. If a solid carrier is used, the preparation can be an enteric coated tablet, enteric pellets placed in a hard capsule or in the form of a lozenge or troche. The amount of the solid carrier varies to a great extent, but preferably from about 25 mg to about 1.0 g. If a liquid carrier is used, the preparation can be a syrup, an emulsion, a soft capsule, a sterile injectable solution or suspension in an ampoule or vial or a non-aqueous liquid suspension.

Various release systems are known and can be used for the administration of compounds or other various preparations, including tablets, capsules, injectable solutions, capsules in liposomes, microparticles, microcapsules, and the like. Methods of introduction include but are not limited to cutaneous, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and (usually preferred) oral routes. Compounds can be administered by any convenient or other appropriate route, such as by injection or bolus injection, absorption through epithelial or mucosal routes (e.g., oral mucosa, rectal and intestinal mucosa, etc.) or by drug-loaded stents and can be administered with other bioactive agents. Can be administered systemically or topically.

In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

Preparation of compounds of formula II:

Example 1: Isopropyl ((((2R,3R,5R)-5-(4-(2-((tert-butoxycarbonyl)amino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphono)-L-alaninate (7a).

Boc-glycine (207.5 mg, 1.19 mmol) was dissolved in anhydrous DCM, HOBt (181.3 mg, 1.34 mmol) and EDCI (257.5 mg, 1.34 mmol) were added under ice bath, and stirred at 0°C for 0.5 h. Then, a mixed solution of 6a (500 mg, 0.79 mmol) and triethylamine (159.6 mg, 1.58 mmol) in DCM was added dropwise to the reaction solution, and stirred at room temperature for 16 h. TLC monitored that all the raw materials were completely reacted. The reaction solution was washed three times with 1N citric acid, saturated sodium bicarbonate, and brine in sequence, dried over anhydrous sodium sulfate, filtered and evaporated to obtain a white solid 7a (540 mg, yield: 86.5%), which was used directly in the next step without purification. ¹ H NMR (500 MHz, DMSO-d ₆ )δ11.07(s,1H),8.01(dd,J＝26.4,7.7Hz,1H),7.38-7.34(m,2H),7.27-7.15(m,4H),7.09(t,J＝6.0Hz,1H),6.33(t,J＝8.5Hz,1H),6.19-6.08(m,1H),5. 27(s,1H),4.92-4.81(m,1H),4.54-4.45(m,1H),4.42-4.30(m,2H),3.81(d,J＝6.0Hz,2H),1.45(s,9H),1.38(s,9H),1.26-1.21(m,4H),1.16-1.1 4(m,6H).MS(ESI ⁺ ):Calcd for C ₃₃ H ₄₆ F ₂ N ₅ O ₁₃ P[M+H] ⁺ 790.28, found 790.45.

Example 2: Isopropyl ((((2R,3R,5R)-5-(4-(2-aminoacetamido)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate trifluoroacetate (8a).

7a (540 mg, 0.68 mmol) was dissolved in anhydrous DCM, trifluoroacetic acid (2 mL) was slowly added dropwise under ice bath, and the temperature was slowly raised to room temperature and stirred for 2 h. After TLC monitoring, all the raw materials reacted completely, the solvent was evaporated, ether was added to precipitate the solid, and the white solid 8a (454 mg, yield: 95%) was obtained by suction filtration, which was used directly in the next step without purification. ¹ H NMR (500 MHz, DMSO-d ₆ )δ11.46(s,1H),8.34-8.18(m,3H),8.06(dd,J＝26.1,7.6Hz,1H),7.36(dd,J＝14.8,7.3Hz,2H),7.27-7.15(m,3H),6.23(dd,J＝17.2,8.8Hz,1H),6.19-6. 10(m,1H),4.89-4.82(m,1H),4.46-4.09(m,5H),3.92(s,2H),3.80(ddd,J＝17.0,10.1,3.1Hz,1H),3.59(t,J＝6.2Hz,1H),1.22(t,J＝7.8Hz,3H),1.17- 1.11(m,6H).MS(ESI ⁺ ):Calcd forC ₂₅ H ₃₁ F ₅ N ₅ O ₁₁ P[M+H] ⁺ 590.04,found 590.04.

Example 3: Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-((tert-butyloxycarbonyl)amino)-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (12a)

11 (469.2 mg, 1.15 mmol) was dissolved in anhydrous DCM, HOBt (186.3 mg, 1.38 mmol) and EDCI (264.5 mg, 1.38 mmol) were added under ice bath, and stirred at 0°C for 0.5 h. A mixed solution of 8a (677 mg, 1.15 mmol) and triethylamine (232 mg, 2.30 mmol) in DCM was added dropwise, and stirred at room temperature for 16 h. TLC monitored that all the raw materials reacted completely, and the reaction solution was washed three times with 1N citric acid, saturated sodium bicarbonate, and brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness, and white solid 12a (350 mg, yield: 31%) was obtained by column separation.

Example 4: Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate hydrochloride (13a)

12a (120 mg, 0.12 mmol) was dissolved in DCM, and a saturated hydrochloric acid ethyl acetate solution (2 mL) was added. The mixture was stirred at room temperature for 1.5 h. After the reaction of all the raw materials was completed as monitored by TLC, part of the ethyl acetate was removed by concentration, and solids were precipitated. After ultrasonic dispersion and filtration, white powder 13a (70 mg, yield: 62.5%) was obtained. ¹ H NMR (500 MHz, DMSO-d ₆ )δ10.99(s,1H),8.50(t,J＝5.7Hz,1H),8.14(d,J＝7.9Hz,1H),8.07-8.01(m,3H),7.97(d,J＝7.6Hz,1H),7.41-7.10(m,11H),6.21(td,J＝9.2,8.0Hz,1H ),6.13(dt,J＝14.8,7.5Hz,1H),4.90-4.81(m,1H),4.45- 4.19(m,4H),4.18-4.07(m,2H),4.07-4.00(m,2H),3.96-3.91(m,2H),3.56(s,1H),2.98-2.86(m,2H),1.66(td,J＝13.2,6.7Hz,1H),1.57-1.53(m ,2H),1.26-1.19(m,3H),1.16-1.13(m,6H),0.91-0.87(m,6H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ173.18,173.01,172.73,171.28,170.69,163.10,154.47,151.07,136.78,130.09(2C),129.90(2C),129.05(2C),127.33,125.07,120.55,120 .51,96.53,6 9.72,68.60,68.46,64.74,54.67,51.64,50.44,50.28,43.54,41.18,35.11,24.56,23.34,22.38,21.88,21.82,20.20,20.15,20.10(m).HRMS (ESI ):m/z(M+H) ⁺ :found 880.3466; Calcd for C ₃₉ H ₅₃ ClF ₂ N ₇ O ₁₂ P(M+H) ⁺ :880.3380.

