EP2285376A1

EP2285376A1 - 6-aminonicotinamide derivatives as potent and selective histone deacetylase inhibitors

Info

Publication number: EP2285376A1
Application number: EP09747249A
Authority: EP
Inventors: Xian-Ping Lu; Zhi-bin LI; Ning ZHI-QIANG
Original assignee: Chipscreen Biosciences Ltd
Current assignee: Chipscreen Biosciences Ltd
Priority date: 2008-05-16
Filing date: 2009-05-08
Publication date: 2011-02-23
Also published as: WO2009140164A1; EP2285376A4; JP2011520891A

Abstract

The present invention relates to certain 6-aminonicotinamide derivatives which are capable of inhibiting histone deacetylases. The compounds of this invention are therefore useful in treating diseases associated with abnormal histone deacetylase activities. Pharmaceutical compositions comprising these compounds, methods of treating diseases utilizing pharmaceutical compositions comprising these compounds, and methods of preparing these compounds are also disclosed.

Description

6-AMINONICOTINAMIDE DERIVATIVES AS POTENT AND SELECTIVE HISTONE DEACETYLASE INHIBITORS

FIELD OF INVENTION

BACKGROUND OF THE INVENTION

Histone deacetylase (HDAC) proteins play a critical role in regulating gene expression in vivo by altering the accessibility of genomic DNA to transcription factors. Specifically, HDAC proteins remove the acetyl group of acetyl-lysine residues on histones, which can result in nucleosomal remodelling (Grunstein, M., 1997, Nature, 389: 349-352). Due to their governing role in gene expression, HDAC proteins are associated with a variety of cellular events, including cell cycle regulation, cell proliferation, differentiation, reprogramming of gene expression, and cancer development (Ruijter, A-J-M., 2003, Biochem. J., 370: 737-749; Grignani, F., 1998, Nature, 391 : 815-818; Lin, R-J. ,1998, 391 : 811 -814; Marks, P-A., 2001 , Nature Reviews Cancer, 1 : 194). The aberrant deacetylation resulting from the misregulation of histone deacetylases (HDACs) has been linked to clinical disorders such as Rubinstein-Taybi syndrome, fragile X syndrome, leukemia, and various cancers (Langley B et al., 2005, Current Drug Targets - CNS & Neurological Disorders, 4: 41-50). In fact, HDAC inhibitors have been demonstrated to reduce tumor growth in various human tissues and in animal studies, including lung, stomach, breast, and prostrate (Dokmanovic, M. ,2005, J. Cell Biochenm., 96: 293-304).

Aberrant histone deacetylase activity has also been linked to various neurological and neurodegenerative disorders, including stroke, Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer's disease. HDAC inhibition may induce the expression of anti-mitotic and anti-apoptotic genes, such as p21 and HSP-70, which facilitate survival. HDAC inhibitors can act on other neural cell types in the central nervous system, such as reactive astrocytes and microglia, to reduce inflammation and secondary damage during neuronal injury or disease. HDAC inhibition is a promising therapeutic approach for the treatment of a range of central nervous system disorders (Langley B et al., 2005, Current Drug Targets - CNS & Neurological Disorders, 4: 41 -50).

Mammalian HDACs can be divided into three classes according to sequence homology. Class I consists of the yeast Rpd3-like proteins (HDAC 1 , 2, 3, 8 and 11 ). Class Il consists of the yeast HDA1-like proteins (HDAC 4, 5, 6, 7, 9 and 10). Class III consists of the yeast SIR2-like proteins (SIRT 1 , 2, 3, 4, 5, 6 and 7). The activity of HDAC1 has been linked to cell proliferation, a hallmark of cancer. Particularly, mammalian cells with knock down of HDAC1 expression using siRNA were antiproliferative (Glaser, K-B., 2003, Biochem. Biophys. Res. Comm., 310: 529-536). While the knock out mouse of HDAC1 was embryonic lethal, the resulting stem cells displayed altered cell growth (Lagger, G., 2002, £M60 J., 21 : 2672-2681 ). Mouse cells overexpressing HDAC1 demonstrated a lengthening of G₂ and M phases and reduced growth rate (Bartl. S., 1997, MoI. Cell Biol., 17: 5033-5043). Therefore, the reported data implicate HDAC1 in cell cycle regulation and cell proliferation.

HDAC2 regulates expression of many fetal cardiac isoforms. HDAC2 deficiency or chemical inhibition of histone deacetylase prevented the re-expression of fetal genes and attenuated cardiac hypertrophy in hearts exposed to hypertrophic stimuli. Resistance to hypertrophy was associated with increased expression of the gene encoding inositol polyphosphate-5-phosphatase f (Inppδf) resulting in constitutive activation of glycogen synthase kinase 3β (Gsk3β) via inactivation of thymoma viral proto-oncogene (Akt) and 3-phosphoinositide-dependent protein kinase-1 (Pdk1 ). In contrast, HDAC2 transgenic mice had augmented hypertrophy associated with inactivated Gsk3β. Chemical inhibition of activated Gsk3β allowed HDAC2-deficient adults to become sensitive to hypertrophic stimulation. These results suggest that HDAC2 is an important molecular target of HDAC inhibitors in the heart and that HDAC2 and Gsk3β are components of a regulatory pathway providing an attractive therapeutic target for the treatment of cardiac hypertrophy and heart failure (Trivedi, C-M., 2007, Nat. Med,. 13: 324-331 ).

HDAC3 is maximally expressed in proliferating crypt cells in the intestine. Silencing of HDAC3 expression in colon cancer cell lines resulted in growth inhibition, a decrease in cell survival, and increased apoptosis. Similar effects were observed for HDAC2 and, to a lesser extent, for HDAC1. HDAC3 gene silencing also selectively induced expression of alkaline phosphatase, a marker of colon cell maturation. Concurrent with its effect on cell growth, overexpression of HDAC3 inhibited basal and butyrate-induced p21 transcription in a Sp1/Sp3-dependent manner, whereas silencing of HDAC3 stimulated p21 promoter activity and expression. These findings identify HDAC3 as a gene deregulated in human colon cancer and as a novel regulator of colon cell maturation and p21 expression (Wilson, A-J., 2006, J. Biol. Chem., 281 : 13548-13558).

HDAC6 is a subtype of the HDAC family that deacetylates alpha-tubulin and increases cell motility. Using quantitative real-time reverse transcription polymerase chain reaction and Western blots on nine oral squamous cell carcinoma (OSCC)-derived cell lines and normal oral keratinocytes (NOKs), HDAC6 mRNA and protein expression were commonly up-regulated in all cell lines compared with the NOKs. Immunofluorescence analysis detected HDAC6 protein in the cytoplasm of OSCC cell lines. Similar to OSCC cell lines, high frequencies of HDAC6 up-regulation were evident in both mRNA (74%) and protein (51 %) levels of primary human OSCC tumors. Among the clinical variables analyzed, the clinical tumor stage was found to be associated with the HDAC6 expression states. The analysis indicated a significant difference in the HDAC6 expression level between the early stage (stage I and II) and advanced -stage (stage III and IV) tumors (P=O.014). These results suggest that HDAC6 expression may be correlated with tumor aggressiveness and offer clues to the planning of new treatments (Sakuma, T., 2006, Int. J. Oncol., 29: 117-124).

