Title of Invention

ACTIVATORS OF HISTONE ACETYL TRANSFERASES

Abstract Disclosed is a process for producing zeaxanthin and ß-cryptoxanthin which comprises cultivating a recombinant microorganism which is expressing a ß-carotene hydroxylase gene and belonging to the genus Xanthophyllomyces (Phaffia) in an aqueous nutrient medium under aerobic conditions, and isolating the resulted carotenoids from the cells of said recombinant microorganism or from the cultured broth.
Full Text

FIELD OF THE INVENTION:
This invention relates to the field of novel anticancer agents for therapeutic application in human medicine.
BACKGOUND AND PRIOR ART:
The eukaryotic genome is organized as a highly complex nucleoprotein structure called chromatin, the unit of which is the nucleosome. The nucleosome is composed of two copies each of four different histones, H3, H2B, H2A, and H4, constituting a scaffold, which is wrapped around by 146 base pairs of DNA. Therefore, for any process that requires access to the DNA (e. g. transcription, replication, recombination and repair), the chromatin needs to be opened by the remodeling systems. There are two different biochemical processes to modify chromatin structure, namely the covalent modifications of histone tails and the ATP-dependent chromatin remodeling. Among the several covalent modifications of histones known, the reversible acetylation of key lysine residues in histones holds a pivotal position in transcriptional regulation, Acetylation of histones is a distinctive feature of the transcriptionally active genes, whereas deacetylation indicates the repressed state of a gene. A balance between the acetylation and deacetylation states of histones regulates transcription. Dysfunction of the enzymes involved in these events, the histone acetyltransferases (HATs) and histone deacetylases (HDACs), is often associated with the manifestation of cancer. These enzymes thus become potential new targets for antineoplastic therapy.
A wide repertoire of transcriptional co-activator proteins is now recognized to possess histone acetyltransferase activity. These include p300/CBP-associated factor (PCAF), which is similar to GCN5, nuclear hormone receptor cofactors such as steroid receptor cofactor 1 (SRCl) and activator of thyroid and retinoid receptor (ACTR) and the multifunctional p300/CBP. The p300/CBP is a global transcriptional coactivator, which plays a critical role in a variety of cellular process including cell cycle control, differentiation and apoptosis. Mutations in p300/CBP are associated with different human cancers and other human diseases. It is one of the most potent histone acetyltransferases,

which can acetylate all four-core histones within nucleosomes as well as tree nistone forms. The HAT activity of p300 is regulated by several other factors. For example, the viral oncoprotein El A binds to p300 and inhibits its activity, whereas phosphorylation of CBP by cyclin E/Cdk2 kinase activates its HAT activity. During the process of transcription, p300 is recruited onto the chromatin template through the direct interaction with the activator and enhances the transcription by acetylation of promoter proximal nucleosomal histones.
Although significant progress has been made in the field of histone deacetylase inhibitors as antineoplastic therapeutics, and some of the compounds are already in human trials, the reports of HAT inhibitors/activators are scanty. Prior to the molecular characterization of HAT enzymes, several polyamine-CoA conjugates were found to block HAT activity in cell extracts. However, the target enzyme (s) tbr these conjugates was not known. Recently, two peptide-CoA conjugates, namely Lysyl CoA (Lys-CoA) and H3-CoA-20, were synthesized that specifically inhibit the HAT activity of p300 and PCAF, respectively. Thus, there is an urgent medical need to identify new drugs having modulating activity (inhibitors/activators) towards histone acetyltransferases which can be used to treat diseases in which histone acetyltransferase play an important role in treatment of diseases due to defects in gene regulation predominantly cancer.
In accordance with the present invention, there is therefore provided the use of a compound of general formula I for activator molecules of histone acetyltransferases and the use of a compound of general formula 11 for inhibitor molecules of histone acetyltransferases.
OBJECTS OF THE INVENTION:
The main object of the present invention is to obtain compounds of structural formula (1) as activators of histone acetyltransferases, containing ring A derived from substituted benzoic acid moiety and ring B is substituted anilide.
Another main object of the present invention is to obtain compounds of structural formula (II) for ring A of formula (I) as inhibitors of histone acetyltransferases.




trifluromethyl"phenyl)-2-isopropoxy-6-pentadecyl"benzamide.

The present invention also relates to a compound of structural formula (IT) for ring A of formula (I) as inhibitors of histone acetyltransferases wherein
Formula H
The present invention also relates to a process of preparing compounds of formula I and formula II as modulators of histone acetyltransferases wherein the process comprising steps of:
> alkylating the aromatic acids to yield dialkylated aromatic acid derivatives;
> converting dialkylated aromatic acid derivatives to its acid halides; and

> condensing the resulting aromatic acid halides with different substituted anilines to yield corresponding benzamide derivatives.

Formula II
In still another embodiment of the present invention, aromatic acids are selected from a group comprising benzoic acid, salicyclic acid and anacardic acid.
In still another embodiment of the present invention, the compound of formula (I) containing ring A derived from substituted benzoic acid moiety and ring B is substituted anilide wherein:


In still another embodiment of the present invention, the compounds are selected from the
group comprising N-(4-nitro-3-trifluromethyl-phenyl)-2-ethoxy-benzamide; N-(4-nitro-3-
trifluromethyl-phenyl)-2-methoxy-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)"2-
propoxy-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-isopropoxy-benzamide; N-(4-
chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide; N-(4-cyano-3-
trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide; N-(4-chloro-3-
trif[uoromethyI-phenyI)-2-methoxy-6-pentadecyl-benzamide; N-(4-cyano-3-
trifluoromethyl-phenyl)-2-methoxy-6"pentadecyl-benzamide; N-(4-chloro-3-
trifluoromethyl-phenyI)-2-n-propoxy-6-pentadecyl-benzamide; N-(4-chloro-3-
trifluoromethyi-phenyl)-2-isopropoxy-6-pentadecyl-benzamide- ; N-(4-cyano-3-
trifluoromethyl-phenyl)-2-n-propoxy-6-pentadecyl-benzamide; N-(4-cyano-3-
trifluoromethyl-phenyl)-2-isopropoxy-6-pentadecyI-benzamide; N-(4-chloro-3-
trifluoromethyl-phenyl)-2"ethoxy-benzamide; N-(4"Cyano-3"trifluoromethyl-phenyl)-2-
ethoxy-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-methoxy-benzamide; N"(4-
cyano-34rifluoromethyl-phenyl)-2-methoxy-benzamide; N-(4-chloro-3-trifluoromethyl-
phenyl)-2-n-propoxy-benzamide; N-(4-cyano-34rifluoromethyl-phenyl)-2-isopropoxy-
benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-ethoxy-6"pentadecyl-benzamide; N"(4-
mtrO'3-trifluromethyl-phenyl)-2-methoxy-6-pentadecyl-benzamide; N-(4-nitro-3-

trifluromethyl-phenyl)-2-propoxy-6-pentadecyl-benzamide; and N-(4-nitro-3-
trifluromethyl-phenyl)-2-isopropoxy-6-pentadecyl-benzamide.
In still another embodiment of the present invention, the compound of formula (II)