The same synthetic method was used to obtain compounds 13b, 13c, 13d, 13e and 13f.

Example 5: Benzyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (13b)

White powder 13b, yield: 80%. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ8.55 (t, J=5.7 Hz, 1H), 7.97 (dd, J=11.2, 7.7 Hz, 1H), 7.89 (d, J=8.3 Hz, 1H), 7.38-7.12 (m, 15H), 6.53 (dd, J=16.4, 5.9 Hz, 1H), 6.26-6.15 (m, 2H), 5.13-5.03 (m, 2H), 4.44-4.15 (m, 4H), 4.15-4.06 (m, 1H), 3 .98-3.87(m,2H),3.83(d,J＝2.4Hz,1H),3.23(td,J＝7.1,2.7Hz,1H),2.82(dd,J＝13.3,6.7Hz,1H),2.58(dd,J＝13.3,7.5Hz,1H),1.68-1.60(m,1H),1.5 8-1.48(m,2H),1.29-1.21(m,3H),0.91-0.85(m,6H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ173.48(m),172.71,172.47,171.26,171.07,170.68,163.09,154.46,151.04,136.67,136.31,130.08(2C),129.88(2C),129.07(2C),128.85, 128.47,128.24,128.20,127.36,125 .08,120.56,120.52,68.58,66.44,54.68,51.59,51.43,50.40,50.24,43.52,41.17(t,1C),35.15,29.52,29.23,27.00,25.55,24.56,23.34,22 .34,20.05(m).HRMS(ESI):m/z(M+H) ⁺ :found 928.3465; Calcd forC ₄₃ H ₅₂ F ₂ N ₇ O ₁₂ P(M+H) ⁺ :928.3380.

Example 6: Methyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate hydrochloride (13c)

White powder 13c, yield: 75%. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 8.50 (t, J = 5.8 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 8.07-7.93 (m, 4H), 7.40-7.11 (m, 10H), 6.27-6.03 (m, 2H), 4.44-4.37 (m, 1H), 4.44-4.37 (m, 1H), 4.35-4.26 (m, 3H), 4. 24-4.08(m,4H),4.05-4.01(m,2H),3.91-3.84(m,2H),3.60-3.53(m,3H),2.98-2.84(m,2H),1.67(dt,J＝13.3,6.7Hz,1H),1.59-1.50(m,2H),1.25 -1.21(m,3H),0.91-0.87(m,6H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ174.04(m),172.74,172.48,171.37,171.28,171.09,170.69,163.10,154.49,151.04,136.78,130.10(2C),129.90(2C),129.05(2C),127.33, 125.08,120.55 (m,2C),96.56,95.40,68.60,64.63,54.67,52.39,51.64,51.49,50.27,50.12,43.55,41.17(t,1C),35.11,24.57,23.35,22.37,20.08(m).HRMS(ES) I):m/z(M+H) ⁺ :found 852.3151; Calcd forC ₃₇ H ₄₉ ClF ₂ N ₇ O ₁₂ P(M+H) ⁺ :852.3067.

Example 7: Cyclohexyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate hydrochloride (13d)

White powder 13d, yield: 74%. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 11.00 (s, 1H), 8.49 (t, J = 5.3 Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 8.07-7.97 (m, 4H), 7.40-7.07 (m, 10H), 6.25-6.09 (m, 2H), 4.63 (d, J = 5.6 Hz, 1H), 4.44-4.10 (m, 5H), 4.05-3.99 (m, 2H), 3.96- 3.89(m,2H),3.87-3.67(m,6H),2.99-2.81(m,2H),1.75-1.67(m,2H),1.66-1.59(m,2H),1.57-1.51(m,2H),1.46-1.40(m,1H),1.36-1.27(m,3H) ,1.23(t,J＝8.0Hz,3H),0.91-0.86(m,6H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ173.01(m),172.72,172.48,171.37,171.27,171.09,170.68,163.11,154.48,151.05,136.77,130.09(2C),129.90(2C),129.05(2C),127.33, 125.06,120.55(m,2C),96.52 ,95.42,72.96,68.60,64.75,54.67,51.63,51.48,50.47,50.31,43.54,41.19(t,1C),35.11,31.28,31.23,25.26,24.56,23.40,23.35,22.35, 20.19(m).HRMS(ESI):m/z(M+H) ⁺ :found 920.3772.

Example 8: Benzyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphonyl)-L-alaninate (13e)

White powder 13e, yield: 85%. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ8.48 (s, 1H), 8.07 (dd, J=22.6, 7.6 Hz, 2H), 7.41-7.20 (m, 12H), 6.52 (dd, J=12.4, 6.0 Hz, 1H), 6.22 (d, J=6.8 Hz, 1H), 5.97-5.81 (m, 1H), 5.12 (q, J=12.5 Hz, 2H), 4.56-4.42 (m, 2H), 4.38-4.25 (m, 2H), 4.25- 4.13(m,2H),4.13-4.04(m,1H),4.01-3.86(m,3H),3.66(d,J＝5.2Hz,3H),3.53-3.64(m,1H),2.96-2.86(m,1H),2.85-2.72(m,1H),1.67-1.45(m, 4H), 1.35 (s, 3H), 1.30 (d, J＝7.0Hz, 3H), 0.92-0.82 (m, 6H). ¹³ C NMR (125MHz, DMSO-d ₆ )δ173.74(m),171.47,170.67,168.95,163.12,154.51,139.63,136.99,136.33,129.85(2C),129.04(2C),128.86(2C),128.48,128.23(2C),127 .27,125.34,122.91,1 07.42,96.58,79.25,68.98,66.47,62.39(m),54.82,52.37,51.55,50.09(m),43.54,41.19,35.69,34.82,30.87,24.57,23.35,22.28,20.08(m) ).HRMS(ESI):m/z(M+H) ⁺ :found 924.3361; Calcd for C ₄₀ H ₅₂ F ₂ N ₇ O ₁₄ P(M+H) ⁺ :924.3278.