Epigenetic silencing of functional chromosomes by HDAC is one of the major mechanisms that occurrs in pathological processes in which functionally critical genes are repressed or reprogrammed by HDAC activities leading to the loss of phenotypes in terminal differentiation, maturation and growth control, and the loss of functionality of tissues. For example, tumor suppressor genes are often silenced during development of cancer and chemical inhibitors of HDAC can derepress the expression of these tumor suppressor genes, leading to growth arrest and differentiation (Glaros S et al., 2007, Oncogene June 4 Epub ahead of print; Mai, A, et al., 2007, lnt J. Biochem Cell Bio., April 4, Epub ahead of print; Vincent A. et al., 2007, Oncogene, April 30, Epub ahead of print; our unpublished results). Repression of structural genes such as FXN in Friedreich's ataxia and SMN in spinal muscular atrophy can be reversed by HDAC inhibitors, leading to re-expression and resumption of FXN and SMN gene function in tissues (Herman D et al., 2006, Nature Chemical Biology, 2(10):551-8; Avila AM et al., 2007, J Clinic Investigation, 117(3)659-71 ; de Bore J, 2006, Tissue Eng. 12(10):2927-37); Induction of the entire MHC Il family gene expression through reprogramming of HDAC "hot spot" in chromosome 6p21 -22 by HDAC inhibitors further extends epigenetic modulation of immune recognition and immune response (Gialitakis M et al., 2007, Nucleic Acids Res., 34(1 );765-72).

Several classes of HDAC inhibitors have been identified, including (1 ) short-chain fatty acids, e.g. butyrate and phenylbutyrate; (2) organic hydroxamic acids, e.g. suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); (3) cyclic tetrapeptides containing a 2-amino-8-oxo 9,10-expoxydecanoyl (AOE) moiety, e.g. trapoxin and HC-toxin; (4) cyclic peptides without the AOE moiety, e.g. apicidin and FK228; and (5) benzamides, e.g. MS-275 (EP0847992A1 , US2002/0103192A1 , WO02/26696A1 , WO01/70675A2, WO01/18171A2). HDAC represents a very promising drug target especially in the context of epigenic biology;; for example, in terms of preferential apoptosis-induction in malignant cells but not normal cells, differentiation of epithelia in cancer cells, anti-inflammatory and immunomodulation, and cell cycle arrest.

The use of HDAC inhibitors can be considered as "neo-chemotherapy" having a much improved toxicity profile over existing chemotherapy options. The success of SAHA from Merck is currently only limited to the treatment of cutaneous T cell lymphoma. No reports exist indicating that SAHA treatment is effective against major solid tumors or for any other indications. Therefore, there is still a need to discover new compounds with improved profiles, such as stronger HDAC inhibitory activity and anti-cancer activity, more selective inhibition on different HDAC subtypes, and lower toxicity; There is a continuing need to identify novel HDAC inhibitors that can be used to treat potential new indications such as neurological and neurodegenerative disorders, cardiovascular disease, metabolic disease, and inflammatory and immunological diseases.

SUMMARY OF THE INVENTION

The present invention is directed to certain 6-aminonicotinamide derivatives which exhibit selective histone deacetylase inhibition activity and are therefore useful in treating diseases associated with aberrant histone deacetylase activities, such as Rubinstein-Taybi syndrome, fragile X syndrome, leukemia, cardiac hypertrophy, metabolic disease, cancer and various neurological and neurodegenerative disorders.

DETAILED DESCRIPTION OF THE INVENTION

Various publications are cited throughout the present application. The contents of these publications and contents of documents cited in these publications are herein incorporated herein by reference.

Accordingly, the present invention provides a compound having the structure represented by formula (I), or its stereoisomer, enantiomer, diastereomer, hydrate, or pharmaceutically acceptable salts thereof:

(I) wherein

R¹ , R², R³ and R⁴ are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl;

-_>5 ;

R is pyridyl, which optionally substituted with one or more halogen, alkyl, alkoxy or trifluoromethyl; or R⁵ is

X is a bond, -CH₂CH₂-, -CH=CH-, O or S;

Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring, which optionally substituted with one or more halogen, nitro, alkyl or alkoxy; n is an integer ranging from 2 to 6. In the above structural formula (I) and throughout the present specification, the following terms have the indicated meaning:

The term "halo" as used herein means fluorine, chlorine, bromine or iodine.

The term "alkyl" as used herein includes methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl and the like.

The term "alkoxy" as used herein includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy and the like.

In one embodiment of a compound of formula (I), R¹, R², R³ and R⁴ are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; R⁵ is pyridyl, which optionally substituted with one or more halogen, alkyl, alkoxy or trifluoromethyl; and n is an integer ranging from 2 to 4.

In another embodiment of a compound of formula (I), R¹, R², R³ and R⁴ are independently H or F; R⁵ is pyridyl; and n is an integer ranging from 2 to 4.

In another embodiment of a compound of formula (I),, R¹, R², R³ and R⁴ are independently

hydrogen, halo, alkyl, alkoxy or trifluoromethyl; R⁵ is wherein X is a bond, -CH₂CH₂-, -CH=CH-, O or S; Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring optionally substituted with one or more of halogen, nitro, alkyl or alkoxy; and n is an integer ranging from 2 to 4.

In another embodiment of a compound of formula (I)₁R¹, R², R³ and R⁴ are independently H

or F; R⁵ is , wherein X is a bond; Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring; n is an integer ranging from 2 to 4.

The compounds of this invention are prepared as described below:

(a) 6-Chloronicotinic acid is condensed with compound 1 to give compound 2;

( 1 ) ( 2 )

(b) Compound 2 is condensed with compound 3 to give compound 4;

( 2 ) ( 4 )

(c) Compound 4 is condensed with compound 5 to give compound 6.

( 5 ) ( 6 )

Condensation reactions (a) and (c) are conducted by using a peptide condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide (DCC), N,N'-carbonyldiimidazole (CDI), etc. The reaction may be conducted at 0 to 80 ⁰C for 4 to 72 hours. Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc. If necessary, a base such as sodium hydroxide, triethylamine and pyridine may be added to the reaction system.

Condensation reaction (b) is conducted at 40 to 120 ⁰C for 1 to 24 hours. Solvents which may be used are normal solvents such as benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, N, N-dimethylformamide, etc. If necessary, a base such as sodium hydroxide, triethylamine and pyridine may be added to the reaction system.

The compounds represented by formula (I) and the intermediate (2) and (4) may be purified or isolated by the conventional separation method such as extraction, recrystallization, column chromatography and the like.

The compounds represented by formula (I) are capable of inhibiting histone deacetylases and are therefore useful in treating diseases associated with abnormal histone deacetylase activities, such as Rubinstein-Taybi syndrome, fragile X syndrome, leukemia, cardiac hypertrophy, metabolic disease, cancer and various neurological and neurodegenerative disorders. Thus, the invention also provides a method for treating diseases associated with abnormal deacetylase activitites to a mammal, including man and mammmals, comprising administering a compound of formula (I) or apharmaceutically acceptable salt thereof to a mammal in need of such treatment.

