Screening of fasn small molecule inhibitors and their applications

Abstract

The application belongs to the field of tumor diagnosis and treatment, and relates to a potential inhibitor of fatty acid synthase (FASN) and application of the potential inhibitor in treating various tumors such as colorectal cancer, breast cancer, ovarian cancer and the like. The small-molecule inhibitor is a compound shown in formula I or II, or a stereoisomer of the compound shown in formula I or II, or a pharmaceutically acceptable salt, a solvate, a hydrate, a polymorph, a co-crystal, a tautomer, an isotopically labeled derivative and a prodrug thereof. The compound is mainly used for inhibiting the enzyme activity of FASN and then inhibiting the proliferation of various tumors, for example, the growth and proliferation of colorectal cancer, breast cancer, ovarian cancer and the like, and is expected to become a novel broad-spectrum tumor treatment drug and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of tumor diagnosis and treatment, and particularly relates to a kind of as potential fatty acid synthase (FASN) inhibitor Compound screening and the use of compounds for treating proliferative diseases such as cancer (for example, colorectal cancer, breast cancer, ovarian cancer, etc.). BACKGROUND

[0002] Cancer is a great threat to human health at present, so the research and development of antitumor drugs is the top priority in the current medical research and development.

[0003] Tumor cells often have metabolic abnormalities (Rafael Moreno-Sanchez, Sara Rodriguez-Enriquez, Alvaro Marin-Hernandez and Emma Saavedra. Energy metabolism in tumor cells [J]. FEBS J. 2007, 274(6): 1393-418), and many signals that promote tumorigenesis also drive the reprogramming of tumor metabolism, such as the Warburg effect, glutamine genesis, enhanced lipid metabolism, and so on. Tumors adjust their metabolic pathways for sugar and fatty acids in response to the environmental adaptability of hypoxia and acid metabolite accumulation during development. This unique metabolic feature that distinguishes it from normal groups enables energy metabolism to become an important direction for the development of anti-tumor drugs (Claudia Fumarola, Pier Giorgio Petronini, Roberta Alfieri. Impairing energy metabolism in solid tumors through agents targeting oncogenic signaling pathways [J]. Biochem Pharmacol. 2018, 151: 114-125). Lipid metabolism is often up-regulated in tumors. This is often achieved by activating the expression of genes related to lipid synthesis, such as FASN, ACC, ACLY, etc. Among them, FASN is one of the popular targets for drug development. FASN is the only key enzyme for de novo fatty acid synthesis. The source of human fatty acids is divided into two important pathways: the exogenous pathway of intake from food and the endogenous pathway of de novo synthesis by the body itself. Fatty acid synthesis is an important physiological process in living organisms and exists in all cells. Under normal circumstances, the body mostly supplements and utilizes lipids through the exogenous pathway, so the expression of FASN is not high. Compared with normal tissues, 90% of fatty acids in tumor cells are derived from de novo synthesis, which can provide more β-oxidation substrates for tumor cells to cope with their energy stress state (Luo Jingtao Li Qiang. Fatty acid de novo synthesis metabolic reprogramming and tumor development [J]. China Oncology, 2017, 44: 939-943). Tumor cells depend more on de novo synthesis of fatty acids, while normal cells rely more on exogenous fatty acid supply, so the malignant transformation of cells is often accompanied by the up-regulation of FASN, and FASN inhibitors can selectively kill tumor cells, which is one of the most promising targets for anti-tumor therapy.

