NEW HDAC INHIBITORS

Field of the invention

The present invention relates to novel compounds of the general formula (I), having histone deacetylase (HDAC) inhibiting enzymatic activity, their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, solvates, pharmaceutically acceptable salts and compositions, metabolites and prodrugs thereof. The present invention more particularly provides novel compounds of the general formula (I).

H (i)

The present invention also provides a process for the preparation of the above said novel compounds of the general formula (I), their derivatives, analogs, stereoisomers, polymorphs, hydrates, solvates, pharmaceutically acceptable salts and compositions, metabolites and prodrugs thereof.

The novel compounds (I) of the present invention are useful for the treatment of cancer, which is one of the leading causes of death in the present society. A great deal of effort has been underway to treat various forms of cancer for decades and until recently, chemoprevention of cancer is receiving its due share of attention.

The first isolation of histone deacetylase was described in 1964 from crude nuclear extracts of cells, but the molecular characterization of isoforms of the enzyme has been achieved only recently. Inhibitors of histone deacetylase (HDACs) are zinc hydrolases responsible for the deacetylation of N-acetyl lysine residues of histone and non-histone protein substrates. Human HDACs are classified into two distinct classes, the HDACs and Sirtuins. The HDACs are divided into two subclasses based on their similarity to yeast histone deacetylases, RPD 3 (class I includes HDAC 1, 2, 3, 8, and 11) and Hda 1 (class II includes HDAC 4, 6, 7, 9, and 10). All of the HDACs have a highly conserved zinc dependent catalytic domain. There is growing evidence that the

acetylation state of proteins and thus the HDAC enzyme family plays a crucial role in the modulation of a number of biological processes, including transcription and cell cycle.

Transcriptional regulation is a major event in cell differentiation, proliferation and apoptosis. Transcriptional activation of a set of genes determines cell destination and for this reason transcription is tightly regulated by a variety of factors. One of its regulatory mechanisms involved in the process is an alteration in the tertiary structure of DNA, which affects transcription factors to their target DNA regimens. Nucleosomal integrity is regulated by the acetylating status of the core histone, with the result being permissiveness to transcription. The acetylating status of the histone is governed by the balance of activities of the histone acetyl transferase (HAT) and histone deacetylase (HDAC). (See, for example Wang, A. H.; Yang, X. J. MoI. Cell Biol. 1999, 19, 7816- 7827). Recently, HDAC inhibitors have been found to arrest growth and apoptosis in several types of cancer cells, including colon cancer, t-cell lymphoma and erythroleukemic cells. (J. E. Bolden, M. J. Peart, R. W. Johnstone, Nature Review Drug Discovery, 5, 2006, 769-784)

Given that apoptosis is a crucial factor for cancer progression, HDAC inhibitors are promising reagents for cancer therapy as effective inducers of apoptosis.

Several structural classes of HDAC inhibitors have been identified and are reviewed in Marks, P.A. et al., J. Natl. Cancer Inst., 92, (2000), 1210-1215. More specifically WO 98/55449 and US patent 5,369,108 report alkanoyl hydroxamates with HDAC inhibitory activity.

Background of the invention

The present invention relates to potentially pharmaceutical compositions and in particular to new molecules as active ingredients, that are used in particular as anticancer agents. Compounds of the general formula (I) or pharmaceutically acceptable salts thereof according to the present invention have an ability of inhibiting histone deacetylating enzyme and of inducing differentiation and are useful as therapeutic or ameliorating agent for diseases that are involved in cellular growth such as malignant tumors, autoimmune diseases, skin diseases, infections etc.

Few prior art references, which disclose the closest compounds, are given here:

I. US 2005/0143385 discloses compounds of formula I,

(I)

wherein Cy is Cyclyl group (C _3-2 O carbocyclyl, C _3-20 heterocyclyl or C _5-20 aryl and is optionally substituted), Qi represents covalent bond or cyclyl leader group (Ci _-7 alkylene, Ci _-7 alkylene-X- Ci _-7 alkylene, -X- Ci _-7 alkylene, or Ci _-7 alkylene-X-), wherein X is -O- or -S- and is optionally substituted; Ji represents covalent bond or -C(=O)- ;

J ₂ is independently -C(=O)- or -S(=O) ₂ -; Q ₂ is independently optionally substituted C ₄ .

8 alkylene, Cs _-20 arylene, Cs _-20 arylene- Ci _-7 alkylene, Ci _-7 alkylene- C _5-20 arylene, or Q-

₇ alkylene- Cs _-20 arylene- Ci _-7 alkylene.

This invention pertains to pharmaceutical compositions containing such compounds and their use both in vitro and in vivo, to inhibit HDAC and in the treatment of conditions mediated by HDAC, cancer, proliferative conditions, psoriasis, etc.

II. US 2004/0092598 discloses certain active carbamic acid compounds which inhibit

HDAC activity and which have the formula I,

(I) wherein A is an aryl group; Qi is an aryl leader group having a backbone of at least two carbon atoms; J is an amide linkage selected from:

Wherein Ri is an amido substituent; and Q ₂ is an acid leader group.

This invention pertains to pharmaceutical compositions comprising such compounds and the use of such compounds and compositions both in vitro and in vivo to inhibit HDAC and to inhibit proliferative conditions, such as cancer and psoriasis. III. US 2004/0092558 discloses compounds of formula I 5

(I) wherein Y is R ¹ NHC(O) or R ² C(O)NR ³ ; R ² is an optionally substituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aryloxy, arylalkyloxy, or alkyl, where the aryl, etc. cyclic

10. systems can be bicyclic; R ³ is H, alkyl or C(O)R ⁴ ; R ⁴ is an optionally substituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, aryloxy, arylalkyloxy, or alkyl, where the aryl, etc. cyclic systems can be bicyclic; R is (CH ₂ ) _n or CH(A-R ⁵ )~(CH ₂ ) _n- i; n is 3-8; A is NH, O, S, CH ₂ , NHCO, or NHCO ₂ ; and R ⁵ is an optionally substituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or alkyl, where the aryl, etc. cyclic systems can be

15 bicyclic. Compounds of the formula I are preferred HDAC inhibitors and in this patent ophthalmic compositions containing HDAC inhibitors and their use for treating ocular neovascular or edematous diseases and disorders are disclosed.