The compounds represented by formula (I) useful as a drug may be used in the form of a general pharmaceutical composition. The pharmaceutical compositions may be in forms normally employed, such as tablets, capsules, powders, syrups, solutions, suspensions, aerosols, and the like, may contain flavorants, sweeteners etc. in suitable solids or liquid carriers or diluents, or in suitable sterile media to form injectable solutions or suspensions. Such composition typically contains from 0.5 to 70%, preferably 1 to 20% by weight of active compound, the remainder of the composition being pharmaceutically acceptable carriers, diluents or solvents or salt solutions.

The compounds represented by formula (I) are clinically administered to mammals, including man and animals, via oral, nasal, transdermal, pulmonary, or parenteral routes. Administration by the oral route is preferred, being more convenient and avoiding the possible pain and irritation of injection. By either route, the dosage is in the range of about 0.0001 to 200 mg/kg body weight per day administered singly or as a divided dose. However, the optimal dosage for the individual subject being treated will be determined by the person responsible for treatment, generally smaller dose being administered initially and thereafter increments made to determine the most suitable dosage.

Representative compounds of the present invention are shown in Table 1 below. The compound numbers correspond to the "Example numbers" in the Examples section. That is, the synthesis of the compound 3 as shown in the Table 1 is described in "Example 3" and the synthesis of the compound 51 as shown in the Table 1 is described in "Example 51 ". The compounds presented in the Table 1 are exemplary only and are not to be construed as limiting the scope of this invention in any manner.

TABLE 1

Example Structure Name

N-(2-Aminophenyl)-6-(2-(2-(9H- fluoren-9-ylidene)acetamido)- ethylamino)nicotinamide Example Structure Name

N-(2-Aminophenyl)-6-(2-

(nicotinamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(3-(2-(9H- fluoren-9-ylidene)acetamido)- propylamino)nicotinamide

N-(2-Aminophenyl)-6-(3-

(nicotinamido)propylamino)-

Nicotinamide

N-(2-Aminophenyl)-6-(4-(2-(9H- fluoren-9-ylidene)acetamido)- butylamino)nicotinamide

N-(2-Aminophenyl)-6-(4-

10 (nicotinamido)butylamino)-

Nicotinamide Example Structure Name

N-(2-Amino-4-fluorophenyl)-6- (2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Amino-4-fluorophenyl)-6-

(2-(nicotinamido)ethylamino)- nicotinamide

N-(2-Amino-4-fluorophenyl)-6- (3-(2-(9H-fluoren-9-ylidene)- acetamido)propylamino)- nicotinamide

N-(2-Amino-4-fluorophenyl)-6- (3-(nicotinamido)propylamino)- Nicotinamide

N-(2-Amino-4-fluorophenyl)-6- (4-(2-(9H-fluoren-9-ylidene)- acetamido)butylamino)- nicotinamide Example Structure Name

N-(2-Amino-4-fluorophenyl)-6-

(4-(nicotinamido)butylamino)-

Nicotinamide

N-(2-Amino-4-chlorophenyl)-6- (2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Amino-4-chlorophenyl)-

6-(2-(nicotinamido)ethylamino)- nicotinamide

N-(2-Amino-4-methylphenyl)-6- (2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Amino-4-methylphenyl)-6-

(2-(nicotinamido)ethylamino)-

Nicotinamide Example Structure Name

N-(2-Amino-4-methoxyphenyl)-6- (2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Amino-4-methoxyphenyl)-6-

(2-(nicotinamido)ethylamino)-

Nicotinamide

N-(2-Amino-4-trifluoromethyl- phenyl)-6-(2-(2-(9H-fluoren-9- ylidene)acetamido)ethylamino)- nicotinamide

N-(2-Amino-4-trifluoromethyl- phenyl)-6-(2-(nicotinamido)ethyl- amino)nicotinamide

N-(2-Aminophenyl)-6-(2-(2-(2,7- dichloro-9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide Example Structure Name

N-(2-Aminophenyl)-6-(2-(2-(2,7- dinitro-9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(2-(2,7- dimethoxy-9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(2-(4,5- dimethyl-9H-fluoren-9-ylidene)- acetamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(2-(5H- dibenzocyclohepten-5-ylidene)-

41 acetamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(2-(10,1 1-dihydro-5H-dibenzocyclohepte

42 n-5-ylidene)acetamido)ethyl- amino)nicotinamide Example Structure Name

N-(2-Aminophenyl)-6-(2-(2-(9H-

43 xanthen-9-ylidene)acetamido)- ethylamino)nicotinamide

N-(2-Aminophenyl)-6-(2-(6-

45 chloronicotinamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(6-

46 methylnicotinamido)ethylamino)- nicotinamide

N-(2-Aminophenyl)-6-(2-(2-

47 methoxynicotinamido)ethyl- amino)nicotinamide Example Structure Name

N-(2-Aminophenyl)-6-(2-(6-

48 trifluoromethylnicotinamido)- ethylamino)nicotinamide

N-(2-Aminophenyl)-6-(6-(2-(9H-

50 fluoren-9-ylidene)acetamido)- hexylamino)nicotinamide

nyl)-6-(6- exylamino)-

Further, all parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified. Any range of numbers recited in the specification or paragraphs hereinafter describing or claiming various aspects of the invention, such as that representing a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers or ranges subsumed within any range so recited. The term "about" when used as a modifier for, or in conjunction with, a variable, is intended to convey that the numbers and ranges disclosed herein are flexible and that practice of the present invention by those skilled in the art using temperatures, concentrations, amounts, contents, carbon numbers, and properties that are outside of the range or different from a single value, will achieve the desired result. Example 1 Preparation of N-(2-aminophenyl)-6-chloronicotinamide

6-Chloronicotinic acid (158 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and o-phenylenediamine (216 mg, 2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (138 mg, 56% yield) as a brown solid. LC-MS (m/z) 248 (M+1 ).

Example 2 Preparation of N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Aminophenyl)-6-chloronicotinamide (248 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (150 mg, 55% yield) as a brown solid. LC-MS (m/z) 272 (M+1 ).

Example 3

Preparation of N-(2-aminophenyl)-6-(2-(2-(9H-fluoren-9-ylidene)acetamido)- ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (280 mg, 59%) as an off-white solid. ¹ H NMR (DMSO-d₆)δ3.48 (m, 4H, CH₂CH₂), 4.86 (s, 2H, benzene-NH₂), 6.55 (m, 2H), 6.75 (d, J= 7.6 Hz, 1 H), 6.94 (t, J= 7.6 Hz, 1 H), 7.09 (s, 1 H), 7.12 (d, J= 7.6 Hz, 1 H), 7.28-7.34 (m, 3H), 7.42 (t, J= 7.2 Hz, 2H), 7.76 (d, J= 7.6 Hz, 1 H), 7.82 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.68 (m, 2H), 8.74 (d, J= 7.6 Hz, 1 H), 9.39 (s, 1 H, benzene-NH). LC-MS (m/z) 476 (M+1 ).