[0004] FASN is a homodimer with two identical 272 kDa polypeptide subunits, with seven catalytic domains: acyl carrier protein (ACP), acyltransferase (MAT), β-ketoacyl synthase (KS), β-ketoacyl reductase (KR), β-hydroxyacyl dehydratase (DH), enoyl reductase (ER), and thioesterase (TE) (Anne P. L. Jensen-Urstad, Haowei Song, Irfan J. Lodhi, Katsuhiko Funai, Li Yin, Trey Coleman, Clay F. Semenkovich. Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPAR [J]. J Lipid Res. 2013, 54(7): 1848-1859.), responsible for catalyzing the entire process of de novo synthesis of long-chain fatty acid palmitate from acetyl-CoA and malonyl-CoA. The TE domain is the seventh functional domain of this macromolecular enzyme complex, and the remaining domains of FASN mainly assist in the de novo synthesis of palmitate. Among them, the MAT, KS, KR, DH, and ER domains are directly involved in the elongation of the fatty acid chain, the ACP domain participates in the transmission by carrying the elongating fatty acid chain between the domains, and the TE domain releases the elongated palmitate from the ACP by catalyzing the hydrolysis of the thioester bond between palmitate and the 4'-phosphopantetheinyl group on the ACP. Inhibiting the hydrolysis reaction of the TE domain, the fatty acid chain connected to the ACP cannot be smoothly detached after elongation to 16 carbons, thereby preventing the ACP from continuing to participate in the transmission of fatty acid biosynthesis intermediates between the various catalytic domains, and further preventing the synthesis process of the new fatty acid chain.FASN is located in the cytoplasm, can be phosphorylated and activated by mTOR and HER-2, induces endoplasmic reticulum stress and tumor cell death, inhibits tumor growth and prevents angiogenesis, thereby regulating the biological behavior of various tumor cells including colorectal cancer (Zaytseva YY, Rychahou PG, Gulhati P, et al. Inhibition of Fatty Acid Synthase Attenuates CD44-Associated Signaling and Reduces Metastasis in Colorectal Cancer [J]. Cancer Res. 2012 72(6): 1504-1517.), ovarian cancer (Dirk Bauerschlag, Nicolai Maass, et al. Fatty acid synthase overexpression: target for therapy and reversal of chemoresistance in ovarian cancer [J]. J Transl Med. 2015 7; 13: 146.), breast cancer (Sarah Crunkhorn. Breast cancer: FASN inhibitor increases survival [J]. Nat Rev Drug Discov. 2016 29; 15(8): 532.) and the like. At present, there are many researches on FASN inhibitors, but there is no FASN inhibitor that can be applied to the clinic, and the side effects of some inhibitors are serious, which cannot meet the diverse clinical needs. Therefore, it is of great significance to explore and develop new FASN small molecule inhibitors. SUMMARY

[0005] Although it has been proved that inhibiting the activity of FASN in various tumors helps the treatment of tumors, there is still a lack of FASN inhibitors that can be used in clinical. In view of the problems existing in the prior art, the purpose of the present application is to propose a new potential FASN small molecule compound inhibitor with high FASN inhibitory activity and high anti-tumor activity.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions.

[0007] The present application provides a FASN small molecule inhibitor, characterized in that the small molecule inhibitor comprises N'-[(1Z)- (3-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-4-methoxyphenyl)methylene]-3,4,5-trihydroxybenzhydrazide or a pharmaceutically acceptable salt thereof, and the chemical structure is as follows:

[0008] .

[0009] The present application also provides a FASN small molecule inhibitor, characterized in that the small molecule inhibitor comprises compound II 5-({[2-hydroxy-3-(prop-2-yn-1-yloxy)propyl][2-(morpholin-4-yl)ethyl]amino}methyl)-8-thia-4,6-diazatricyclo[7.4.0.0(2),]trideca-1(9),2(7),5-trien-3-one or a pharmaceutically acceptable salt thereof, and the chemical structure is as follows:

[0010] .

[0011] Further, the small molecule inhibitor according to any one of the above is used for preparing a product for inhibiting the activity of FASN.

[0012] Further, the small molecule inhibitor according to any one of the above is used for preparing a medicament for treating tumors.

[0013] Preferably, the tumors include human colorectal cancer, human breast cancer, human ovarian cancer.

[0014] The present application also provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the compound according to any one of the above and at least one of pharmaceutically acceptable carriers and excipients.

[0015] Further, the pharmaceutical composition is used for preparing a medicament for treating tumors.

[0016] Preferably, the tumors include human colorectal cancer, human breast cancer, human ovarian cancer.

[0017] Further, the dose of the pharmaceutical composition is any pharmaceutically acceptable dose.

[0018] Further, the administration mode of the pharmaceutical composition includes intravenous injection, oral administration, and inhalation.

