WO2017007301A1

WO2017007301A1 - Compound and method for inhibiting sirtuin activities

Info

Publication number: WO2017007301A1
Application number: PCT/MY2016/050029
Authority: WO
Inventors: Soo Choon Tan; Mohamed Ashraf Ali; Keng Yoon YEONG; Chern Ein OON; Chee Wei Ang
Original assignee: Universiti Sains Malaysia
Priority date: 2015-07-06
Filing date: 2016-05-06
Publication date: 2017-01-12

Abstract

A compound of formula wherein R₁ is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, alkyl of up to 5 carbon atoms, imidazolyl, piperazinyl, morpholinyl, benzyl, R₄OH or R4COOH, where R₄ is (CH₂)_m and m is an integer of from 1 to 4; R₂ is selected from the group consisting of hydrogen, phenyl or 3-(2-oxopyrrolidin-l-yl) propyl; and R₃ is hydrogen or alkyl of from 1-4 carbon atoms.

Description

Compound And Method For Inhibiting Sirtuin Activities Field of Invention

This present invention relates to new compounds, more particularly new benzimidazole derivatives, and their uses for the production of pharmaceutical agent for treatment and/or prevention of diseases such as cancer and neurodegenerative disorders.

Background of the Invention

Sirtuins, also know as Sir2 enzymes, belong to the third class of histone deacetylase enzymes (HDACs). Up to date, there are seven human sirtuins identified, namely SIRTl -7. All sirtuins contain a conserved catalytic core domain and have a stoichiometric requirement for its co-factor, nicotinamide adenine dinucleotide (NAD⁺), to deacetylate substrates ranging from histones to transcriptional regulators. Nevertheless, not all sirtuins are NAD+-dependent deacetylase. For example, SIRT4 is a ADP-ribosyl transferase. Sirtuins have been found in various subcellular locations, including nucleus, cytoplasm, mitochondria, heterochromatin and nucleolus. Each type of sirtuins has different main substrates and functions. Examples of main substrates for sirtuins are histones, acetyl-CoA synthetases, FOXO family, succinate or glutamate dehydrogenase, transcription factor p53 and tumor necrosis factor TNF-a.

Sirtuins have been associated with a wide variety of biological processes. Particularly, this family of proteins is required for transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Moreover, these enzymes have been linked to development, inflammation and neuroprotection.

Nevertheless, sirtuins are also associated with certain diseases such as cancer, Alzheimer's disease and Parkinson's disease. Cancer cells tend to require sirtuins to survive, proliferate, repair the otherwise catastrophic genomic events and evolve. For example, SIRTl promotes cancer genomic instability and cancer evolution. Over- expression of SIRTl increases incidence of thyroid cancer and lung cancer, indicating that SIRT1 promotes tumor progression. Pharmacological inhibition of SIRT1 is able to inhibit leukemia development. Similar to SIRT1, SIRT2 may promoto oncogenic phenotypes. It shows an increased expression in acute myeloid leukemid (AML) cells as compared to normal bone barrow cells. SIRT2 also promotes apoptosis of cancer cells. Further, inhibition of SIRT2 activity confers neuroprotection and thus can help to prevent or treat neurodegenerative disorders such as Parkinson's disease.

The connection between sirtuins and cancer or neurodegenerative disorder should be readily understood. Hence, there exist a clear need for a method/therapeutic agent capable of inhibiting sirtuins and might aids in the prevention or treatment of cancer and neurodegenerative disorders. Several compounds have been identified to be potential sirtuin inhibitors, including splitomicin, sirtinol and AGK2. In more particular, phenylsplitomicin illustrates antiproliferative effect against MCF-7 breast cancer cells. Sirtinol induces senescence-like growth arrest in MCF-7 breast cancer cells and enhances chemosensitivity of MCF-7 cells to camptothecin and cisplantin, leading to enhanced apoptotic cell death.

