WO2009076234A2

WO2009076234A2 - Synthesis methods of histone deacetylase inhibitors (hdacis)

Info

Publication number: WO2009076234A2
Application number: PCT/US2008/085735
Authority: WO
Inventors: Vincent C. O. Njar; Lalji K. Gediya
Original assignee: University Of Maryland, Baltimore
Priority date: 2007-12-07
Filing date: 2008-12-05
Publication date: 2009-06-18
Also published as: WO2009076234A3

Abstract

Simple and efficient procedures for the synthesis of histone deacetylase inhibitors. The procedure may provide CI-994 in 80% overall yields.

Description

TITLE OF INVENTION:

SYNTHESIS METHODS OF HISTONE DEACETYLASE INHIBITORS (HDACIs) CROSS REFERENCE TO RELATED APPLICATIONS:

[01] This Application claims benefit to U.S. Provisional Application 61/012,374, filed

December 7, 2007 and claims benefit to U.S. Provisional Application 61/012,736, filed

December 10, 2007, each of which are hereby incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT:

[02] This invention was made with the support of the U.S. government under Grant

Number CAl 17991 from the National Institutes of Health and the National cancer Institute

(NCI) and Grant Number W81XWH-04-1-0101 from the U.S. Department of Defense. The

U.S. government has certain rights in this invention.

NAMES OF PARTIES OF A JOINT RESEARCH AGREEMENT:

[03] Not Applicable

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A

COMPACT DISC:

[04] Not Applicable

BACKGROUND OF THE INVENTION

Prostate Cancer

[05] Prostate cancer (PCA) is the most common malignancy and age-related cause of cancer death worldwide. Apart from lung cancer, PCA is the most common form of cancer in men and the second leading cause of death in American men. In the United States in 2007, an estimated 218,890 new case of prostate cancer will be diagnosed and about 27,050 men will die of this disease. (Jemal et al, Cancer statistics, CA Cancer J CHn 2007, 57, (1), 43-66).

The growth of most prostate tumors depends on androgens during the initial stages of tumor development, and thus, anti-hormonal therapy by surgical or medical suppression of androgen action remains a major treatment option of the disease. (Denmeade and Isaacs, A history of prostate cancer treatment. Nat Rev Cancer 2002, 2, (5), 389-96). Although this treatment may be initially successful, most tumors eventually recur due to the expansion of an androgen-refractory population of PCA cells. (Barry et al, A nationwide survey of practicing urologists: current management of benign prostatic hyperplasia and clinically localized prostate cancer, J Urol 1997, 158, (2), 488-91; discussion 492). Treatment of androgen-independent tumors with cytotoxic agents is generally unsatisfactory, and, at this stage, the disease is usually fatal to the patient (Singh et al., Combinatorial androgen receptor targeted therapy for prostate cancer, Endocr Relat Cancer, 13: 653-666, 2006.). Metastatic disease that develops even after potentially curative surgery remains a major clinical challenge. Therapeutic treatments for patients with metastatic PCA are limited because current chemotherapeutic and radiotherapeutic regimens are largely ineffective. (Feldman and Feldman, The development of androgen-independent prostate cancer, Nat Rev Cancer 2001, 1, (1), 34-45).

[06] Since androgen-independent, metastatic prostate cancer is incurable up to now, there exist a need for treatments that block proliferation and induce differentiation and/or apoptosis of prostate cancer cells.

[07] There is an urgent need to develop new therapeutic agents with defined targets to prevent and treat PCA. [08] Histone Deacetylases