Example 9: Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphonyl)-L-alaninate (13f)

White powder 13f, yield: 78%. ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 8.48 (s, 1H), 8.08 (dd, J=19.1, 7.1 Hz, 2H), 7.33-7.27 (m, 6H), 6.87 (s, 1H), 6.58-6.46 (m, 1H), 6.23 (s, 1H), 5.87-5.73 (m, 1H), 4.95-4.82 (m, 1H), 4.62-4.47 (m, 2H), 4.40-4.18 (m, 4 H),4.11(s,1H),4.05-3.87(m,3H),3.79-3.63(m,4H),3.00-2.87(m,1H),2.86-2.74(m,1H),1.75-1.45(m,4H),1.35(s,4H),1.31-1.23(m,3H),1. 22-1.13(m,5H),1.00-0.71(m,6H). ¹³ C NMR(125MHz,DMSO-d ₆ )δ173.42(m),172.76,171.49,170.68,168.94(m),163.14,154.51,151.90,139.63,137.02,129.85(2C),129.03(2C),127.26,125.34,96.57,79 .26,69. 04,68.48,62.449(m),54.83,52.37,51.53,50.13,50.06,43.52,41.20,35.78,34.82,30.87,24.57,23.36,22.28,21.87,20.11(m).HRMS(ESI):m/ z(M+H) ⁺ :found 876.3347; Calcd forC ₃₆ H ₅₂ F ₂ N ₇ O ₁₄ P(M+H) ⁺ :876.3278.

Preparation of another class of compounds of general formula II:

Example 10: tert-Butyl (S)-(1-((3-hydroxypropyl)amino)-4-methyl-1-oxopentyl-2-yl)carbamate (16a)

Compound 14 (6.0 g, 25.95 mmol) was dissolved in anhydrous DCM, TBTU (10 g, 31.44 mmol) and triethylamine (3.94 g, 38.91 mmol) were added at 0°C, stirred at 0°C for 30 min, and then compound 15a (3 g, 38.91 mmol) was added, and stirred at room temperature for 12 h. TLC monitored that all the raw materials reacted completely, and the organic layer was washed three times with 1N citric acid, saturated sodium bicarbonate, and brine in sequence, dried over anhydrous sodium sulfate, filtered, evaporated, and purified by column chromatography to obtain an oily liquid 16a (6.94 g, yield: 80.2%). 1H NMR(400MHz,Chloroform)δ6.64(s,1H),4.96(s,1H),4.06-4.05(m,1H),3.66-3.54(m,2H),3.53-3.39(m,2H),3.37(s,1H),1.66(tq,J＝11.4,5.8Hz,4H), 1.53-1.46(m,1H),1.42(s,9H),0.97-0.88(m,6H).

Example 11: tert-Butyl (S)-(4-methyl-1-((3-(((4-nitrophenoxy)carbonyl)oxy)propyl)amino)-1-oxopentyl-2-yl)carbamate (18a)

16a (2.1 g, 7.28 mmol) was dissolved in anhydrous DCM, triethylamine (0.92 g, 9.1 mmol) and 17 (3.04 g, 8.74 mmol) were added, and the mixture was stirred at room temperature for 12 h. TLC monitored that all the raw materials were completely reacted, and the organic layer was washed three times with 1N citric acid, saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated, and purified by column chromatography to obtain an oily liquid 18a (2.5 g, yield 75.8%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.32 (d, J = 9.1 Hz, 2H), 7.93 (t, J = 5.6 Hz, 1H), 7.56 (d, J = 9.1 Hz, 2H), 6.84 (d, J = 8.2 Hz, 1H), 4.24 (t, J = 6.3 Hz, 2H), 3.90 (dd, J = 14 .4,8.6Hz,1H),3.24-3.08(m,2H),1.91-1.71(m,2H),1.62-1.52(m,1H),1.46-1.31(m,11H),0.85(t,J＝7.2Hz,6H).

Example 12: Benzyl ((((2R,3R,5R)-5-(4-(((3-((S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoylamino)propoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-3-((tert-butoxycarbonyl)oxy)-4,4-difluorotetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (19a)

Compound 18a (900 mg, 2 mmol), 6 (1088 mg, 1.6 mmol) and triethylamine (202 mg, 2 mmol) were dissolved in DCE, and then DMAP (192 mg, 1.6 mmol) was added to the reaction solution, and the reaction solution was stirred at 60°C for 12 h. After the reaction was completed by TLC monitoring, DCE was evaporated, and the residue was extracted with ethyl acetate. The organic layer was washed with saturated brine three times, dried over anhydrous sodium sulfate, filtered and evaporated, and purified by column chromatography to obtain oily liquid 19a (636.4 mg, yield 40%). NMR (400MHz, DMSO-d6) δ10.88(s,1H),7.92(dd,J＝18.0,7.7Hz,1H),7.82(t,J＝5.7Hz,1H),7.40-7.21(m,7H),7.19-7.10(m,3H),7.09-6.99(m,1H),6.80-6 .78(m,1H),6.29-6.28(m,1H),6.20(d,J＝4.8Hz,1H),5.22(s,1H),5.11(s,2H ),4.40(d,J＝7.2Hz,1H),4.37-4.22(m,2H),3.97-3.76(m,2H),3.34(d,J＝3.6Hz,1H),3.11-3.09(m,2H),1.76-1.64(m,2H),1.50(dt,J＝12.9,6.5Hz,2 H), 1.40-1.36 (m, 9H), 1.33 (s, 10H), 1.23 (d, J = 7.1Hz, 3H), 0.81 (d, J = 9.0Hz, 6H). ESI MS:995.99([M+H] ⁺ ).

Example 13: Benzyl ((((2R,3R,5R)-5-(4-(((3-((S)-2-amino-4-methylpentanoylamino)propoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (20a)

19a (636 mg, 0.64 mmol) was dissolved in anhydrous DCM, trifluoroacetic acid (2 mL-3 mL) was slowly added dropwise under ice bath, and the temperature was slowly raised to room temperature and stirred for 2 h after the addition was complete. After TLC monitoring, the reaction of all the raw materials was complete, the solvent was evaporated, ether was added to precipitate the solid, and the white solid 20a (600 mg, yield: 93%) was obtained by suction filtration, which was used directly in the next step without purification.

Example 14: Benzyl ((((2R,3R,5R)-5-(4-(((3-((S)-2-((2S,3R)-3-((tert-butyloxycarbonyl)amino)-2-hydroxy-4-phenylbutanamide)-4-methylpentanoylamino)propoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (21a)

9 (212.5 mg, 0.72 mmol) was dissolved in anhydrous DCM, HOBt (116.6 mg, 0.86 mmol) and EDCI (164.3 mg, 0.86 mmol) were added under ice bath, and stirred at 0°C for 0.5 h. A mixed solution of 20a (600 mg, 0.6 mmol) and triethylamine (86.9 mg, 0.86 mmol) in DCM was added dropwise, and stirred at room temperature for 16 h. TLC monitored that all the raw materials reacted completely, and the reaction solution was washed three times with 1N citric acid, saturated sodium bicarbonate, and brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness, and white solid 21a (327.7 mg, yield: 51%) was obtained by column separation.