Brief Description of the Figures:
Figure 1: Different unsaturated anacardic acids present in CNSL.
Figure 2: General formulae of the compounds claimed for the patent.
Figure 3: Effect of increasing concentration of Anacardic acid on the histone acetyltransferase activity of p300 and PCAF.
Figure 4: X-ray crystal structure of N- (4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy- 6-pentadecyl-benzamide (CTPB)
Figure 5: X-ray crystal structure of N-(4-Chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide (CTB)
Figure 6: Efl^ct of increasing concentrations of CTPB and CTB on the HAT activity of p300.

Figure 7: Specificity of CTPB to activate the histone transferase activity of p300 and not that of PCAF.
Cashew nut shell liquid (CNSL) possessed inhibitory activity towards p300. The systematic bio-activity guided fractionation of CNSL yielded unsaturated anacardic acids mixture, namely, the 8'Z-monoene, the 8'Z, H'Z-diene, and the 8'Z, ll'Z, 14'Z-triene, which are the chief constituents (--75%) of cashew nutshell liquid (18), having maximum HAT inhibitory activity. The hydrogenation of unsaturated anacardic acids mixture yielded a single compound, Anacardic acid (2-hydroxy-6' pentadecylbenzoic acid) showing an equally potent inhibitory activity towards p300. This data indicated that absence of unsaturation in anacardic acid did not alter its HAT inhibitory property. Neither salicylic acid nor benzoic acid shown any inhibitory activity against histone acetyltransferases.
The rate of the acetylation reaction at different concentrations of the inhibitors (and in its absence) was recorded with increasing concentrations of [3H]-acetyl CoA and a constant amount of core histones. The double reciprocal plot for each inhibitor concentration and in its absence (1/c.p.m vs. 1/ [Acetyl CoA]) was plotted as shown in Figure 2E, The results suggest that anacardic acid is a non-competitive type of p300- HAT inhibitor.
The acidic group on both salicylic acid and anacardic acid was moditled to respective different amide derivatives using substituted anilides. Most of these compounds, with different anilide moieties on anacardic acid and salicylic acid, when tested in vitro HAT assay (filter binding), surprisingly showed an enhancement in the p300 HAT activity, while keeping the PCAF HAT activity mostly unperturbed. The interchanging of substitution pattern on the ring B in formula I was found to be affecting activation profile of molecule.
Deacetylation of the core histones in the presence or absence of the HAT activating compounds, at 100 µM or 500µM, shows no difference in the deacetylase enzyme activity indicating no affect on deacetylation activity and the HAT enzyme specificity.
The addition of increasing concentration of either inhibitors or activators does not produce any variation in the transcript levels as compared to the DMSO control

indicating that the compounds do not atTect any component of the basal transcription machinery.
The template pG5ML-array (8) was assembled into chromatin using the NAPl mediated assembly method (Experimental Procedures). Addition of activators to the HAT-dependent transcription reaction along with the p300 and acetyl CoA after allowing tor 30 min of acetylation either in the presence or absence of the compound. Under these conditions, we found the addition of DMSO produced a slight drop in the transcript levels while the addition of activators enhanced the levels of transcription 1.6 fold over the DMSO control. Thus this result indicates that CTPB (Figure 6 and 7) specifically enhances the HAT activity of p300, a function that is reflected even at the transcriptional level In order to explain the L6-tbld increase in transcription levels, in contrast to the -5-fold increase in the histone acetylation levels ; Inhibitors did not affect the transcription from the DNA template, but the HAT-dependent transcription from chromatin template was inhibited by addition of inhibitors at lOjiM concentration.
Methods:
A: Chemical Methods
The cashew nut shell liquid (CNSL) is also known as cashew nut shell oil. CNSL is a dark brown viscous liquid reported to be 15-20% by weight of the unshelled nut in Africa, 25-30% by weight in India and ca. 25% overall, CNSL contains 90% anacardic acid and 10% cardol.
Methods reported to extract CNSL from cashew shells are:
1. Roasting nuts and collecting expelled liquid (Indian native method, yield 50%).
2. Extract with hot CNSL without charring the kernels (yield 85-90%).
3. Super heated steam treatment and collect condensate (used to improve yields of 2).
4. Solvent extraction with hexane leading to more percentage of anacardic acid.
5. Supercritical fluid extraction.

CNSL can also be extracted from cashew nuts that are soaked in water or humidified in piles and then held in a humid atmosphere so that the shell has set moisture content from 15-45% depending on the methods. CNSL is used in the manufacture of brake linings, industrial belting and clutches, reinforcing synthetic rubber, for oil and acid resistance, in lacquers, in electrical insulation material, as a metal anti-corrosive material, for waterproofing and as an adhesive. The use of CNSL in varnishes, lacquers, paints and brake linings requires distillation and further refinement.
Anacardic acid (6-pentadecylsalicylic acid), a major component of cashew nut shell liquid (CNSL), is obtained by solvent extraction of cashew nut shells. It exists as a heterogeneous mixture of monoenes, dienes and trienes. More specifically Cold processed CNSL was purchased from commercial source. The Anacardic acid present in the CNSL was purified as calcium anacardate by adding Calcium hydroxide to CNSL dissolved in isopropyl alcohol. The pure calcium salt of anacardic acid was dried and treated with IN HCl to release free anacardic acid ene mixture (containing n=0, 2,4, 6) (General food corporation (Rye, NY) Indian patent; 34671,1946). The ene mixture obtained by above method was hydrogenated in ethylacetate far 4hrs over 10% palladium-carbon using a Parr hydrogenator. The catalyst was filtered off and the solvent evaporated in vacuo to yield saturated anacardic acid. The alkylation with dimethyl and diethyl sulphates using potassium carbonate gave the dialkylated derivative. Di isopropyl anacardic acid was obtained by using isopropyl bromide in presence of K2CO3 with phase transfer catalyst in MIBK for 36 hrs,
Dialkylated anacardic acids were treated with potassium tertiary butoxide in DMSO to yield respective 0-alkyl anacardic acids. The 0-alkyl anacardic acids on treatment with thionyl chloride in the presence of a catalytic amount of DMF yield corresponding O-alkyl anacardic acid chlorides. The resultant acid chlorides condensed with different substituted anilines yielded respective benzamide derivatives.
All compounds were characterized by using FABMS. TLC was done using precoated silica gel GF2M plates (Merck, Darmstadt, Germany) with hexanerEthyl acetate (7: 3) as the developing solvent and visualized after spraying with vanillin sulphuric acid reagent.