Example 4 Preparation of N-(2-aminophenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (256 mg, 68%) as a brown solid. ¹ H NMR (DMSO-d₆)δ3.48 (m, 4H, CH₂CH₂), 4.86 (s, 2H, benzene-NH₂), 6.52 (d, J= 8.8 Hz, 2H), 6.57 (m, 1 H), 6.75 (dd, J= 1.2 & 8.0 Hz, 1 H), 6.94 (t, J= 8.0 Hz, 1 H), 7.12 (d, J= 8.0 Hz, 1 H), 7.20 (s, 1 H), 7.50 (m, 1 H), 7.91 (d, J= 8.0 Hz, 1 H), 8.18 (d, J= 8.0 Hz, 1 H), 8.65 (s, 1 H), 8.69 (m, 1 H), 8.83 (m, 1 H), 9.01 (s, 1 H), 9.39 (s, 1 H, benzene-NH). LC-MS (m/z) 377 (M+1 ).

Example 5 Preparation of N-(2-aminophenyl)-6-(3-aminopropylamino)nicotinamide

N-(2-Aminophenyl)-6-chloronicotinamide (248 mg, 1 mmol) and 6 ml of 1 ,3-propanediamine were heated to 8O⁰C for 3 hours. The excess 1 ,3-propanediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (168 mg, 59% yield) as a brown solid. LC-MS (m/z) 286 (M+1 ).

Example 6 Preparation of N-(2-aminophenyl)-6-(3-(2-(9H-fluoren-9-ylidene)acetamido)- propylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(3-aminopropylamino)nicotinamide (299 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (269 mg, 55%) as an off-white solid. ¹H NMR (DMSO-d₆)δ1.82 (m, 2H, CH₂), 3.38 (m, 4H, 2xCH₂), 4.86 (s, 2H, benzene-NH₂), 6.51 -6.57 (m, 2H), 6.76 (d, J= 7.6 Hz, 1 H), 6.94 (t, J= 7.6 Hz, 1 H), 7.10 (s, 1 H), 7.10-7.13 (d, J= 8.0 Hz, 1 H), 7.20 (s, 1 H), 7.29-7.39 (m, 2H), 7.41 (t, J= 7.2 Hz, 2H), 7.78 (d, J= 7.6 Hz, 1 H), 7.83 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.60 (s, 1 H), 8.69 (s, 1 H), 8.70 (d, J= 7.6 Hz, 1 H), 9.36 (s, 1 H, benzene-NH). LC-MS (m/z) 490 (M+1 ).

Example 7 Preparation of N-(2-aminophenyl)-6-(3-(nicotinamido)propylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(3-aminopropylamino)nicotinamide (299 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (281 mg, 72%) as a brown solid. ¹ H NMR (DMSO-d₆)δ1.81 (m, 2H, CH₂), 3.38 (m, 4H, 2xCH₂), 4.85 (s, 2H, benzene-NH₂), 6.50 (d, J= 8.0 Hz, 2H), 6.57 (m, 1 H), 6.75 (d, J= 8.0 Hz, 1 H), 6.93 (t, J= 8.0 Hz, 1 H), 7.12 (d, J= 8.0 Hz, 1 H), 7.19 (t, J= 4.0 Hz, 1 H), 7.50 (m, 1 H), 7.91 (d, J= 8.0 Hz, 1 H), 8.18 (d, J= 8.0 Hz, 1 H), 8.63 (s, 1 H), 8.68-8.72 (m, 2H), 9.00 (s, 1 H), 9.36 (s, 1 H, benzene-NH). LC-MS (m/z) 391 (M+1 ). Example 8 Preparation of N-(2-aminophenyl)-6-(4-aminobutylamino)nicotinamide

N-(2-Aminophenyl)-6-chloronicotinamide (248 mg, 1 mmol) and 7 ml of 1 ,4-butanediamine were heated to 8O⁰C for 3 hours. The excess 1 ,4-butanediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (158 mg, 53% yield) as a brown solid. LC-MS (m/z) 300 (M+1 ).

Example 9

Preparation of N-(2-aminophenyl)-6-(4-(2-(9H-fluoren-9-ylidene)acetamido)- butylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(4-aminobutylamino)nicotinamide (314 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (307 mg, 61 %) as an off-white solid. ¹H NMR (DMSO-d₆)δ1 .61 (m, 4H, CH₂CH₂), 3.29 (m, 4H, 2xCH₂), 4.85 (s, 2H, benzene-NH₂), 6.49 (d, J= 8.8 Hz, 1 H), 6.57 (t, J= 8.8 Hz, 1 H), 6.76 (d, J= 7.6 Hz, 1 H), 6.93 (t, J= 7.6 Hz, 1 H), 7.09 (s, 1 H), 7.09-7.13 (d, J= 8.0 Hz, 1 H), 7.18 (m, 1 H), 7.28-7.35 (m, 3H), 7.42 (t, J= 7.2 Hz, 2H), 7.76 (d, J= 7.2 Hz, 1 H), 7.82 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.57 (s,1 H), 8.63 (s, 1 H), 8.69 (d, J= 7.6 Hz, 1 H), 9.35 (s, 1 H, benzene-NH). LC-MS (m/z) 504 (M+1 ). Example 10 Preparation of N-(2-aminophenyl)-6-(4-(nicotinamido)butylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(4-aminobutylamino)nicotinamide (314 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (307 mg, 76%) as a brown solid. ¹ H NMR (DMSO-d₆)δ1.60 (m, 4H, CH₂CH₂), 3.30 (m, 2H, CH₂), 3.36 (m, 2H, CH₂), 4.85 (s, 2H, benzene-NH₂), 6.48 (d, J= 8.8 Hz, 2H), 6.57 (m, 1 H), 6.75 (d, J= 8.0 Hz, 1 H), 6.93 (t, J= 8.0 Hz, 1 H), 7.12 (d, J= 8.0 Hz, 1 H), 7.17(t, J= 4.0 Hz, 1 H), 7.48 (m, 1 H), 7.91 (d, J= 8.0 Hz, 1 H), 8.18 (d, J= 8.0 Hz, 1 H), 8.63 (s, 1 H), 8.68 (m, 2H), 8.99 (s, 1 H), 9.36 (s, 1 H, benzene-NH). LC-MS (m/z) 405 (M+1 ).

Example 11 Preparation of N-(2-amino-4-fluorophenyl)-6-chloronicotinamide

6-Chloronicotinic acid (157.5 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-fluoro-o-phenylenediamine (151 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (193 mg, 73% yield) as a brown solid. LC-MS (m/z) 266 (M+1 ). Example 12 Preparation of N-(2-amino-4-fluorophenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Amino-4-fluorophenyl)-6-chloronicotinamide (266 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (176 mg, 61 % yield) as a brown solid. LC-MS (m/z) 290 (M+1 ).