Objective of the invention 0

Cancer is now believed to be the number one cause of premature death in industrialized nations. The market for anti-cancer agents was estimated at about US $ 10 billion in 1997 and continues to escalate. Because of the need and the value of these drugs, many laboratories are intensively investigating the nature and vulnerability of

25 cancerous cells, resulting in the development of novel screens and approaches.

Our sustained efforts have resulted in novel anticancer agents of the formula (I). Histone acetylation and deacetylation play an essential role in modifying chromatin structure and regulating gene expression in eukaryotic cells. Hyperacetylated histones are generally found in transcriptionally active genes and in transcriptionally silent

30 regions of the genome. Key enzymes, which modify histone proteins and thereby regulate gene expression, are histone acetyl transferases (HATs) and histone

deacetylases (HDACs). Compounds able to inhibit HDAC activity i.e. HDAC inhibitors such as Trichostatin A (TSA), Trapoxin (TPX), Suberoylanilide hydroxamic acid (SAHA), Sodium butyrate (NaB), Sodium valproate (VPA), Cyclic hydroxamic acid containing peptides (CHAPs), Depsipeptide FK-228 and MS-275 (Benzamide) can - de-repress these genes, resulting in antiproliferative effects in vitro and anti tumor effects in vivo.

Summary of the invention

The present invention relates to novel substituted HDAC inhibitors of the

. general formula (I),

(I) their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvates, pharmaceutically acceptable salts and compositions, metabolites and prodrugs thereof wherein A represents substituted or unsubstituted groups selected from aryl, aralkyl, heterocyclyl, heteroaryl, ^' heteroarylmethyl, benzofused heteroarylmethyl and benzofused heteroaryl; wherein X represents oxygen, sulphur or NH; wherein B represents the substituted or unsubstituted hydroxamic acid group, the thioate group, the heterocyclyl groups and n is an integer in the range of 1 to 7.

Detailed description of the invention

The present invention relates to novel substituted HDAC inhibitors of the general formula (I),

(I)

their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, solvates, pharmaceutically acceptable salts and compositions, metabolites and prodrugs thereof wherein, suitable groups represented by A are substituted or unsubstituted groups selected from aryl groups such as phenyl, naphthyl and the like; arylalkyl groups such as, phenylmethyl, naphthylmethyl, and the like; heterocyclyl groups such as pyrrolidinyl, thiazolidinyl, oxazolidinyl, morpholinyl, thiomorpholinyl, piperidinyl,

, piperazinyl and the like; heteroaryl groups such as pyridyl, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isooxazolyl, oxadiazolyl, triazolyl, thiadiazolyl, tetrazolyl, pyrimidinyl, pyrazinyl, pyridazinyl and the like; benzofused heteroaryl groups such as indolyl, indolinyl, benzothiazolyl, quinolinyl, quinoxalinyl, quinazolinyl, acridinyl, phenazinyl, pteridinyl, phenoxazinyl, phenothiazinyl, carbazolyl and the like; heteroarylalkyl groups such as pyridylmethyl, thienylmethyl, furylmethyl, pyrrolylmethyl, oxazolylmethyl, thiazolylmethyl, imidazolylmethyl, isooxazolylmethyl, oxadiazolylmethyl, triazolylmethyl, . thiadiazolylmethyl, - tetrazolylmethyl, pyrimidinylmethyl, pyrazinylmethyl, pyridazinylmethyl and the like; benzofused heteroarylalkyl groups such as indolylmethyl, indolinylmethyl, benzothiazolylmethyl, quinolinylmethyl, isoquinolinylmethyl, quinoxalinyl methyl, quinazolinylmethyl, acridinylmethyl, phenazinemethyl, pteridinylmethyl, phenoxazinylmethyl, phenothiazinylmethyl, carbazolyl methyl and the like. The group X is represented by oxygen, sulphur or NH.

B represents the substituted or unsubstituted hydroxamic acid group (O-acyl, N- acyl, O-alkyl, N-alkyl, O-pivaloyl, O-alkoxy carbonyl, N-alkoxy carbonyl, O-aralkyl, both or either of any two combination), thioate group such as ethane thioate and the like, heterocyclyl groups such as morpholine and thiomorpholine and the like. n is an integer in the range of 1 to 7.

Suitable groups substituted on A may be selected from halogens such as fluorine, chlorine, bromine or iodine, hydroxy, nitro, cyano, azido, nitroso, amino, hydrazine, hydrazide, hydroxamate, formyl, alkyl, haloalkyl, haloalkoxy, cycloalkyl, aryl, alkoxy, aryloxy, acyl, acyloxy, acyloxyacyl, heterocyclyl, heteroaryl, monoalkylamino, dialkylamino, acylamino, alkylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, heterocyclylcarbonyl, arylsulfonyl, alkylsulfinyl, arylsulfinyl,

alkylthio, arylthio, sulfamoyl, alkoxyalkyl groups and carboxylic acids or its derivatives such as carboxamide and carboxamidoalkyl.

Furthermore A which is a cyclic ring represents substituted or unsubstituted 5 to 10 membered ring systems, and also the rings may be monocyclic or bicyclic, saturated 5 or partially saturated or aromatic containing 1 to 4 hetero atoms selected from O, S, N and the like.