The following are preparation of starting materials used for synthesis of activator molecules and inhibitor molecules.
Isolation of Anacardic Acid from CNSL: Commercially available solvent-extracted CNSL (IKg) was dissolved in 5% aqueous methanol (6.2L), and calcium hydroxide (510 g) was added in portions under stirring- After complete addition of calcium hydroxide, the temperature of the reaction mixture was raised to 54 °C and stirring was continued for 4h. The supernatant solution was monitored by TLC for the absence of anacardic acid. After completion of the reaction, the precipitated calcium anacardate was filtered and washed thoroughly with methanol (2,5L) and the cake was dried under vacuum at 45-54 oC for 3h (dry weight 1.08 Kg). The filtrate was preserved for subsequent isolation of cardoi and cardanol. Calcium anacardate (l.OSKg) was suspended in distilled water (4,5 L) and 11 M HCl (600ml) was added and stirred for L5 h. The resultant solution was extracted with ethyl acetate (2x2 L), The combined organic layer was washed with distilled water (2 x 2L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 600g of mixture of anacardic acid (monoene, diene and triene).
Preparation of Saturated Anacardic Acid: To a solution of an ene mixture of anacardic acid (500gj 1300 mmol) in methanol (2.0 L) was added 5% palladium- carbon (25 g), and then hydrogen gas was passed through the solution at 2.5 kg/cm2 until consumption of hydrogen gas ceased (4-5 h). The reaction mass was filtered over a Celite bed and washed with methanol (100 mL). The filtrate was concentrated under reduced pressure to give an off-white solid (477g), which on recrystalligation from hexane yielded 450g.
Preparation of Methyl 2-methoxy-6-pentadecylbenzoate: To a stirred solution of anacardic acid mentioned above (50g, 143 mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80g, 580 mmol). Dimethyl sulfate (44.25g, 290 mmol) was added in portions for about 10min at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200m]) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with

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distilled water (2 x 200 ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Methyl 2-methoxy-6-pentadecylbenzoate (45g, 80%).
Preparation of 2-Methoxy-6-pentadecylbenzoic Acid; To a stirred solution of Methyl 2-methoxy-6-pentadecylbenzoate (10g, 240 mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (10g, 890 mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8: 2) solvent system. The reaction mass was cooled to 10 °C, poured into ice waten and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in hexane (50ml) to yield an off-white solid of 2-Methoxy-6-pentadecylbenzoic Acid (7.6g, 80%).
Preparation of 2-Methoxy-6-pentadecylbenzoyl Chloride: To a stirred solution of 2-Methoxy-6-pentadecylbenzoic Acid (6,5g, 16 mmol) in hexane (60ml) were added thionyl chloride (2,5g, 21 mmol) and N,N-dimethylformamide (0.5 ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Methoxy"6- pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anilides.
Preparation of Ethyl 2-Ethoxy-6-pentadecyIbenzoate: To a stirred solution of anacardic acid mentioned above (50g, 143 mmol) in acetone (300m]) was added anhydrous powdered potassium carbonate (80g, 580 mmol). Diethyl sulfate (44.25g. 290 mmol) was added in portions tbr about 10 min at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with distilled water (2 x 200ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Ethyl 2-Ethoxy-6-pentadecylbenzoate (45g, 80%).
Preparation of 2"Ethoxy-6-pentadecylbenzoic Acid: To a stirred solution of Ethyl 2-Ethoxy-6"pentadecylbenzoate (20g, 240 mmol) in dimethyl sulfoxide (80ml) was added

potassium tert-butoxide (20g, 890 mmol) in portions. The solution was heated to 68oC on a water bath for 3h, the reaction was monitored by TIX using a hexane-ethyl acetate (9: 1) solvent system. The reaction mass was cooled to 15°C, poured into ice water, and then acidified with 10% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in petroleum ether (50ml) to yield an off-white solid of 2-Ethoxy-6-pentadecylbenzoic Acid (15g, 80%).
Preparation of 2-Ethoxy-6-pentadecylbenzoyl Chloride: To a stirred solution of 2-Ethoxy-'6-pentadecylbenzoic Acid (6.5g, 16 mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21 mmol) and N,N-dimethylformamide (0.5 mL). The reaction mixture was heated to reflux for 1 h. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Ethoxy-6-pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anilides.
Preparation of Propyl 2-propoxy-6-pentadecyIbenzoate: To a stirred solution of anacardic acid mentioned above (50g, 143 mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80g, 580 mmol). N-propyl iodide (44.25g, 290 mmol) was added in portions for about 24hrs at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml), The organic layer was washed with distilled water (2 x 200ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Propyl 2-propoxy-6-pentadecylbenzoate (45g, 80%).
Preparation of 2-Propoxy-6-pentadecylbenzoic Acid: To a stined solution of Propyl 2-propoxy-6-pentadecylbenzoate (10g, 240mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (10g, 890mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8: 2) solvent system. The reaction mass was cooled to 10°C, poured into ice water, and then acidified with 5% dilute hydrochloric acid. The precipitated solid

was filtered and washed thoroughly with distilled water, and the crude mass was rccrystallized in hexane (50ml) to yield an off-white solid of 2-Propoxy-6-pentadecylbenzoic Acid (7.6g, 80%),
Preparation of 2-Propoxy-6-pentadecylbenzoyI Chloride: To a stirred solution of 2-Ethoxy-6-pentadecylbenzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21mmol) and N,N-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Propoxy-6-pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anilides
Preparation of Isopropyl 2-isopropoxy-6-pentadecyIbenzoate: To a stirred solution of anacardic acid mentioned above (50g, 143 mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80g, 580 mmol) isopropoxyiodide (44.25g, 290mmol) was added in portions for about 10 min at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with distilled water (2 x 200ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Isopropyl 2-isopropoxy-6- pentadecylbenzoate (45g, 80%).
Preparation of 2-Isopropoxy-6-pentadecylbenzoic Acid: To a stirred solution of Isopropyl 2-isopropoxy-6-pentadecylbenzoate (10g, 240 mmol) in dimethyl sulfoxide (40 ml) was added potassium tert-butoxide (10g, 890mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8: 2) solvent system. The reaction mass was cooled to 10°C , poured into ice waten and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was rccrystallized in hexane (50ml) to yield an off-white solid of 2-Isopropoxy-6-pentadecylbenzoic Acid (7.6g, 80%).