[09] Histone Deacetylases (HDACs) are the catalytic subunits of multiprotein complexes responsible for deacetylation of histones and nonhistone proteins. Lysine acetylation, i.e., the transfer of an acetyl moiety from acetyl-coenzyme A to the ε-amino group of a specific lysine residue, has emerged as the major form of posttranslational modification of histones, and other proteins have been correlated with transcription, chromatin assembly, DNA repair, and recombinatorial events (Marks et al., Histone deacetylases and cancer, causes and therapies, Nat Rev Cancer, 1: 194-202, 2001.). Histone acetylation in vivo is a dynamic, reversible process governed by the opposite actions of histone acetyltransferases (HATs) and HDACs. Aberrant acetylation of histone tails, emerging from either HAT mutation or abnormal recruitment of HDACs, has been linked to carcinogenesis (Pandolfi, P. P. Transcription therapy for cancer. Oncogene, 20: 3116-3127, 2001). In various cases, altered HAT or HDAC activity has been identified in a variety of cancers. It has recently been demonstrated that the expression and activity of HDACl is up-regulated in prostate cancer compared to benign prostatic hyperplasia (BPH) (Patra et al., Histone deacetylase and DNA methyltransferase in human prostate cancer, Biochem Biophys Res Commun, 287: 705-713, 2001).

[10] Histone deacetylase inhibitors (HDACIs) have been found to be useful for the activation of genes responsive to hormone receptors. HDACIs are potent inducers of growth arrest, differentiation and/or apoptosis of several cell lines, and they constitute a novel class of chemotherapeutic agents initially identified by their ability to reverse the malignant phenotype of transformed cells. They have been shown to activate differentiation programs, inhibit cell cycle, and induce apoptosis in a wide range of tumor-derived cell lines and to block angiogenesis and stimulate the immune system in vivo (Marks et al., Histone deacetylases and cancer, causes and therapies, Nat Rev Cancer, 1: 194-202, 2001 ; Johnstone, R. W. Histone-deacetylase inhibitors, novel drugs for the treatment of cancer. Nat Rev Drug Discov, 1: 287-299, 2002.). Whereas the mechanisms through which HDACIs exert these anti -tumor activities have not been fully delineated, induction of histone hyperacetylation and modulation of gene transcription through chromatin remodeling are thought to be primarily responsible, leading to the selective activation of genes associated with cell growth and survival. Suberoylanilide hydroxamic acid (SAHA, Vorinostat^® or Zolinza^®) was approved in 2006 for the treatment of patients with relapsed or refractory cutaneous T-cell lymphoma (Marks et al, Dimethyl sulfoxide to vorinostat, development of this histone deacetylase inhibitor as an anticancer drug, Nat Biotechnol, 25: 84-90, 2007.).

[11] One of the early HDACIs discovered is TV-hydroxy-iV'-phenylactanediamide, also called suberanilide hydroxamic acid (SAHA). (Richon et al., Second generation hybrid polar compounds are potent inducers of transformed cell differentiation, Proc Natl Acad Sci USA 1996, 93, (12), 5705-8; Kelly et al., Phase I clinical trial of histone deacetylase inhibitor, suberoylanilide hydroxamic acid administered intravenously, CHn Cancer Res 2003, 9, (10 Pt 1), 3578-88.) This compound (trade name: Vorinostat^®) was approved in 2006 by the U.S. Food and Drug Administration (FDA) for the treatment of advanced cutaneous T-cell- lymphoma. (Bolden et al., Anticancer activities of histone deacetylase inhibitors, Nat Rev Drug Discov 2006, 5, (9), 769-84.)