Example 15: Benzyl ((((2R,3R,5R)-5-(4-(((3-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)propoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (22a)

21a (327 mg, 0.31 mmol) was dissolved in DCM, and a saturated hydrochloric acid ethyl acetate solution (2 mL) was added. The mixture was stirred at room temperature for 1.5 h. After the reaction of all the raw materials was completed as monitored by TLC, part of the ethyl acetate was removed by concentration, and solids were precipitated. After ultrasonic dispersion and filtration, white powder 22a (265.5 mg, yield: 85%) was obtained. ¹ H NMR (400 MHz, DMSO-d ₆ )δ10.89(s,1H),8.18(t,J＝5.4Hz,1H),8.05(d,J＝8.0Hz,1H),7.99-7.91(m,4H),7.39-7.26(m,13H),7.18(dd,J＝15.6,8.1Hz,3H),7.07(dd,J＝14.6,7. 5Hz,1H),6.32-6.13(m,2H),5.16-4.99(m,2H),4.43-4.18(m,4H),4.09(t ,J＝6.0Hz,3H),4.01-3.88(m,2H),3.19-3.08(m,2H),2.93(dd,J＝13.4,8.1Hz,1H),2.83(dd,J＝13.8,6.4Hz,1H),1.79-1.66(m,2H),1.61-1.40(m,3H) , 1.26 (t, J = 7.3Hz, 4H), 0.87 (dd, J = 8.9, 6.5Hz, 6H). LC-MS (ESI): m/z (M+H) ⁺ : found 673.25; Calcd for C ₄₅ H ₅₇ ClF ₂ N ₇ O ₁₃ P (M + H) ⁺ : 972.36.

The same synthetic method was used to obtain compounds 22b and 22c.

Example 16: Benzyl ((((2R,3R,5R)-5-(4-(((2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)ethoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (22b)

White powder 22b, yield: 88%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.87 (s, 1H), 8.27 (t, J = 5.3 Hz, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.98-7.90 (m, 4H), 7.38-7.26 (m, 13H), 7.23-7.15 (m, 3H), 7.11-7.02 (m, 1H), 6.70-6.46 (m, 1H), 6.30-6.14 (m, 2H), 5.14-5.04 (m, 2H), 4.41 -4.21(m,4H),4.20-4.05(m,4H),4.03-3.87(m,3H),3.31-3.20(m,2H),2.97-2.87(m,1H),2.83(dd,J＝14.2,6.3Hz,1H),1.63-1.38(m,3H),1.30-1. 20 (m, 3H), 0.84 (t, J = 5.6Hz, 6H). LCMS (ESI): m/z (M+H) ⁺ : found 958.25; Calcd for C ₄₄ H ₅₅ ClF ₂ N ₇ O ₁₃ P (M+H) ⁺ : 958.35.

Example 17: Benzyl ((((2R,3R,5R)-5-(4-(((4-(((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)butoxy)carbonyl)amino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonyl)-L-alaninate (22c)

White powder 22c, yield: 76%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.88 (s, 1H), 8.15 (t, J = 5.5 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.97-7.91 (m, 4H), 7.41-7.25 (m, 13H), 7.18 (dd, J = 15.5, 8.0 Hz, 3H), 7.12-7.04 (m, 1H), 6.29-6.16 (m, 2H), 5.14-5.03 (m, 2H), 4.41-4.17 (m, 4H), 4.15-4.05 (m, 3 H),4.03-3.88(m,3H),3.40–3.35(m,2H),3.06(td,J＝13.1,6.6Hz,2H),2.93(dd,J＝13.8,8.0Hz,1H),2.87–2.77(m,1H),1.63-1.52(m,3H),1.52-1.39 (m,3H),1.26(t,J＝7.4Hz,3H),0.94–0.73(m,6H).LCMS(ESI):m/z(M/2+H) ⁺ :found 493.66; Calcd for C ₄₆ H ₅₉ ClF ₂ N ₇ O ₁₃ P(M/2+H) ⁺ :493.69.

Preparation of compounds of formula III:

Example 18: 4-((Bis(4-methoxyphenyl)(phenyl)methyl)amino)-1-((2R,4R,5R)-3,3-difluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one (25)

Gemcitabine hydrochloride (1 g, 3.34 mmol) was suspended in pyridine. At -5 °C, TMSCl (2.17 g, 20 mmol) was slowly added dropwise to the above reaction system, stirred for 1 h, and heated to room temperature and stirred for 30 min. TLC monitored the reaction to be complete. DMAP and DMTrCl were directly added, and the system was heated to 55 °C for overnight reaction. The solvent was evaporated, ethyl acetate and water were added, and after stratification, the ethyl acetate layer was washed 3 times with 1N citric acid, saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated to obtain 2.7 g of compound 24, which was directly used in the next step. The above product 24 (2.4 g) was dissolved in methanol, ammonium fluoride (377.4 mg, 10.2 mmol) was added, heated to (55-60) °C for 30 min, the reaction was monitored to be complete, the organic solvent was evaporated, DCM and water were added for washing, and column chromatography was purified to obtain compound 25 (1.2 g, yield: 63.8%). ¹ H NMR (500MHz, DMSO-d ₆ ) δ8.52 (s, 1H), 7.59 (d, J = 7.6Hz, 1H), 7.27 (t, J = 7.5Hz, 2H), 7.23-7.15 (m, 3H), 7.13 (d, J = 8.9Hz, 4H), 6.84 (d, J = 8.7Hz, 4H), 6.2 9(d,J＝7.6Hz,1H),6.20(d,J＝6.6Hz,1H),5.98(t,J _H-2F ＝8.2Hz,1H,),5.14(t,J＝5.2Hz,1H),4.16-4.05(m,1H),3.72(d,J＝12.7Hz,9H),3.63-3.51(m,1H ).MS(ESI ⁺ ):Calcd for C ₃₀ H ₂₉ F ₂ N ₃ O ₆ 565.20,found[M+Na] ⁺ 588.07.