[0019] The pharmaceutical composition of the present application can be prepared according to the methods known in the art using the compound of the present application as an active ingredient. The pharmaceutical composition can be prepared in any dosage form suitable for human or animal use by combining the compound of the present application with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The pharmaceutical composition can be used for treating proliferative diseases including cancer, for example, colorectal cancer, breast cancer, ovarian cancer, etc.

[0020] The compound of the present application or the pharmaceutical composition containing the same can be administered in unit dosage form and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, injection, etc.

[0021] The compound of the present application can be prepared in ordinary formulation, sustained release formulation, controlled release formulation, targeted formulation, and various microparticle drug delivery systems.

[0022] To prepare tablets of the compound of the present application, various excipients known in the art can be widely used, including diluents, wetting agents, binders, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropyl alcohol, etc.; the binders can be starch paste, dextrin, sugar syrup, honey, glucose solution, microcrystalline cellulose, acacia paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecylsulfate, etc.; the lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0023] The tablets can be further prepared into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0024] To prepare capsules of the compound of the present application, the active ingredient, the compound of the present application, can be mixed with diluents and glidants, and the mixture can be directly placed in hard or soft capsules. Alternatively, the active ingredient, the compound of the present application, can be first mixed with diluents, binders, and disintegrants to prepare granules or pellets, and then placed in hard or soft capsules. The various diluents, wetting agents, binders, disintegrants, and glidants used for preparing tablets of the compound of the present application can also be used for preparing capsules of the compound of the present application.

[0025] For injection, the compounds of the present application can be formulated in aqueous solutions, such as water, ethanol, isopropanol, propylene glycol, or mixtures thereof, and include appropriate amounts of nontoxic, parenterally acceptable diluent or solubilizer, such as polyol, glycerol, sorbitol, mannitol, propylene glycol, or polyethylene glycol, or mixtures thereof, and pH adjusting agents, such as sodium chloride, sodium acetate, citric acid, or mixtures thereof. The diluent or solubilizer can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, or the like; the pH adjusting agent can be phosphate, acetate, hydrochloric acid, sodium hydroxide, or the like; and the osmotic pressure adjusting agent can be sodium chloride, mannitol, glucose, phosphate, acetate, or the like. If a freeze-dried powder is desired, mannitol, dextrose, or other suitable excipient can be added.

[0026] In addition, if desired, coloring, preserving, flavoring, and / or other additives can be included in the pharmaceutical formulations.

[0027] To achieve the intended purpose, enhance the therapeutic effect, the pharmaceutical or pharmaceutical composition of the present application can be administered by any known administration method.

[0028] The compound or composition of the present application can be taken alone, or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present application has a synergistic effect with other therapeutic drugs, the dosage thereof should be adjusted according to the actual situation.

[0029] The term "pharmaceutically acceptable salt" as used herein refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of this application include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acid, or organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acid, or base addition salts derived from inorganic bases such as ammonium, potassium, and sodium, and organic bases such as isopropylamine, caffeine, and particularly, those bases that are compatible with the formation of pharmaceutically acceptable salts with such organic acids as ascorbic, maleic, succinic, and citric acid. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bicarbonate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutamate, glycolate, hibenzate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-4alkyl)4salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and basci amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0030] The term "treatment" in some embodiments refers to ameliorating the disease or condition (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In other embodiments, "treatment" refers to alleviating or ameliorating at least one physical parameter, including possible subjective parameters that are not directly observable. In other embodiments, "treatment" refers to modulating the disease or condition either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In other embodiments, "treatment" refers to preventing or delaying the onset or development or worsening of the disease or condition.

[0031] The present application has the following advantages over the prior art.

[0032] 1. The application is first proposed by the applicant through the construction of pharmacophore and docking model, and the small molecule compound inhibitor obtained by multi-layer virtual screening has good inhibitory effect on the proliferation of tumor cells through the experiment of expanding its inhibition on tumor cell growth. The small molecule inhibitor can be used as a new drug for treating proliferative diseases including cancer (such as colorectal cancer, breast cancer, ovarian cancer, etc.).