[12] Histone acetyltransferase and histone deacetylase (HDAC) have opposing effect on transcription (Ito et al., Histone acetylation and histone deacetylation, MoI Biotechnol, 20: 99-106, 2002; Kuo et al., Roles of histone acetyltransferases and deacetylases in gene regulation, Bioessays, 20: 615-626, 1998. ). Often, DNA methylation and histone deacetylation of tumor suppressor genes occur in many human cancers, leading to suppression of function of these genes thereby conferring a growth advantage for the tumor cells (Macaluso et al., A. How does DNA methylation mark the fate of cells?, Tumori, 90: 367-372, 2004; Robertson et al. DNA methylation: past, present and future directions. Carcinogenesis, 21: 461-467, 2000.). It has recently been demonstrated that the expression and activity of HDACl is up-regulated (2-4-fold) in prostate cancer compared to benign prostatic hyperplasia (Patra et al., Histone deacetylase and DNA methyltransferase in human prostate cancer, Biochem Biophys Res Commun, 287: 705-713, 2001.). HDACIs, such as SAHA, and jV-(2-aminophenyl)4-[jV-(pyridine-3-yl-methoxy-carbonyl)aminomethyl] benzamide (MS -275) can directly interact with the HDAC enzymes at the catalytic site and inhibit their function (Bolden et al, Anticancer activities of histone deacetylase inhibitors, Nat Rev Drug Discov, 5: 769-784, 2006; Marks et al., Histone deacetylase inhibitors: inducers of differentiation or apoptosis of transformed cells, J Natl Cancer Inst, 92: 1210-1216, 2000; Minucci et al., Histone deacetylase inhibitors and the promise of epi genetic (and more) treatments for cancer, Nat Rev Cancer, 6: 38-51, 2006.). This leads to acetylation of histones which opens-up the chromatin structure allowing transcription of anti-growth and pro- apoptotic genes to occur. MS-275 is now in phase I/II clinical trials for various solid tumors and hematological malignancies (Hess-Stumpp et al., MS-275, a potent orally available inhibitor of histone deacetylases-The development of an anticancer agent. Int J Biochem Cell Biol, 39: 1388-1405, 2007.).

[13] Suberoylanilide hydroxamic acid (SAHA) is a modest HDACI and has been used extensively in vitro and in vivo in cancer models (Butler et al., Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo. Cancer Res, 60: 5165-5170, 2000.) and it is the only HDACI currently approved for clinical use (Marks et al, Dimethyl sulfoxide to vorinostat, development of this histone deacetylase inhibitor as an anticancer drug, Nat Biotechnol, 25: 84-90, 2007; Richon et al., Histone deacetylase inhibitors, development of suberoylanilide hvdroxamic acid

(SAHA) for the treatment of cancers. Blood Cells MoI Dis, 27: 260-264, 2001.). The HDACI benzamide compound MS-275 is in several clinical trials as a potential therapy for a variety of cancers (Hess-Stumpp et al, MS-275, a potent orally available inhibitor of histone deacetylases-the development of an anticancer agent, Int J Biochem Cell Biol, 39: 1388-

1405, 2007.).

[14] Figure 1 shows structures of SAHA, MS-275 and CI-994 HDACIs and their IC50 values.

[15] Synthesis Methods

[16] N-(2'-Aminophenyl)-benzamide derivatives.

[17] Weiershausen et al in U.S. Patent 5,137,918 discloses a synthesis method for

N-(2'-Aminophenyl)-benzamide derivatives. Weiershausen et al U.S. Patent 5,137,918 is hereby incorporated herein by reference in its entirety. An example of the synthesis method of Weiershausen et al to produce 7V-(2aminophenyl)-4-acetylaminobenamide (CI-994) is outlined in Scheme 1 below. To the best of Applicants' knowledge the method of

Weiershausen et al in U.S. Patent 5,137,918 is the only method that has been reported for synthesis of CI-994.

Scheme 1 : Previous Procedure for Synthesis of CI-994

[Overall yield: 0.20 x 0.69 x 100 = 13.8%]

[18] The 3-step procedure of Weiershausen et al involves the reaction of oxalyl chloride with 4-acetamidobenzoic acid to yield the corresponding acid chloride that was coupled with

2-nitroaniline in situ to afford iV-(2¹-nitrophenyl)-4-acetylaminobenzamide in 20.0% yield.

This was then hydrogenated in THF using 10% palladium on activated charcoal to produce

CI-994 (3) in 69.0% yield. The overall yield was only 13.8%. In addition to the low yield achieved, this method is tedious and requires special hydrogenation conditions.

[19] Thus, there continues to be a need for efficient and high yield production of HDACIs.

BRIEF SUMMARY OF THE INVENTION

[20] METHODS OF SYNTHESES

[21] Applicants have discovered improved syntheses of HDACIs.

[22] Applicants have developed simple and efficient one-step procedures for the high yield synthesis of benzamide derivatives having histone deacetylase inhibitory activity of structural formula C-I :

C-I

[23] Where X is a hydrogen or an alkyl group. X may be optionally substituted with one or more hydroxyl groups. The alkyl group may have one to three carbon atoms. The alkyl groups may be linear or branched and substituted or unsubstituted. [24] n is 0-4.