Nevertheless, the abovementioned sirtuin inhibitors have relatively high half-maximal inhibitory concentration, IC₅o. For instance, sirtinol has a IC₅o for SIRT1 of 99 μΜ and a IC₅o for SIRT2 of 43 μΜ. A sirtuin inhibitor having a relatively low IC₅o is desirable so that the concentration of sirtuin inhibitor used will not be toxic to the cells other than the targeted cells. The present invention provides such sirtuin inhibitor. Summary of The Invention

The primary object of the present invention is to provide a compound having carboxylate functional group and a benzimidazole group as core. Particularly, this compound can be used in the production of sirtuin inhibitor medicament. Another object of the present invention is to provide a method of producing the abovementioned compound. Specifically, the compound can be synthesized in a vessel without any purification step until the final product is formed. At least one of the preceding objects is met, in whole or in part, by the present invention, in which the embodiment of the present invention describes a compound of formula

wherein ¾ is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, alkyl of up to 5 carbon atoms, imidazolyl, piperazinyl, morpholinyl, benzyl, R4OH or R4COOH, where R4 is (CH₂)_m and m is an integer of from 1 to 4; R₂ is selected from the group consisting of hydrogen, phenyl or 3-(2-oxopyrrolidin-l-yl) propyl; and R₃ is hydrogen or alkyl of from 1-4 carbon atoms.

Preferably, the compound comprises a hydrogen at position R₂ and/or a methyl group at position R₃. The present invention also describes the use of a compound of the above formula in the manufacture of a sirtuin inhibitor medicament for treatment of cancer or neurodegenerative disorders. Particularly, the compound inhibits activities of SIRT2 and to a lesser extent SIRT1. Further, a method for producing a compound of the above formula is disclosed. The method comprises the steps of a) reacting 4-halo-3-nitrobenzoic acid with alcohol to produce an alkyl 4-halo-3-nitrobenzoate; b) adding ammonium hydroxide or an amine to the alkyl 4-halo-3-nitrobenzoate to produce a nitroalkyl ester; c) adding a reducing agent to the nitroalkyl ester to produce a diamino alkyl ester; and d) reacting the diamino alkyl ester with an aryl aldehyde of formula

Ri Preferably, steps a) to d) are carried out under reflux. The preferred alcohol for use in step (a) is ethanol. Amines suitable for use in step (b) may be selected from the group consisting of aniline, l-(3-aminopropyl)-2-pyrrolidinone, 4-(2- aminoethyl)morpholine, N-(3-aminopropyl)imidazole or hydroxylamine. The reducing agent used in step (c) can be selected from the group consisting of tin (II) chloride, or palladium on activated charcoal. Step d) can be carried out in the presence of a catalyst, wherein the catalyst is selected from the group consisting of bisulfite or metabisulfite salts.

In the preferred embodiment, 4-halo-3-nitrobenzoic acid and alcohol are present in a molar ratio of 1:1000 to 1 :2000. Alkyl 4-halo-3-nitrobenzoate and amine are maintained in a molar ratio of 1 :1 to 1: 1.5 in step (b). A molar ratio of 1 :4 to 1:8 of nitroalkyl ester to reducing agent in step (c) is preferred. For step (d), diamino alkyl ester and aryl aldehyde are present in a molar ratio of 1 : 1 to 1 : 1.5.

Brief Description of the Drawings

For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawing the preferred embodiments from an inspection of which when considered in connection with the following description, the invention, its construction and operation and many of its advantages would be readily understood and appreciated.

Figure 1 shows synthetic scheme for ethyl 2-phenyl-lH-benzo[d] imidazole carboxylate (compound i).

Detailed Description of The Invention

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiment described herein is not intended as limitations on the scope of the invention.

The present invention provides a compound of formula

wherein Ri is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, alkyl of up to 5 carbon atoms, imidazolyl, piperazinyl, morpholinyl, benzyl, R4OH or R4COOH, where R4 is (CH₂)_m and m is an integer of from 1 to 4; R₂ is selected from the group consisting of hydrogen, phenyl or 3-(2-oxopyrrolidin-l-yl) propyl; and R₃ is hydrogen or alkyl of from 1-4 carbon atoms. In the preferred embodiment of the invention, the compound comprises a hydrogen at position R₂ and/or a methyl group at position R₃.

Compound of the above formula comprises a benzimidazole core. Benzimidazole is an important pharmacophore in medicinal chemistry. Its nucleus substitutes an important part of the vitamin B12 structure. However, substituted benzimidazoles, especially at positions 1 and 2, are generally found to be more potent than benzimidazoles. In the preferred embodiment of the invention, benzene ring on the position-2 of the benzimidazole core of the compound can be stabilized though π-π stacking interactions between the imidazole group of His 187 amd benzene ring of Phel l9 of a sirtuin enzyme, particularly SIRT2.