Example 13

Preparation of N-(2-amino-4-fluorophenyl)-6-(2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(2-aminoethylamino)nicotinamide (303 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (261 mg, 53%) as an off-white solid. ¹ H NMR (DMSO-d₆)δ3.48 (m, 4H, CH₂CH₂), 5.19 (s, 2H, benzene-NH₂), 6.34 (m, 1 H), 6.53 (m, 2H), 7.06 (m, 2H), 7.28-7.38 (m, 3H), 7.42 (t, J= 7.2 Hz, 2H), 7.76 (d, J= 7.6 Hz, 1 H), 7.82 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.67 (m, 2H), 8.74 (d, J= 7.6 Hz, 1 H), 9.32 (s, 1 H, benzene-NH). LC-MS (m/z) 494 (M+1 ).

Example 14

Preparation of

N-(2-amino-4-fluorophenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(2-aminoethylamino)nicotinamide (303 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (236 mg, 60%) as a brown solid. ¹H NMR (DMSO-d₆)δ3.46~3.52 (m, 4H, CH₂CH₂), 5.18 (s, 2H, benzene-NH₂), 6.33 (m, 1 H), 6.50-6.53 (m, 2H), 7.06 (m, 1 H), 7.30 (s, 1 H), 7.49 (dd, J= 4.8 & 8.0 Hz, 1 H), 7.91 (d, J= 8.0 Hz, 1 H), 8.18 (d, J= 8.0 Hz, 1 H), 8.64(s, 1 H), 8.69 (dd, J= 1.2 & 4.8 Hz, 1 H), 8.81 (m, 1 H), 9.00 (s, 1 H), 9.31 (s, 1 H, benzene-NH). LC-MS (m/z) 395 (M+1 ).

Example 15 Preparation of N-(2-amino-4-fluorophenyl)-6-(3-aminopropylamino)nicotinamide

N-(2-Amino-4-fluorophenyl)-6-chloronicotinamide (266 mg, 1 mmol) and 6 ml of 1 ,3-propanediamine were heated to 8O⁰C for 3 hours. The excess 1 ,3-propanediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (158 mg, 52% yield) as a brown solid. LC-MS (m/z) 304 (M+1 ).

Example 16

Preparation of N-(2-amino-4-fluorophenyl)-6-(3-(2-(9H-fluoren-9-ylidene)- acetamido)propylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(3-aminopropylamino)nicotinamide (318 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (248 mg, 49%) as an off-white solid. ¹ H NMR (DMSO-d₆)δ1.81 (m, 2H, CH₂), 3.38 (m, 4H, 2xCH₂), 5.18 (s, 2H, benzene-NH₂), 6.33 (m, 1 H), 6.51 (m, 2H), 7.06-7.10 (m, 2H), 7.20 (s, 1 H), 7.29-7.36 (m, 2H), 7.42 (t, J= 7.2 Hz, 2H), 7.77 (d, J= 7.6 Hz, 1 H), 7.82 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.60 (s, 1 H), 8.64 (s, 1 H), 8.69 (d, J= 7.6 Hz, 1 H), 9.29 (s, 1 H, benzene-NH). LC-MS (m/z) 508 (M+1 ).

Example 17

Preparation of

N-(2-amino-4-fluorophenyl)-6-(3-(nicotinamido)propylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(3-aminopropylamino)nicotinamide (318 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (314 mg, 77%) as a brown solid. ¹H NMR (DMSO-d₆)δ1.81 (m, 2H, CH₂), 3.38 (m, 4H, 2xCH₂), 5.18 (s, 2H, benzene-NH₂), 6.33 (m, 1 H), 6.52 (m, 2H), 7.06 (m, 1 H), 7.18 (s, 1 H), 7.49 (dd, J= 4.8 & 8.0 Hz, 1 H), 7.89 (d, J= 8.0 Hz, 1 H), 8.17 (d, J= 8.0 Hz, 1 H), 8.63 (s, 1 H), 8.69 (m, 2H), 9.00 (s, 1 H), 9.29 (s, 1 H, benzene-NH). LC-MS (m/z) 409 (M+1 ).

Example 18 Preparation of N-(2-amino-4-fluorophenyl)-6-(4-aminobutylamino)nicotinamide

N-(2-Amino-4-fluorophenyl)-6-chloronicotinamide (266 mg, 1 mmol) and 7 ml of 1 ,4-butanediamine were heated to 8O⁰C for 3 hours. The excess 1 ,4-butanediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (149 mg, 47% yield) as a brown solid. LC-MS (m/z) 318 (M+1 ).

Example 19

Preparation of N-(2-amino-4-fluorophenyl)-6-(4-(2-(9H-fluoren-9-ylidene)- acetamido)butylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(4-aminobutylamino)nicotinamide (333 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (344 mg, 66%) as an off-white solid. ¹H NMR (DMSO-d₆)δ1.60 (m, 4H, CH₂CH₂), 3.29 (m, 4H, 2xCH₂), 5.18 (s, 2H, benzene-NH₂), 6.33 (m, 1 H), 6.49 (m, 2H), 7.06-7.08 (m, 2H), 7.18 (m, 1 H), 7.28-7.35 (m, 3H), 7.41 (t, J= 7.2 Hz, 2H), 7.76 (d, J= 7.6 Hz, 1 H), 7.82 (m, 2H), 7.93 (d, J= 7.6 Hz, 1 H), 8.56 (s, 1 H), 8.63 (s, 1 H), 8.69 (d, J= 7.6 Hz, 1 H), 9.27 (s, 1 H, benzene-NH). LC-MS (m/z) 522 (M+1 ).

Example 20

Preparation of

N-(2-amino-4-fluorophenyl)-6-(4-(nicotinamido)butylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-fluorophenyl)-6-(4-aminobutylamino)nicotinamide (333 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (291 mg, 69%) as a brown solid. ¹H NMR (DMSO-d₆)δ1.59 (m, 4H, CH₂CH₂), 3.33 (m, 4H, 2xCH₂), 5.17 (s, 2H, benzene-NH₂), 6.33 (m, 1 H), 6.50 (m, 2H), 7.06 (m, 1 H), 7.15 (s, 1 H), 7.49 (m, 1 H), 7.88 (d, J= 8.0 Hz, 1 H), 8.16 (d, J= 8.0 Hz, 1 H), 8.61 (s, 1 H), 8.67 (m, 2H), 9.89 (s, 1 H), 9.27 (s, 1 H, benzene-NH). LC-MS (m/z) 423 (M+ 1 ).

Example 21 Preparation of N-(2-amino-4-chlorophenyl)-6-chloronicotinamide

6-Chloronicotinic acid (157.5 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-chloro-o-phenylenediamine (171 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (135 mg, 48% yield) as a brown solid. LC-MS (m/z) 282 (M+1 ).

Example 22 Preparation of N-(2-amino-4-chlorophenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Amino-4-chlorophenyl)-6-chloronicotinamide (282 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (180 mg, 59% yield) as a brown solid. LC-MS (m/z) 306 (M+1 ).

Example 23

Preparation of N-(2-amino-4-chlorophenyl)-6-(2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-chlorophenyl)-6-(2-aminoethylamino)nicotinamide (321 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (326 mg, 64%) as an off-white solid. LC-MS (m/z) 510 (M+ 1 ).

Example 24 Preparation of N-(2-amino-4-chlorophenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-chlorophenyl)-6-(2-aminoethylamino)nicotinamide (321 mg, 1.05 mmol)were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (258 mg, 63%) as a brown solid. LC-MS (m/z) 411 (M+1 ).