As used throughout the specification and the appended claims the following terms have the following meanings:

The term "alkyl" as used herein, means a straight or branched chain 10 hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t- butyl and the like.

The term "alkoxy" as used herein, means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative 15 examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, iso- propoxy, ?-butoxy and the like.

The term "alkoxycarbonyl" as used herein, includes groups such as methoxy carbonyl, ethoxy carbonyl and the like.

The term analog includes a compound, which differs from the parent structure 20 by one or more C, N, O or S atoms. Hence, a compound in which one of the N atoms in the parent structure is replaced by an S atom is an analog of the former.

The term stereoisomer includes isomers that differ from one another in the way the atoms are arranged in space, but whose chemical formulas and structures are otherwise identical. Stereoisomers include enantiomers and diastereoisomers. 25 The term tautomers include readily interconvertible isomeric forms of a compound in equilibrium. The keto- enol tautomerism is an example.

The term polymorphs include crystallographically distinct forms of compounds with chemically identical structures.

The term pharmaceutically, acceptable solvates includes combinations of solvent 30. molecules with molecules or ions of the solute compound.

The term derivative refers to a compound obtained from a compound according to formula (I), an analog, tautomeric form, stereoisomer, polymorph, hydrate,

pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, by a simple chemical process converting one or more functional groups, such as, by oxidation, hydrogenation, alkylation, esterification, halogenation, and the like.

Pharmaceutically acceptable salts of the present invention include alkali metals like Li, Na, and K, alkaline earth metals like Ca and Mg, salts of organic bases such as diethanolamine, α-phenylethylamine, benzylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, choline hydroxyethylpiperidine, and the like, ammonium or substituted ammonium salts, aluminum salts. Salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine etc. Salts may include acid addition salts where appropriate, which are, sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like. Pharmaceutically acceptable solvates may be hydrates or comprising other solvents of crystallization such as alcohols.

A term once described, the same meaning applies for it, throughout the patent

Particularly useful compounds according to invention include:

1. Ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyl]amino}-l,3-benzothiazole- 6- carboxylate;

2. Ethyl 2-({8-[(acetyloxy) amino]-8-oxooctanoyl} amino)-l,3-benzothiazole-6- carboxylate;

3. Ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyl]amino}-4-methyl-l,3-thiaz ole-5- carboxylate;

4. iV-Hydroxy-N-l,3-thiazol-2-yloctanediamide;

5. N-Hydroxy-N-(5-nitro-l,3-thiazol-2-yl)octanediamide;

6. N- l,3-Benzothiazol-2-yl-N'-hydroxyoctanediamide;

7. ^-(Acetyloxy)-^- 1 ,3-benzothiazol-2-yloctanediamide; ^' 8. iV-(Acetyloxy)-N'-l,3-thiazol-2-yloctanediamide;

9. N-[(Methoxycarbonyl)oxy]-N ¹ -l,3-benzothiazol-2-yIoctanediamide;

10. Ethyl 2-({8-[(benzyloxy) amino]-8-oxooctanoyl} amino)- 1,3-benzothiazole -6- carboxylate;

11. Ethyl 2-({8-[hydroxy(methyl)amino]-8-oxooctanoyl}amino)-4-methyl-l ,3- thiazole-5-carboxyJate;

12. Ethyl 2-{[8-(methoxyamino)-8-oxooctanoyl]amino}-4-methyl-l,3-thiaz ole -5- carboxylate; 13. 2- { [8-(Benzyloxyamino)-8-oxooctanoyl]amino} -6-(morpholin-4-ylcarbonyl)-

1 ,3 -benzothiazole;

14. 2-{[8-(Benzyloxyamino)-8-oxooctanoyl]amino}-6-[(methylamino) carbonyl)]- . 1,3-benzothiazole;

15. S-(6-{[5-(Morpholin-4-ylcarbonyl)-2-furyl]amino}-6-oxohexyl) ethanethioate; 16. S-(6-{[4-Methyl-5-(morpholin-4-ylcarbonyl)-2-thiazolyl]amino }-6-oxohexyl) ethanethioate;

17. S-(6- { [5-(Morpholin-4-ylcarbonyl)-2-benzothiazolyl]amino} -6-oxohexyl) ethanethioate;

18. N-Methyl-5-[(6-morpholin-4-ylhexanoyl)amino]-2-furamide and 19. N-Methyl-5-[(6-thiomorpholin-4-ylhexanoyl)amino]-2-furamide.

According to another feature of the present invention, there is provided a process as shown in the following schemes, for the preparation of compounds of the formula (I), wherein all the groups are as defined earlier.

Scheme 1:

* ^a ' A = Heterocyclic Amine

X ₁ Y = O ₁ OR ₁ S n is 0 - 4 Step-2

1 1(d)

Condensation of the compounds of formula l(a) and l(b) using BOP reagent, hydroxy benzotriazole and DIPEA, yielded a compound of formula l(c), wherein A, X,

and Y are as defined. Further the compound of formula l(c) is converted to the compound of formula l(d) with BOP reagent, hydroxy benzotriazole, DIPEA and hydroxylamine hydrochloride. The compound of formula l(d) was further converted to the compound of formula 1 by reaction with acetyl chloride & base.

Scheme -2

(1)

Condensation of compound of formula (Ia) and compound of formula (Ib) using EDCI, hydroxy benzotriazole and triethylamine, yielded a compound of formula (Ic). Compound (Ia) and the reagent used in the Step-V are secondary amines, wherein R ¹ , R ² and R ³ , R ⁴ may be same or different and are selected from alkyl, aryl, or R ¹ and R ² , and also R ³ and R ⁴ form, together with the nitrogen to which they are attached, a cyclic group selected from azetidino, pyrrolidino, piperidino, piperazino and morpholino, wherein the said cyclic group may be optionally substituted; A represents substituted or unsubstituted groups selected from aryl, aralkyl, heterocyclyl, heteroaryl, heteroarylmethyl, benzofused heteroaryl and benzofused heteroarylmethyl; and X represents oxygen, sulphur or NH. Further the compound of formula (Ic) is reduced to the compound of formula (Id) using 10% Pd/C. The compound of formula (Id) was converted to the compound of formula (Ie) by reaction with halo alkanoylchloride. The compound of the formula (Ie) was treated with potassium thioacetate in the presence of a suitable base like triethylamine, NaOH, KOH, pyridine, or potassium carbonate i.e.