Pursuant to the preferred embodiment of the invention, compounds of the above formula are relatively stable to heat. Theompounds show very low or no degradation of compounds when stored at about 37 °C for about 4 weeks or around 4 °C for about 10 months. Moreover, the present invention provides the use of a compound as set forth in the preceding description in the manufacture of a sirtuin inhibitor medicament for treatment of cancer and/or neurodegenerative disorders. Examples of neurodegenerative disorders are Alzheimer's disease and Parkinson's disease. Particularly, the compound inhibits the activities of sirtuins via competitive inhibition, wherein binding of the compound to the active site on the sirtuin prevents binding of sirtuin substrate to the active site of sirtuin. Preferably, the compound inhibits activities of SIRT2 and to a lesser extent SIRTl . In the present invention, inhibitory activity refers to the half maximal inhibitory concentration (IC50). In the preferred embodiment of the present invention, the inhibitory activity of compound of the above formula against sirtuins is in the range of 13.00 μΜ to 87.97 μΜ. More specifically, the inhibitory activity of the compound against SIRTl is in the range of 20.00 μΜ to 87.97 μΜ wherein the inhibitory activity of the compound against SIRT2 is in the range of 13.00 μΜ to 63.00 μΜ. Table 1 shows the inhibitory activity of different compounds of the above formula.

Table 1

Compound Ri R₂ Ra SIRT1 SIRT2 inhibition, inhibition, IC₅₀ (μΜ) ICso (μΜ)

Cambinol - - - 56.0 59.0

Tenovin-6 - - - 27.0 48.0

In an examplary embodiment, a compound (compound ix) having a phenyl group at Ri position, Ν,Ν-dimethylamino group (-N(CH₃)₂) at R2 position and a methyl group (-CH₃) at R3 position is shown to have docked within the binding site of SIRT2 for its substrate, ADP-ribose (ADPr). In more particular, the compound interacts with the receptor of SIRT2 due to hydrogen bonding as well as π-π stacking interactions. Lone pair oxygen-π interactions between the Val233 of SIRT2 and phenyl ring of dimethyl aminobenzene substituent as well as the N of dimethylamino group with benzene ring from Phell9 of SIRT2 can be observed. Besides, oxygen from the ester chain of this compound forms a strong hydrogen bond with the N-H group of Ser263 of SIRT2. Also, hydrogen bonds are observed between this compound with amino acids Thr262, Leu264, Gln265, Arg97 and Glnl67 of SIRT2. This observation is in agreement with the hydrogen bonds observed between SIRT2 and ADPr. Hence, it is apparent that the compound is able to compete with ADPr for binding sites of SIRT2, thus preventing SIRT2 activities. As a result, progression of cancer and neurodegenerative disorders due to SIRT2 activity may be inhibited.

Accordingly, the compound disclosed herein possesses antitumor activity. Specifically, the compound inhibits proliferation of cancerous cells. Cancer cells may be derived from tumor in colon, breast or blood. In one preferred embodiment of the invention, a compound (compound ix) having a phenyl group at Ri position, N,N- dimethylamino group (-N(CH₃)₂) at R₂ position and a methyl group (-CH₃) at R₃ position possess cytotoxic activity against cancer cells derived from colon tumor, breast tumor and blood cancer. Cell viability of colon cancer cells, breast cancer cells and blood cancer cells after treating with 50 μΜ of this compound for 72 hours are 40%, 53.2% and 27.2%, respectively. A method of producing compound of the above formula is also provided herein. The method of production comprises 4 steps: (a) reacting 4-halo-nitrobenzoic acid with an alcohol to produce an alkyl 4-halo-3-nitrobenzoate; (b) adding ammonium hydroxide or an amine to the alkyl 4-halo-3-nitrobenzoate to produce a nitroalkyl ester; (c) adding a reducing agent to the nitroalkyl ester to produce a diaminoalkyl ester; (d) reacting the aminoalkyl ester with a aryl aldehyde to produce a compound of the above formula. In the preferred embodiment, the alcohol used is ethanol.