Example 19: Methyl 2-(((4aR,6R,7aR)-6-(4-((bis(4-methoxyphenyl)(phenyl)methyl)amino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-oxotetrahydro-4H-furo[3,2-d][1,3,2]dioxophosphinylidene-2-yl)oxy)acetate (28a)

Phosphorus oxychloride (337 mg, 2.2 mmol) was dissolved in anhydrous DCM and placed in cold hydrazine at -15°C. Methyl glycolate and triethylamine were dissolved in anhydrous DCM and slowly added dropwise to the above reaction. The reaction was stirred for 3 hours to produce a white precipitate. Filter and set aside. 25 (360 mg, 0.64 mmol) and triethylamine were dissolved in DCM and slowly added dropwise to the filtrate of the filtered phosphate solution. After the addition was complete, it was stirred at room temperature for 1-2 hours. TLC detection and column chromatography purification were used to prepare compound 28a (200 mg, yield 44.7%). ¹ HNMR(400MHz,DMSO-d ₆ )δ8.67(s,1H),7.79-7.64(m,1H),7.31-7.24(m,2H),7.21-7.18(m,3H),7.12(d,J＝8.8Hz,4H),6.88(d,J＝8.8Hz,4H),6.45-6.25(m,2H),5.42-4.94(m,1H),4.87-4.62(m,4H),4.49-4.25(m,1H),3.74-3.71(m,9H).MS(ESI ^- ):Calcd forC ₃₃ H ₃₂ F ₂ N ₃ O ₁₀ P 699.18,found[MH] ^- 698.19。

Example 20: Methyl 2-(((4aR,6R,7aR)-6-(4-amino-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-oxotetrahydro-4H-furo[3,2-d][1,3,2]dioxophosphanylidene-2-yl)oxy)acetate (29a)

28a (560 mg, 0.8 mmol) was dissolved in anhydrous DCM, triethylsilane (650 mg, 5.6 mmol) was added, and trifluoroacetic acid (912 mg, 8 mmol) was slowly added dropwise at room temperature. The color of the system turned red, and the red color slowly faded after 30 minutes of reaction. TLC detected that all the raw materials reacted completely, and a relatively polar spot was produced. The solvent was evaporated and purified by silica gel column chromatography to obtain compound 29a (200 mg, yield 63%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.78 (s, 1H), 8.45 (d, J = 10.0 Hz, 1H), 8.07-7.87 (m, 1H), 6.42 (s, 1H), 6.06-5.93 (m, 1H), 5.50-5.13 (m, 1H), 4.86-4.78 (m, 3H) ),4.73-4.63(m,1H),4.55-4.49(m,1H),4.42-4.30(br,1H),3.83-3.60(m,3H).MS(ESI ⁺ ):Calcd forC ₁₂ H ₁₄ F ₂ N ₃ O ₈ P 397.05,found[M+H] ⁺ 397.85.

Example 21: Methyl 2-(((4aR,6R,7aR)-6-(4-(2-((tert-butyloxycarbonyl)amino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-oxotetrahydro-4H-furo[3,2-d][1,3,2]dioxophosphinilidene-2-yl)oxy)acetate (31a)

Boc-glycine (262 mg, 1.5 mmol) was dissolved in anhydrous DCM, and EDCI (325 mg, 1.7 mmol) and HOBt (607 mg, 1.7 mmol) were added under ice bath. After 30 min of reaction, 29a (397 mg, 1 mmol) and triethylamine (303 mg, 3 mmol) were added, and the reaction solution was stirred at room temperature for 3 h. After the reaction was complete, the organic layer was washed three times with 1N citric acid, saturated sodium bicarbonate, and saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated to obtain an oily substance, which was purified by column chromatography to obtain compound 31a (200 mg, yield 36%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.14(s,1H),8.38-8.07(m,1H),7.26-7.23(m,1H),7.11(t,J＝5.9Hz,1H),6.46(s,1H),5.54-5.10(m,1H),4.91-4.68(m,4 H),4.61-4.36(m,1H),3.80(d,2H),3.72(s,3H),1.38(s,9H).MS(ESI ⁺ ):Calcd for C ₁₉ H ₂₅ F ₂ N ₄ O ₁₁ P 554.12,found[M+H] ⁺ 554.98.

Example 22: Methyl 2-(((4aR,6R,7aR)-6-(4-(2-aminoacetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-oxotetrahydro-4H-furo[3,2-d][1,3,2]dioxophosphanylidene-2-yl)oxy)acetate trifluoroacetate (32a)

31a (200 mg, 0.36 mmol) was dissolved in anhydrous DCM, trifluoroacetic acid (2-3 mL) was added, and the mixture was stirred at room temperature for 1.5 h. The solvent was evaporated and ether was added to precipitate 200 mg of white solid 32a, which was used directly in the next step.

Example 23: Methyl 2-(((4aR,6R,7aR)-6-(4-((6R,7S,10S)-6-benzyl-7-hydroxy-10-isobutyl-2,2-dimethyl-4,8,11-trioxy-3-oxa-5,9,12-triazatetradec-14-amine)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-oxotetrahydro-4H-furo[3,2-d][1,3,2]dioxophosphinilidene-2-yl)oxy)acetate (33a)

Boc-Bestatin (159 mg, 0.39 mmol) was dissolved in anhydrous DCM, and EDCI (75 mg, 0.39 mmol), HOBt (53 mg, 0.39 mmol), and NMM (78.8 mg, 0.78 mmol) were added under ice bath, and stirred for 30 min. 33a (150 mg, 0.26 mmol) was dissolved in DMF, and then added to the reaction solution, and stirred at room temperature for 14 h. After the reaction was completed, DCM was added to dilute the reaction solution, and the solution was washed three times with water, 0.5 N citric acid, saturated sodium bicarbonate and saturated brine, dried over anhydrous sodium sulfate, filtered and evaporated to obtain an oily substance, which was purified by column chromatography to obtain a white solid 33a (125 mg, yield 57%).

Example 24: Methyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofuran[3,2-d][1,3,2]diphosphinylidene-2-yl)oxy)acetate (34a)

33a (120 mg, 0.142 mmol) was dissolved in anhydrous DCM, and trifluoroacetic acid (2 mL) was added. The mixture was stirred at room temperature for 1-2 h. After all the raw materials reacted completely, the solvent was evaporated. A small amount of THF was added to dissolve the product, and the pH value was adjusted to alkaline with saturated sodium bicarbonate. The product was extracted with ethyl acetate and directly dried over anhydrous sodium sulfate. The product was filtered and evaporated to obtain a white solid 34a (70 mg, yield 66.7%). ¹ HNMR (400 MHz, DMSO-d ₆ )δ8.59(t,J＝5.3Hz,1H),8.29-8.23(m,1H),7.85(d,J＝8.5Hz,1H),7.35-7.14(m,8H),6.46(s,1H),5.50-5.13(m,1H),4.90-4.69(m,5H),4.66-4.51 (m,1H),4.39-4.34(m,2 H),3.98-3.91(m,2H),3.79(d,J＝2.3Hz,1H),3.72(d,3H),3.20-3.12(m,1H),2.79(dd,J＝13.3,6.5Hz,1H),2.57-2.52(m,1H),1.69-1.40(m,4H),0.9 3-0.77(m,6H).MS(ESI ⁺ ): Calcd for C ₃₀ H ₃₉ F ₂ N ₆ O ₁₂ P 744.23, found[M+H] ⁺ 745.33.