Step IV to get the compounds of the formula (If) and the compound of the formula (Ie) is also further reacted with either primary or secondary amines in the presence of a suitable base like triethylamine, NaOH, KOH, pyridine, or potassium carbonate i.e. Step V to get the compounds of the formula (1). It is appreciated that in any of the above-mentioned reactions any reactive functional group in the substrate molecule may be protected according to the conventional chemical practice. Suitable protecting groups in any of these reactions are those used conventionally in the art and the methods of formation and removal of such protecting groups are those conventional methods appropriate to the molecule being protected.

Pharmaceutically acceptable salts of the present invention include alkali metals like Li, Na, and K, alkaline earth metal like Ca and Mg, salts of organic bases such as diethanolamine, α-phenylethylamine, benzylamine, piperidine, morpholine, pyridine, hydroxyethylpyrrolidine, hydroxyethylpiperidine, choline and the like, ammonium or substituted ammonium salts, aluminum salts. Salts also include amino acid salts such as glycine, alanine, cystine, cysteine, lysine, arginine, phenylalanine, guanidine etc. Salts may include acid addition salts where appropriate like sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartarates, maleates, citrates, succinates, palmoates, methanesulphonates, tosylates, benzoates, trifluoroacetates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, glycerophosphates, ketoglutarates and the like. Compounds of the formula (I) may form solvates of DMF, hydrates and the like.

It should be noted that compounds of the invention may contain groups that may exist in tautomeric forms and though one form is named, described, displayed and/or claimed herein, all the forms are intended to be inherently included in such name, description, display and/or claim.

The stereoisomers of the compounds forming part of this invention may be prepared by using reactants in their single enantiomeric form, in the process wherever possible or by conducting the reaction in the presence of reagents or catalysts in their single enantiomeric form or by resolving the mixture of stereoisomers by conventional methods. Some of the preferred methods include use of microbial resolution, resolving the diastereomeric salts formed with chiral acids such as mandelic acid,

camphorsulfonic acid, tartaric acid, lactic acid, and the like wherever applicable or by using chiral bases such as brucine, cinchona alkaloids, their derivatives and the like.

Prodrugs of the compounds of formula (I) are also contemplated by this invention. A prodrug is an active or inactive compound that is modified chemically through in-vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of the invention following administration of the prodrug to a patient. The suitability and techniques involved in making/using prodrugs are well known to those skilled in the art.

Various polymorphs of the compounds of the general formula (I), forming part of this invention may be prepared by crystallization of the compounds of formula (I) under different conditions. For example, using different commonly used solvents, or their mixtures for recrystallization; crystallizations at different temperatures; various modes of cooling, ranging from very fast to very slow cooling during crystallizations. Heating or melting the compounds followed by cooling gradually or immediately, one can also obtain polymorphs. The presence of polymorphs may be determined by solid probe NMR spectroscopy, IR spectroscopy, differential scanning calorimetry and powder X-ray diffraction or other such techniques.

Pharmaceutically acceptable solvates of the compounds of the formula (I) forming part of this invention may be prepared by conventional methods such as dissolving the compounds of the formula (I) in solvents such as water, methanol, ethanol, mixture of solvents such as acetone: water, dioxane:water, N ₉ N- dimethylformamide:water and the like, preferably water and recrystallization by using different crystallization techniques.

The present invention also provides a pharmaceutical composition, containing one or more of the compounds of the general formula (I) as defined above, their derivatives, analogs, tautomeric forms, stereoisomers, polymorphs, hydrates, metabolites, prodrugs, pharmaceutically acceptable salts, pharmaceutically acceptable solvates in combination with the usual pharmaceutically employed carriers, diluents and the like, useful for the treatment of cancer, psoriasis, proliferative conditions and conditions mediated by HDAC .

The derivatives provided by the present invention can be employed as pharmaceutical compositions, for example, in the form of pharmaceutical compositions

containing the derivatives together with appropriate, pharmaceutically acceptable carriers. The products in accordance with the invention can be administered, for example, perorally, such as in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions, or rectally, such as in the form of suppositories, etc. The compositions may be sterilized and may contain auxiliary agents generally employed in the pharmaceutical art, such as sodium hydrogen carbonate, citric acid, propylene glycol, tween 80, etc. The compounds can be used orally or parenterally.

Pharmaceutical compositions containing these compounds can be prepared using conventional procedures familiar to those skilled in the art, such as by combining the ingredients into a dosage form together with suitable, non-toxic, inert, therapeutically compatible solid or liquid carrier materials and if desired, the usual pharmaceutical adjuvants. The amount of the active ingredient in the composition may be less than 70% by weight. Such compositions typically contain from 1 to 25%, preferably 1 to 15% by weight of active compound, the remainder of the composition being pharmaceutically acceptable carriers, diluents, excipients or solvents.