Preferably, steps (a) to (d) are carried out under reflux with alcohol as solvent. Ethanol as solvent is preferred as it is readily recyclable and has very low toxicity, allowing this method of producing the compound suitable for scale-up production. In the referred embodiment, the aryl aldehyde has a formula of

In accordance with the present invention, steps (a) to (d) are conducted in a same vessel/flask. No purification of material is required until the last step. Step (a) is a esterification reaction between halo-substituted nitrobenzoic acid and alcohol, preferably ethanol, to produce a halo-substituted nitrobenzoate. Preferably, step (a) is performed in the presence of a catalyst such as sulfuric acid. Halo-substituted nitrobenzoic acid and alcohol are present in a preferred molar ratio of 1:1000 to

1:2000. Following step (a), amine is added to the vessel to perform a N-arylation/substitution reaction of the halo-substituted nitrobenzoate. Preferably, molar ratio of halo- substituted benzoate to amine is 1 :1 to 1:1.5. A Meisenheimer adduct is thus created. The intermediate complex is generated through the intermolecular hydrogen bonding of halo-substituted benzoate with the amine and ethanol solvent. The halogen atom can form a strong hydrogen bond with ethanol and is in agreement with the predicted hydrogen bond acceptor properties of aromatically bound halogens (F » CI » Br » I). The alkyl carboxylate group at the para-position of the benzimidazole ring, which withdraws electrons, helps to activate the C-X bond and facilitates the N-arylation. Nitroalkyl ester is formed. Suitable amines can be selected from the group consisting of aniline, l-(3-aminopropyl)-2-pyrrolidinone, 4-(2-aminoethyl)morpholine, N-(3- aminopropyl)imidazole or hydroxylamine. These amines are highly soluble in the ethanol solvent, hence enhancing the efficiency of the N-arylation/substitution reaction.

Step (b) is followed by the reduction step (c) of nitroalkyl carboxylate group. Reducing agent contemplated within the invention may be, but not limited to, tin (II) chloride or palladium on activated charcoal. The molar ratio of nitroalkyl ester to reducing agent is preferably 1:4 to 1:8. This process is relatively cost-effective as the use of expensive metal catalysts such as platinum is eliminated. Successful reduction of the nitro group into amino group is indicated by a color change of the solution in the vessel from yellowish to colorless.

Step (d) is the condensation of diamino alkyl ester produced in step (c) with various aryl aldehydes to produce a compound of the above formula. It is preferable to maintain a mole ratio of diamino alkyl ester and aryl aldehye from 1:1 to 1:1.5. Preferably, step (d) is carried out in the presence of a catalyst selected from the group consisting bisulfate and metabisulfite salts to shorten the reaction time of condensation process by at least twofold. The compound of the invention is removed from the solvent by evaporating the solvent under pressure.

Example

An example is provided below to illustrate different aspects and embodiments of the invention. The example is not intended in any way to limit the disclosed invention, which is limited only by the claims.

Example 1

Preparation of ethyl 2-phenyI-lH-benzo[d]imidazole-5-carboxylate (compound i)

4-fluoro-3-nitrobenzoic acid (0.05g, 0.27 mmol) was esterified in the presence of catalytic sulfuric acid in ethanol (lOmL) by refluxing at 65 °C for 6 hours. Ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 hour, treated with tin (II) chloride (190mg, 1 mmol) and stirred for a further 0.5 hour. The resulting mixture was then treated with benzaldehyde (0.3 mmol) and sodium bisulfite (57 mg, 0.3 mmol) and left to stir for another 3 hour. The solution was cooled to room temperature and subsequently evaporated under reduced pressure. It was resuspended in ethyl acetate (lOmL), washed with 10% sodium carbonate (20mL) and water (20mL x2), dried over sodium sulfate and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70-230 mesh; CHCl₃-MeOH 9:1) to obtain the final products (70-90%). The synthetic scheme for this compound is shown in Figure 1.

Example 2

Preparation of ethyl 2-(4-bromophenyl)-lH-benzo[d]imidazole-5-carboxylate

This compound is prepared using the method as described in example 1 except bromobenzaldehyde is used instead of benzaldehyde.

4-fluoro-3-nitrobenzoic acid (0.05g, 0.27 mmol) was esterified in the presence of catalytic sulfuric acid in ethanol (lOmL) by refluxing at 65 °C for 6 hours. Ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 hour, treated with tin (II) chloride (190mg, 1 mmol) and stirred for a further 0.5 hour. The resulting mixture was then treated with bromobenzaldehyde (0.3 mmol) and sodium bisulfite (57 mg, 0.3 mmol) and left to stir for another 3 hour. The solution was cooled to room temperature and subsequently evaporated under reduced pressure. It was resuspended in ethyl acetate (lOmL), washed with 10% sodium carbonate (20mL) and water (20mL x2), dried over sodium sulfate and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70-230 mesh; CHCl₃-MeOH 9:1) to obtain the final products (70-90%).