Example 25 Preparation of N-(2-amin-4-methylophenyl)-6-chloronicotinamide

6-Chloronicotinic acid (157.5 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-methyl-o-phenylenediamine (146 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 imL of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (164 mg, 63% yield) as a brown solid. LC-MS (m/z) 262 (M+1 ).

Example 26 Preparation of N-(2-amino-4-methylphenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Amino-4-methyl-phenyl)-6-chloronicotinamide (261 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (145 mg, 51 % yield) as a brown solid. LC-MS (m/z) 286 (M+1 ).

Example 27

Preparation of N-(2-amino-4-methylphenyl)-6-(2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-methylphenyl)-6-(2-aminoethylamino)nicotinamide (299 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (279 mg, 57%) as an off-white solid. LC-MS (m/z) 490 (M+1 ).

Example 28 Preparation of N-(2-amino-4-methylphenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-methylphenyl)-6-(2-aminoethylamino)nicotinamide (299 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (281 mg, 72%) as a brown solid. LC-MS (m/z) 391 (M+1 ). Example 29 Preparation of N-(2-amino-4-methoxyphenyl)-6-chloronicotinamide

6-Chloronicotinic acid (157.5 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-methoxy-o-phenylenediamine (166 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml. of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (144 mg, 52% yield) as a brown solid. LC-MS (m/z) 278 (M+1 ).

Example 30 Preparation of N-(2-amino-4-methoxyphenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Amino-4-methoxyphenyl)-6-chloronicotinamide (277 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (144 mg, 48% yield) as a brown solid. LC-MS (m/z) 302 (M+1 ).

Example 31

Preparation of N-(2-amino-4-methoxyphenyl)-6-(2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-methoxyphenyl)-6-(2-aminoethylamino)nicotinamide (316 mg, 1.05 mmol)were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (278 mg, 55%) as an off-white solid. LC-MS (m/z) 506 (M+1 ).

Example 32

Preparation of

N-(2-amino-4-methoxyphenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-methoxyphenyl)-6-(2-aminoethylamino)nicotinamide (316 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (244 mg, 60%) as a brown solid. LC-MS (m/z) 407 (M+1 ).

Example 33 Preparation of N-(2-amino-4-trifluoromethylphenyl)-6-chloronicotinamide

6-Chloronicotinic acid (157.5 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and 4-trifluoromethyl-o-phenylenediamine (211 mg, 1.2 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 imL of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum. To the residue was added 5 ml of absolute ethanol. The solids were collected by vacuum filtration, washed with absolute ethanol and dried under vacuum to give the title compound (418 mg, 42% yield) as a brown solid. LC-MS (m/z) 316 (M+1 ). Example 34

Preparation of

N-(2-amino-4-trifluoromethylphenyl)-6-(2-aminoethylamino)nicotinamide

N-(2-Amino-4-trifluoromethylphenyl)-6-chloronicotinamide (316 mg, 1 mmol) and 5 ml of ethylenediamine were heated to 8O⁰C for 3 hours. The excess ethylenediamine was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (159 mg, 47% yield) as a brown solid. LC-MS (m/z) 340 (M+1 ).

Example 35

Preparation of N-(2-amino-4-trifluoromethylphenyl)-6-(2-(2-(9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-trifluoromethylphenyl)-6-(2-aminoethylamino)nicotinamide (356 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (255 mg, 47%) as an off-white solid. LC-MS (m/z) 544 (M+1 ).

Example 36

Preparation of

N-(2-amino-4-trifluoromethylphenyl)-6-(2-(nicotinamido)ethylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-amino-4-trifluoromethylphenyl)-6-(2-aminoethylamino)nicotinamide (356 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (275 mg, 62%) as a brown solid. LC-MS (m/z) 445 (M+1 ).

Example 37

Preparation of N-(2-aminophenyl)-6-(2-(2-(2,7-dichloro-9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(2,7-Dichloro-9H-fluoren-9-ylidene)acetic acid (291 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (358 mg, 66%) as an off-white solid. LC-MS (m/z) 544 (M+1 ).

Example 38

Preparation of N-(2-aminophenyl)-6-(2-(2-(2,7-dinitro-9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(2,7-Dinitro-9H-fluoren-9-ylidene)acetic acid (312 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (379 mg, 67%) as an yellow solid. LC-MS (m/z) 566 (M+1 ).

Example 39

Preparation of N-(2-aminophenyl)-6-(2-(2-(2,7-dimethoxy-9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(2,7-Dimethoxy-9H-fluoren-9-ylidene)acetic acid (282 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (289 mg, 54%) as an off-white solid. LC-MS (m/z) 536 (M+1 ).

Example 40

Preparation of N-(2-aminophenyl)-6-(2-(2-(4,5-dimethyl-9H-fluoren-9-ylidene)- acetamido)ethylamino)nicotinamide

2-(4,5-Dimethyl-9H-fluoren-9-ylidene)acetic acid (250 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (302 mg, 60%) as an off-white solid. LC-MS (m/z) 504 (M+1 ).

Example 41

Preparation of N-(2-aminophenyl)-6-(2-(2-(5H-dibenzocyclohepten-5-ylidene)- acetamido)ethylamino)nicotinamide

2-(5H-Dibenzocyclohepten-5-ylidene)acetic acid (248 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (255 mg, 51 %) as an off-white solid. LC-MS (m/z) 502 (M+1 ).

Example 42 Preparation of

N-(2-aminophenyl)-6-(2-(2-(10,11 -dihydro-δH-dibenzocyclohepten-δ-ylidene)- acetamido)ethylamino)nicotinamide

2-(10, 11 -Dihydro-5H-dibenzocyclohepten-5-ylidene)acetic acid (250 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)- nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (211 mg, 42%) as an off-white solid. LC-MS (m/z) 504 (M+1 ).

Example 43 Preparation of N-(2-aminophenyl)-6-(2-(2-(9H-xanthen-9-ylidene)acetamido)- ethylamino)nicotinamide

2-(9H-Xanthen-9-ylidene)acetic acid (238 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (240 mg, 49%) as an off-white solid. LC-MS (m/z) 492 (M+1 ).

Example 44

Preparation of N-(2-aminophenyl)-6-(2-(2-(9H-thioxanthen-9-ylidene)acetamido)- ethylamino)nicotinamide

2-(9H-Thioxanthen-9-ylidene)acetic acid (254 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (289 mg, 57%) as an off-white solid. LC-MS (m/z) 508 (M+1 ). Example 45

Preparation of

N-(2-aminophenyl)-6-(2-(6-chloronicotinamido)ethylamino)nicotinamide

6-Chloronicotinic acid (158 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (296 mg, 72%) as a brown solid. LC-MS (m/z) 411 (M+1 ).

Example 46

Preparation of

N-(2-aminophenyl)-6-(2-(6-methylnicotinamido)ethylamino)nicotinamide

6-Methylnicotinic acid (137 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (288 mg, 74%) as a brown solid. LC-MS (m/z) 391 (M+1 ).