The same procedure was used to obtain compounds 34b and 34c.

Example 25: Ethyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofuran[3,2-d][1,3,2]diphosphinylidene-2-yl)oxy)acetate (34b)

The yield of white solid 34b was 70%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.50 (s, 1H), 8.35-8.21 (m, 1H), 8.11 (d, J=7.5 Hz, 1H), 7.85-7.70 (m, 1H), 7.60-7.49 (m, 1H), 7.39-7.08 (m, 6H), 6.45 (s, 1H), 4.89-4.63 (m, 4H), 4.52-4.27 (m, 2H) ,4.19(dd,J＝13.8,6.8Hz,2H),3.52-3.44(m,1H),3.97(s,3H),2.99-2.85(m,1H),2.83-2.71(m,1H),1.69-1.42(m,3H),1.27-1.16(m,3H),0.97-0 .77(m,6H).MS(ESI ⁺ ):Calcd for C ₃₁ H ₄₁ F ₂ N ₆ O ₁₂ P 758.25,found[M+H] ⁺ 759.47.

Example 26: Phenyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofuran[3,2-d][1,3,2]dioxophosphinylidene-2-yl)oxy)acetate (34c)

The yield of white solid 34c was 72%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.58 (s, 1H), 8.27 (s, 1H), 7.92 (s, 1H), 7.52-7.01 (m, 10H), 6.45 (s, 1H), 5.22 (s, 2H), 5.00-4.62 (m, 3H), 4.37 (s, 2H), 4.04-3.71 (d, 3H), 2.81 (s, 2H), 2.58 (s, 1H), 1.78-1.10 (m, 9H), 0.87 (s, 7H). MS (ESI ⁺ ): Calcd for C ₃₆ H ₄₃ F ₂ N ₆ O ₁₂ P 820.26, found [M+H] ⁺ 821.57.

Preparation of compounds of formula IV:

Example 27: (2R,3R,4R,5R)-2-(5-fluoro-4-(((S)-8-isobutyl-12,12-dimethyl-7,10-dioxo-2,11-dioxa-6,9-diazotridecanoyl)amino)-2-oxypyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3,4-diacetate (36a)

Compound 18a (5.66 g, 12.5 mmol), 35 (3.3 g, 10 mmol) and triethylamine (1.25 g, 12.5 mmol) were dissolved in DCE, and then DMAP (1.2 g, 10 mmol) was added to the reaction solution, and the reaction solution was stirred at 60°C for 12 h. After the reaction was completed by TLC monitoring, DCE was evaporated, and the residue was extracted with ethyl acetate. The organic layer was washed with saturated brine three times, dried over anhydrous sodium sulfate, filtered and evaporated, ^and purified by column chromatography to obtain an oily liquid 36a (2.19 g, yield 34%). NMR (400MHz, DMSO-d6) δ10.61(s,1H),8.29(s,1H),7.84(d,J＝5.7Hz,1H),6.79(d,J＝8.3Hz,1H),5.77(d,J＝4.5Hz,1H),5.42(dd,J＝6.3,4.5Hz,1H),5.07(t,J＝ 6.3Hz, 1H),4.07(d,J＝9.2Hz,2H),3.86(td,J＝8.8,5.6Hz,1H),3.10(d,J＝18.9,2H),2.02(s,6H),1.71-1.68(m,2H),1.50-1.47(m,2H),1.33-1.28(m,10H) ,1.32(d,J＝5.2Hz 3H),0.81(d,J＝9.4,6H).ESI-MS m/z:666.21[M+Na] ⁺ .

Example 28: (2R,3R,4R,5R)-2-(4-(((3-((S)-2-amino-4-methylpentanamide)propoxy)carbonyl)amino)-5-fluoro-2-oxypyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3,4-diacetate trifluoroacetate (37a)

35a (2.19 g, 3.4 mmol) was dissolved in anhydrous DCM, trifluoroacetic acid (2 mL-3 mL) was slowly added dropwise under ice bath, and the temperature was slowly raised to room temperature and stirred for 2 h after the addition was complete. After TLC monitoring, the reaction of all the raw materials was complete, the solvent was evaporated, ether was added to precipitate the solid, and the white solid 37a (2.0 g, yield: 90%) was obtained by suction filtration, which was used directly in the next step without purification.

Example 29: (2R,3R,4R,5R)-2-(4-(((8S,11S,12R)-12-benzyl-11-hydroxy-8-isobutyl-16,16-dimethyl-7,10,14-trioxy-2,15-dioxa-6,9,13-triazaheptadecanoyl)amino)-5-fluoro-2-oxypyrimidin-1(2H)-yl)-5-methyltetrahydrofuran-3,4-diacetate (38a)

9 (1.21 g, 4.1 mmol) was dissolved in anhydrous DCM, HOBt (630 mg, 5.0 mmol) and EDCI (960 mg, 5.0 mmol) were added under ice bath, and stirred at 0°C for 0.5 h. A mixed solution of 37a (2.15 g, 3.28 mmol) and triethylamine (414.5 mg, 4.1 mmol) in DCM was added dropwise, and stirred at room temperature for 16 h. TLC monitored that all the raw materials reacted completely, and the reaction solution was washed three times with 1N citric acid, saturated sodium bicarbonate, and brine in sequence, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness, and white solid 38a (1.08 g, yield: 40%) was obtained by column separation.

Example 30: (6R,7S,10S)-6-Benzyl-7-hydroxy-10-isobutyl-2,2-dimethyl-4,8,11-trioxy-3-oxa-5,9,12-triazapentadecanoyl-15-yl (1-((2R,3R,4S,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (39a)

38a (500 mg, 0.61 mmol) was dissolved in methanol, followed by the addition of K ₂ CO ₃ (84 mg, 0.61 mmol), and the reaction solution was stirred at 25°C for 20 min. HCl/MeOH (1 M, 0.61 mL) was added to quench the reaction, the solvent was evaporated at room temperature, tetrahydrofuran was added to the residue, filtered, concentrated, and separated by silica gel column to obtain compound 39a (395 mg, yield: 88%).

