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HK1078473B - Inhibitors of histone deacetylase - Google Patents

Inhibitors of histone deacetylase Download PDF

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Publication number
HK1078473B
HK1078473B HK05110453.4A HK05110453A HK1078473B HK 1078473 B HK1078473 B HK 1078473B HK 05110453 A HK05110453 A HK 05110453A HK 1078473 B HK1078473 B HK 1078473B
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Hong Kong
Prior art keywords
alkyl
compound
compounds
amino
formula
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HK05110453.4A
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Chinese (zh)
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HK1078473A1 (en
Inventor
Kristof Van Emelen
Marc Gustaaf Celine Verdonck
Sven Franciscus Anna Van Brandt
Patrick René ANGIBAUD
Lieven Meerpoel
Alexey Borisovich Dyatkin
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Janssen Pharmaceutica N.V.
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Priority claimed from PCT/EP2003/002515 external-priority patent/WO2003075929A1/en
Publication of HK1078473A1 publication Critical patent/HK1078473A1/en
Publication of HK1078473B publication Critical patent/HK1078473B/en

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Description

Histone deacetylase inhibitors
The present invention relates to compounds having enzymatic activity that inhibit Histone Deacetylase (HDAC). The invention further relates to processes for their preparation, compositions comprising them and their use in inhibiting HDACs in vitro and in vivo and as medicaments, for example as medicaments for inhibiting proliferative conditions, such as cancer and psoriasis.
In all eukaryotic cells, genomic DNA in chromatin binds to histones to form nucleosomes. Each nucleosome is composed of protein octamers formed by two groups of H2A, H2B, H3, and H4 histones. DNA is wound around the protein core and the basic amino acids of the histone interact with the negatively charged phosphate groups of DNA. The most common post-translational modification of core histones is reversible acetylation of the epsilon-amino group of a conserved, strongly basic N-terminal lysine residue. The steady state of histone acetylation is established by a dynamic equilibrium between competing histone acetyltransferases and histone deacetylases, referred to herein as "HDACs". Acetylation and deacetylation of histones has long been implicated in transcriptional regulation. Recent gene clones encoding different histone acetyltransferases and histone deacetylases have provided a possible explanation for the relationship between histone acetylation and transcriptional regulation. Reversible acetylation of histones can lead to chromatin remodelling and thus become a regulatory mechanism for gene transcription. Generally, histone hyperacetylation favors gene expression, while histone deacetylation is associated with transcriptional repression. Histone acetyltransferases show the role of transcriptional co-activators, while histone deacetylases belong to the transcriptional repression pathway.
A dynamic balance between histone acetylation and deacetylation is essential for normal growth of cells. Inhibition of histone deacetylase results in cell cycle arrest, cell differentiation, apoptosis and reversal of the transformed phenotype. HDAC inhibitors therefore have strong therapeutic potential in the treatment of cell proliferative diseases or conditions. (Marks et al, Nature Review: Cancer 1: 194-202, 2001).
Studies with Histone Deacetylase (HDAC) inhibitors have shown that in fact these enzymes play an important role in cell proliferation and differentiation. The inhibitor trichostatin a (tsa) causes a arrest in the cell cycle both at the G1 and G2 stages, reverses the transformed phenotype of the different cell lines, and induces differentiation of Friend leukemia cells and other cells. TSA (and suberoylanilide hydroxamic acid SAHA) has been reported to inhibit cell growth, induce terminal differentiation, and prevent tumor formation in mice (Finnin et al, Nature, 401: 188-193, 1999).
Trichostatin a has also been reported to be useful in the treatment of fibrosis, such as liver fibrosis and cirrhosis (gerts et al, european patent application EP 0827742, published on 11/3/1998).
The general formula Cy-L is disclosed in the patent application WO01/38322 published 5/31/2001, together with other inhibitors1-Ar-Y1-C (O) -NH-Z histone deacetylase inhibitors, compositions and methods for treating cell proliferative diseases and conditions are provided.
Published patent application WO01/70675 at 9/27 mesh 2001 discloses Cy-X-Y1-W and Cy-S (O)2-NH-Y3-inhibitors of histone deacetylase, and further provides compositions and methods for treating cell proliferative diseases and conditions.
The problem to be solved is to provide histone deacetylase inhibitors with high enzymatic activity, which also show superior properties such as cellular activity and increased bioavailability, preferably oral bioavailability, with little or no side effects.
The novel compounds of the present invention solve the above problems. These compounds differ from the prior art structures.
The compounds of the present invention show excellent in vitro inhibition of histone deacetylase enzymatic activity. The present compounds have superior properties in terms of cell activity and have specific properties of inhibiting cell cycle processes at the G1 and G2 checkpoints (p21 induction capacity). The compounds of the invention show good metabolic stability and high bioavailability, in particular they show oral bioavailability.
The invention relates to compounds of formula (I)
The N-oxide forms, the pharmaceutically acceptable addition salts and the stereochemically isomeric forms thereof, wherein
n is 0, 1, 2 or 3, and when n is 0, is a direct bond;
each Q is nitrogen or
Each X is nitrogen or
Each Y is nitrogen or
Each Z is nitrogen or
R1Is C (O) NR5R6、-N(H)C(O)R7、-C(O)-C1-6Alkanediyl (alkanediyl) SR7、-NR8C(O)N(OH)R7、-NR8C(O)C1-6Alkanediyl SR7、-NR8C(O)C＝N(OH)R7Or another Zn-chelating group, wherein R5And R6Each independently selected from hydrogen, hydroxy, C1-6Alkyl, hydroxy C1-6Alkyl, amino C1-6Alkyl or aminoaryl;
R7independently selected from hydrogen, C1-6Alkyl radical, C1-6Alkylcarbonyl, aryl C1-6Alkyl radical, C1-6An alkyl pyrazinyl, a pyridine cyclic ketone (pyridinone), a pyrrolidine cyclic ketone (pyrrolidinone), or a methylimidazolyl group;
R8independently selected from hydrogen or C1-6An alkyl group;
R2is hydrogen, halogen, hydroxyl, amino, nitro, C1-6Alkyl radical, C1-6Alkoxy, trifluoroMethyl, di (C)1-6Alkyl) amino, hydroxyamino, or naphthalenesulfonylpyrazinyl;
R3is hydrogen, C1-6Alkyl, aryl C2-6Alkenediyl, furancarbonyl, naphthylcarbonyl, -C (O) phenyl R9、C1-6Alkylaminocarbonyl, aminosulfonyl, arylaminosulfonyl, aminosulfonylamino, di (C)1-6Alkyl) aminosulfonylamino, arylaminosulfonamino, aminosulfonylamino C1-6Alkyl, di (C)1-6Alkyl) aminosulfonylamino C1-6Alkyl, arylaminosulfonylamino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl, trihalo C1-6Alkylsulfonyl, di (aryl) C1-6Alkylcarbonyl, phenylthio C1-6Alkylcarbonyl, pyridylcarbonyl or aryl C1-6An alkyl-carbonyl group, a carboxyl group,
wherein each R9Independently selected from phenyl; is one, two or three independently selected from halogen, amino, C1-6Alkyl radical, C1-6Alkoxy, hydroxy C1-4Alkyl, hydroxy C1-4Alkoxy, amino C1-4Alkoxy, di (C)1-4Alkyl) amino C1-4Alkoxy, di (C)1-6Alkyl) amino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical (C)1-6Alkyl) amino C1-6Alkyl, hydroxy C1-4Alkyl piperazinyl C1-4Alkyl radical, C1-4Alkoxypiperidyl radical C1-4Alkyl, hydroxy C1-4Alkoxy radical C1-4Alkyl piperazinyl, C1-4Alkyl piperazinyl C1-4Alkyl, di (hydroxy C)1-4Alkyl) amino C1-4Alkyl, pyrrolidinyl C1-4Alkoxy, morpholinyl C1-4Alkoxy, or morpholinyl C1-4Phenyl substituted with a substituent of alkyl; a phenylthio group; or is divided into two (C)1-4Alkyl) amino C1-4Alkoxy, di (C)1-6Alkyl) amino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical (C)1-6Alkyl) amino C1-6Alkyl, pyrrolidinyl C1-4Alkoxy radical, C1-4Alkylpiperidinyl C1-4Alkyl, di (hydroxy C)1-4Alkyl) amino C1-4Alkyl or morpholinyl C1-4Alkoxy-substituted phenylthio.
R4Is hydrogen, hydroxy, amino, hydroxy C1-6Alkyl radical, C1-6Alkyl radical, C1-6Alkoxy, aryl C1-6Alkyl, aminocarbonyl, hydroxycarbonyl, amino C1-6Alkyl, aminocarbonyl C1-6Alkyl, hydroxy carbonyl C1-6Alkyl, hydroxyaminocarbonyl, C1-6Alkoxycarbonyl group, C1-6Alkylamino radical C1-6Alkyl or di (C)1-6Alkyl) amino C1-6An alkyl group;
when R is3And R4When on the same carbon atom, R3And R4Taken together may form a divalent radical of the formula
-C(O)-NH-CH2-NR10- (a-1)
Wherein R is10Is hydrogen or aryl;
when R is3And R4When on adjacent carbon atoms, R3And R4Taken together may form a divalent radical of the formula
＝CH-CH＝CH-CH＝ (b-1)；
The aryl is phenyl, or is selected from one or more of halogen and C1-6Alkyl radical, C1-6Phenyl substituted with substituents of alkoxy, trifluoromethyl, cyano or hydroxycarbonyl.
The term "histone deacetylase inhibitor" or "inhibitor of histone deacetylase" is used to denote a compound which is capable of interacting with a histone deacetylase and inhibiting its activity, more specifically its enzymatic activity. Inhibiting the enzymatic activity of histone deacetylase means reducing the ability of histone deacetylase to remove acetyl groups from histone. Preferably, such inhibition is specific, i.e., the histone deacetylase inhibitor is capable of reducing the ability of the histone deacetylase to remove acetyl groups from histones at a concentration that is lower than the concentration of the inhibitor required to produce some other unrelated biological effect.