Example 3

Preparation of ethyl 2-(4-(piperidin-l-yl)phenyl-lH-benzo[d]imidazole-5- carboxylate

This compound is prepared using the method as described in example 1 except 4-(l- Piperidinyl)benzaldehyde is used instead of benzaldehyde.

4-fluoro-3-nitrobenzoic acid (0.05g, 0.27 mmol) was esterified in the presence of catalytic sulfuric acid in ethanol (lOmL) by refluxing at 65 °C for 6 hours. Ammonium hydroxide 28% (0.26 mmol) was subsequently added to the solution, stirred for 0.5 hour, treated with tin (II) chloride (190mg, 1 mmol) and stirred for a further 0.5 hour. The resulting mixture was then treated with 4-(l- Piperidinyl)benzaldehyde (0.3 mmol) and sodium bisulfite (57 mg, 0.3 mmol) and left to stir for another 3 hour. The solution was cooled to room temperature and subsequently evaporated under reduced pressure. It was resuspended in ethyl acetate (lOmL), washed with 10% sodium carbonate (20mL) and water (20mL x2), dried over sodium sulfate and concentrated under reduced pressure. The crude products were purified by column chromatography (silica gel, 70-230 mesh; CHCl₃-MeOH 9:1) to obtain the final products (70-90%).

Claims

1. A compound of formula

wherein Ri is selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, alkyl of up to 5 carbon atoms, imidazolyl, piperazinyl, morpholinyl, benzyl, R₄OH or R4COOH, where R4 is (CH₂)_m and m is an integer of from 1 to 4;

R₂ is selected from the group consisting of hydrogen, phenyl or 3-(2-oxopyrrolidin-l- yl) propyl; and

R3 is hydrogen or alkyl of from 1-4 carbon atoms.

2. A compound according to claim 1 , wherein R₂ is hydrogen.

3. A compound according to claim 1 or 2, wherein R₃ is a methyl group.

4. A method for producing a compound according to any of claims 1 to 3 comprising the steps of

a) reacting 4-halo-3-nitrobenzoic acid with alcohol to produce an alkyl 4-halo-3- nitrobenzoate;

b) adding ammonium hydroxide or an amine to the alkyl 4-halo-3-nitrobenzoate to produce a nitroalkyl ester;

c) adding a reducing agent to the nitroalkyl ester to produce a diamino alkyl ester; and d) reacting the diamino alkyl ester with an aryl aldehyde of formula

5. A method according to claim 4, wherein steps a) to d) are carried out under reflux.

6. A method according to claims 5 or 6, wherein the alcohol is ethanol.

7. A method according to any of claims 4 to 6, wherein the reducing agent is selected from the group consisting of tin (II) chloride or palladium on activated charcoal.

8. A method according to any of claims 4 to 7, wherein step d) is carried out in the presence of a catalyst.

9. A method according to any of claims 4 to 8, wherein the amine is selected from the group consisting of aniline, l-(3-aminopropyl)-2-pyrrolidinone, 4-(2- armnoethyl)morpholine, N-(3-aminopropyl)imidazole or hydroxylamine.

10. A method according to any of claims 4 to 9, wherein 4-halo-3-nitrobenzoic acid and alcohol are present in a molar ratio of 1:1000 to 1 :2000.

11. A method according to any of claims 4 to 10, wherein alkyl 4-halo-3-nitrobenzoate and amine are present in a molar ratio of 1 : 1 to 1 : 1.5.

12. A method according to any of claims 4 to 11, wherein nitroalkyl ester and reducing agent are present in a molar ratio of 1 :4 to 1 :8.

13. A method according to any of claims 4 to 12, wherein diamino alkyl ester and aryl aldehyde are present in a molar ratio of 1 : 1 to 1 : 1.5.

14. A method according to any of claims 8, wherein the catalyst is selected from the group consisting of bisulfite or metabisulfite salt.

15. Use of a compound prepared by the method according to any of claims 4 to 14 in the manufacture of a sirtuin inhibitor medicament.