[0033] 2. The small molecule inhibitor of the application has a wide anti-cancer spectrum and has good growth inhibition effect on various cancer cells such as colorectal cancer, breast cancer, ovarian cancer, etc.

[0034] In summary, the compound disclosed in the application has good application prospect in preventing or treating proliferative diseases, especially cancer, in terms of structure and mechanism of action. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 It is the detection spectrum of FASN small molecule inhibitor compound I of the application and the targeting analysis of FASN protein. Among them, A is the detection spectrum of compound I; B is the Western blot graph of FASN protein targeting analysis of compound I cell heat transfer experiment; C is the thermal melt curve graph of FASN protein targeting analysis of compound I cell heat transfer experiment.

[0036] Figure 2 It is the detection spectrum of FASN small molecule inhibitor compound II of the application and the targeting analysis of FASN protein. Among them, A is the detection spectrum of compound II; B is the detection spectrum of compound II; C is the Western blot graph of FASN protein targeting analysis of compound II cell heat transfer experiment; D is the thermal melt curve graph of FASN protein targeting analysis of compound II cell heat transfer experiment.

[0037] Figure 3 It is the change of the proliferation of various tumor cells such as colorectal cancer, breast cancer and ovarian cancer inhibited by FASN small molecule inhibitor compound I of the application. Among them, A is the change of the proliferation of colorectal cancer inhibited by compound I; B is the change of the proliferation of breast cancer inhibited by compound I; C is the change of the proliferation of ovarian cancer inhibited by compound I.

[0038] Figure 4 It is the change of the proliferation of various tumor cells such as colorectal cancer, breast cancer and ovarian cancer inhibited by FASN small molecule inhibitor compound II of the application. Among them, A is the change of the proliferation of colorectal cancer inhibited by compound I; B is the change of the proliferation of breast cancer inhibited by compound I; C is the change of the proliferation of ovarian cancer inhibited by compound I. DETAILED DESCRIPTION

[0039] The application will be further described in connection with the following non-limiting examples. This should not be interpreted as limiting the scope of the above subject matter of the application to the following examples, any technology realized based on the content of the application falls within the scope of the application.

[0040] Unless otherwise specified, the reagents and materials used in the application are commercially available.

[0041] Example 1: Screening and structural identification of FASN inhibitors.

[0042] Applicants apply the method of computer-aided drug design to obtain FASN inhibitors with high affinity and binding stability to target proteins through multi-layer virtual screening. The pharmacophore screening and molecular docking are constructed by using molecular simulation drug design software MOE. Ten superior structures are obtained through layer-by-layer screening from 1767416 small molecule compounds without known FASN inhibitory activity in ChemDiv, Specs database, and experimental verification is carried out to obtain the FASN inhibitors of the application.

[0043] Construction of pharmacophore and preliminary screening of FASN small molecule inhibitors.

[0044] The FASN TE domain protein X-ray diffraction model is obtained from the RCSB PDB database, and the pharmacophore is established with the ligand provided with the model. The key positions of the ligand interacting with the crystal model are found and selected by using the pharmacophore Query Editor in the MOE software, and four pharmacophores of Acc, Don&Acc, Acc, and Don (Don: H-bond donor; Acc: H-bond acceptor) are obtained for preliminary screening. After screening the compounds in the compound library by the pharmacophore model, 775 compounds matching the same are obtained.

[0045] Molecular docking to obtain FASN inhibitor compounds in superior conformation

[0046] The Dock in MOE software is used for molecular docking of 775 highly matched compounds and the kinase activity domain of FASN protein tertiary structure. 300 conformations are selected for each molecule, and the highest scoring conformation is output. In the final output of 775 compound docking results, the compounds with higher scores (S < -8.0) are selected, and the compounds with gene toxicity and not meeting the 5 principles of drug-like are deleted. Finally, 10 superior conformations with good affinity to the FASN TE domain are obtained as potential FASN small molecule inhibitors. The applicant entrusts TOPSCIENCE to synthesize the above-mentioned 10 FASN small molecule inhibitors, and finally screens 2 small molecule inhibitors with significant antitumor activity in the present application through MTT antitumor activity experiment. The targeting effect of the small molecule inhibitor on FASN protein is analyzed by cell heat transfer experiment (CESTA), and the structure of the FASN small molecule inhibitor is as follows:

[0047] .