The pharmaceutical composition may be in the forms normally employed, such as tablets, capsules, powders, syrups, solutions, suspensions and the like, may contain flavorants, sweeteners etc. in suitable solid or liquid carriers or diluents, or in suitable sterile media to form injectable solutions or suspensions. Suitable pharmaceutically acceptable carriers include solid fillers or diluents and sterile _j aqueous or organic solutions. The active compound will be present in such pharmaceutical compositions in the amounts sufficient to provide the desired dosage in the range as described above. Thus, for oral administration, the compounds can be combined with a suitable solid or liquid carrier or diluent to form capsules, tablets, powders, syrups, solutions, suspensions and the like. The pharmaceutical compositions, may, if desired, contain additional components such as flavorants, sweeteners, excipients and the like. For parenteral administration, the compounds can be combined with sterile aqueous or organic media to form injectable solutions or suspensions. For example, solutions in sesame or peanut oil, aqueous propylene glycol and the like can be used, as well as aqueous solutions of water-soluble pharmaceutically-acceptable acid addition salts or alkali or alkaline earth metal salts of the compounds. The injectable solutions prepared

in this manner can then be, administered intravenously, intraperitoneally, subcutaneously, or intramuscularly, with intramuscular administration being preferred in humans.

Generally, the effective dose for treating a particular condition in a patient may be readily determined and adjusted by the physician during treatment to alleviate the symptoms or indications of the condition or disease. Generally, a daily dose of the active compound in the range of about 0.01 to 1000 mg/kg of body weight is appropriate for administration to obtain effective results. The daily dose may be administered in a single dose or divided into several doses. In some cases, depending upon the individual response, it may be necessary to deviate upwards or downwards from the initially prescribed daily dose. Typical pharmaceutical preparations normally contain from about 0.2 to about 500 mg of active compound of formula I and/or its pharmaceutically active salts or solvates per dose.

While the compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the invention or other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition. The term "therapeutically effective amount" or "effective amount" refers to that amount of a compound or mixture of compounds of Formula (I) that is sufficient to effect treatment, when administered alone or in combination with other therapies to a mammal in need of such treatment.

The term "animal" as used herein is meant to include all mammals, and in particular humans. Such animals are also referred to herein as subjects or patients in need of treatment. The therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the particular compound of Formula (I) chosen, the dosing regimen to be followed, timing of administration, the manner of administration and the like, all of which can readily be determined by one of ordinary skilled in the art. The term "treatment" or "treating" means any treatment of a disease in a mammal, including:

a) Preventing the disease, that is, causing the clinical symptoms of the disease not to develop; b) Inhibiting the disease, that is, slowing or arresting the development of clinical symptoms; and/or c) Relieving the disease, that is, causing the regression of clinical symptoms.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, make various changes and modifications of the invention to adapt it to various usages and conditions. The present invention is provided by the examples given below, which are provided by the way of illustration only, and should not be considered to limit the scope of the invention. Variation and changes, which are obvious to one skilled in the art, are intended to be within the scope and nature of the invention, which are defined in the appended claims.

Example: 1

Synthesis of ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyl]amino}-l,3-benzo- thiazole-6-carboxylate.

Step: 1 Preparation of ethyl 2-amino-l,3-benzothiazole-6-carboxylate.

A mixture of ethyl-4-amino benzoate (1.65g, lOmmol), KCNS (9.72g, 100 mmol) and CuSO ₄ (8g, 50mmol) in methanol (3OmL) was stirred for 5 hours at 7O ⁰ C. Subsequently, the suspension was cooled and filtered; the filtrate was diluted with water (4OmL) and heated to boiling. Ethanol was added to the boiling filtrate until a clear, although slightly yellowish solution was formed. Cooling of this solution resulted in crystallization of the product (l.lg, 50% yield) as a pale yellow solid. NMR

o

(DMSO-de) ¹ H δ (ppm): 8.34 (IH, d, Ar-H), 7.87 (IH, dd, Ar-H), 7.42 (IH, d, Ar-H), 4.30 (2H, q, CH ₂ ), and 1.32 (3H, t, CH ₃ ). MS m/z: 223 (M+l)

Step: 2 Preparation of 8-{[6-(ethoxycarbonyl)-l,3-benzothiazol-2-yl]amino}-8-oxo octanoic acid

To an emulsion of suberic acid (0.87g, 5mmol) in dry THF (2OmL) was added

BOP reagent (2.65g, 6mmol) and hydroxy benzotriazole (0.86g, 6mmol) and the reaction mixture was stirred for 5 minutes at 3O ⁰ C. Subsequently diisopropyl ethylamine (2.55mL, 15mmol) was added and the reaction mixture was stirred for a further 10 minutes. Finally ethyl 2-amino-l,3-benzothiazole-6-carboxylate (l.lg, 5mmol) from step-1 was added and the stirring was continued for 5-8 hours. The reaction mixture was later poured into ice water to give a white precipitate, which was filtered. The precipitate was dissolved in 20% NaHCO ₃ solution, and filtered again. The filterate was neutralized with IN HCl to give a white colored precipitate, which was finally filtered, washed consecutively with hexane (15OmL x 2) and diisopropyl ether (5OmL) to give the product (0.93g, 47.5 % yield) as a white colored solid. Step: 3 Preparation of ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyI]amino}-l,3- benzothiazole-6-carboxylate.

To an emulsion of 8-{[6-(ethoxycarbonyl)-l,3-benzothiazol-2-yl]amino}-8-oxo octanoic acid (0.37g, lmmol) from step-2 in dry THF (2OmL) was added BOP reagent (0.53g, 1.2mmol) and hydroxy benzotriazole (0.16g, 1.2mmol) and the mixture was stirred for 5 minutes at 3O ⁰ C. Subsequently diisopropylethyl amine (0.5ImL, 3mmol) was added and the mixture was stirred for a further 10 minutes. Finally hydroxylamine hydrochloride (0.13g, 2mmol) was added and the reaction mixture was stirred for 5-8 hours. The reaction mixture was later poured into ice water to give a white precipitate, which was filtered and washed consecutively with hexane (15OmL x 2), dichloromethane (5OmL) to give the product (0.33g, 84% yield) as a white colored

solid. ¹ H NMR (DMSO-d ₆ ) δ (ppm): 1.27-1.28 (4H, m, -CH ₂ ), 1.32-1.36 (4H, m, -

CH ₂ ), 1.47-1.50 (2H, t, -CH ₂ ), 1.59-1.63 (2H, t, -CH ₂ ), 1.91-1.95 (3H, t, -CH ₃ ), 4.31-

4.36 (2H, q, -CH ₂ ), 7.79-7.81 (IH, d, Ar-H), 7.99-8.01 (IH, dd, Ar-H), 8.62-8.63 (IH ₅ d, Ar-H), 8.65 (IH, s, OH), 10.32 (IH, s, NH), 13.02 (IH, s, NH). MS m/z: 394.1

(M+l).