[25] Each R¹ is independently selected from the group consisting of H, -NHAc, -OH, alkyl, alkoxy, halogen, an amino group, a nitro group, a cyano group, an aminoalkyl group, an alkylamino group, an acyl group, an acylamino group, a thiol, thiolurea, an alkylthio group, a perfluoroalkyl group, a perfluoroalkyloxy group, a carboxyl group and an alkoxycarbonyl group.

[26] The alkyl group, the alkoxy group, the aminoalkyl group, the alkylamino group, the acyl group, the acylamino group, the alkylthio group, the perfluoroalkyl group, and the perfluoroalkyloxy group substituents may contain a lower alkyl group (C 1-4). The alkyl groups may be linear or branched and substituted or unsubstituted.

[27] The alkyl may be -CH₃. The alkoxy may be -OCH₃. Preferably the halogen may be

F or Cl.

[28] Preferably R¹ is H.

[29] Some examples of benzamide derivatives of Formula C-I are 4-Acetamino-N-(2'- aminophenyl)-benzamide, 4-(β-Hydroxypropanoyl)-amino-N-(2'-aminophenyl)-benzamide,

4-Isobutyrylamino N (2'-aminophenyl) benzamide, 4-Glycoloylamino-N-(2'-aminophenyl)- benzamide, N-(2'Aminophenyl)-4-formylamino-benzamide.

[30] Preferably structural formula C-I is of structural formula CI-994:

CI-994 Synthesis of benzamide derivatives having histone deacetylase inhibitory activity

[31] The simple and efficient one-step procedure for the synthesis of compounds of structural formula C-I is as follows in Scheme 2a:

Scheme 2A:

6B

[32] Where X, n and R¹ are as defined in Formula C-I . In Scheme 2 A Compound 6A may be converted into its imidazolide derivative by reaction with N, iV'-carbonyldiimidazole (CDI) in THF at ambient temperature. This may be further reacted with compound 6B in the presence of TFA to obtain compounds of formula C-I.

[33] Other known solvents may be used in place of THF (tetrahydrofuran), such as aprotic solvents, electron donating solvents, such as aceonitrile (ACN) and dimethylformaide (DMF) solvents. Known weak acids may also be used.

[34] Other known reagents in organic synthesis, such as peptide type coupling agents, may be used in place of CDI.

[35] Other known acids be used in place of TFA, for example, other strong carboxylic acids.

[36] The compounds of structural formula C-I may be synthesized using one transformation such as described in Scheme 2a above. [37] While Applicants' new synthesis method may be preformed in one-step, the synthesis may include other steps.

[38] The product may be recrystallized from THF/solvent to purify.

[39] The synthesis of C-994 is shown below in Scheme 2 as an example of Applicants invention of a simple and efficient one-step procedure:

Scheme 2: New synthesis of CI-994

TFA, rt, 16 hrs

[40] Acetamidobenzoic acid (6) was converted into its imidazolide derivative by reaction with N, jV'-carbonyldiimidazole (CDI) in THF at ambient temperature. This was further reacted with 1,2-phenylenediamine in the presence of TFA to obtain CI-994 (3) in 85.0% yield.

[41] The product was recrystallized from THF/methanol to yield pure CI-994 (2) in

80.0% yield.

[42] Other known solvents may be used in place of methanol. The solvents may be alcohols or short-chain hydrocarbons. The solvent may be pentane, hexane or ether, including pet ether. The solvent may be aceonitrile (ACN). The volume ratio of solvent to water of about 1 :1, 1 :2, 1 :3, 1 :4, 1 :5, 1:6, 1 :7, 1:8, 1:9, 1 :10, 2:1, 2:2, 2:3, 2:4, 2:5, 2:6, 2:7,

2:8, 2:9, 2:10 or the like. [43] While the reagents disclosed herein are named specifically, the skilled artisan is aware of equivalent reagents that function in substantially the same manner as the reagents disclosed herein. A non-limiting example is that although CDI is employed, any compound that converts alcohols and amines into carbamates, esters and/or ureas is contemplated. [44] The compound may be used in a pharmaceutical composition. The pharmaceutical composition may be formulated for oral administration, parentral administration or for injectable administration.