Example 31: 3-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)propyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40a)

39a (395 mg, 0.537 mmol) was dissolved in anhydrous DCM, and ethyl acetate (2 mL) saturated with hydrochloric acid was added. The mixture was stirred at room temperature for 1-2 h. After all the starting materials reacted completely, the solvent was evaporated and ether was added to precipitate a white solid 40a (334 mg, yield: 92%). ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.22(t,J＝5.1Hz,1H),8.07-7.89(m,5H),7.31-7.22(m,5H),5.63(d,J＝3.2Hz,1H),4.22(q,J＝8.0Hz,1H),4.03-3.97(m,5H),3. 88-3.82(m,1H),3.65(t,J＝5.2Hz,2H),3.11-3.07(m,3H),2.90(d,J＝7.1Hz,2H),1.71-1.68(m,2H),1.58-1.39(m,3H),1.28(d,J＝5.52Hz,3H),0.85-0 .76(m,6H). ESI-MS m/z: 637.15 [M+H] ⁺ .

Compounds 40b, 40c and 40d were synthesized using the same synthetic method.

Example 32: 2-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)ethyl (1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40b)

The yield of white solid 40b was 82%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.24 (t, J = 3.8 Hz, 1H), 8.04-8.00 (m, 5H), 7.36-7.26 (m, 5H), 5.68 (s, 1H), 4.30 (q, J = 5.0 Hz, 2H), 4.11-4.04 (m, 5H), 3.91 (t, J = 4.2 Hz, 1H), 3.71 (t, J = 4.0 Hz, 1H), 3.58 (s, 1H), 3.41-3.31 (m, 3H), 2.99-2.87 (m, 2H), 1.62-1.49 (m, 3H), 1.32 (d, J = 4.2 Hz, 3H), 0.89-0.85 (m, 6H).

Example 33: 4-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)butyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40c)

The yield of white solid 40c was 87%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.18(t,J＝5.2Hz,1H),8.06-7.99(m,5H),7.32-7.20(m,5H),5.62(d,J＝3.1Hz,1H),4.22(q,J＝7.6Hz,1H),4.03-3.97(m,5H),3. 88-3.82(m,1H),3.65(t,J＝5.7Hz,1H),3.06-2.96(m,2H),2.90(d,J＝7.1Hz,2H),1.55-1.40(m,7H),1.27(d,J＝6.3Hz,3H),0.85-0.81(m,6H).ESI-MS m/z :651.25[M+H] ⁺ .

Example 34: 5-((R)-2-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)pentyl(1-((3R,4R,5R)-3,4-dihydroxy-5-methyltetrahydrofuran-2-yl)-5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl)carbamate (40d)

The yield of white solid 40d was 89%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.15 (t, J = 5.2 Hz, 1H), 8.07-7.99 (m, 5H), 7.32-7.22 (m, 5H), 5.63 (d, J = 3.3 Hz, 1H), 4.22 (q, J = 7.7 Hz, 1H), 4.04-3.97 (m, 5H), 3.88-3.82 (m, 1H), 3.65 (t, J = 5.1 Hz, 2H), 3.03-2.93 (m, 2H), 2.89 (d, J = 7.2 Hz, 2H), 1.56-1.33 (m, 7H), 1.27 (d, J = 6.28 Hz, 5H), 0.85-0.80 (m, 6H). ESI-MS m/z:665.27[M+H] ⁺ .

Example 35: Activity of target compounds on CD13:

CD13 enzyme and substrate L-leucyl-p-nitroanilide were purchased from Sigma

Preparation of phosphate buffer: Dissolve Na ₂ HPO ₄ .12H ₂ O (12.89 g) and NaH ₂ PO ₄ .2H ₂ O (2.18 g) in freshly boiled and cooled distilled water, dilute to 1000 mL to obtain a 50 mM phosphate buffer solution with a pH of 7.2, and place at room temperature for use.

The CD13 enzyme was dissolved in the prepared buffer to prepare a 0.1 IU/mL solution for later use.

The substrate was dissolved in DMSO to prepare a solution with a concentration of 16 mmol/mL, and each solution was placed in a refrigerator for later use.

The positive control and target compound were prepared into solutions with different concentration gradients using buffer.

40 μL of target compound with different gradient concentrations, 5 μL of substrate, and 10 μL of APN enzyme solution were added to a 96-well plate, and the phosphate buffer solution was added to 200 μL. The 100% group did not contain inhibitors, but only contained 5 μL of substrate and 10 μL of APN enzyme solution, which were filled to 200 μL with buffer. The blank group did not contain enzymes and inhibitors, but only contained 5 μL of substrate, which was filled to 200 μL with buffer. Incubate at 37°C for 0.5 h, and measure the absorbance at a wavelength of 405 nm. The compound inhibition rate was calculated according to the following formula:

According to the concentration of the compound and the corresponding inhibition rate, the curve was fitted using Origin7.5 software to calculate the IC ₅₀ of the tested compound.

Table 1. Inhibitory activity of target compounds on CD13 in vitro (IC ₅₀ : μM)

^aThe values in the table are the mean values of three experiments, SD < 20%.

The data in Table 1 show that the designed and synthesized compounds all have significant CD13 inhibitory activity, and the inhibitory activity is significantly better than that of the positive drug Ubenimex, indicating that these compounds have a certain targeting activity on CD13.

Example 36: Determination of the activity of the target compound in inhibiting cell proliferation in vitro:

The target compound was tested for its in vitro inhibitory cell proliferation activity using the resazurin method. Human multiple myeloma MM.1S and U266 were taken and cultured conventionally. Logarithmic growth phase cells were used in the experiment. The cell suspension of the above cells was taken to count the number of cells under an inverted microscope, and the culture medium was added to adjust the cell number to 1×10 ⁵ /mL. A 96-well cell culture plate was used for cell inoculation and drug experiments. The peripheral wells were not used (filled with sterile PBS). A blank control group, a negative control group, a positive control group and a drug experimental group were set up. The blank control group only added 150μL/well of cell culture medium, the negative control group was inoculated with 100μL/well of cell suspension and added with 50μL/well of cell culture medium, and the positive control group was inoculated with 100μL/well of cell suspension and added with positive control drugs (NUC1031, ubenimex, NUC 1031 + ubenimex (1:1)) solution 50μL/well, the drug experimental group was inoculated with cell suspension 100μL/well and added with the test compound (respectively 13a, 13b, 13c, 13d, 13e, 13f, 22b, 22a, 34a, 34b, 34c) solution 50μL/well, the positive control group and the drug experimental group were set up with 8 different final drug concentrations: 0.03, 0.1, 0.3, 1, 3, 10, 30, 100μmol·L ^-1 , and 3 parallel wells were set up for each drug concentration. After the drug was added, the 96-well cell culture plate was cultured at 37°C, 5% _CO2 and saturated humidity for 72h, 10μL of resazurin (1mg/mL) was added to each well, and the incubation continued for 3h, and then the fluorescence value was measured at ex.560/em.590nm using an ELISA reader. The inhibition rate was calculated. GI ₅₀ is the inhibition rate of 8 different concentrations. The value obtained after software fitting is shown in Tables 2-3.

Table 2: The results of the inhibitory activity of the target compounds on the proliferation of multiple myeloma cells MM1.S (GI ₅₀ , μM ^a )

^aThe values in the table are the mean values of three experiments, with SD < 20%.

Table 3: The results of the inhibitory activity of the target compounds on the proliferation of multiple myeloma cells MM1.S (GI ₅₀ , μM ^a )

^aThe values in the table are the mean values of three experiments, with SD < 20%.

The test data in the above table show that the target compounds 13a, 13b, 13c, 13d, 13e, 13f, 22b, 22a, 22c, 34a, 34b, and 34c all showed significant inhibitory activity against prostate cancer 22RV1 and multiple myeloma MM1.S, and the inhibitory IC ₅₀ of some compounds was in the low nanomolar level. In addition, the inhibitory activity of 13e, 13f, 34a, 34b, and 34c was better than that of positive drugs Ubenimex and NUC-1031. The inhibitory activity of compound 13b against multiple myeloma MM1.S was not only better than the two parent drugs Ubenimex and NUC-1031, but also significantly better than the combination of Ubenimex and NUC-1031, indicating that a significant synergistic effect can be achieved by designing a small molecule coupling molecule 13b.

Example 37: Preliminary stability of the target compound in human plasma:

1. Experimental materials and protocols: Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc.), Agilent 1260 UV-visible detector (Agilent Technologies, Inc.), high-speed centrifuge, Mettler-Toledo AL104-IC electronic balance (Mettler, Switzerland), microporous filter membrane (organic membrane 0.22 μm), human plasma, chromatographic grade methanol, and chromatographic grade acetonitrile.

The test compound was prepared into a 4 mg/mL aqueous solution with acetonitrile/water for standby use. 25 μL of the compound of this concentration was taken from each group and incubated with 225 μL of human plasma at 37°C. Samples were taken at the corresponding set time points, 300 μL of acetonitrile and 300 μL of aqueous solution were added, vortexed for 30 seconds and centrifuged in a high-speed centrifuge at 12,000 rpm. After centrifugation, the supernatant was filtered with a 0.22 μm microporous filter membrane, and 20 μL was taken for injection analysis. The liquid phase detection conditions were: Alltima C ₁₈ column (specification: 5 μm, 4.6 mm × 250 mm); detection wavelength: 262 nm; mobile phase: 25% acetonitrile/75% water (containing 0.1% triethylamine and 0.15% trifluoroacetic acid); injection volume was 20 μL; column temperature was 25°C, flow rate was 1 mL/min, and detection time was 15 min.

2. Test results:

As shown in Figure 1, compound 13b has good stability in plasma and is degraded by less than 50% within 24 h.

Example 38: 13a and 13b inhibit the growth of H22 liver cancer transplanted tumors in mice.

(1) Establishment of mouse transplanted tumor model

Materials: Kunming mice (female, 4-5 weeks old); mice bearing liver cancer tumors were purchased from the Institute of Materia Medica, Shandong Academy of Medical Sciences. They were kept in a constant temperature and humidity environment according to standard requirements and controlled by a 12-hour light-dark cycle. Animal experiments were approved by the Experimental Animal Committee of Weifang Medical College and complied with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised in 1996).

A certain number of Kunming mice were purchased according to the experimental requirements and placed in the laboratory for balanced feeding for 1 week. The H22 ascites cell suspension of the mice was taken and counted with a counting plate under a microscope to adjust the number of cells. Under strict sterile conditions, the cells were uniformly inoculated under the right armpit of the mice, and the cell inoculation amount was 1.0-2.0×10 ⁷ /mL, 0.2mL/mouse. After inoculation, the tumor formation of the mice was observed, and the mice were randomly grouped when the tumor grew to nearly 100mm ^3. The drug was administered according to the designed dosage and route, and the drug was administered continuously for 5 days, with two days of drug withdrawal, for a total of two weeks. The weight of the mice was recorded every 2-3 days during the entire drug administration period. After two weeks of administration, the mice were killed by dislocation of the neck, the tumor tissue was completely removed, the tumor mass was weighed, and the tumor inhibition rate was calculated.

Tumor inhibition rate (%) = (1-average tumor weight of treatment group/average tumor weight of control group) × 100%.

(2) Test results:

As shown in Figures 2 and 3, compounds 13a and 13b have significant inhibitory activity on the growth of mouse liver cancer cell line H22 transplanted tumors. The activity of 13a and 13b at a dose of 0.15 mmol/kg is comparable to the anti-tumor activity of the positive drug NUC1031 at a dose of 0.19 mmol/kg.

The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (5)

1. A novel small molecule coupling compound based on aminopeptidase N/CD13, characterized in that the novel small molecule coupling compound based on aminopeptidase N/CD13 is at least one of the compounds shown in 1) to 5) below: 1) Benzyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphonyl)-L-alaninate, the structural formula of which is: ; 2) Isopropyl ((((2R,3R,5R)-5-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(2-methoxy-2-oxoethoxy)phosphonyl)-L-alaninate, the structural formula of which is: ; 3) Methyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy4H-tetrahydrofuran[3,2-d][1,3,2]diphosphinediylidene-2-yl)oxy)acetate, the structural formula of which is: ; 4) Ethyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy-4H-tetrahydrofuran[3,2-d][1,3,2]diphosphinediylidene-2-yl)oxy)acetate, the structural formula of which is: ; 5) Phenyl 2-(((4aR,6R,7aR)-6-(4-(2-((S)-2-((2S,3R)-3-amino-2-hydroxy-4-phenylbutyrylamino)-4-methylpentanoylamino)acetylamino)-2-oxypyrimidin-1(2H)-yl)-7,7-difluoro-2-hydroxy-4H-tetrahydrofuran[3,2-d][1,3,2]diphosphinediylidene-2-yl)oxy)acetate, the structural formula of which is: . 2. Use of the compound according to claim 1 in the preparation of a drug for preventing or treating tumors, characterized in that the tumor is selected from multiple myeloma, leukemia or solid tumors. 3. The use according to claim 2, characterized in that the solid tumor is lymphoma. 4. A pharmaceutical composition, characterized in that the compound according to claim 1 is used as an active ingredient, and the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers or excipients. 5. Use of the pharmaceutical composition according to claim 4 in the preparation of a pharmaceutical preparation for treating tumor diseases.