Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
  • C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
  • C07C255/58—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton
  • C07C255/60—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton at least one of the singly-bound nitrogen atoms being acylated
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
  • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
  • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
  • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
  • C07C235/64—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring

Definitions

• This invention relates to the field of novel anticancer agents for therapeutic application in human medicine.
• 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.
• biochemical processes to modify chromatin structure namely the covalent modifications of histone tails and the ATP-dependent chromatin remodeling.
• HATs histone acetyltransferases
• HDACs histone deacetylases
• PCAF p300/CBP-associated factor
• SRC1 nuclear hormone receptor cofactor 1
• ACTR activator of thyroid and retinoid receptor
• 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.
• HAT activity of p300 is regulated by several other factors.
• 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.
• p300 is recruited on to the chromatin template through the direct interaction with the activator and enhances the transcription by acetylation of promoter proximal nucleosomal histones.
• HAT inhibitors/activators are scanty.
• polyamine-CoA conjugates were found to block HAT activity in cell extracts.
• target enzyme(s) for these conjugates was not known.
• 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.
• Cashew nut shell liquid 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, ll'Z-diene, and the 8'Z, 11 '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.
• salicylic acid nor benzoic acid shown any inhiribitory activity against histone acetyltransferases.
• the acidic group on the both salicylic acid and anacardic acid was modified to respective different amide derivatives using substituted anilides.
• the interchanging of substitution pattern on the ring B in formula I was found to be affecting activation profile of molecule.
• the template pG 5 ML-array (8) was assembled into chromatin using the NAP1 mediated assembly method (Experimental Procedures). Addition of activators to the HAT-dependent transcription reaction along with the p300 and acetyl CoA after allowing for 30 in 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 specifically enhances the HAT activity of p300, a function that is reflected even at the transcriptional level.
• 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.
• 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 ene mixture obtained by above method was hydrogenated in ethylacetate for 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 K 2 CO 3 with phase transfer catalyst in M1BK for 36 hrs.
• Dialkylated anacardic acids were treated with potassium tertiary butoxide in DMSO to yield respective O-alkyl anacardic acids.
• the O-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.
• the solution was heated to 70 °C on a water bath for 2 h, 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 (50 mL) to yield an off-white solid of 2-Methoxy-6-pentadecylbenzoic Acid (7.6 g, 80%).
• Human core histones were purified from HeLa nuclear pellet as described previously (13).
• the FLAG epitope tagged Human Topoisomerase I, histone deacetylase 1 (HDACl) and PCAF, were purified from the recombinant baculovirus infected insect cell line, Sf2l, by the im unoaffinity purification using M2-agarose (SIGMA) (14).
• Full-length p300 was also purified from the recombinant baculovirus infected S£21 cells as a His ⁇ -tagged protein through the Ni-NTA affinity column (Qiagen) as described previously (13).
• NAP1 His 6 -tagged nucleosome assembly protein 1
• PC4 Human positive transcriptional coactivator
• the peptide substrate a 45 residue core histone H3 N-terminal peptide (N- CARTKQTARKSTGGKAPRQLASKAARKSAPSTGGNKKPHRYKPG-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 50 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride, 0.1 mM EDTA, pH 8.0, 10 mM sodium butyrate at 30 °C for 10 min in presence or absence of compound followed by the addition of 1 ⁇ l of 6.2 Ci/mmol [ 3 H]-acetyl Coenzyme A (acetyl-CoA) and were further incubated for another 10 min. The final reaction volume was 30 ⁇ l. The reaction mixture was then blotted onto P-81 (Whatman
• the HAT p300 was pre-incubated with indicated amounts of inhibitor on ice for 20 min., following which it was added to the acetylation reaction in the transcription assay.
• the Lysyl CoA step was omitted.
• HeLa nuclear extract (5 ⁇ l, which contains ⁇ 8 mg ml protein) was added to initiate the pre-initiation complex formation. Transcription reaction was started by the addition of ⁇ TP-mix and ⁇ -[ 32 P]-UTP, after the pre-initiation complex formation. The incubation was continued for 40 min at 30°C.
• 2-ethoxy-6-pentadecyl-benzoyl chloride was condensed with 5-amino-2-chloro benzenetriflouride in dichloromethane in presence of triethylamine as acid scavenger to yield N-(4-Chloro-3 -trifluoromethyl-phenyl)-2-ethoxy-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.
• 2-ethoxy-6-pentadecyl-benzoyl chloride was condensed with 4-Amino-2- trifluromefhyl benzonitrile in dichloromethane in presence of trietiiylamine as acid scavenger to t yield N-(4-Cyano-3-trifluoromethyl-phenyl)-2-ethoxy-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.
• 2-ethoxy-6-pentadecyl-benzoyl chloride was condensed with 5-amino-2-chloro benzenetriflouride in dichloromethane in presence of triethylamine as acid scavenger to 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.
• 2-ethoxy-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-trifluoromethyl-phenyl)-2-e1hoxy-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.
• 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-Isopropoxy-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.
• 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-nitro-3-trifluromethyl-phenyl)-6-pentadecyl- benza ide.
• 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.
• Anacardic alcohol was oxidized to corresponding anacardic aldehyde using pyridinium chloro chromate.
• 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.
• Figure 1 Different unsaturated anacardic acids present in CNSL.

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• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
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• 2003
  • 2003-12-12 EP EP03780608A patent/EP1590318A2/de not_active Withdrawn
  • 2003-12-12 WO PCT/IN2003/000389 patent/WO2004053140A2/en not_active Application Discontinuation

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