[0048] The detection spectrum of the above two compounds and the targeting analysis of the FASN protein are shown in Figure 1 and 2 .

[0049] Example 2: Antitumor activity of potential FASN small molecule inhibitors.

[0050] Experimental materials.

[0051] Cells and reagents.

[0052] Human colorectal cancer and intestinal epithelial cell lines: HCT116, SW620, HT29, SW480, RKO, LoVo; human breast cancer and breast epithelial cell lines: MDA-MB-231, MDA-MB-468, SKBR3, MCF7, T47D; human ovarian cancer and ovarian epithelial cell lines: SKOV3, A2780, CAOV3, OVCAR3. DMEM medium, L-15 medium, 1640 medium, McCOY's 5A medium, DMEM / F12 medium, MEM medium, trypsin, fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), MTT (thiazolyl blue).

[0053] Experimental methods and conclusions.

[0054] Cell culture.

[0055] The cells were taken out from the -80℃ deep freezer or liquid nitrogen storage tank, quickly placed in a 37℃ water bath to melt, then transferred to a 2mL centrifuge tube, centrifuged at 1200rmp for 5min, the supernatant containing the freezing solution was discarded, 1mL of the corresponding medium containing 10% fetal bovine serum was added to completely resuspend the cells, and the cell suspension was transferred to a 25cm 2 culture bottle, 4mL of complete medium containing 10% fetal bovine serum was added, and the culture bottle was placed in a 37℃, 5% CO2 incubator for culture. The next day, the original culture medium was discarded, 2mL of 1×PBS was washed three times, and 5mL of complete culture medium was added for continuous culture, and the cells were subcultured after 1-2 days.

[0056] The cell proliferation was detected by MTT method.

[0057] The well-digested and centrifuged cells were resuspended and counted, and then inoculated into a 96-well plate, configured as 100μL of cell suspension, and 5×10 3 cells per well. After pre-culturing the culture plate in an incubator (37℃, 5% CO2) for 24h, different concentrations of compounds were given for treatment for 48h; 6 replicate wells were set for the experimental group and the control group, and zero adjustment holes (pure culture medium) and blank holes (DMSO solvent control) were set. After treatment, 20μL of MTT (0.5%) solution was added to each well, the culture plate was incubated in an incubator for 2-4h, the culture solution and MTT reagent in the culture plate were aspirated, 150μL of DMSO was added to each well, and the plate was placed in a 37℃ constant temperature shaking bed at 100rpm for 10min. The absorbance at 570nm was measured by using an enzyme-labeled instrument.

[0058] The cell survival rate (%) was calculated as follows: [A (drug added)-A (blank)] / [A (0 drug added)-A (blank)]×100%.

[0059] A (drug added): the absorbance of the well with cells and drug solution; A (blank): the absorbance of the well with medium but without cells; A (0 drug added): the absorbance of the well with cells but without drug solution).

[0060] Three repeated experiments were performed, and the SPSS software was used for statistics.

[0061] Experimental results and conclusions.

[0062] The above two compounds have anti-tumor activity, and the MTT proliferation results are shown in Figure 3 and 4 The compounds have obvious inhibitory effect on the proliferation of various tumor cells. From the above experiments, it can be seen that the compounds of the present application have good proliferation inhibition effect on various tumor cells, and are very promising as new tumor treatment drugs.

[0063] Although the above has been described in detail for the preferred embodiments of the present application, those skilled in the art understand that various modifications and changes can be made to the present application without departing from the spirit and scope of the present application. The scope of the present application is not limited to the above detailed description, any modification, replacement, improvement in the same spirit and meaning is within the scope of the present application.

Claims

1. Use of a FASN small molecule inhibitor in the manufacture of a medicament for treating a tumor, wherein the FASN small molecule inhibitor is any one of the following compounds or a pharmaceutically acceptable salt thereof: ###0001### ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ### 。 2. Use according to claim 1, wherein ​ 3. The use according to claim 1, wherein ​ 4. The use according to claim 1, wherein ​

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