Preparation of 2-Isopropoxy-6-pentadecylbenzoyl Chloride: To a stirred solution of 2-2-Isopropoxy-6-pentadecylbenzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21mmol) and N, N-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Isopropoxy- 6-pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for condensation with anilides.
Preparation of Methyl 2-rtiethoxy-benzoate: To a stirred solution of salicylic acid (50 g, 143mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80g, 580mmol). Dimethyl sulfate (44.25g, 290 mol) was added in portions for about 10 min at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200 ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200 ml). The organic layer was washed with distilled water (2 x 200 ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Methyl 2-methoxy-benzoate (45g, 80%).
Preparation of 2-Methoxy-benzoic Acid: To a stirred solution of V (lOg, 240 mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (lOg, 890mmol) in portions. The solution was heated to 10°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8:2) solvent system. The reaction mass was cooled to 10°C , poured into ice water, and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in hexane (50ml) to yield an off-white solid of 2-Methoxy-benzoic Acid (7.6g, 80%).
Preparation of 2-Methoxy-benzoyl Chloride: To a stirred solution of 2-Methoxy-benzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21 mmol) and M A^-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced

pressure to yield the desired 2-Methoxy-benzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anil ides.
Preparation of Ethyl 2-Ethoxy-benzoate: To a stirred solution of Salicylic acid (50g, 143mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80 g, 580mmol). Diethyl sulfate (44.25g, 290mmol) was added in portions for about 10 min at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with distilled water (2 x 200ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Ethyl 2-Ethoxy-benzoate (45g, 80%).
Preparation of 2-Ethoxy-bcnzoic Acid: To a stirred solution of Ethyl 2-Ethoxy-benzoate (10g, 240mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (10g, 890mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8:2) solvent system. The reaction mass was cooled to 10°C, poured into ice water, and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in hexane (50ml) to yield an oft-white solid of 2-Ethoxy-benzoic Acid (7.6 g, 80%).
Preparation of 2-Ethoxy-benzoyl Chloride: To a stirred solution of 2-Ethoxy-6-pentadecylbenzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21mmol) and N,N-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Ethoxy-benzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anilides.
Preparation of Propyl 2-propoxy-6"pentadecylbenzoate: To a stirred solution of Salicylic acid mentioned above (50g, 143mmol) in ACETONE (300ml) was added anhydrous powdered potassium carbonate (80g, 580mmol). n-propyl iodide (44.25g, 290 mmol) was added in portions for about 24hrs at room temperature. After the addition was

complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with distilled water (2 x 200ml), dried over anhydrous sodium sulfate, and concentrated to yield the title Propyl 2-propoxy-benzoate (45g, 80%).
Preparation of 2-Propoxy-benzoic Acid: To a stirred solution of Propyl 2-propoxy-benzoate (lOg, 240mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (lOg, 890mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8:2) solvent system. The reaction mass was cooled to 10°C , poured into ice water, and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in hexane (50ml) to yield an off-white solid of 2-Propoxy-benzoic Acid (7.6g, 80%).
Preparation of 2-Propoxy-benzoyl Chloride: To a stirred solution of 2-Propoxy-benzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2.5g, 21 mmol) and N,N-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-"Propoxy-6-pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for the condensation with anilides.
Preparation of Isopropyl 2-isopropoxy-benzoate: To a stirred solution of anacardic acid mentioned above (50g, 143mmol) in acetone (300ml) was added anhydrous powdered potassium carbonate (80g, 580mmol). fsopropyl iodide (44.25g, 290mmol) was added in portions for about lOmin at room temperature. After the addition was complete, the solution was heated to reflux temperature on a water bath and maintained for 3h. The solution was cooled to room temperature and then concentrated under reduced pressure. Distilled water (200ml) was added to the reaction mixture, which was then extracted with ethyl acetate (200ml). The organic layer was washed with distilled

water (2 x 200m]), dried over anhydrous sodium sulfate, and concentrated to yield the title Isopropyl 2-isopropoxy-benzoate (45g, 80%).
Preparation of 2-Isopropoxy-benzoic Acid; To a stirred solution of Isopropyl 2-isopropoxy-benzoate (01g, 240mmol) in dimethyl sulfoxide (40ml) was added potassium tert-butoxide (10g, 890mmol) in portions. The solution was heated to 70°C on a water bath for 2h, and the progress of the reaction was monitored by TLC using a hexane-ethyl acetate (8:2) solvent system. The reaction mass was cooled to I0°C , poured into ice water, and then acidified with 5% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in hexane (50ml) to yield an off-white solid of 2-Isopropoxy-benzoic Acid (7.6g, 80%).
Preparation of 2-lsopropoxy-benzoyl Chloride: To a stirred solution of 2- Isopropoxy-benzoic Acid (6.5g, 16mmol) in hexane (60ml) were added thionyl chloride (2,5g, 21 mmol) and N,N-dimethylformamide (0.5ml). The reaction mixture was heated to reflux for Ih. After the reaction was complete, the solvent was evaporated under reduced pressure to yield the desired 2-Isopropoxy-6-pentadecylbenzoyl Chloride, which was redissolved in dichloromethane (50ml) and used for condensation with following anilides to yield respective benzamides,
B: Biochemical Methods:
Purification of Human Core Histones and Recombinant Proteins-Human core histones were purified from HeLa nuclear pellet as described previously (13). The FLAG epitope tagged Human Topoisomerase h histone deacetylase 1 (HDACl) and PCAF, were purified from the recombinant baculovirus infected insect cell line, Sf21 by the immunoaftmity purification using M2-agarose (SIGMA) (14). Full-length p300 was also purified from the recombinant baculovirus infected Sf21 cells as a His6-tagged protein through the Ni-NTA affinity column (Qiagen) as described previously (13). The His6-tagged nucleosome assembly protein 1 (NAPl), used for the in vitro chromatin assembly was purified from E. coli cells as previously reported (13) and the FLAG-tagged chimeric activator Gal4-VP16, expressed in E. coli and purified by immunoaffinity purification