Example: 2

Synthesis of ethyl 2-({8-[(acetyloxy)amino]-8-oxooctanoyI}amino)-l,3- benzothiazole-6-carboxylate

To an emulsion of ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyl]amino}-l,3- benzothiazole-6-carboxylate (0.39g, lmmol) in dry DCM (2OmL) was added acetyl chloride (0.078mL, 1. lmmol) and the mixture was stirred for 5 minutes at 0 ⁰ C then diisopropyl ethylamine (0.5ImL, 3mmol) was added and the mixture was stirred for 2 hours. The reaction mixture was later poured into ice water to give a white precipitate, which was filtered, washed consecutively with hexane (100ml x 2), and dichloromethane (25ml) to give the title compound as a white colored solid (0.37g, 90% yield) with m.p.: 168-171 ⁰ C. ¹ H NMR (DMSOd ₆ ) δ (ppm): 1.30-1.36 (4H, m, - CH ₂ ), 1.42-1.46 (4H, m, -CH ₂ ), 1.59-1.63 (2H, t, -CH ₂ ), 1.91-1.95 (3H, t, -CH ₃ ), 2.10- 2.13 (2H, t, -CH ₂ ), 2.32 (3H, s, -COCH ₃ ), 4.31-4.34 (2H, q, -CH ₂ ), 7.78-7.80 (IH, d, Ar-H), 7.95-7.99 (IH, dd, Ar-H), 8.62-8.63 (IH, d, Ar-H), 10.30 (IH, s, NH), 13.05 (IH, s, NH). MS m/z: 436.1 (M+l) Example: 3

Synthesis of ethyl 2-{[8-(hydroxyamino)-8-oxooctanoyl] amino}-4-methyl-l,3- thiazole-5-carboxylate

-

Step; 1 Preparation of ethyl 2-amino-4-methyl-l,3-thiazoIe-5-carboxylate

To a solution of 2-chloro-ethyl acetoacetate (0.82g, 5mmol) in EtOH (25ml) was added thiourea (0.38gm, 5mmol) and the reaction mixture was stirred for 5 minutes at 3O ⁰ C. Pyridine (0.08g, lOmmol) was later added to the reaction mixture at 3O ⁰ C and the stirring was continued at reflux temperature for 6 hours. Subsequently the reaction mixture was diluted with ethyl acetate: hexane mixture (1:1, 10OmL) and filtered. After washing with diethyl ether (5OmL x 3), the crude material was purified by column chromatography using 1% MeOH in dichloromethane as an eluent to give the product (0.61g, 64.8% yield) as a white colored solid. Remaining steps were all performed according to the procedure given in example 1 to afford the title compound.

The following compounds were prepared according to the procedure given in Example: 1

-

t, -

- t,

Example: 13

Synthesis of 2-{[8-(benzyloxyamino)-8-oxooctanoyI]amino}-6(morpholin-4- ylcarbonyl)-l,3-benzothiazole.

To an emulsion of ethyl 2-{[8-(O-benzyl hydroxyamino)-8- oxooctanoyl]amino}-l,3-benzothiazole-6-carboxylate (0.37g, lmmol, prepared as described in example 1) in methanol (2OmL) was added KOH (0.1 Ig, 2mmol) and the mixture was stirred for 5 minutes at 3O ⁰ C. Subsequently morpholine (0.17g, 2mmol) was added and the mixture was stirred for a further 4 hours. Subsequently the reaction mixture was poured into ice water to give a white precipitate, which was filtered, washed consecutively with hexane (15OmL x 2) and chloroform (5OmL) to give the product (0.33g, 78.7% yield) as a white colored solid. ¹ H NMR (DMSOd ₆ ) δ (ppm): 1.26-1.27 (6H, m, -CH ₂ ), 1.48-1.49 (2H, m, -CH ₂ ), 1.60-1.61 (2H, t, -CH ₂ ), 1.94-1.98 (2H, t, -CH ₂ ), 3.56-3.60 (8H, bs, -CH ₂ ), 4.76 (2H, s, -OCH ₂ ), 7.36-7.40 (5H, m, Ar-H), 7.45-7.47 (IH, d, Ar-H), 7.74-7.76 (IH, dd, Ar-H), 8.07 (IH, d, Ar-H), 10.92 (IH, s, NH), 12.12 (IH, s, NH). MS m/z: 525.3 (M+l).