Example 47

Preparation of

N-(2-aminophenyl)-6-(2-(2-methoxynicotinamido)ethylamino)nicotinamide

2-Methoxynicotinic acid (153 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (207 mg, 51 %) as a brown solid. LC-MS (m/z) 407 (M+1 ).

Example 48

Preparation of

N-(2-aminophenyl)-6-(2-(6-trifluoromethylnicotinamido)ethylamino)nicotinamide

6-(Trifluoromethyl)nicotinic acid (162 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1.2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(2-aminoethylamino)nicotinamide (284 mg, 1.05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (204 mg, 46%) as a brown solid. LC-MS (m/z) 445 (M+1 ).

Example 49 Preparation of N-(2-aminophenyl)-6-(6-aminohexylamino)nicotinamide

N-(2-Aminophenyl)-6-chloronicotinamide (248 mg, 1 mmol) and 1 ,6-diaminohexane (5.80 g, 50 mmol) were heated to 8O⁰C for 3 hours. The excessi ,6-diaminohexane was removed under vacuum. To the residue was added 5 ml of 0.20 M NaOH. The mixture was extracted with 100 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (219 mg, 67% yield) as a brown solid. LC-MS (m/z) 328 (M+1 ). Example 50

Preparation of N-(2-aminophenyl)-6-(6-(2-(9H-fluoren-9-ylidene)acetamido)- hexylamino)nicotinamide

2-(9H-Fluoren-9-ylidene)acetic acid (222 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(6-aminohexylamino)nicotinamide (343 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine. The solids were collected by vacuum filtration, washed with water and dried under vacuum to give the title compound (324 mg, 61 %) as an off-white solid. LC-MS (m/z) 532 (M+1 ).

Example 51 Preparation of N-(2-aminophenyl)-6-(6-(nicotinamido)hexylamino)nicotinamide

Nicotinic acid (123 mg, 1 mmol) and 8 ml of DMF were stirred at room temperature while 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (384 mg, 2 mmol), hydroxybenzotriazole (162 mg, 1 .2 mmol), triethylamine (404 mg, 4 mmol) and N-(2-aminophenyl)-6-(6-aminohexylamino)nicotinamide (343 mg, 1 .05 mmol) were added. The mixture was stirred for 20 hours at room temperature. The mixture was diluted with 400 ml of brine and extracted with 200 ml of ethyl acetate. The ethyl acetate was removed under vacuum to give the title compound (311 mg, 72%) as a brown solid. LC-MS (m/z) 433 (M+1 ).

Example 52

In vitro inhibition of total HDAC enzyme activity and in vivo inhibition of HDAC subtype activity by compounds of formula (I) Class I HDAC HDAC3 HDAC4/5 HDAC7

(P21 reporter (GDF11 reporter (MEF2 reporter (Nur77 reporter assay) assay) assay) assay)

% inhibition of

Example total HDAC -

(compound) enzyme activity % Max % Max

% Max % Max at 30μM Res p Resp EC₅₀ EC₅₀ Resp of EC₅₀ EC₅₀ Resp of of of μM μM CS055 μM μM CS055 CS055 CS055 at 3μM at 3μM at 3μM at 3μM

CS055 46.2 3.5 100.0 3.2 100.0 15.1 100.0 6.8 100.0

SAHA 95.7 0.5 304.1 0.8 317.9 1.2 427.3 3.0 514.9

3 56.8 3.3 159.7 3.2 133.3 4.6 177.0 4.7 139.9

4 39.0 14.5 3.0 14.5 1.6 17.0 13.2 15.9 0.0

6 56.2 3.9 110.5 3.5 99.3 4.5 74.8 4.6 110.5

7 45.1 13.2 21.4 11.0 20.6 12.0 20.3 15.5 30.4

9 52.6 4.8 67.1 3.9 75.3 4.6 28.3 3.8 71.9

10 47.9 14.1 14.7 13.2 10.7 16.6 15.8 16.2 34.1

13 56.4 4.0 105.8 3.9 88.8 5.3 71.2 4.6 84.6

14 46.9 14.8 4.8 15.5 2.1 11.0 14.2 15.1 0.0

16 55.8 3.9 62.9 4.2 64.0 4.0 37.8 5.0 50.6

17 44.8 14.1 9.7 13.2 5.9 11.5 20.4 13.8 2.7

19 43.3 7.1 10.8 7.1 8.3 11.8 17.2 8.9 15.6 Class I HDAC HDAC3 HDAC4/5 HDAC7

% inhibition of

Example total HDAC -

(compound) enzyme activity % Max % Max

% Max % Max at 30μM Res p Resp EC₅₀ EC₅₀ Resp of EC₅₀ EC₅₀ Resp of μM μM CS055 μM μM CS055 CS055 CS055 at 3μM at 3μM at 3μM at 3μM

20 40.1 14.5 6.6 15.1 3.9 11.8 13.0 14.5 4.1

CS055: Chidamide is a HDACi currently in clinical development with good efficacy and toxicity profile in oncology indication from Chipscreen Biosciences

Measurement of in vitro inhibition of total HDAC enzyme activity:

The in vitro inhibition of total HDAC enzyme was determined by HDAC Fluorimetric Assay/Drug Discovery Kit (BIOMOL) according to manufacture's instruction. 1. Add Assay buffer, diluted trichostatin A or test inhibitor to appropriate wells of the microtiter plate. Following table lists examples of various assay types and the additions required for each test.

2. Add diluted HeLa extract or other HDAC sample to all wells except those that are to be "No Enzyme Controls" (Blank).

3. Allow diluted Fluor de Lys™ Substrate and the samples in the microtiter plate to equilibrate to assay temperature (25⁰C).

4. Initiate HDAC reactions by adding diluted substrate (25 μl) to each well and mixing thoroughly.

5. Allow HDAC reactions to proceed for desired length of time and then stop them by addition of Fluor de Lys™ Developer (50 μl). Incubate plate at room temperature (25⁰C) for 10-15 min.

6. Read samples in a microtiter-plate reading fluorimeter capable of excitation at a wavelength in the range 350- 380 nm and detection of emitted light in the range 440- 460 nm. Measurement of in vivo inhibition of HDAC subtype activity:

HDAC subtype selectivity inhibition assay of tested compounds was carried out by several reporter gene assays experiments. Briefly, HeLa cells were seeded in 96-well plates the day before transfection to give a confluence of 50-80%. Cells were transfected with one of reporter gene plasmid containing a promoter sequence or response element upstream of a luciferase gene construct using FuGeneδ transfection reagent according to the manufacturer's instruction (Roche). The promoters or response elements including p21-promoter, gdf11 -promoter, MEF-binding element (MEF2), Nur77-promoter were fused upstream to the luciferase gene reporter construct. For normalizing the transfection efficiency, a GFP expression plasmid was cotransfected. Cells were allowed to express protein for 24 hours followed by addition of individual compounds or the vehicle (DMSO). 24 hours later the cells were harvested, and the luciferase assay and GFP assay were performed using corresponding assay kits according to the manufacturer's instructions (Promega).