with M2 agarose. Human positive transcriptional coactivator, PC4, was expressed in E. coli and purified as described earlier (15).
The peptide substrate, a 45 residue core histone H3 N-terminal peptide (N-
CARTKQTARKSTGGKAPRQLASKAARKSAPSTGGVKKPHRYKPG-C) was
synthesized.
HAT Assay: Each of the compounds of Examples 1 to 26 were assessed for inhibition /activation of histone acetyltransferase activity of enzymes p300, PCAF and CBP. HAT assays were performed as described elsewhere (13). Briefly, indicated amounts of proteins/peptide were incubated in HAT-assay buffer containing 50mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, ImM dithiothreitol, ImM phenylmethyl sulfonyl fluoride, 0.1 mM EDTA, pH 8.0, 10 mM sodium butyrate at 30°C tor 10 min in presence or absence of compound followed by the addition of Ijxl of 6.2 Ci/mmol [3H]-acetyl Coenzyme A (acetyl-CoA) and were further incubated for another lOmin.The final reaction volume was 30uL The reaction mixture was then blotted onto P-81 (Whatmann) filter papers and radioactive counts were recorded on a Wallac 1409 liquid scintillation counter.
In order to characterize the inhibition kinetics of anacardic acid, filter-binding assays were done using constant amount of HeLa core histones in the presence or absence of AA with increasing concentrations of [ H]-acetyl CoA (see Figure Legends, 2E), To visualize the radiolabeled acetylated histones, the reaction mixtures were resolved on 15% SDS-PAGE and processed for fluorography as described elsewhere (15).
Histone deacetylase assay: Deacetylation assays were performed in the HAT assay buffer without sodium butyrate. 2µg of core histones were incubated with 20ng of p300 and 1µl of 6.2 Ci/mmol [3H]-acetyl CoA for ]5min at 30°C. The activity of p300 was inhibited by incubating the reaction mixture with lOnM p300-HAT specific inhibitor, Lysyl-CoA (10), for 10min atter which 50ng of HDACl was added, in the presence or absence of the compounds, and incubated further for 45min. The samples were analysed by tlurography.
In Vitro Chromatin assembly: Chromatin template for in vitro transcription experiments was assembled and characterized as described earlier (8).

In Vitro Transcription Assay: Transcription assays were essentially carried out as described elsewhere (8), with minor modifications. The scheme of transcription is enumerated in Figure 5A. Briefly, 30ng of DNA/equivalent amount of chromatin template was incubated with 30ng of activator (Gal4-VP16) in a buffer containing 4mM ITEPES (pH 7.8), 20mM KCl, 2mM DTT, 0.2mM PMSF, lOmM sodium butyrate, 0.1 mg/ml BSA, 2% glycerol (8). The activating compounds were added to the acetylation reaction along with p300 and acetyl-CoA, and incubated for 30min at 30'C. This was followed by addition of the p300 specific inhibitor Lysyl CoA (5|am) to quench the acetylation reaction.
For inhibitors, the HAT p300 was pre-incubated with indicated amounts of inhibitor on ice for 20min, following which it was added to the acetylation reaction in the transcription assay. For the DNA transcription assays and chromatin transcription inhibition assays, the Lysyl CoA step was omitted. After acetylation, HeLa nuclear extract {5\\\, which contains-8mg/ml protein) was added to initiate the pre-initiation complex formation. Transcription reaction was started by the addition of NTP-mix and a-['"P]"UTP, after the pre-initiation complex formafion. The incubation was continued for 40min at 30°C, Transcription was terminated by the addition of 250|,d stop buffer (20 mM Tris-HCl pH 8.0, ImM EDTA, lOOmM NaCI, 1% SDS and 0.025 ng/|,il tRNA). The 32P-radiolabeled transcript was extracted with phenol-chloroform, ethanol precipitated, dried pellet dissolved in loading dye (8M Urea, 0.005% bromophenol blue and xylene cyanol) and analyzed on 5% urea-polyacrylamide gel. Gels were then dried and subjected to autoradiography at -70oC. Quantification of transcription was done by Fuji BAS system. Quantitaion of DNA and chromatin transcription data represents three independent experiments.
The invention is further elaborated with the help of following examples. However, these examples should not be construed to limit the scope of the invention.
The following Examples illustrate the preparation of compounds of formula I using the starting materials described above:

EXAMPLE 1:
2-Ethoxy-N- (4-nitro-3-tritluromethyl"phenyl)-benzamide :
2-ethoxy—benzoyl chloride was condensed with 5-Amino-2-nitrobenzotritluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Ethoxy-N~ (4-nitro-3-lrinuromethyl-'phenyl)-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
IH-NMR: 66.95-8.10 (7H, aromatic), 53.98 (2H,t,OCH2), 61.33 (3H,t,Methyl)
EXAMPLE 2:
-Methoxy-N-(4-nitrO""3-trifluromethyl"phenyl)-benzamide:
2-Methoxy-benzoyl chloride was condensed with 5-Amino-2-nhrobenzotrifluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Methoxy-N" (4-nitro-3-trifluromethyl-phenyl)-benzaniide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product,
IH-NMR: 86,95-8.10 (7H, aromatic), 63. 73 (3H,t,Methyl).
EXAMPLE 3:
2-Propoxy-N- (4-nitro-3-trifluromethyl-phenyl)'benzamide:
2-Propoxy-benzoyl chloride was condensed with 5-Amino-2-nitrobenzotrifluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Propoxy-N-(4-nitro-3-trifluromethyl-phenyl)-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was



EXAMPLE 6:
N-(4-Cyano-3-tritluoromethyl-phenyl)-2-ethoxy-6"pentadecyl-benzamide;