The following compound was prepared according to the procedure given in Example: 13

t, - -

Example: 15

Synthesis of S-(6-{[5-(morpholin-4-ylcarbonyl)-2-furyl]amino}-6-oxohexyl) ethanethioate

Preparation of 4-(5-nitro-2-furoyl) morpholine

5-Nitro-2-furoic acid (5.Og, 31.8mmol) was dissolved in DMF (10OmL); EDCI (12.2g, 63.6mmol), hydroxy benzotriazole (1.7g, 12.6mmol) and morpholine (5.5g, 63 mmol) were added to it, finally triethylamine (9.65 g, 96 mmol) was added and the reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with water (50OmL) and extracted with EtOAc (1 x 200 mL, 2 x 10OmL); the combined organic layers were washed with brine solution (10OmL), dried over anhydrous sodium sulphate and evaporated in vacuum to yield the crude compound viz., 4-(5-nitro-2-furoyl) morpholine, which was purified by column chromatography using 1% MeOH: DCM as eluent to afford the pure product (3.Og, 41.7% yield). Step-2 Preparation of 5-(morpholin-4-yIcarbonyl)furan-2-amine

4-(5-nitro-2-furoyl)morpholine (3.Og, 13mmol) was dissolved in methanol (20OmL); 10% Pd/C (300mg) was added to the above and the reaction mixture was hydrogenated in a Parr shaker at 80 psi for 3 hours. The reaction mass was filtered through celite and the filtrate was concentrated in vacuum to get 5-(morpholin-4- ylcarbonyl) furan-2-amine (2.Og, 76.9% yield).

Step-3 Preparation of 6-bromo-iV-[5-(morphoIin-4-yIcarbonyI)-2-furyl] hexanamide

5. 5-(morpholin-4-ylcarbonyl) furan-2-amine (3.Og, 15mmol) was dissolved in

THF (10OmL) and cooled to 1O ⁰ C. 6-Bromohexanoyl chloride (4.9g, 23mmol) and triethylamine (3.Og, 30mmol) were added and the reaction mixture was stirred at room temperature for 5 hours. Subsequently the reaction mass was diluted with water (50OmL) and extracted with EtOAc (1 x 200 niL, 2 x 100 niL); the combined organic 0 layers were washed with brine solution (10OmL), dried over anhydrous sodium sulphate and concentrated in vacuum to get 6-bromo-N-[5-(morpholin-4-ylcarbonyl)-2-furyl] hexanamide (3.2g, 56.0% yield).

Step-4 Preparation of S-(6-{[5-(morpholin-4-yIcarbonyI)-2-furyI]amino}-6- oxohexyl) ethanethioate 5

6-bromo-N-[5-(morpholin-4-ylcarbonyl)-2-furyl]hexanamide(3.O g, 8mmol) was dissolved in EtOH (10OmL), potassium thioacetate (1.8g, lόmmol) was added and the 0 reaction mixture was heated to 7O ⁰ C and maintained at the same temperature for 3 hours. Subsequently the solvent was removed by evaporation and the resulting residue was dissolved in water (200ml) and extracted with EtOAc (2 x 10OmL, 1 x 5OmL); the combined organic layers were washed with brine solution (10OmL), dried over anhydrous sodium sulphate and evaporated in vacuum to yield the crude material, 5 which was purified by column chromatography using 1% MeOH: DCM as eluent to afford the title compound as a pale yellow crystalline solid (0.6Og, 20.3% yield) with m.p: 68-68.5 ⁰ C. ¹ H NMR (DMSO-d6) δ ppm: 1.31-1.33 (2H, m, -CH ₂ ), 1.49-1.56 (4H, m, -CH ₂ ), 2.3 (3H, s, -SCH ₃ ), 2.50 (2H, t, -CH ₂ ), 2.80-2.84 (2H, t, -CH ₂ ), 3.62-3.65

(8H, m, -CH ₂ ), 6.28-6.29 (IH, d, Ar-H), 7.00-7.01 (IH, d, Ar-H), 11.21 (IH, bs, NH), MS m/z: 369.1 M+l.

The following compounds are prepared according to the procedure given in Example: 15

Example: 18

Synthesis of iV-methyl-5-[(6-morpholin-4-ylhexanoyI)amino]-2-furamide

Step-1 Preparation of N-methyI-5-nitro-2-furamide

5-Nitro-2-furoic acid (3.Og, 19mmol) was dissolved in THF (10OmL); EDCI

5 (7.3g, 38mmol), hydroxy benzotriazole (1.Og, 7.4mmol) and methylamine hydrochloride (2.58g, 38mmol) were added followed by triethylamine (5.8g, 57mmol).

The reaction mixture was stirred at room temperature for 4 hours. Subsequently it was diluted with water (40OmL) and extracted with EtOAc (1 x 20OmL, 2 x 10OmL); the combined organic layers were washed with brine solution (10OmL), dried over

10 anhydrous sodium sulphate and evaporated to yield the crude compound which was purified by column chromatography using 0.5% MeOH: DCM as the eluent to afford the title compound as a yellow solid (2.1g, 64.65% yield).

Step-2 Preparation of 5-amino-N-methyl-2-furamide

N-methyl-5-nitro-2-furamide (1.Og, 5.9mmol) was dissolved in methanol

(20OmL), 10% Pd/C (lOOmg) was added and the reaction mixture was hydrogenated in a Parr shaker at 80 psi for 5 hours. Subsequently the reaction mixture was filtered through celite and the filterate was evaporated to give 5-amino-N-methyl-2-furamide 20. (300mg, 36.45% yield).

Step-3 Preparation of 5-[(6-bromohexanoyI) amino]-iV-nietliyI-2-furamide

5-amino-N-methyl-2-furamide (l.Og, 7mmol) was dissolved in tetrahyrofuran

25 (10OmL) and cooled to 10 ⁰ C. 6-bromohexanoylchloride (2.3g, lO.Smmol) and triethylamine (1.43g, 14mmol) were added and the reaction mixture was stirred at room temperature for 5 hours. Subsequently the reaction mixture was diluted with water

(20OmL) and extracted with EtOAc (2 x 10OmL, 1 x 5OmL); the combined organic

layers were washed with brine solution (10OmL), dried over anhydrous sodium sulphate and evaporated to give 5-[(6-bromohexanoyl)amino]-N-methyl-2-furamide (l.lg, 48.67% yield). Step-4 Preparation of ν-methyl-5-[(6-morpholin-4-ylhexanoyl)amino]-2-furamide