[45] In making compositions with the compounds of the present invention, the compound can be mixed with a pharmaceutically acceptable carrier or an excipient, diluted by an excipient or enclosed within such a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the compound. Thus, the compositions can be in the form of tablets, pills, powers, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, soft and hard gelatin capsules, and other orally ingestible formulations.

[46] The pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule or the like, prepared according to methods well known in the art. It is also contemplated that administration of such compositions may be by the oral, injectable and/or parenteral routes depending upon the needs of the artisan. The novel compound can be administered by nasal or oral inhalation, oral ingestion, injection (intramuscular, intravenous, and intraperitoneal), transdermally, or other forms of administration.

[47] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propyl-hydroxybenzoates, sweetening agents; and flavoring agents. The compositions of the present invention can also be formulated so as to provide quick, sustained or delayed release of the novel compound after administration to the patient by employing procedures known in the art.

[48] The term "pharmaceutically acceptable carrier" refers to those components in the particular dosage form employed which are considered inert and are typically employed in the pharmaceutical arts to formulate a dosage form containing a particular active compound. This may include without limitation solids, liquids and gases, used to formulate the particular pharmaceutical product. Examples of carriers include diluents, flavoring agents, solubilizers, suspending agents, binders or tablet disintegrating agents, encapsulating materials, penetration enhancers, solvents, emolients, thickeners, dispersants, sustained release forms, such as matrices, transdermal delivery components, buffers, stabilizers, and the like. Each of these terms is understood by those of ordinary skill.

[49] Aerosol formulations for use in this invention typically include propellants, such as a fluorinated alkane, surfactants and co-solvents and may be filled into aluminum or other conventional aerosol containers which are then closed by a suitable metering valve and pressurized with propellant, producing a metered dose inhaler. Aerosol preparations are typically suitable for nasal or oral inhalation, and may be in powder or solution form, in combination with a compressed gas, typically compressed air. Additionally, aerosols may be useful topically.

[50] Generally, the amount of the novel compound used in the treatment methods is that amount which effectively achieves the desired therapeutic result in animals. Naturally, the dosages of the various novel compounds will vary somewhat depending upon the parent compound, rate of in vivo hydrolysis, etc. Those skilled in the art can determine the optimal dosing of the novel compound selected based on clinical experience and the treatment indication. Preferably the amount of the novel compound is 0.1 to 100 mg/kg of body weight, more preferably, 5 to 40 mg/kg.

[51] Suitable solid carriers are known, e.g., magnesium carbonate, magnesium stearate, talc, lactose and the like. These carriers are typically used in oral tablets and capsules.

[52] Suitable carriers for oral liquids include, e.g., water, ethanol, propylene glycol and others.

[53] Topical preparations useful herein include creams, ointments, solutions, suspensions and the like. These may be formulated to enable one to apply the appropriate dosage topically to the affected area once daily, up to 3-4 times daily as appropriate. Topical sprays may be included herein as well.

[54] Depending upon the particular compound selected, transdermal delivery may be an option, providing a relatively steady state delivery of the medication which is preferred in some circumstances. Transdermal delivery typically involves the use of a compound in solution, with an alcoholic vehicle, optionally a penetration enhancer, such as a surfactant and other optional ingredients. Matrix and reservoir type transdermal delivery systems are examples of suitable transdermal systems. Transdermal delivery differs from conventional topical treatment in that the dosage form delivers a systemic dose of medication to the patient.

[55] The compounds can also be converted into a pharmaceutically acceptable salt or pharmaceutically acceptable solvate or other physical forms (e.g., polymorphs by way of example only and not limitation) via known in the art field methods.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS:

[56] Figure 1 shows structures of SAHA, MS-275 and CI-994 HDACIs and their IC50 values.