EXAMPLE 11:


yield N- (4-Chloro-3-"trifluoromethyl-phenyl)-2-ethoxy-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
m/z: 344.4 (M+1), 239J49, 121,102, 82,57
EXAMPLE 14:
N- (4-Cyano-3-tritluoromethyl-phenyl)-2-ethoxy-benzamide;
2-ethoxy'6-pentadecyl-benzoyl chloride was condensed with 4-Amin0"2- trifluromethyl benzonitrile in dichloromethane in presence of triethylamine as acid scavenger to yield N- (4-Cyano-3-tritluoromethyl'phenyl)-2-ethoxy-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and fmally concentrated in vacuoThe residue obtained was chromatographed over silica gel to afford the desired product.
IH-NMR: 86.95-8.01 (7H, aromatic), 83.98 (2H, m, 0CH2), 8T33 (3H, t, Methyl)
EXAMPLE 15:
N- (4-Chloro-3"'trifluoromethyl-phenyl)-2-methoxy-benzamide:
2-Methoxy-6-pentadecyl-benzoyl chloride was condensed with 5'amino-2-chloro benzenetritlouride in dichloromethane in presence of triethylamine as acid scavenger to yield N-(4"Chloro-3-trifluoromethyl-phenyl)"2-methoxy-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product,
IH-NMR: 86.95-7.40 {7H, aromatic), 83. 73 (3H, s, OCH3)

EXAMPLE 16:
N-(4-Cyano-3-trinuoromethyl-phenyl)"2-methoxy-benzamide:
2-Methoxy-6-pentadecyl-benzoyl chloride was condensed with 4-Amino-2-trifluromethyl benzonitrile in dichloromethane in presence of triethylamine as acid scavenger to yield N"(4-Cyano-3-tritluoromethyl"phenyl)-2-methoxy-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
IH-NMR: 66.95-8.01 {7H, aromatic), 53.73 (3H, 0CH3)
EXAMPLE 17:
N" (4-Chloro-3-trifluoromethy]-phenyl)-2-n-propoxy-benzamide:
2-Propoxy-benzoyl chloride was condensed with 5-amino-2-chloro benzenetritlouride in dichloromethane in presence of triethylamine as acid scavenger to yield N- (4- Chloro-3-trifluoromethyl-phenyl)-2"n-propoxy"benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and fmally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
IH-NMR: 86.95-8.01 (7H, aromatic), 53.94 (2H, t, 0CH2). 51.75 (2H, m, Methylene), 60. 96 (3H, t, Methyl)
EXAMPLE 18;
N- (4-Cyano-3-tritluoromethyl-phenyl)-2-lsopropoxy-benzamide:
2-Isopropoxy-benzoyl chloride was condensed with 4-Amino-2-trifluromethyl benzonitrile in dichloromethane in presence of triethylamine as acid scavenger to yield N-(4-CyanO"3-trifluoromethyl-phenyl)"2-lsopropoxy-benzamide. The reaction mixture

was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
IH-NMR: 86.95-8.01 (7H, aromatic), 54. 04 (IH, m, OCH), 51.38 (61-1, d. Methyl)
EXAMPLE 19:
2-Ethoxy-N- (4-nitro-3-trifluromethyl-pheny!)-6-pentadecyl-benzamide:
2-ethoxy-6-pentadecyl-benzoyl chloride was condensed with 5-Amino-2-nitrobenzotritluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2"Ethoxy'-N-(4"nitro-3-trifluromethyl-phenyl)-6-pentadecyl-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
m/z : 565 (because of N02), 359,175, 149,136, 107,77
EXAMPLE 20:
2-Methoxy-N-(4-nitro-3-trifluromethyl-phenyl)-6-pentadecyl-benzamide:
2-Methoxy-6-pentadecyl-benzoyl chloride was condensed with 5-Amino-2-nitrobenzotrifluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Methoxy-N-(4-nitro-3-trifluromethyl-phenyl)-6-pentadecyl-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to aftbrd the desired product.
m/z: 551 (because of N02), 345,161. 149,121,91

EXAMPLE 21:
2~Propoxy-N- (4-nitro-3-tritluromethyl-phenyl)'-6-pentadecyl-benzaniide:
2-Propoxy-6-pentadecyl-benzoyl chloride was condensed with 5-Amino-2-nitrobenzotrifluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Propoxy-N-(4-nitro-3-trifluromethyl-phenyl)"6-pentadecyl-benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to afford the desired product.
m/z: 580 (because of N02), 537,373, 331.189, 161,147, 91
EXAMPLE 22:
2-Isopropoxy-N- (4-nitro-3-trifluromethyl-phenyl)-6-pentadecyl-"benzamide:
2-Isopropoxy-6-pentadecyl"benzoyl chloride was condensed with 5-Amino-2-nitrobenzotrifluoride in dichloromethane in presence of triethylamine as acid scavenger to yield 2-Isopropoxy-N- (4-nitroo-trifluromethyl-phenyl)-6-pentadecyl- benzamide. The reaction mixture was then concentrated in vacuo and the residue was extracted into ethyl acetate. The ethyl acetate layer was washed with water and with cold aqueous hydrochloric acid, then dried over sodium sulphate and finally concentrated in vacuo. The residue obtained was chromatographed over silica gel to atTord the desired product.
m/z: 580 (because of N02), 537,373, 331,189, 16K147, 107,91
EXAMPLE 23:
Anacardic acid
Isolation of Anacardic Acid from CNSL: Commercially available solvent-extracted CNSL (IKg) was dissolved in 5% aqueous methanol (6.2L), and calcium hydroxide (510 g) was added in portions under stirring. After complete addition of calcium hydroxide, the temperature of the reaction mixture was raised to 54°C and stirring was continued for

4h. The supernatant solution was monitored by TLC for the absence of anacardic acid. After completion of the reaction, the precipitated calcium anacardate was filtered and washed thoroughly with methanol (2.5L), and the cake was dried under vacuum at 45-54 °C for 3h (dry weight 1.08 Kg). The filtrate was preserved for subsequent isolation of cardol and cardanol. Calcium anacardate (l,08Kg) was suspended in disfilled water (4.5 L) and 11 M HCl (600 ml) was added and stirred for 1.5 h. The resultant solution was extracted with ethyl acetate (2 x 2 L). The combined organic layer was washed with disfilled water (2 x 2L), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to yield 600 g of mixture of anacardic acid (monoene, diene, and triene).
Preparation of Saturated Anacardic Acid: To a solufion of an ene mixture of anacardic acid (500g, 1300mmol) in methanol (2.0 L) was added 5% palladium- carbon (25 g). and then hydrogen gas was passed through the solufion at 2.5 kg/cnr until consumption of hydrogen gas ceased (4-5 h). The reaction mass was filtered over a Celite bed and washed with methanol (100ml). The filtrate was concentrated under reduced pressure to give an off-white solid (477g), which on recrystallizafion from hexane yielded 450g.
EXAMPLE 24:
Anacardic alcohol
To a stirred solution of Ethyl 2-ethoxy-6-pentadecylbenzoate was reduced to alcohol using lithium aluminum hydride.
m/z: 334.9 (M+1) 317,163, 147,133, 121,107, 91,69, 55
EXAMPLE 25:
Anacardic aldehyde
Anacardic alcohol was oxidized to corresponding anacardic aldehyde using pyridinium chloro chromate,
m/z: 334.9 (M+1) 317,163, 147,133, 121,107, 91,69, 55