As used in the above definitions and below, halogen is a generic term for fluorine, chlorine, bromine and iodine; c1-4Alkyl is defined as a straight or branched chain saturated hydrocarbon group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, butyl, 1-methylethyl, 2-methylpropyl, and the like; c1-6The alkyl group comprising C1-4Alkyl and its higher homologues having 5 to 6 carbon atoms, such as pentyl, 2-methyl-butyl, hexyl, 2-methylpentyl and the like; c1-6Alkanediyl is defined as a divalent straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms, such as methylene, 1, 2-ethanediyl, 1, 3-propanediyl, 1, 4-butanediyl, 1, 5-pentanediyl, 1, 6-hexanediyl and branched chain isomers thereof, such as 2-methylpentanediyl, 3-methylpentanediyl, 2-dimethylbutanediyl, 2, 3-dimethylbutanediyl and the like; trihalo C1-6Alkyl is defined as C containing three identical or different halogen substituents1-6Alkyl groups such as trifluoromethyl; c2-6Alkenediyl is defined as a divalent straight or branched chain hydrocarbon group having one double bond and having 2 to 6 carbon atoms, such as ethenediyl, 2-propenediyl, 2-butenediyl, 2-pentenediyl, 3-methyl-2-butenediyl, and the like; and aminoaryl is defined as aryl substituted with amino.
The term "another Zn-chelating group" refers to a group capable of interacting with a Zn-ion that may be located at an enzyme binding site.
Pharmaceutically acceptable addition salts include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The meaning of the above pharmaceutically acceptable acid addition salts includes the therapeutically active non-toxic acid addition salt forms which the compounds of formula (I) are able to form. Compounds of formula (I) having basic properties may be converted into their pharmaceutically acceptable acid addition salts by treating the basic form with a suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids, e.g., hydrochloric or hydrobromic acid; sulfuric acid; nitric acid; acids such as phosphoric acid; organic acids such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid (i.e., succinic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-methylbenzenesulfonic acid, cyclohexanesulfamic acid, salicylic acid, p-amino-salicylic acid, pamoic acid (pamoic acid), and the like.
The compounds of formula (I) having acidic properties may be converted into their pharmaceutically acceptable base addition salts by treating the acid form with a suitable organic or inorganic base. Suitable basic salt forms include, for example, ammonium, alkali metal and alkaline earth metal salts, such as lithium, sodium, potassium, magnesium, calcium and the like, salts with organic bases, such as benzathine, N-methyl-D-glucamine, hydrabamine, and salts with amino acids, such as arginine, lysine and the like.
The term "acid or base addition salt" also includes the hydrates and solvent addition forms which the compounds of formula (I) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
The term "stereochemically isomeric forms of a compound of formula (I)" as used herein is defined as all possible compounds consisting of the same atoms with the same bond sequence but having different possible three-dimensional structures which the compounds of formula (I) may have, said three-dimensional structures being not interconvertible. Unless otherwise indicated or indicated, the chemical designation of a compound encompasses the mixture of all possible stereochemically isomeric forms which said compound possesses. The mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of the compounds of formula (I) either in pure form or as an admixture with each other are intended to be embraced within the scope of the present invention.
The meaning of the N-oxide forms of the compounds of formula (I) includes compounds of formula (I) wherein one or more nitrogen atoms are oxidized to the so-called N-oxide, in particular N-oxides wherein one or more piperidine-, piperazine-or pyridazinyl-nitrogens are N-oxidized.
Some of the compounds of formula (I) may also exist in their tautomeric form. Such forms, although not explicitly indicated in the above formula, are also included within the scope of the present invention.
The term "compound of formula (I)" as used in any of the following text also includes the pharmaceutically acceptable addition salts and all stereoisomeric forms.
The terms "histone deacetylase" and "HDAC" as used herein refer to any member of the family of enzymes that remove an acetyl group from the epsilon-amino group of a lysine residue located at the N-terminus of histone proteins. Unless otherwise indicated in context, the term "histone" refers to any histone protein derived from any species, including H1, H2A, H2B, H3, H4, and H5. Human HDAC proteins or gene products include, but are not limited to, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9, and HDAC-10. Histone deacetylases may also be derived from protozoan or fungal sources.
The first group of compounds of interest comprises applying one or more compounds of formula (I) as defined below:
a) n is 0 or 1;
b) each Q is
c)R1is-C (O) NH (OH) or-NHC (O) C1-6An alkanediyl SH;
d)R2is hydrogen or nitro;
e)R3is C1-6Alkyl, aryl C2-6Alkenediyl, furancarbonyl, naphthylcarbonyl, C1-6Alkylamino radicalCarbonyl, aminosulfonyl, di (C)1-6Alkyl) aminosulfonylamino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl, trihalo C1-6Alkylsulfonyl, di (aryl) C1-6Alkylcarbonyl, phenylthio C1-6Alkylcarbonyl, pyridylcarbonyl or aryl C1-6An alkylcarbonyl group;
f)R4is hydrogen;
g) when R is3And R4When on the same carbon atom, R3And R4Taken together may form a divalent radical of formula (a-1) wherein R10Is aryl;
when R is3And R4When on adjacent carbon atoms, R3And R4Together, a divalent group of formula (b-1) may be formed.
A second group of compounds of interest comprises applying one or more compounds of formula (I) as defined below:
a) n is 1;
b) each Q is
c) Each Z is nitrogen;
d)R1is-C (O) NH (OH);
e)R2is hydrogen;
f)R3is naphthyl carbonyl, C1-12Alkylsulfonyl or di (aryl) C1-6An alkylcarbonyl group;
g)R4is hydrogen;
a third group of interesting compounds comprises compounds of formula (I), wherein R2Is hydrogen.
Fourth group of compounds of interestThe compounds include compounds of formula (I) wherein R1is-C (O) NH (OH).
A fifth group of compounds of interest includes compounds of formula (I), wherein R2Is hydrogen and R1is-C (O) NH (OH).
A sixth group of compounds of interest comprises applying one or more compounds of formula (I) as defined below:
a)R1is C (O) NR5R6、-C(O)-C1-6Alkanediyl SR7、-NR8C(O)N(OH)R7、-NR8C(O)C1-6Alkanediyl SR7、-NR8C(O)C＝N(OH)R7Or another Zn-chelating group, wherein R5And R6Each independently selected from hydrogen, hydroxy C1-6Alkyl or amino C1-6An alkyl group;
b)R2is hydrogen, halogen, hydroxyl, amino, nitro, C1-6Alkyl radical, C1-6Alkoxy, trifluoromethyl or di (C)1-6Alkyl) amino;
c)R3is hydrogen, C1-6Alkyl, aryl C2-6Alkenediyl, furancarbonyl, naphthylcarbonyl, -C (O) phenyl R9、C1-6Alkylaminocarbonyl, aminosulfonyl, arylaminosulfonyl, aminosulfonylamino, di (C)1-6Alkyl) aminosulfonylamino, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl or pyridylcarbonyl wherein each R is9Independently selected from phenyl; is one, two or three independently selected from halogen, C1-6Alkyl radical, C1-6Phenyl substituted with a substituent of alkoxy; or a phenylthio group;
d)R4is hydrogen, hydroxy, amino, hydroxy C1-6Alkyl radical, C1-6Alkyl radical, C1-6Alkoxy, aryl C1-6Alkyl, aminocarbonyl, amino C1-6Alkyl radical, C1-6Alkylamino radical C1-6Alkyl or di (C)1-6Alkyl) amino C1-6An alkyl group.
One preferred group of compounds includes compounds of the formula (I) wherein
R1Is C (O) NR5R6、-C(O)-C1-6Alkanediyl SR7、-NR8C(O)N(OH)R7、-NR8C(O)C1-6Alkanediyl SR7、-NR8C(O)C＝N(OH)R7Or another Zn-chelating group, wherein R5And R6Each independently selected from hydrogen, hydroxy C1-6Alkyl or amino C1-6An alkyl group;
R2is hydrogen, halogen, hydroxyl, amino, nitro, C1-6Alkyl radical, C1-6Alkoxy, trifluoromethyl or di (C)1-6Alkyl) amino;
R3is hydrogen, C1-6Alkyl, aryl C2-6Alkenediyl, furancarbonyl, naphthylcarbonyl, -C (O) phenyl R9、C1-6Alkylaminocarbonyl, aminosulfonyl, arylaminosulfonyl, aminosulfonylamino, di (C)1-6Alkyl) aminosulfonylamino, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl or pyridylcarbonyl wherein each R is9Independently selected from phenyl; is one, two or three independently selected from halogen, C1-6Alkyl radical, C1-6Phenyl substituted with a substituent of alkoxy; a phenylthio group; and
R4is hydrogen, hydroxy, amino, hydroxy C1-6Alkyl radical, C1-6Alkyl radical, C1-6Alkoxy, aryl C1-6Alkyl, aminocarbonyl, amino C1-6Alkyl radical, C1-6Alkylamino radical C1-6Alkyl or di (C)1-6Alkyl) amino C1-6An alkyl group.
A further preferred group of compounds comprises the compounds of the following formula (I),
wherein n is 0 or 1; each Q isR1is-C (O) NH (OH) or-NHC (O) -C1-6An alkanediyl SH; r2Is hydrogen or nitro; r3Is C1-6Alkyl, aryl C2-6Alkenediyl, furancarbonyl, naphthylcarbonyl, C1-6Alkylaminocarbonyl, aminosulfonyl, di (C)1-6Alkyl) aminosulfonylamino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl, trihalo C1-6Alkylsulfonyl, di (aryl) C1-6Alkylcarbonyl, phenylthio C1-6Alkylcarbonyl, pyridylcarbonyl or aryl C1-6An alkylcarbonyl group; r4Is hydrogen; when R is3And R4When on the same carbon atom, R3And R4Taken together may form a divalent radical of formula (a-1) wherein R10Is aryl; or when R is3And R4When on adjacent carbon atoms, R3And R4Together, a divalent group of formula (b-1) may be formed.
A more preferred group of compounds comprises the compounds of formula (I),
wherein n is 1; each Q isEach Z is nitrogen; r1is-C (O) NH (OH); r2Is hydrogen; r3Is naphthyl carbonyl, C1-12Alkylsulfonyl or di (aryl) C1-6An alkylcarbonyl group; r4Is hydrogen.