DETAILED DESCRIPTION OF THE INVENTION [57] ABBREVIATIONS

[58] CDI = 1 , 1 '-Carbonyldiimidazole (C₃H₃N₂)2CO)

[59] THF = Tetrahydrofuran (C₄H₈O)

[60] DBU = l,8-Diazabicyclo[5.4.0]undec-7-ene

[61 ] TFA = Trifluoroacetic acid (CF₃CO₂H)

[62] Et₃N = triethylamine

Experimental Section

[63] iV-(2-Aminophenyl)-4-acetylaminobenzamide (3, CI-994)

[64] To the suspension of acetamido benzoic acid (6, 7.5 gm, 0.041 mol) in 75 ml THF, added CDI (7.5 gm, 0.046 mol) portion wise at ambient temperature. The reaction mixture was stirred for one hour to form acylimidazole followed by addition of 1 ,2-phenylenediamine

(36.2 gm, 0.335 mol) and TFA (4.19 gm, 0.036 mol, 2.8 ml) and stirring for 16 hours. The reaction mixture was then filtered to afford crude CI-994 which was re-crystallized from

THF/methanol to yield 9.0 gm of CI-994 (3) as white crystals (yield, 80%). HPLC analysis showed purity >99%. mp 207-208 ⁰ C; IR (Nujol): cm^"1; ¹H-NMR: δ 2.08 (s, 3H, CH₃), 4.86

(s, 2H, NH₂), 6.59 (s, IH, Ar-H), 6.78 (d, IH, J= 7.5 Hz, Ar-H), 6.96 (s, IH, Ar-H), 7.16 (d,

IH, J = 7.5 Hz, Ar-H), 7.69 (d, 2H, J = 7.5, Ar), 7.94 (d, 2H, J = 8.0, Ar-Hs), 9.55 (s, IH,

NH), 10.188 (s, IH, NH). HRMS calcd 269.1164 (Ci₅H₁₅N₃O₂), found 269.1161. These spectral and analytical data are as previously reported in Weiershausen U.S. Patent

5,137,918.

Claims

[65] We Claim:

Claim 1. A method of making a benzamide derivative having histone deacetylase inhibitory activity of structural formula C-I :

C-I said method comprising converting Compound 6A into its imidazolide derivative by reaction with N, V-carbonyldiimidazole (CDI); and

reacting said imidazolide derivative with Compound 6B to obtain a compound of structural formula C-I,

6B wherein in formulae C-I, 6A and 6b X is a hydrogen or an alkyl group; n is 0-4; each R¹ is independently selected from the group consisting of H, -NHAc, -OH, alkyl, alkoxy, halogen, an amino group, a nitro group, a cyano group, an aminoalkyl group, an alkylamino group, an acyl group, an acylamino group, a thiol, thiolurea, an alkylthio group, a perfluoroalkyl group, a perfluoroalkyloxy group, a carboxyl group and an alkoxycarbonyl group.

Claim 2. The method of claim 1, wherein each R¹ is H.

Claim 3. The method of claim 1, wherein compound 6 A is acetamidobenzoic acid and compound 6B is 1 ,2-phenylenediamine and compound C-I has structural formula CI-994:

CI-994

Claim 4. The method of claim 1, wherein converting Compound 6 A into its imidazolide derivative is conducted at ambient temperature and reacting said imidazolide derivative with compound 6B is conducted at ambient temperature.

Claim 5. The method of claim 1, wherein said converting Compound 6A into its imidazolide derivative by reaction with N, N'-carbonyldiimidazole (CDI) is conducted in the present of THF.

Claim 6. The method of claim 1, wherein said reacting said imidazolide derivative with Compound 6B to obtain a compound of structural formula C-I is conducted in the present of TFA.

Claim 7. A method of making a benzamide derivative having histone deacetylase inhibitory activity of structural formula CI-994 comprising converting acetamidobenzoic acid into its imidazolide derivative by reaction with N, JV'-carbonyldiimidazole (CDI) in THF at ambient temperature; and further reacting with 1 ,2-phenylenediamine in the presence of TFA at ambient temperature to obtain CI-994,

CI-994