EXAMPLE 26:
2-Ethoxy-6"-pentadecyl-benzoic acid:
To a stirred solution of Etliyl 2-Ethoxy-6-pentadecylbenzoate (20g, 240inmol) in dimethyl sulfoxide (80ml) was added potassium tert-butoxide {20g, 890mmol) in portions. The solution was heated to 68°C on a water bath for 3h, the reaction was monitored by TLC using a hexane-ethyl acetate (9:1) solvent system. The reaction mass was cooled to 15°C, poured into ice water, and then acidified with 10% dilute hydrochloric acid. The precipitated solid was filtered and washed thoroughly with distilled water, and the crude mass was recrystallized in petroleum ether (50ml) to yield an off-white solid of 2-Ethoxy-6-pentadecylbenzoic Acid (15g, 80%).
IH-NMR: 86.80-7.44 (3H, aromatic), 83.98 (2H, m, 0CH2), 52.55 (2H, m, Ar-CH2), 1.29- 1.63 (26H, m. methylene), 81.33 (3H t, methyl next to 0CH2), 50. 96 (3H, t. Methyl)
EXAMPLE 27:
Cardanol:
Commercially available solvent-extracted CNSL (IKg) was dissolved in 5% aqueous methanol (6.2L) and calcium hydroxide (510 g) was added in portions under stirring. After complete addition of calcium hydroxide, the temperature of the reaction mixture was raised to 54°C and stirring was continued for 4h. The supernatant solution was monitored by TLC for the absence of anacardic acid. After completion of the reaction, the precipitated calcium anacardate was filtered and washed thoroughly with methanol (2. 5L). The methanol layer was collected and concentrated in vacuo. The concentrate was further purified on silica gel (100-200 mesh) by column chromatography by using increasing amounts ethyl acetate in hexane to fractionate cardanol.
m/z: 304.7 (M+1) 149.121, 107,95, 71,57

EXAMPLE 28:
Cardol:
Commercially available solvent-extracted CNSL (IKg) was dissolved in 5% aqueous methanol (6.2L) and calcium hydroxide (510 g) was added in portions under stirring. After complete addition of calcium hydroxide, the temperature of the reaction mixture was raised to 54°C and stirring was continued for 4h. The supernatant solution was monitored by TLC tbr the absence of anacardic acid. After completion of the reaction, the precipitated calcium anacardate was filtered and washed thoroughly with methanol (2. 5L). The methanol layer was collected and concentrated in vacuo. The concentrate was further purified on silica gel (100-200 mesh) by column chromatography by using increasing amounts ethyl acetate in hexane to fractionate cardol.
m/z: 321 (M+l), 149,123, 107,95, 69,55
Results:
We have identified a small molecule compound, anacardic acid, from Cashew Nut Shell Liquid, know to have anti-tumor activity, which inhibits HAT activity of p300 and PCAF (Figure 1),
Surprisingly, the amide derivatives (Figure 2 and 3) of the same compound show an enhancement of p300 HAT activity with human core histones (Figure 4). These compounds are found to be specific for p300 since even at high concentration it cannot affect the HAT activity of PCAF (Figure 5).
The inhibitor anacardic acid also inhibits p300 HAT activity dependent transcription from the chromatin template but not DNA transcription. These results indicate the HAT specific activity of anacardic acid. As expected, the amide derivatives enhance HAT-dependent chromatin transcription whereas transcription from the DNA template remained unaffected.

Most of the analogs of the amide compounds showed similar activity with regards to activation of histone acetylation, except one of the CN-derivatives, predominantly enhanced the acetylation of histone H3.
References:
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We claim:
1. A compound of structural formula (I) as activators of histone acetyltransferases, containing ring A derived from substituted benzoic acid moiety and ring B is substituted anilide wherein: Rl is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, C8H18, C15H26,
C15H28, C15H30, C15H32;
R2 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl;
R3 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCI3, CI3, F,
CI, I, NO2, CN;
R4 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCI3, CI3, F,
CI, I, NO2, CN;
R5 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCI3, CI3, F,
CI, I, NO2;
R6 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCI3, CI3, F,
CI, I, NO2, CN; and
R7 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCI3, CI3, F,
CI, I, NO2, CN;

Formula I

2. The compounds as claimed in claim 1, wherein the compounds are selected from the group comprising N-(4-nitro-3-trifluromethyl-phenyl)-2"ethoxy-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-methoxy-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-propoxy-benzamide; N-(4-nitrO'3-trifluromethyl-phenyl)-2-isopropoxy-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl"benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)'2-ethoxy-6-pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-methoxy-6-pentadecyl-benzamide; N-(4-cyano-3-trifluoromethyl-phenyI)-2-methoxy-6-pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-n-propoxy-6-pentadecyl-benzamide; N-(4-chloro-34rifluoromethyl-phenyl)-2-isopropoxy-6-pentadecyl-benzamide- ; N-(4-cyano-3"trifluoromethyl-phenyl)-2-n-propoxy-6-pentadecyl-benzamide; N-(4-cyano-34rifluoromethyl-phenyl)-2-isopropoxy-6-pentadecyl-benzamide; N"(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide; N'(4-chloro-3-trifluoromethyl-phenyl)-2-methoxy-'benzamide; N-(4-cyano-3-trifluoromethyl-phenyI)-2-methoxy-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-n-propoxy-benzamide; N-(4-cyano-3-trifluoromethyl'-phenyl)-2-isopropoxy-benzamide; N-(4-nitro-3"trifluromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-methoxy-6' pentadecyl-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-propoxy-6-pentadecyl-benzamide; and N-(4-nitro-3-trifluromethyl-phenyl)-2-isopropoxy-6-pentadecyl-benzamide.
3. A compound of structural formula (11) for ring A of formula (I) as inhibitors of histone acetyltransferases wherein
Rl is H, CH3, hydroxyl, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-isopropoxy, n-butoxy, t-butoxy, C 8H18, C15H26, C15H28, C15H30, C15H32; R2 is H, CH3, hydroxyl, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-isopropoxy, n-butoxy, t-butoxy, CKHIH, C15H26, C15H28, C15H30. C15H32;