5-[(6-bromohexanoyl) amino] -N-methyl-2-furamide (l.Og, 3mmol) was dissolved in DCM (10OmL); morpholine (0.55g, 6mmol) and triethylamine (0.95g, 9mmol) were added and the reaction mixture was stirred at room temperature for 8

10 hours. Subsequently the reaction mixture was diluted with water (20OmL) and extracted with EtOAc (2 x 10OmL, 1 x 5OmL); the combined organic layer was washed with brine solution (10OmL), dried over anhydrous sodium sulphate and concentrated in vacuum to give crude iV-methyl-5-[(6-morpholin-4-ylhexanoyl)amino]-2-furamide which was purified by column chromatography using 1% MeOH: DCM as eluent to

15. afford the title compound as brown colored sticky compound (0.15g, 15% yield). ¹ H νMR (DMSO-de) δ (ppm): 1.26-1.30 (4H, m, -CH ₂ ), 1.46-1.49 (2H, m, -CH ₂ ), 1.57- 1.61 (4H, m, -CH ₂ ), 2.70-2.71 (3H, s, -CH ₃ ), 3.58-3.60 (8H, bs, -CH ₂ ), 6.25-6.26 (IH, d, Ar-H), 7.07-7.08 (IH, d, Ar-H), 8.03 (IH, s, NH), 11.18 (IH, s, NH). MS m/z: 324.1 (M+l).

20 The following compound was prepared according to the procedure given in Example: 18

Anti-cancer experimental methods

Anti-cancer screen;

Experimental drugs are screened for anti-cancer activity in three cell lines for

5 their GI ₅₀ , TGI and LC ₅ 0 values (using five concentrations for each compound). The cell lines are maintained in DMEM containing 10% fetal bovine serum. 96 well micro titer plates are inoculated with cells in 100 μL for 24 hours at 37°C, 5% CO ₂ , 95% air and 100% relative humidity. 5000 HCT 116 cells/well, 5000 NCIH 460 cells/well and

5000 U251 or 5000 MDAMB 231 cells/well are plated. A separate plate with these cell

10 lines is also inoculated to determine cell viability before the addition of the compounds

(To).

Addition of experimental drugs:

Following 24-hour incubation, experimental drugs are added to the 96 well

15 plates. Each plate contains one of the above cell lines and the following in triplicate: five different concentrations (0.01, 0.1, 1, 10 and 100 μM) of four different compounds, appropriate dilutions of a cytotoxic standard and,control (untreated) wells. Compounds are dissolved in DMSO to make 20 niM stock solutions on the day of drug addition and frozen at -2O ⁰ C. Serial dilutions of these 20 mM stock solutions are made in complete

20 growth medium such that 100 μL of these drug solutions in medium, of final concentrations equaling 0.01, 0.1, 1, 10 and 100 μM can be added to the cells in triplicate. Standard drugs whose anti-cancer activity has been well documented and which are regularly used are doxorubicin and SAHA.

25. End-point measurement:

For T ₀ measurement, 24 hours after seeding the cells, 10 μL of 3-(4,5-Dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) solution per well is added and incubation carried out for 3 hours at 37°C, 5% CO ₂ , 95% air and 100% relative humidity, protected from light. Cells incubated with compounds for 48 hours are 30 treated similarly except with the addition of 20 μL MTT solution per well and a subsequent incubation under the same conditions. After 3 hours of MTT incubation, well contents are aspirated carefully followed by addition of 150 μL DMSO per well.

Plates are agitated to ensure solution of the formazan crystals in DMSO and absorbance read at 570 nm.

Calculation of Gk _n , TGI and LCsn; Percent growth is calculated for each compound's concentration relative to the control and zero measurement wells (To; viability right before compound addition). If a test well's O.D. value is greater than the T ₀ measurement for that cell line % Growth = (test - zero) / (control - zero) X lOO

If a test well's O.D. value is lower than the To measurement for that cell line, then, % Growth = (test - zero) / zero X 100

Plotting % growth versus experimental drug concentration, GI ₅₀ is the concentration required to decrease growth by 50%; TGI is the concentration required to decrease growth by 100% and LC50 is the concentration required to decrease growth by 50%.

HDAC Activity screening:

Histone Deacetylase (HDAC) Inhibition Assay using Boc-Lys(Ac)-AMC Substrate:

Inhibition of HDAC has been implicated to modulate transcription and to induce apoptosis or differentiation in cancer cells. The fluorometric assay provides a fast and fluorescence based method that eliminates radioactivity, extractions or chromatography, as used in traditional assays. The assay is based on two steps. First, the HDAC fluorometric substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (Mouse Liver Extract). Deacetylation of the substrate sensitizes the substrate, in the second step; treatment with the Trypsin stop solution produces a fluorophore that can be easily analyzed using fluorescence plate reader.

Assay was done in 96 well black microplate and total volume of the assay is 100 μL. Mouse liver enzyme is diluted 1:6 with HDAC buffer. Enzyme cocktail is made of 10 μL of diluted enzyme and 30 μL of HDAC buffer. 40 μL of enzyme cocktail is dispensed into each well. 10 μL of different concentrations of inhibitor is added into each well, except enzyme control well. Preincubated the plate at 30 ⁰ C for 5 minutes. The HDAC reaction is started by adding 50 μL of HDAC substrate (Boc- Lys(Ac)-AMC Substrate) solution. Incubated the plate at 30 ⁰ C for 30 minutes. Adding 100 μL of Trypsin stop solution stops the reaction. The plate is incubated again at 30 ⁰ C

for 20-30 minutes. The release of AMC is monitored by measuring the fluorescence at an excitation wavelength of 365 or 360 nm and Emission wavelength of 440 or 460 nm. Buffer and substrate alone kept for blank subtraction. (Dennis Wegener et, al., Anal. Biochem, 321, 2003, 202-208).