The most preferred compounds are compounds No. 18, 5 and 24.
The compounds of formula (I), their pharmaceutically acceptable salts and the N-oxides and stereochemically isomeric forms thereof may be prepared in a conventional manner.
General synthetic routes include, as examples:
a) wherein R is1Hydroxamic acids of formula (I) that are-C (O) NH (OH), referred to as compounds of formula (I-a), can be prepared by reacting an intermediate of formula (II) with a suitable acid, such as trifluoroacetic acid. The reaction is carried out in a suitable solvent, such as methanol.
b) Intermediates of formula (II) may be prepared by reacting an intermediate of formula (III) with an intermediate of formula (IV) in the presence of a suitable reagent such as N' - (ethylcarbodiimide)) -N, N-dimethyl-1, 3-propanediamine, monohydrochloride (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). The reaction may be carried out in a suitable solvent such as a mixture of DCM and THF.
c) Intermediates of formula (III) may be prepared by reacting an intermediate of formula (V) with a suitable base such as NaOH in the presence of a suitable solvent such as ethanol.
The compounds of formula (I) may also be advantageously prepared using solid phase synthesis techniques. Typically, solid phase synthesis involves reacting an intermediate with a polymeric support during synthesis. The polymer-supported intermediate can then be subjected to a number of synthetic steps. After each step, the resin was filtered and washed several times with various solvents to remove impurities. In each step, the resin can be split to react with various intermediates in the next step to synthesize a large number of compounds. After the final step of the method is performed, the resin is treated by a reagent or procedure to remove the resin from the sample. A more detailed explanation of The techniques used in solid phase chemistry is described, for example, in The composite Index (B.Bunin, academic Press) and Novabiochem's 1999 Catalogue & Peptide Synthesis handbook (Novabiochem AG, Switzerland), both of which are incorporated herein by reference.
The compounds of formula (I) and some intermediates may have at least one chiral center in their structure. The chiral center may exist in either the R or S configuration.
The compounds of formula (I) prepared by the above methods are typically racemic mixtures of enantiomers, which can be separated from each other by analytical methods known in the art. Racemic compounds of formula (I) can be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. The diastereomeric salt forms are subsequently separated, for example, by selective or fractional crystallization, and the enantiomers are separated therefrom by a base. Another method for separating the enantiomeric forms of the compounds of formula (I) includes liquid chromatography using a chiral stationary phase. The pure stereochemically isomeric forms may also be prepared from the corresponding pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction proceeds stereospecifically. Preferably, if a particular stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These processes will advantageously employ enantiomerically pure starting materials.
The compounds of formula (I), their pharmaceutically acceptable acid addition salts and their stereoisomeric forms have important pharmacological properties because they have an inhibitory effect on Histone Deacetylases (HDACs).
The present invention provides a method of inhibiting the abnormal growth of cells, including amebocytes, by administering an effective amount of a compound of the present invention. Abnormal growth of a cell refers to growth that is not associated with normal regulatory mechanisms (e.g., loss of contact inhibition). It includes direct inhibition of tumor growth by promoting growth arrest, terminal differentiation, and/or apoptosis of cancer cells, as well as indirect inhibition of tumor growth by inhibiting the formation of new blood vessels by the tumor.
The invention also provides a method of inhibiting tumor growth by administering to a subject, such as a mammal (particularly a human) in need of such treatment an effective amount of a compound of the invention. In particular, the invention provides a method of inhibiting tumor growth by administering an effective amount of a compound of the invention. Examples of tumors that can be inhibited include, but are not limited to, lung cancer (e.g., pancreatic cancer and including non-small cell lung cancer), pancreatic cancer (e.g., pancreatic cancer such as exocrine pancreatic cancer), colon cancer (colorectal cancer such as colon adenocarcinoma and colon adenoma), prostate cancer including advanced diseases, lymphohematopoietic tumors (e.g., acute lymphocytic leukemia, B-cell lymphoma, Burkitt's lymphoma), myeloid leukemia (e.g., Acute Myelogenous Leukemia (AML)), thyroid follicular cancer, myelodysplastic syndrome (MDS), mesenchymal cell-derived tumors (e.g., fibrosarcoma and rhabdomyosarcoma), melanoma, teratocarcinoma, neuroblastoma, glioma, benign tumors of the skin (e.g., keratoacanthoma), breast cancer (e.g., advanced breast cancer), renal cancer, ovarian cancer, bladder cancer, and epidermal carcinoma.
The compounds of the invention may be used for other therapeutic purposes, for example:
a) administering a compound according to the invention before, during and after irradiation of a tumor to treat cancer to promote sensitivity of the tumor;
b) treating joint diseases and bone conditions such as rheumatoid arthritis, osteoarthritis, juvenile arthritis, gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus;
c) inhibiting smooth muscle cell proliferation including vascular proliferative disorders, atherosclerosis, and restenosis (restenosis), among others;
d) treating inflammatory and dermatological diseases such as ulcerative colitis, crohn's disease, allergic rhinitis, graft versus host reaction disease, conjunctivitis, asthma, ARDS, behcet's disease, transplant rejection, utiaria, allergic dermatitis, alopecia areata, scleroderma, exanthema, eczema, dermatophytosis, acne, diabetes, systemic lupus erythematosus, kawasaki disease, multiple sclerosis, emphysema, cystic fibrosis and chronic bronchitis;
e) treating endometriosis, uterine fibroids, functional uterine bleeding and endometrial hyperplasia;
f) treating ocular vascularization such as vascular disease damage to the retina and choroidal vasculopathy;
g) treating cardiac dysfunction;
h) inhibiting immunosuppressive diseases such as treating HIV infection;
i) treating renal dysfunction;
j) inhibiting endocrine disorders;
k) inhibition of gluconeogenic dysfunction;
1) treating neurological diseases such as parkinson's disease or neurological diseases leading to cognitive disorders such as alzheimer's disease or neurological disorders related to polyglutamine;
m) inhibiting neuromuscular diseases such as amyloid lateral sclerosis (amylotropic laterals);
n) treating spinal muscular atrophy;
o) treatment of other conditions capable of receiving enhanced gene expression;
p) enhanced gene therapy.
The present invention therefore discloses the use of a compound of formula (I) as a medicament and the use of a compound of formula (I) for the manufacture of a medicament for the treatment of one or more of the above conditions.
The compounds of formula (I), their pharmaceutically acceptable acid addition salts and stereoisomeric forms thereof have important diagnostic properties because they are useful for detecting or identifying HDACs in a biological sample, including detecting and measuring the formation of a complex between a marker compound and HDACs.
The detection or identification method may use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like. Examples of radioactive isotopes include125I、131I、3H and14C. enzymes are typically detectable by binding to a suitable substrate, which in turn catalyzes a detectable reaction. Examples thereof include, for example, β -galactosidase, β -glucosidase, alkaline phosphoglucomutase, peroxidase, and malate dehydrogenase, preferably horseradish peroxidase. Luminescent substances include, for example, luminol derivatives, luciferin, aequorin, and luciferase.
A biological sample may be defined as a body tissue or a body fluid. Examples of body fluids are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva, etc.
In view of their useful pharmacological properties, the test compounds may be formulated into a variety of pharmaceutical dosage forms for administration purposes.
To prepare the pharmaceutical compositions of this invention, an effective amount of the particular compound, in base or acid addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. It is desirable that these pharmaceutical compositions take a suitable, preferably oral, rectal, transdermal, or parenteral, unit dosage form for administration. For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, e.g., water, glycols, oils, alcohols, and the like for oral liquid preparations such as suspensions, syrups, elixirs, and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like for powders, pills, capsules and tablets.
Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will typically comprise sterile water as at least the major part, although other ingredients may also be included, for example to aid solubility. For example, injectable solutions may be prepared in which the carrier comprises a physiological saline solution, a glucose solution or a mixture of physiological saline and glucose solution. Injectable suspensions may also be prepared in which case appropriate liquid carriers, suspending agents and the like may be employed. In compositions suitable for transdermal administration, the carrier optionally includes a penetration enhancer and/or a suitable wetting agent, optionally in minor proportions, in combination with any naturally occurring suitable additive which does not have a significant deleterious effect on the skin. The additives may facilitate dermal administration and/or aid in the preparation of the desired composition. These compositions may be administered in a variety of ways, such as transdermal patches, spot-on or ointments.
The above pharmaceutical compositions are advantageously formulated in dosage unit dosage forms for convenient administration and uniform dosage. Dosage unit dosage form as used in the specification and claims herein refers to physically discrete units suitable as unitary dosages, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including scored and sugar-coated tablets), capsules, pills, powder packets, wafers, injectable solutions or suspensions, teaspoonful, tablespoonful and the like, and segregated multiples thereof.
The effective amount is readily determined by one skilled in the art by the assay results provided below. A therapeutically effective amount is generally considered to be from 0.05mg/kg body weight to 100mg/kg body weight, in particular from 0.05mg/kg body weight to 10mg/kg body weight. It may be appropriate to administer the required dose in 2, 3, 4 or more sub-doses at appropriate intervals throughout the day. The small doses may be formulated as unit dosage forms, for example containing from 5 to 500mg, especially from 10mg to 500mg, of active ingredient per unit dosage form.
As a further aspect of the invention, the combination of an HDAC-inhibitor with another anti-cancer agent is contemplated, in particular for use as a medicament, more particularly for the treatment of cancer or related diseases.
For the treatment of the above conditions, the compounds of the present invention may be advantageously combined with one or more other pharmaceutical agents, more particularly, with other anti-cancer agents. Examples of anticancer agents are:
-platinum coordination compounds, such as cisplatin, carboplatin, oxalyplatin;
-taxanes, such as paclitaxel or docetaxel;
topoisomerase I inhibitors, for example camptothecin compounds such as irinotecan or topotecan;
topoisomerase II inhibitors, for example anti-tumour podophyllotoxin derivatives such as etoposide or teniposide;
-an anti-tumour vinca alkaloid, such as vinblastine, vincristine or vinorelbine;
-antineoplastic nucleoside derivatives, such as 5-fluorouracil, gemcitabine or capecitabine;
alkylating agents, for example nitrogen mustards or nitrosoureas such as cyclophosphamide, chlorambucil, carmustine or lomustine;
anti-tumor anthracycline derivatives, such as daunorubicin, doxorubicin, idarubicin, or mitoxantrone;
-HER2 antibodies, such as trastuzumab;
-estrogen receptor antagonists or selective estrogen receptor modulators, such as tamoxifen, toremifene, droloxifene, faslodex or raloxifene;
aromatase inhibitors, such as exemestane, anastrozole, letrozole and vorozole;
differentiation inducing agents, such as retinoids, vitamin D and Retinoic Acid Metabolism Blockers (RAMBA), such as isotretinoin (accutane);
-DNA methyltransferase inhibitors, such as azacytidine;
kinase inhibitors, such as flavoperidol, imatinib mesylate or gefitinib;
-inhibitors of farnesyl transferase (farnesyl transferase); or
-other HDAC inhibitors.
The term "platinum coordination compound" as used herein refers to any platinum coordination compound that inhibits tumor cell growth, which provides platinum in ionic form.
The term "taxane" refers to a class of compounds having a taxane ring system, which are related to, or derived from, extracts of certain species of taxus species (taxus spp.).
The term "topoisomerase inhibitor" is used to refer to an enzyme capable of altering the topology of DNA in a eukaryotic cell. They are critical for important cellular functions and cell proliferation. There are two types of topoisomerases in eukaryotic cells, i.e., type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of about 100,000. The enzyme binds to DNA and causes transient single strand separation, unfolding the double helix (or allowing it to unfold), and then resealing the cleft prior to detaching from the DNA strand. Topoisomerase II has a similar mechanism of action, including induction of DNA strand separation or formation of free radicals.
The term "camptothecin compound" is used to refer to a compound related to or derived from the parent camptothecin, which is a water-insoluble alkaloid derived from the tree camptothecinum grown in china and the tree Nothapodytes foetida grown in india.
The term "podophyllotoxin compound" is used to refer to a compound that is related to or derived from the parent podophyllotoxin, which is extracted from the mandrake (mandrake) plant.
The term "anti-tumor vinca alkaloids" is used to refer to compounds related to or derived from extracts of the genus vinca (vinca).
The term "alkylating agent" includes classes of chemical substances that share the common characteristic of being able to supply alkyl groups to biologically important macromolecules such as DNA under physiological conditions. For most of the more important drugs such as nitrogen mustards and nitrosoureas, reactive alkylated residues are produced in vivo after degradation of the coordination compound, some of which are enzymatic. The most important pharmacological role of alkylating agents is to perturb the fundamental mechanisms associated with cell proliferation, particularly DNA synthesis and cell division. Alkylating agents interfere with the function and integrity of DNA in rapidly proliferating tissues is the basis for their therapeutic use and for many of their toxicities.
The term "antineoplastic anthracycline derivative" includes antibiotics obtained from the fungus strep. peuticus var. caesius and their derivatives, characterized by having a tetracycline cyclic structure with a specific sugar hexaamino sugar linked by glycosidic linkages.
Amplification of the human epidermal growth factor receptor 2 protein (HER 2) in primary breast cancer has been shown to be associated with poor prognosis in some patients. Trastuzumab is a highly purified recombinant DNA-derived human monoclonal IgG1 kappa antibody that binds with high affinity and high selectivity to the extracellular HER2 receptor region.
Many breast cancers have estrogen receptors and the growth of these tumors can be stimulated by estrogen. The terms "estrogen receptor antagonist" and "selective estrogen receptor modulator" are used to refer to competitive inhibitors of estradiol binding to the Estrogen Receptor (ER). Selective estrogen receptor modulators, when bound to the ER, induce a change in the three-dimensional shape of the receptor, inhibiting its binding to the Estrogen Response Element (ERE) on the DNA.
In postmenopausal women, circulating estrogen is derived primarily from the conversion of adrenal and ovarian androgens (androstenedione and testosterone) to estrogens (estrone and estradiol) by aromatase enzymes of the surrounding tissues. Removal of estrogen by aromatase inhibition or inactivation is an effective and selective treatment for postmenopausal hormone-dependent breast cancer patients.
The term "anti-estrogen agents" as used herein includes not only estrogen receptor antagonists and selective estrogen receptor modulators, but also aromatase inhibitors as described above.
The term "differentiation-inducing agent" includes compounds that inhibit cell proliferation and induce differentiation in various ways. Vitamin D and retinoids are known to have important roles in regulating the growth and differentiation of a variety of normal and malignant cells. Retinoic acid metabolism blockers (RAMBA's) increase endogenous retinoic acid levels by inhibiting cytochrome P450-mediated catabolism of retinoic acid.
DNA methylation changes are the most common abnormalities in human neoplasia. Hypermethylation of the promoter of a selection gene is often associated with inactivation of the gene of interest. The term "DNA methyltransferase inhibitor" is used to refer to compounds that act by pharmacologically inhibiting DNA methyltransferases and reactivating tumor suppressor gene expression.
The term "kinase inhibitor" includes strong kinase inhibitors associated with cell cycle processes and programmed cell death (apoptosis).
The term "farnesyl transferase inhibitor" is used to refer to compounds designed to prevent farnesylation of Ras and other intracellular proteins. They have been shown to affect malignant cell proliferation and survival.
The term "other HDAC inhibitors" includes, but is not limited to:
short-chain fatty acids, such as butyric acid, 4-phenylbutyric acid or valproic acid (valproic acid);
hydroxamic acids, such as suberoylanilide hydroxamic acid (SAHA), diarylhydroxamic acid a-161906, bicyclic-aryl-N-hydroxy-carboxamide, pyroxamide, CG-1521, PXD-101, sulfonamide hydroxamic acid, LAQ-824, trichostatin a (tsa), oxamflatin, scriptaid, m-carboxycinnamic acid, or trapoxin-hydroxamic acid analogs;
cyclic tetrapeptides such as trapoxin, apicidin or depsipeptide (depsipeptide);
benzamides, e.g. MS-275, or C1-994, or
-depudecin。
For the treatment of cancer, the compounds of the present invention may be administered to a patient as described above in conjunction with radiation therapy. Radiation refers to ionizing radiation, particularly gamma radiation, especially radiation emitted by linear accelerators or radioactive nuclei commonly used today. The radionuclear irradiation of the tumor may be located outside the tumor or inside the tumor.
The invention also relates to a combination of an anti-cancer agent and an HDAC inhibitor of the invention.
The invention also relates to the use of a compound according to the invention in medical treatment, for example to inhibit the growth of tumour cells.
The invention also relates to the use of a compound according to the invention for inhibiting the growth of tumor cells.
The invention also relates to a method of inhibiting the growth of tumor cells in a subject comprising administering to the subject an effective amount of a compound of the invention.
The invention further provides a method of inhibiting the abnormal growth of cells, including amebocytes, by administering an effective amount of a compound of the invention.
The further agent and the HDAC inhibitor may be administered simultaneously (e.g. in separate or unit compositions) or sequentially in any order. In the latter case, the two compounds are administered in a dosage and manner over a period of time sufficient to ensure that a beneficial or synergistic effect is achieved. It will be appreciated that the preferred method and sequence of administration and the respective dosages and modes of administration of the individual components of the compound will depend on the particular alternative drug and HDAC inhibitor being administered, their route of administration, the particular tumour being treated and the particular host being treated. The optimal method and sequence of administration, as well as the dosages and modes of administration, may be readily determined by those skilled in the art by reference to the information provided herein, in a conventional manner.
The platinum coordination compound is preferably administered in an amount of 1 to 500mg per square meter (mg/m) in one course of treatment2) Body surface area, e.g. 50 to 400mg/m2In particular about 75mg/m for cisplatin doses2And about 300mg/m for carboplatin2。
The taxane is preferably administered in a dosage of 50 to 400mg per square meter (mg/m) during a course of treatment2) Body surface area, e.g. 75 to 250mg/m2In particular about 175 to 250mg/m for paclitaxel doses2And about 75 to 150mg/m for docetaxel2。
The camptothecin compound is preferably administered in an amount of 0.1 to 400mg per square meter (mg/m) in one course of treatment2) Body surface area, e.g. 1 to 300mg/m2In particular about 100 to 350mg/m for irinotecan doses2And about 1 to 2mg/m for topotecan2。
The antitumor podophyllotoxin derivative is preferably administered in an amount of 30 to 300mg per square meter (mg/m) in one course of treatment2) Body surface area, e.g. 50 to 250mg/m2In particular about 35 to 100mg/m for etoposide doses2And for teniposide about 50 to 250mg/m2。
The anti-tumor vinca alkaloids are preferably administered in a dosage of 2 to 30mg per square meter (mg/m) during a course of treatment2) Body surface area, toolThe dose of the extract is about 3 to 12mg/m for vinblastine2For vincristine doses, about 1 to 2mg/m2And about 10 to 30mg/m for vinorelbine dosage2。
The antitumor nucleoside derivative is preferably administered in a dose of 200 to 2500mg per square meter (mg/m) in one treatment course2) Body surface area, e.g. 700 to 1500mg/m2In particular about 200 to 500mg/m for 5-FU2About 800 to 1200mg/m for gemcitabine dose2And about 1000 to 250mg/m for capecitabine2。
In one course of treatment, the alkylating agent such as a nitrogen mustard or a nitrosourea is preferably administered in an amount of 100 to 500mg per square meter (mg/m)2) Body surface area, e.g. 120 to 200mg/m2In particular about 100 to 500mg/m for cyclophosphamide doses2About 0.1 to 0.2mg/kg for chlorambucil and about 150 to 200mg/m for carmustine2And about 100 to 150mg/m for lomustine doses2。
The anthracycline derivative is preferably administered in an amount of 10 to 75mg per square meter (mg/m) during a course of treatment2) Body surface area, e.g. 15 to 60mg/m2In particular about 40 to 75mg/m for doxorubicin doses2For daunorubicin, the dosage is about 25 to 45mg/m2And about 10 to 15mg/m for idarubicin (idarubicin) dosage2。
Preferably, trastuzumab is administered at a dose of 1 to 5mg per square meter (mg/m) during a course of treatment2) Body surface area, in particular 2 to 4mg/m2。
The preferred dosage of antiestrogens is from about 1 to about 100mg per day, depending on the particular drug and condition being treated. A preferred oral dosage of tamoxifen is from 5 to 50mg, preferably from 10 to 20mg, twice daily for a sufficient treatment period to achieve and maintain the therapeutic effect. A preferred oral dosage of toremifene is about 60mg once daily for a sufficient treatment period to achieve and maintain the therapeutic effect. A preferred oral dosage of anastrozole is about 1mg, once a day. A preferred oral dosage of droloxifene is about 20 to 100mg once a day. A preferred oral dose of raloxifene is about 60mg, once daily. A preferred oral dosage of exemestane is about 25mg once daily.
These doses may be administered, for example, once, twice or more per course of treatment, which may be repeated every 7, 14, 21 or 28 days.
In view of their valuable pharmacological properties, the compounds according to the invention, i.e. the further drug and the HDAC inhibitor, may be formulated in various dosage forms for administration purposes. These components may be formulated individually into respective pharmaceutical compositions, or into a single pharmaceutical composition containing both components.
The invention therefore also relates to a pharmaceutical composition comprising another drug and an HDAC inhibitor together with one or more pharmaceutical carriers.
The invention also relates to a compound according to the invention in the form of a pharmaceutical composition comprising an anti-cancer agent and an HDAC inhibitor of the invention and one or more pharmaceutical carriers.
The invention further relates to the use of a compound according to the invention for the preparation of a pharmaceutical composition for inhibiting the growth of tumor cells.
The invention further relates to a pharmaceutical product comprising an HDAC inhibitor of the invention as a first active ingredient and an anti-cancer agent as a second active ingredient as a combined preparation for simultaneous, separate or sequential use in the treatment of a patient suffering from cancer.
Experimental part
The following embodiments are provided for exemplary purposes.
"BSA" refers to bovine serum albumin, "DCM" refers to dichloromethane, "DIEA" refers to diisoPropylethylamine, "DMF" refers to dimethylformamide, "DMSO" refers to dimethyl sulfoxide, "EtOAc" refers to ethyl acetate, "Fmoc" refers to fluorenylmethoxycarbonyl, "Hepes" refers to 4- (-2-hydroxyethyl) -1-piperazine-ethanesulfonic acid, "HOBT" refers to 1-hydroxy-1H-benzotriazole, "MeOH" refers to methanol, "PyBop" refers to benzotriazol-1-yl-oxo-tri-pyrrolidine-phosphonium hexafluorophosphate, "PyBrOP" refers to bromo-tri-pyrrolidine-phosphonium hexafluorophosphate, "TEA" refers to triethylamine, "TFA" refers to trifluoroacetic acid, "THF" refers to tetrahydrofuran, "Extrelut" refers to tetrahydrofuranTM"is a product of Merck KgaA, Darmstadt, Germany, and is a short column containing diatomaceous earth.
A. Preparation of intermediates
Example A1
a) Preparation of intermediate 1
Intermediate 1
A solution of 1- (phenylmethyl) -piperazine (0.068mol) in acetonitrile p.a. (135ml) was gradually added to a solution of potassium carbonate (0.18mol) and ethyl 2- (methylsulfonyl) -5-pyrimidinecarboxylate (0.082mol) in acetonitrile p.a. (135ml) and the reaction mixture was stirred at room temperature for 45 minutes. The reaction mixture was then kept overnight. DCM (400ml) was added. Water (300ml) was added again, and the organic layer was separated and dried (MgSO4) The solvent was filtered and evaporated. The residue (28g) was purified by column chromatography on silica gel (eluent: DCM/MeOH 95/5). The pure fractions were collected and the solvent was evaporated. The residue was crystallized from acetonitrile, filtered and dried in vacuo to give 15.1g of intermediate 1.
b) Preparation of intermediate 2
Intermediate 2
A mixture of intermediate 1(0.03mol) and EtOH (250ml) was hydrogenated over Pd/C10% (2g) as catalyst at 50 ℃. Absorption of H2After (1 eq) the catalyst was removed by filtration and the filtrate evaporated. The residue was chromatographed over a column of silica gel (eluent: DCM/(MeOH/NH)3)90/10) is purified. The product fractions were collected and the solvent was evaporated, yielding 6.8g (> 96%) of intermediate 2.
c) Preparation of intermediate 3
Intermediate 3
In N2A solution of dimethyl-aminosulfonyl chloride (0.0015mol) in DCM (1ml) was added at 5 ℃ to a mixture of intermediate 2(0.0012mol) and TEA (0.0017mol) dissolved in DCM (1 ml). The mixture was stirred at room temperature for 18 hours. 10% potassium carbonate was added. The mixture was extracted with DCM. The organic layer was separated and dried (MgSO)4) Filtered and the solvent evaporated to dryness. The residue (0.69g) was extracted with diethyl ether. The precipitate was filtered and dried to yield 0.64g (73%) of intermediate 3, m.p. 193 ℃.
Example A2
Preparation of intermediate 4
Intermediate 4
A solution of ethyl 2- (methylsulfonyl) -5-pyrimidinecarboxylate (0.0434mol) in acetonitrile (100ml) was added dropwise to a solution of 4-piperidinemethylamine (0.0868mol) and potassium carbonate (0.0434mol) in acetonitrile (200ml) at 10 ℃ under a stream of nitrogen. The mixture was stirred at room temperature for 2 hours, poured out into ice water and usedAnd (5) DCM extraction. The organic layer was separated and dried (MgSO)4) The solvent was filtered and evaporated. The residue (14.18g) was purified by column chromatography over silica gel (20-45 μm) (eluent: DCM/MeOH/NH)4OH 90/10/1 to 80/20/2). The pure fractions were collected and the solvent was evaporated, yielding 3.7g (32%) of intermediate 4.
Example A3
a) Preparation of intermediate 5
Intermediate 5
A mixture of intermediate 2(0.0002mol), α -phenylbenzacetylchloride (0.0003mol) and morpholinomethyl-PS-scavenger (supplier Novabiochem catalog No. 01-64-0171: morpholinomethyl polystyrene HL (200-400 mesh), 2% divinylbenzene) (0.150g) in DCM (5ml) was stirred at room temperature for 20 hours, then tris (2-aminoethyl) amine PS-scavenger (supplier Novabiochem catalog No. 01-64-0170: tris- (2-aminomethyl) -amine polystyrene HL (200-400 mesh), 1% divinylbenzene) (0.150g) was added and the reaction mixture was stirred for an additional 4 hours. The scavenger was removed by filtration, washed with DCM and the solvent evaporated to give intermediate 5.
b) Preparation of intermediate 6
Intermediate 6
A mixture of intermediate 5(0.0003mol), THF (4ml) and MeOH (1ml) in sodium hydroxide 1N (1.5ml) was stirred at room temperature for 3 days, then the reaction mixture was neutralized with HCl (1.5ml, 1N). Passing the mixture through an ExtreltTMNT (supplier: Merck) was filtered and dried under a stream of nitrogen to give intermediate 6.
c) Preparation of intermediate 7
Intermediate 7
A mixture of intermediate 6(0.0003mol), HOBT-6-carboxamide methyl-PS-scavenger (0.200 g; Novabiochem catalog No. 01-640425), and N, N-dimethyl-4-pyridylamine (0.00015mol) in DCM/DMF (5ml) was stirred at room temperature for 15 minutes, then N, N' -tetramethylbis (hanettylbis) -2-propylamine (0.070ml) was added and the reaction mixture shaken for 4 hours. The resin was washed 3 times with DCM, 3 times with DMF, 3 times with DCM and 3 times with DMF and finally 6 times with DCM. A solution of O- (tetrahydro-2H-pyran-2-yl) -hydroxylamine (0.00026mol) in DCM (5ml) was added and the reaction mixture shaken for 20H, then PS-linked methyl isocyanate (supplier Novabiochem catalog No. 01-64-0289: methyl isothiocyanate polystyrene HL (200-400 mesh), 2% divinylbenzene) (0.150g) was added and the mixture shaken for 4H. The scavenger was removed by filtration and washed 2 times with DCM and the filtrate taken to give intermediate 7.
B. Preparation of the Final Compounds
Example B1
N-Fmoc-hydroxylamine-2-chlorotrityl resin (Novabiochem, Cat. Nos. 01-64-0165) was deprotected with 50% piperidine DMF solution (room temperature, 24 hours). The resin was washed several times with DCM and DMF and swollen in DMF. 2 equivalents of acid are added in one portion1PyBrOP and 4 equivalents DIEA. The mixture was shaken for 24 h, the liquid was drained and the resin was washed several times with DCM and DMF. The resin was swollen in DMF containing 2 equivalents of amine, shaken at room temperature for 24 h, the liquid was drained and the resin was washed several times with DCM and DMF. The resin was expanded in DMF containing 4 equivalents of TEA and arylsulfonyl chloride (2 equivalents) was added in one portion. The reaction was stirred overnight, the liquid was removed and the resin was washed with DCM and DMF. With 5% TFThe final product was isolated from the DCM solution of a, analysed by hplc and mass spectrometry and evaporated in a pre-weighed tube.
1Resin-based load
The reaction scheme for exemplary purposes is as follows.
Example B2
N-Fmoc-hydroxylamine-2-chlorotrityl-resin (Novabiochem, catalog No. 01-64-0165) was deprotected with 50% piperidine DMF solution (24 h at room temperature)1. The resin was washed with DCM and DMF2Several times and expanded in DMF. 2 equivalents of acid are added in one portion3，PyBrOP4And 4 equivalents of DIEA. The mixture was shaken for 24 h, the liquid was drained and the resin was washed several times with DCM and DMF. The resin was expanded in DMF containing 2 equivalents of amine, shaken at room temperature for 24 h, the liquid was drained and the resin was washed with DCM and DMF. The final product was isolated with 5% TFA in DCM, analyzed by HPLC and mass spectrometry, and evaporated in a pre-weighed tube.
1In one example (compound 1) carboxylic acid methyl mercaptan 4-methoxytrityl-resin (Novabiochem 01-64-0238) was used.
2In one case (compound I), methanol was also used in the different washing steps.
3Resin-based load
4In one case (compound 1), PyBOP is used instead of PyBrOP.
Example B3
Reacting N-Fmoc-hydroxyAmine-2-chlorotrityl-resin (Novabiochem, 01-64-0165) was deprotected with 50% piperidine DMF solution (room temperature, 24 hours)1. The resin was washed with DCM and DMF2Several times and expanded in DMF. 2 equivalents of acid are added in one portion3，PyBrOP4And 4 equivalents of DIEA. The mixture was shaken for 24 h, the liquid was drained and the resin was washed several times with DCM and DMF. The resin was expanded in DMF containing 2 equivalents of amine, shaken at room temperature for 24 h, the liquid was drained and the resin was washed with DCM and DMF. 3 equivalents of acid, DIC and DIEA were shaken with the resin at room temperature overnight. The resin was drained and washed with DCM and DMF. The final product was isolated with 5% TFA in DCM, analyzed by HPLC and mass spectrometry, and evaporated in a pre-weighed tube.
Example B4
a) Preparation of intermediate 8 sodium salt
Intermediate 8 sodium salt
A mixture of intermediate 3(0.0016mol) and sodium hydroxide (0.0033mol) in ethanol (6ml) was stirred and refluxed for 2 hours, then cooled to room temperature. The precipitate was filtered, washed with ethanol and dried to yield 0.59g (> 100%) of intermediate 8 sodium salt.
b) Preparation of intermediate 9
Intermediate 9
Under a stream of nitrogen, N '- (ethylcarbodiimide) -N, N' -dimethyl-1, 3-propanediamine, monohydrochloride (0.0021mol) was added stepwise to a mixture of intermediate 8 sodium salt (0.0016mol), O- (tetrahydro-2H-pyran-2-yl) -hydroxylamine (0.0021mol) and 1-hydroxy-1H-benzotriazole (0.0021mol) in DCM/DMF (10ml), and the mixture was stirred at room temperature over the weekend. A 10% potassium carbonate solution was added. The mixture was extracted with DCM. The organic layer was separated, dried (magnesium sulfate), filtered and the solvent was evaporated to dryness. The residue (0.94 g) was purified by kromasil column chromatography (eluent: DCM/methanol/ammonium hydroxide 97/3/0.1; 15-40 μm). The pure fractions were collected, the solvent was evaporated and the residue (0.45 g, 65%) was extracted with diethyl ether. The precipitate was filtered and dried to yield 0.422g (61%) of intermediate 9, mp 183 ℃.
c) Preparation of Compound 2
Compound 2
0.5 ml of trifluoroacetic acid was added to a solution of intermediate 9(0.0009mol) in methanol (10 ml). The mixture was stirred at room temperature for 18 hours. The precipitate was filtered, washed with DCM and dried. 0.176 g (59%) of compound 2 are obtained, having a melting point of more than 260 ℃.
Example B5
Preparation of intermediate 10
Intermediate 10
A mixture of intermediate 2(0.0019mol) and sulfonamide (0.0021mol) in 1, 2-dimethoxy-ethane (5ml) was stirred and refluxed for 4 days. Water was added. The mixture was filtered and dried to yield 0.51g (83%) of intermediate 10, mp 192 ℃.
Intermediate 10 was treated similarly as described in example [ B4] to give 0.034g (13%) of Compound 3, mp 212 ℃.
Compound 3
Example B6
Preparation of intermediate 11
Intermediate 11
A solution of dimethyl-aminosulfonyl chloride (0.007mol) in DCM (5ml) was added to a solution of intermediate 4(0.0057mol) and TEA (0.0085mol) in DCM (5ml) at 10 ℃ under a stream of nitrogen. The mixture was stirred overnight, then poured into ice water and extracted with DCM. The organic phase was separated, dried over magnesium sulfate, filtered and the solvent was evaporated. The residue is taken up in CH3CN/Ether crystallization, filtration of the precipitate and drying gave 0.492g (24%) of intermediate 11, m.p. 142.
Intermediate 11 was treated similarly as described in example [ B4] to give 0.7g (85%) of Compound 4, mp 182 ℃.
Compound 4
Example B7
Preparation of Compound 5
Compound 5
A mixture of intermediate 7(0.0003mol) with acetic acid, trifluoroacetic acid (5ml, 5% in methanol) was stirred at room temperature for 20 hours, and then the mixture was blown dry to afford compound 5.
Example B8
Preparation of intermediate 12
Intermediate 12
A mixture of intermediate 2(0.0025mol), 2-naphthoyl chloride (0.003mol) and potassium carbonate (0.005mol) in acetonitrile (20ml) was stirred and refluxed overnight, then cooled to room temperature, poured out into ice water and extracted with DCM. The organic phase was separated, dried over magnesium sulfate, filtered and the solvent was evaporated. The residue was crystallized from diethyl ether, the precipitate was filtered off and dried to yield 0.97g (100%) of intermediate 12, m.p. 140 ℃. Intermediate 12 was similarly processed as described in example [ B4] to give 0.338g (86%) of Compound 6, melting point 130 ℃.
Compound 6
Table F-1 lists compounds prepared according to one of the above examples. The following abbreviations are used in the tables. Co.No. represents the compound number, Ex. [ Bn °)]Represents the same process as described in the Bn ° example, C2HF3O2Represents trifluoroacetate, some compounds are characterized by melting point (mp.), and others by mass spectrometry [ MH ]+](ms.) characterization was performed.
C. Pharmacological examples
The inhibitory effect of the compounds of formula (I) on histone deacetylase activity was determined by a histone deacetylase in vitro inhibition assay (see example c.1).
The cell activity of the compounds of the formula (I) on A2780 tumor cells was determined by a colorimetric assay for cytotoxicity or survival (Mosmann Tim, Journal of immunological methods 65: 55-63, 1983) (see example C.2).
The ability of the compounds to dissolve in aqueous solution upon stepwise dilution was determined by dynamic solubility experiments in aqueous media (see example c.3).
DMSO stock solutions of compounds were diluted three times in series with a single aqueous buffer solvent. The turbidity after each dilution was measured by a turbidimeter.
The permeability of a drug reflects its ability to transfer from one medium to or through another medium. And in particular to the ability of the drug to pass through the intestinal membrane to the bloodstream and/or from the bloodstream to a target site. Permeability can be measured by forming a membrane-fixed artificial membrane phospholipid bilayer (example c.4). In the membrane-immobilized artificial membranous phospholipid bilayer experiment, a 96-well microtiter plate and a 96-well filter plate were formed in a "sandwich" configuration such that each composite well was divided into two chambers, with the donor solution at the bottom and the acceptor solution at the top, separated by a 125 micron microfiltration disk (0.45 micron filter well), and coated with a 2% (wt/v) dioleoylphosphatidylcholine (dioleylphosphatidyl-choline) solution in dodecane. Provided that when the system is contacted with an aqueous buffer solution, multi-lamellar bilayers will form inside the filtration channel. The permeability of a compound through the artificial membranous system is measured in centimeters per second. The purpose of this experiment was to examine the ability of the drug to cross parallel artificial membranes at two different pH values (4.0 and 7.4). The compounds were detected by UV-spectrometry at optimal wavelengths of 250-500 nm.
Drug metabolism refers to the process by which oil-soluble xenobiotic or endophytic substances are converted into polar, water-soluble excretable metabolites by enzymatic reactions. The major organ of drug metabolism is the liver. The metabolite is generally less or not active than the parent drug. However, some metabolites have enhanced activity or exhibit toxic effects. Drug metabolism may therefore include both "detoxification" and "toxic" processes. One of the major enzyme systems that determines the ability of the body to cope with drugs and chemicals is cytochrome P450 monooxygenase, which is an NADPH dependent enzyme. The metabolic stability of a compound can be determined by in vitro experiments using human subcellular tissue (see example c.5). Here, the metabolic stability of a compound is evaluated by the percentage of drug metabolized after 15 minutes of incubation of the compound with microsomes. The amount of compound was determined by LC-MS analysis.
The tumor suppressor P53 transcriptionally activates a variety of genes in response to DNA damage, including the WAF1/CIP1 gene. The 21Kda product of the WAF1 gene, which appears as a universal inhibitor of CDK activity, was found in complexes comprising cyclins, Cyclin Dependent Kinases (CDKs) and Proliferating Cell Nuclear Antigen (PCNA) in normal cells but not in transfected cells. One of the consequences of p21WAF1 binding to and inhibiting CDKs is the blocking of CDK-dependent phosphorylation and subsequent inactivation of the Rb protein, which is crucial during the cell cycle. Thus, the induction of p21WAF1 by cell exposure to HDAC inhibitors is a potent specific indicator showing that cell cycle processes are blocked at the G1 and G2 monitoring points.
The ability of a compound to induce p21WAF1 can be measured by p21WAF1 enzyme-linked immunosorbent assay (WAF 1 ELISA for oncogenes). The p21WAF1 assay is a "sandwich" enzyme immunoassay using mouse monoclonal antibodies as well as rabbit polyclonal antibodies. The rabbit polyclonal antibody, a specific antibody for human WAF1 protein, was immobilized on a plastic well surface provided in the kit. Any p21WAF1 in the sample to be tested will bind to the capture antibody. The biotinylated detector monoclonal antibody also recognizes the human p21WAF1 protein and is capable of binding to any p21WAF1 protein that has been bound to the capture antibody. Subsequently, the detector antibody is bound to streptavidin (streptavidin) conjugated to horseradish peroxidase. This horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetramethylbenzidine from a colorless solution to a blue solution (or to a yellow solution upon addition of a terminator), the intensity of which is proportional to the amount of plate-bound p21WAF1 protein in the sample. The colored reaction product can be quantified by a spectrophotometer. A standard curve was prepared by known concentration of p21WAF1 protein (lyophilized) and quantitative data were obtained (see example c.6).
Example C1: in vitro assay method for histone deacetylase inhibition
60 μ g/ml HeLa cell nuclear extract (Biomol) was mixed with 2X 10-8M radiolabeled peptide substrate. As a substrate for determining HDAC activity, 14 to 21 amino acid sequences of histone H4, which is a synthetic peptide, were used. The substrate is biotinylated at the amino terminus with a 6-aminocaproic acid spacer, the carboxy terminus is protected by an amide group, and specifically lysine at position 163H]And (4) acetylation. Subjecting a substrate (biotin- (6-aminocaproic acid) -glycine-alanine- ([ 2 ])3H]-acetyl-lysine-arginine-histidine-arginine-lysine-valine-amino)) to a solution containing 25mM Hepes, 1M sucrose, 0.1mg/ml BSA and 0.01% TritonX-100 in a buffer solution with pH 7.4. After 30 minutes, hydrochloric acid and acetic acid were added to terminate the deacetylation reaction (final concentrations of 0.035mM and 3.8mM, respectively). After the reaction was terminated, the free form was extracted with ethyl acetate3And H, acetate salt. After mixing and centrifugation, an aliquot of the upper (organic) radioactivity was measured by a beta counter.
For each experiment, controls (containing HeLa nuclear extract and DMSO without compound), blanks (containing DMSO alone without HeLa nuclear extract or compound) and sample groups (containing compound dissolved in DMSO and HeLa nuclear extract) were set in parallel. In a first experiment, test 10 was first performed-5Compound at M concentration. When the compound showed activity at this concentration, preparation was at 10-5-10-12Concentration-response curves for test compounds over the range of M concentrations. In each test, blank values were subtracted from control and sample values. The control group represents 100% deacetylation of the substrate. For each sample, the radioactivity was expressed as a percentage of the mean of the control. Calculating IC by probability value analysis of the rating data, where appropriate50(the reduction in metabolite reached the drug concentration required for 50% of the control group). Thereafter the activity of the compounds was determined using pIC50(IC50Negative logarithm of value). All compounds tested were at 10-5M concentration is enzymatic, pIC of 21 compounds50Not less than 5 (see Table F2)
Example c.2: determination of antiproliferative Activity of Compounds on A2780 cells
All compounds tested were dissolved in DMSO and then further diluted with culture medium. The final concentration of DMSO in cell proliferation experiments should not exceed 0.1% (v/v). The control group contained a2780 cells and DMSO but no compound, and the blank group contained DMSO but no cells. MTT was dissolved in phosphate buffer at a concentration of 5 mg/ml. A glycine buffer was prepared consisting of 0.1M glycine and 0.1M sodium chloride and adjusted to pH 10.5 with 1N sodium hydroxide solution (all reagents from Merck).
Human ovarian cancer cells A2780(Dr. T.C. Hamilton [ Fox Chase cancer centre, Pennsylvania, USA)]Gift) medium was RPMI1640 medium supplemented with 2mM L-glutamic acid, 50ug/ml gentamycin and 10% fetal bovine serum. The cells contained 5% CO at 37 ℃ according to the conventional method2Monolayer growth was maintained in humid air. Cells were passaged weekly using trypsin/EDTA solution at a 1: 40 split ratio. All media and supplements were purchased from Life Technologies. The cells were not contaminated with chlamydia as determined by the Gen-probe chlamydia tissue culture kit (supplied by biomerieux).
Seeding cells in NUNCTMThe cells were attached to a 96-well culture plate (provided by Life Technologies) overnight. The density used for plating was 1500 cells/well and the total volume of medium per well was 200 microliters. After the cells were attached to the plate, the medium was changed and the drug and/or solvent was added to a final volume of 200 microliters. After 4 days of incubation, the medium was replaced with 200. mu.l of fresh medium and cell density and viability were determined by MTT. To each well 25 microliters of MTT solution was added and the cells were further incubated at 37 ℃ for 2 hours. The medium was carefully aspirated, 25. mu.l glycine buffer was added followed by 100. mu.l DMSO to dissolve the blue MTT-formazanAnd (3) obtaining the product. The micro-test plates were shaken on a microplate shaker for 10 minutes and the absorbance was measured at a wavelength of 540 nm using an Emax96 well plate spectrophotometer (supplied by Sopachem). In the experiments, the results for each experimental condition were averaged over three replicate wells. As an initial screen, compounds were screened at 10-6M was measured at a single fixed concentration and the experiment was repeated for the active compound to obtain a complete concentration-response curve. For each experiment, a control group (no drug) and a blank group (no cells or drug) must be set in parallel. Blank values were subtracted from control and sample values. For each sample, the average of cell growth (absorbance units) can be expressed as a pairPercentage of mean growth of control cells. Where appropriate, by probability value analysis of the rating data (Finey, d.j., probit analytics, 2)ndChapter 10, Graded Responses, Cambridge university Press, Cambridge 1962) for IC computation50(cell growth reduction to reach the control group 50% required drug concentration). The following pIC was used to test the activity of the compounds50(IC50Negative logarithm of value). The majority of compounds tested were at 10-6Cell viability at M concentration, pIC of 9 Compounds50Not less than 5 (see Table F-2)
Example c.3: dynamic solubility in aqueous media
During the first dilution, 10. mu.l of concentrated stock solution of active compound dissolved in DMSO (5mM) are added to 100. mu.l of phosphate citrate buffer pH 7.4 and mixed until homogeneous. In the second dilution step, an aliquot (20. mu.l) of the first dilution was further dispersed into 100. mu.l of phosphate citrate buffer pH 7.4 and mixed well. Finally, in the third dilution step, 20. mu.l of the second sample was further diluted to 100. mu.l of phosphate citrate buffer pH 7.4 and mixed well. All dilution steps were performed in 96-well plates. Immediately after the end of the last dilution step, the turbidity of the three successive dilution steps was measured with a turbidimeter. Each compound was diluted in triplicate to eliminate occasional errors. The results were scored by turbidity measurements and classified into three categories. The compound with high solubility is 3 points, and the compound is a clear solution when diluted once. The medium solubility compound was 2 points, which was cloudy on the first dilution and clear on the second dilution. The compound with low solubility is 1 point, and the compound is turbid after the first dilution and the second dilution. The solubility of 9 compounds was determined, 7 of which were 3 points and 2 of which were 1 point (see Table F-2)
Example c.4: parallel artificial membrane permeability analysis
A sample of the stock solution (10 μ l aliquot of 5mM stock solution dissolved in 100% DMSO) was diluted into a deep-well plate or pre-mix plate containing 2 ml of an aqueous buffer system (PSR4 system solution concentration (pION)) with pH 4 or 7.4.
Before the sample solution was added to the reference plate, 150 μ l of buffer was first added to the wells and the blank was assayed for uv absorbance. The buffer was then discarded and the plate used as a reference plate. All assays were performed in a uv-resistant plate (supplied by Costar or Greiner).
After blank measurement of the reference plate, 150. mu.l of the diluted sample was added to the reference plate and 200. mu.l of the dilution was added to the donor plate 1. Acceptor filter plate 1 (supplied by Millipore, model: MAIP N45) was coated with 4. mu.l of an artificial membrane forming solution (dodecane solution of 1, 2-dioleoyl-sn-glycerol-3-phosphocholine containing 0.1% 2, 6-di-tert-butyl-4-methylphenol) and placed on top of the donor plate to form a "sandwich" structure. Buffer (200. mu.l) was dispensed into the upper receptor well, the sandwich was covered with a lid and stored in the dark at room temperature for 18 hours.
Add 150. mu.l buffer to the wells and perform a blank UV assay of acceptor filter plate 2. Following a blank assay of the acceptor filter plate 2, the buffer is discarded, 150. mu.l of acceptor solution is transferred from the acceptor filter plate 1 to the acceptor plate 2, and the acceptor filter plate 1 is removed from the "sandwich" structure. After performing a blank assay on donor plate 2 (as above), 150 μ l of donor solution was transferred from donor plate 1 to donor plate 2. The donor plate 2, the acceptor plate 2 and the reference plate were uv-spectrally scanned (using SpectraMAX 190). All spectral data were processed and permeability of the compounds calculated by PSR4p command software (command software). All compounds were assayed in triplicate. In each experiment, compounds were scored and classified into three categories using carbamazepine, griseofulvin, acyclovir, atenolol, furosemide and chlorothiazide as criteria: low permeability (average effect < 0.5X 10)-6cm/s, fraction 1 point), medium permeability (1X 10)-6cm/s > average effect ≥ 0.5X 10-6cm/s, fraction of 2 points) or high permeability (average effect ≥ 0.5X 10-6cm/s, fraction 3 points). Both compounds were scored at 1 point at one pH value measured.
Example c.5: metabolic stability test
Subcellular tissue was prepared according to the method of good et al (Xenobiotica 5: 453-462, 1975) by mechanical homogenization of the tissue followed by centrifugation. Liver tissue was washed with ice-cold 0.1M Tris-HCl buffer (pH 7.4) to wash out excess blood. The tissue was blotted dry, weighed, and roughly minced with surgical scissors. Three volumes of ice cold 0.1M phosphate (Tris-HCl) buffer (pH 7.4) were then added and homogenized 7 times for 10 seconds using a Potter-S (Braun, Italy) or Sorvall Omni-Mix homogenizer equipped with a Teflon pestle. In both cases, the tubes need to be kept in ice throughout the homogenization process.
The homogenate is centrifuged at 4 ℃ using a Sorvall centrifuge or a Beckman ultracentrifuge at 9000Xg for 20 minutes. The supernatant was collected and stored at-80 ℃ and designated "S9".
Further centrifugation of "S9" was carried out using a Beckman ultracentrifuge at 4 ℃ with a centrifuge force of 100000Xg for 60 minutes. The supernatant was carefully aspirated, aliquoted and designated "cytosol". The pellet was resuspended in 0.1M phosphate buffer at pH 7.4 to a final volume of 1ml buffer per 0.5 g of original tissue weight and designated "microsomes".
All subcellular components were aliquoted separately, immediately frozen in liquid nitrogen and stored at-80 ℃ prior to use.
For the samples to be tested, the incubation mixture comprises: 0.1M PBS, 5uM compound, 1mg/ml microsomes and NADPH producing system (0.8mM 6 glucose-6-phosphate, 0.8mM magnesium chloride and 0.8 units glucose-6-phosphate dehydrogenase). The control sample contained the same material, but the microsomes were replaced with heat-inactivated microsomes (heated at 95 ℃ for 10 minutes). Recovery of compound in the control sample was always 100%.
The mixture was preincubated at 37 ℃ for 5 minutes. At 0min, 0.8mM NADP was added, the reaction was started (t ═ 0), and the samples were incubated for 15min (t ═ 15). The reaction was stopped by adding 2 volumes of DMSO. The samples were then centrifuged at 900 Xg for 10 minutes and the supernatants were stored at room temperature for no more than 24 hours before analysis. All incubations were performed in parallel twice. The supernatant was analyzed by LC-MS. Samples (50X 4.6mm, particle size 5 μm, Waters, USA) were eluted on a Xterra MS C18 column. An Alliance2790HPLC system (Waters, USA) was used in this experiment, eluting with buffer A, solution B, C at a flow rate of 2.4 ml/min. And (3) buffer solution A: 25mM ammonium acetate in water/acetonitrile (95/5) (pH5.2), solution B in acetonitrile and solution C in methanol. Gradient setting: the organic phase concentration was increased linearly from 0% to 50% solution B and 50% solution C (5 min) to 100% solution B (1 min) and the organic phase concentration was kept constant for 1.5 min. The total injection volume of the sample was 25 microliters.
A Quattro (Micromass, Manchester, UK) triple quadrupole mass spectrometer was equipped with an ESI source as the detector. The ion source and desolvation temperatures were set at 120 and 350 c, respectively, nitrogen was used as the atomizing agent and the drying gas, and data (single-ion reaction) was obtained in a positive scan mode with a cone voltage set at 10 volts and a residence time of 1 second.
Metabolic stability may be expressed as the percentage of compound metabolism after 15 minutes of incubation with active microsomes (e (act)) (% metabolism 100% - (total ion flow (TIC) at 15min (e (act)))/TIC at 0min (e (act)) x 100). Compounds with a metabolism of less than 20% are defined as being hypermetabolically stable. Compounds with a metabolism between 20-70% are defined as moderately metabolically stable, compounds with a metabolism above 70% are defined as less metabolically stable. For the metabolic stability screening assay used, 3 reference compounds must be included simultaneously. Verapamil was used as a less metabolically stable compound (percent metabolism 73%), cisapride was used as a moderately metabolically stable compound (percent metabolism 45%), and propanol was used as a moderately to highly metabolically stable compound (percent metabolism 25%). These reference compounds can be used to validate metabolic stability experiments.
One compound in the experiment was tested and its percent metabolism was less than 20%.
Example c.6: p21 inducibility
The following protocol is applicable to the determination of the expression level of P21 protein in human A2780 ovarian cancer cells. A2780 cells (cell concentration 20000 cells/180. mu.l) were seeded in 96-well plates in RPMI1640 medium supplemented with 2mM L-glutamic acid, 50ug/ml gentamicin and 10% fetal bovine serum. 24 hours before cell digestion, the compound was added to a final concentration of 10-5，10-6，10-7And 10-8And M. All compounds tested were dissolved in DMSO and further diluted with culture medium. After 24 hours of compound addition, the supernatant was removed from the cells, the cells were washed with 200. mu.l of ice-cold PBS, the well liquid was aspirated, and 30. mu.l of digestion buffer (containing 50mM Tris-HCl (pH 7.6), 150mM sodium chloride, 1% Nonidet P40, and 10% glycerol) was added. The plates were incubated at-70 ℃ overnight.
The appropriate number of microtiter wells are removed from the foil pouch and placed in an empty well holder. A working solution (1-fold concentration) of a washing buffer (20-fold plate concentration: 100ml of a 20-fold concentrated PBS solution and a surfactant containing 2% chloroacetamide) was prepared. The lyophilized p21WAF standard was dissolved in distilled water and further diluted with sample diluent (provided in the kit). The samples were prepared by dilution (1: 4) with sample diluent. Samples (100. mu.l) and p21WAF standards (100. mu.l) were injected into appropriate wells and incubated for 2 hours at room temperature. The wells were washed 3 times with 1 fold wash buffer and 100 μ l of detection antibody reagent (biotinylated p21WAF1 monoclonal antibody solution) was added to each well. Incubate at room temperature for 1 hour, wash wells 3 times with 1 fold wash buffer. The 400 times concentration of the conjugate (peroxidase-streptavidin conjugate: 400 times concentration of the concentrated solution) is diluted, and to each well is added 100 microliter of 1 times concentration of the solution, room temperature in 30 minutes, 1 times concentration of the washing buffer will be washed 3 times, and then distilled water washing 1 time. Substrate solution (chromogenic substrate) (100. mu.l) was added to the wells, the wells were incubated at room temperature for 30 minutes in the dark, and stop solution was added to each well in the order of the addition of the substrate solution. The absorbance of each well was measured using a spectrophotometric plate reader, the operating wavelength was two wavelengths: 450/595 nm.
For each experiment, a control group (no drug) and a blank group (no cells or drug) must be set in parallel. Blank values were subtracted from control and sample values. For each sample, the intensity of induction (in absorbance units) of p21WAF1 by the compound can be expressed as a percentage of the value of p21WAF1 for the control group. A percentage of induction higher than 130% is significant induction. This experimental assay was performed on three compounds, two of which showed significant induction.
TABLE F-2: table F2 shows the results of the compounds tested in examples C.1, C.2 and C.3.
Compound number Enzyme activity pIC50 Cell Activity pIC50 Fraction of solubility
8 ＞5
7 ＞5
9 ＞5
10 ＞5
11 ＞5
12 ＞5
1 ＜5 ＜5 1
18 6.173 6.166 1
5 7.096 6.181
23 6.932 5.796
24 7.073 6.084
25 6.29 ＜5
26 6.984 5.378
27 6.433 ＜5
6 7.104 5.828 3
19 5.536 ＜5 3
20 5.451 ＜5
21 5.679 ＜5 3
22 5.599 5.297 3
2 6.615 5.534 3
3 6.881 ＜5 3
4 7.27 5.528 3
D. Composition examples: film-coated tablet
Tablet core preparation
A mixture of 100g of the compound of formula (I), 570g of lactose and 200g of starch is mixed thoroughly and then moistened with a solution of 5g of sodium lauryl sulfate and 10g of polyvinylpyrrolidone in water (200 ml). The wet powder mixture was sieved, dried and sieved again. 100g of microcrystalline cellulose and 15g of hydrogenated vegetable oil were then added. The whole mixture was mixed well and tabletted to obtain 10000 tablets, each containing 10mg of the compound of formula (I).
Coating film
To a solution of 10g of methylcellulose in denatured ethanol (75ml) was added a solution of 5g of ethylcellulose in methylene chloride (150 ml). Then 75ml of dichloromethane and 2.5 ml of 1, 2, 3-propanetriol are added. 10g of polyethylene glycol was dissolved and dissolved in 75ml of methylene chloride. The latter is added to the former solution, then 2.5g magnesium octadecanoate, 5g polyvinylpyrrolidone and 30ml of concentrated pigment suspension are added, the mixture is mixed well and the tablet cores are coated with the mixture obtained in a coating apparatus.

Claims (10)

1. A compound of the formula (I),
pharmaceutically acceptable addition salts thereof and stereochemically isomeric forms thereof, wherein
n is 0 or 1, and when n is 0, is a direct bond;
each Q is nitrogen or
Each X is nitrogen or
Each Y is nitrogen or
Each Z is nitrogen or
R1is-C (O) NH (OH) or-NHC (O) C1-6An alkanediyl SH;
R2is hydrogen, halogen, hydroxyl, amino, nitro, C1-6Alkyl radical, C1-6Alkoxy, trifluoromethyl, di (C)1-6Alkyl) amino or hydroxyamino;
R3is C1-6Alkyl, phenyl C2-6Alkenediyl, furancarbonyl, C1-6Alkylaminocarbonyl, aminosulfonyl, di (C)1-6Alkyl) aminosulfonylamino C1-6Alkyl, di (C)1-6Alkyl) amino C1-6Alkyl radical, C1-12Alkylsulfonyl, di (C)1-6Alkyl) aminosulfonyl or trihalo C1-6An alkylsulfonyl group;
R4is hydrogen, hydroxy, amino, hydroxy C1-6Alkyl radical, C1-6Alkyl or C1-6An alkoxy group;
when R is3And R4When on the same carbon atom, R3And R4Taken together may form a divalent radical of the formula
-C(O)-NH-CH2-NR10- (a-1)
Wherein R is10Is phenyl;
when R is3And R4When on adjacent carbon atoms, R3And R4Taken together may form a divalent radical of the formula
＝CH-CH＝CH-CH＝ (b-1).
2. The compound of claim 1, wherein
R2Is hydrogen, halogen, hydroxyl, amino, nitro, C1-6Alkyl radical, C1-6Alkoxy, trifluoromethyl or di (C)1-6Alkyl) amino.
3. The compound of claim 1, wherein each Q isR2Is hydrogen or nitro; r4Is hydrogen.
4. The compound of claim 1 or 3, wherein n is 1; each Q isEach Z is nitrogen; r1is-C (O) NH (OH); r2Is hydrogen; r3Is C1-12An alkylsulfonyl group; and R4Is hydrogen.
5. The compound of claim 1, 3 or 4 selected from the group consisting of compound No. 18, 5 and 24:
6. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and, as active ingredient, a therapeutically effective amount of a compound according to any one of claims 1 to 5.
7. A process for preparing a pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is intimately mixed with a compound according to any one of claims 1 to 5.
8. A compound according to any one of claims 1 to 5 for use as a medicament.
9. A process for the preparation of a compound according to claim 1, characterized in that an intermediate of formula (II) is reacted with trifluoroacetic acid to give a hydroxamic acid of formula (I-a), wherein R is1is-C (O) NH (OH),
10. use of a compound as defined in claim 1 for the preparation of a reagent for the detection or identification of histone deacetylase in a biological sample, said detection or identification comprising detecting or determining the formation of a complex between a marker compound as defined in claim 1 and histone deacetylase.
HK05110453.4A 2002-03-13 2003-03-11 Inhibitors of histone deacetylase HK1078473B (en)

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