Example 53 In vivo anti-cell proliferation by Compounds of formula (I)

Exam Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀

-pie μM in μM in μM in μM in μM in μM in μM in μM in μM μM μM in μM

(comHL60 Hut- Raji Jurkat U937 RaA549 HeLa in in MDA-M in pound 78 mos BeI- MCF7 B-231 HCT

) 7402 -8

3 1.17 3.01 7.91 3.37 5.47 27.43 12.83 19.87 10.92 5.47 3.96 13.13

4 32.97 >60 >60 >60 >60 >60 >60 >60 >60 >60 >60 >60

6 3.01 2.23 6.73 5.35 7.21 >60 10.53 22.29 15.07 5.11 3.22 14.39

7 8.48 27.43 >60 41.51 49.91 >60 >60 >60 35.33 >60 >60 >60

9 3.53 1.65 1.41 5.60 9.29 27.43 11.43 22.81 12.25 6.73 4.35 11.17

10 26.19 28.06 54.72 44.48 54.72 3.22 >60 >60 37.86 >60 >60 >60

13 2.81 2.81 3.15 9.51 4.25 9.73 11.17 19.87 9.96 6.89 5.11 12.54 Exam Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀ Gl₅₀

-pie μM in μM in μM in μM in μM in μM in μM in μM in μM μM μM in μM

) 7402

14 32.97 >60 4.55 >60 >60 1.15 >60 >60 >60 >60 >60 >60

16 4.06 6.14 2.33 6.00 6.89 18.12 18.54 >60 12.54 >60 12.83 17.30

17 42.48 >60 6.00 43.47 46.57 11.70 >60 >60 48.77 >60 >60 >60

19 4.06 10.43 28.06 8.67 8.09 10.43 >60 >60 >60 11.97 >60 >60

20 32.97 >60 11.97 >60 49.91 4.66 >60 >60 >60 >60 >60 >60

CS05

1.00 1.69 9.29 3.79 2.50 >60 13.75 21.29 28.06 >60 36.15 >60 5

Sora-

1.28 12.54 4.15 16.91 4.06 1.51 13.75 30.77 9.73 9.51 4.25 5.35 finib

Su-

1.73 4.06 5.47 7.05 8.28 11.97 14.73 9.29 13.13 7.55 4.66 12.25 tent

Note: Chidamide is a HDAC inhibitor currently in clinical development against cancers with preference against class I HDAC enzyme; Suten and Sorafinib are two marketed RTK and Ser/Thr kinase inhibitors with broad activity against many different receptor tyrosine or ser/thr kinases

Measurement of in vivo cell proliferation:

Tumor cells were trypsinized and plated into 96-well plates at 3,000 per well and incubated in complete medium with 10% FBS for 24 hours. Compounds were added over a final concentration range of 100 μmol/L to 100 nmol/L in 0.1 % DMSO and incubated for 72 hours in complete medium. The effects on proliferation were determined by addition of MTS reagent (Promega) according to the instruction, incubation for 2 hours at 37⁰C in CO₂ incubator, and record the absorbance at 490nm using an ELISA plate reader.

Human Cell lines are listed below: HL-60: Acute promyelocytic leukemia

Hut-78: Cutaneous T cell lymphoma

Raji: Burkitt's lymphoma Jurkat: T cell leukemia

U937: Histiocytic lymphoma

Ramos: Burkitt's lymphoma

A549: Non small cell lung carcinoma

HeLa: Cervix adenocarcinoma

Bel-7402: Hepatocellular carcinoma

MCF-7: Mammary gland adenocarcinoma

MDA-MB-231 : Mammary gland adenocarcinoma

HCT-8: Ileocecal colorectal adenocarcinoma

Claims

What is claimed is:

1. An isolated compound of formula I:

(I) or its stereoisomer, enantiomer, diastereomer, or a pharmaceutically acceptable salt thereof, wherein

:>5 :

X is a bond, -CH₂CH₂-, -CH=CH-, O or S;

Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring, which optionally substituted with one or more halogen, nitro, alkyl or alkoxy; n is an integer ranging from 2 to 6.

2. A compound of claim 1 , wherein

R¹ , R², R³ and R⁴ are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; R⁵ is pyridyl, which optionally substituted with one or more halogen, alkyl, alkoxy or trifluoromethyl; n is an integer ranging from 2 to 4.

3. A compound of claim 1 , wherein

R¹ , R², R³ and R⁴ are independently H or F;

R⁵ is pyridyl; n is an integer ranging from 2 to 4.

4. A compound of claim 1 , wherein

R¹ , R², R³ and R⁴ are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; R⁵ Is

X is a bond, -CH₂CH₂-, -CH=CH-, O or S;

Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring, which optionally substituted with one or more halogen, nitro, alkyl or alkoxy; n is an integer ranging from 2 to 4.

5. A compound of claim 1 , wherein

R¹ , R², R³ and R⁴ are independently H or F; R⁵ Is

X is a bond;

Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring; n is an integer ranging from 2 to 4.

6. A process for the preparation of a compound of formula I

(I) wherein

R¹ , R², R³ and R⁴ are independently hydrogen, halo, alkyl, alkoxy or trifluoromethyl; R⁵ is pyridyl, which optionally substituted with one or more halogen, alkyl, alkoxy or trifluoromethyl; or R 5 : is,

X is a bond, -CH₂CH₂-, -CH=CH-, O or S;

Ring A and ring B, fused to the ring containing X, independently of each other represents a benzene ring, which optionally substituted with one or more halogen, nitro, alkyl or alkoxy; n is an integer ranging from 2 to 6; a stereoisomer, enantiomer, diastereomer, or pharmaceutically acceptable salts thereof comprising the steps of:

(a) condensing 6-chloronicotinic acid with compound 1 to give compound 2;

( 1 ) ( 2 )

(b) condensing compound 2 with compound 3 to give compound 4;

( 2 ) ( 4 )

(c) condensing compound 4 with compound 5 to give compound 6;

( 5 ) ( 4 ) ( 6 )

7. The process of claim 6, wherein the condensation reactions of steps (a) and (c) are conducted by using a peptide condensing agent.

8. The process of claim 7, wherein said peptide condensing agent is 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide, dicyclohexylcarbodiimide, or N,N'-carbonyldiimidazole.

9. A pharmaceutical composition useful as a therapeutic and/or improving agent for diseases associated with abnormal histone deacetylase activities comprising an effective amount of a compound according to claim 1 and at least one pharmaceutically acceptable excipient, carrier or diluent.

10. The pharmaceutical composition of claim 9, wherein said disease associated with abnormal histone deacetylase activities is selected from the group consisting essentially of Rubinstein-Taybi syndrome, fragile X syndrome, leukemia, cardiac hypertrophy, metabolic disease, cancer, and various neurological and neurodegenerative disorders.

11. The pharmaceutical composition of claim 10, wherein said various neurological and neurodegenerative disorders is selected from the group consisting essentially of stroke, bipolar, Huntington's disease, Amyotrophic Lateral Sclerosis and Alzheimer's disease.

12. A dosage form unit of the pharmaceutical composition of claim 9 comprising an amount within the range of about 0.0001 to about 200 mg of said compound.

13. A pharmaceutical composition of claim 9 for administration by the oral, nasal, transdermal, pulmonary, or parenteral route.