R3 is H, CH3, hydroxyK carboxylic, O-methoxy, O-ethoxy, n-propoxy, O- ;
isopropoxy, n-butoxy, t-butoxy, CgHig, Ci5H26, C15H28, C15H30, C1SH32;
R4 is M, CH3, hydroxy], carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy. CsHig, C15H26, C15H28, C15H30, C15H32;
R5 is H, CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, CgHis, C15H26, C15H28, C15H30, C15H32;
R6 is fi CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, CgHis. C15H26, C15H28, C15H30. C15H32;

Formula II
4. A process of preparing compounds of formula I and formula II as modulators of histone acetyltransferases wherein the process comprising steps of:
Ø alkylating the aromatic acids to yield dialkylated aromatic acid derivatives;
Ø converting dialkylated aromatic acid derivatives to its acid halides; and
Ø condensing the resulting aromatic acid halides with different substituted anilines to yield corresponding benzamide derivatives.


Formula IX
5. The process as claimed in claim 4, wherein the aromatic acids are selected from a group comprising benzoic acid, salicyclic acid and anacardic acid.
6. The process as claimed in claim 4, wherein the compound of formula (I) containing ring A derived from substituted benzoic acid moiety and ring B is substituted anilide wherein:
Rl is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, C8H18, C15H26,
C15H28, C15H30, C15H32;
R2 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl;

R3 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCl35 Cl3, F,
Cl, I, NO2, CN;
R4 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCl3, Cl3, F,
CI, I, NO2, CN;
R5 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCl3, Cl3, F,
CI, I, NO2;
R6 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCl3, Cl3, F,
CI, I, NO2, CN; and
R7 is H, methyl, ethyl, n-propyl, isopropyl, n-butyl and t-butyl, CF3, CCl3, Cl3, F,
CI, I, NO2, CN;
7. The process as claimed in claim 6, wherein the compounds are selected from the
group comprising N-(4-nitro-3-trifluromethyl-phenyl)-2-ethoxy-ben2amide; N-(4-
nitro-3-trifluromethyl-phenyl)-2-methoxy-benzamide; N-(4-nitro-3-
trifluromethyl-phenyl)-2-propoxy-benzamide; N-(4-nitro-34rifluromethyl-
phenyl)-2-isopropoxy-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-
ethoxy-6-pentadecyl-benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-ethoxy-
6-pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-methoxy-6-
pentadecyl-benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-methoxy-6-
pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-n-propoxy-6-
pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-isopropoxy-6-
pentadecyl-benzamide- ; N-(4-cyano-3-trifluoromethyl-phenyl)-2-n-propoxy'6-
pentadecyl-benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-isopropoxy-6-
pentadecyl-benzamide; N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-
benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-ethoxy-benzamide; N-(4-
chloro-3-trifluoromethyl-phenyl)-2-methoxy-benzamide; N-(4-cyano-3-
trifluoromethyl-phenyl)-2-methoxy-benzamide; N-(4-chloro-3-trifluoromethyl-
phenyl)-2-n-propoxy-benzamide; N-(4-cyano-3-trifluoromethyl-phenyl)-2-
isopropoxy-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-ethoxy'6-
pentadecyl-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-methoxy-6-
pentadecyl-benzamide; N-(4-nitro-3-trifluromethyl-phenyl)-2-propoxy-6-

pentadecyl-benzamide; and N-(4-nitro-3-trifluromethyl-'phenyl)-2-isopropoxy-6-pentadecyl-benzamide.
8, The process as claimed in claim 4, wherein the compound of formula (II)
containing ring A wherein
Rl is H, CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy,C8H18,, C15H26, C15H28, C15H30, C15H32;
R2 is H, CH3, hydroxyl, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, C8H18, C15H26, C15H28, C15H30, C15H32;
R3 is H, CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, C8H18, C15H26. C15H28, C15H30, C15H32;
R4 is II, CH3. hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, CgUn, C15H26, C15H28, C15H30. C15H32;
R5 is H, CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, C8H18,, C15H26, C15H28, C15H30. C15H32;
R6 is H, CH3, hydroxy!, carboxylic, O-methoxy, O-ethoxy, n-propoxy, O-
isopropoxy, n-butoxy, t-butoxy, C8H18,, C15H26, C15U28, C15H30, C15H32;
9, The compound of structural formula (I), the compound of structural formula (II),
the process of preparing compounds of formula (I) and formula (11) as modulators
of histone acetyltransferases as herein described with reference to foregoing


Documents:

0925-mas-2002 abstract-duplicate.pdf

0925-mas-2002 claims-duplicate.pdf

0925-mas-2002 description(complete)-duplicate.pdf

925-mas-2002-abstract.pdf

925-mas-2002-claims filed.pdf

925-mas-2002-claims granted.pdf

925-mas-2002-correspondnece-others.pdf

925-mas-2002-correspondnece-po.pdf

925-mas-2002-description(complete)filed.pdf

925-mas-2002-description(complete)granted.pdf

925-mas-2002-description(provisional).pdf

925-mas-2002-drawings.pdf

925-mas-2002-form 1.pdf

925-mas-2002-form 26.pdf

925-mas-2002-form 3.pdf

925-mas-2002-other documents.pdf


Patent Number 212171
Indian Patent Application Number 925/MAS/2002
PG Journal Number 02/2008
Publication Date 11-Jan-2008
Grant Date 26-Nov-2007
Date of Filing 12-Dec-2002
Name of Patentee JAWAHARLAL NEHRU CENTRE FOR ADVANCED SCIENTIFIC RESEARCH
Applicant Address JAKKUR CAMPUS,JAKKUR POST, BANGALORE - 64,
Inventors:
# Inventor's Name Inventor's Address
1 KARANAM BALASUBRAMNAYAN FLOT NO.1,CHITRAKUT ANNEXE, 55/1A,4TH MAIN,18TH CROSS,MALLESWARAM, BANGALORE-560 055,
PCT International Classification Number A 61 P 3P/02
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA