Bromodomain3 (BRD3) inhibitor compound: 4-((4-chloro-3-(4-OXO-4 h-chromen-2-YL)phenyl)sulfonyl)-6-fluoro-3,4-dihydro-quinoxal in-2(1 h)-one

Abstract

The invention relates to a compound having a bromodomain3 (BRD3) inhibitory effect With its specific BRD3 inhibitory activity, the inventive compound is a candidate as an original active ingredient for the treatment of cardiovascular diseases such as atherosclerosis, type 2 diabetes and high-density lipoprotein (HDL).

Description

[0001] BROMODOMAIN3 (BRD3) INHIBITOR COMPOUND: 4-( (4-CHLORO-3- (4-OXO-4H-CHROMEN-2-YL)PHENYL)SULFONYL)-6-FLUORO-3,4- DIHYDRO-QUINOXAL IN-2(1 / / )-() NE

[0002] Technical Field

[0003] The invention relates to a compound having a bromodomain3 (BRD3) inhibitory effect.

[0004] Prior Art

[0005] Bromodomain and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) play antiapoptotic roles in the cell cycle sequence and act as transcriptional regulators essential for cell growth. By specifically binding acetylated lysine on histones, BET proteins alter enzyme activities, protein integrity, protein-protein interactions and play an active role in the mechanisms underlying common cellular events such as transcriptional activation and chromatin rearrangement, leading to the formation of various types of cancer. Since 2010, studies have shown that bromodomain (BRD) inhibitors offer new hope for developing therapeutic drug molecules that target gene transcription in cancer treatment.

[0006] In recent years, based on the proposal of the dihydroquinoxaline structure as a pharmacophoric model for BRD inhibitors, the aim has been to develop more selective and effective BRD inhibitor compounds by combining the flavone structure (the core ring system of flavonoids found naturally in plants with anticancer properties) with the dihydroquinoxaline pharmacophore through a "sulfonamide" connection.

[0007] RVX-208 molecule (Apabetalon, Formula 1), a structural analog of flavone, which targets the second domain (BD2) of BET proteins BRD2, BRD3 and BRD4, and inhibits the transcriptional interactions of BET proteins with acetylated lysines on histones and transcription factors;2’3is currently undergoing Phase III clinical trials for the treatment of cardiovascular diseases in patients with atherosclerosis, type 2 diabetes, and high-density lipoprotein (HDL) (https: / / www.resverlogix.com / science-and-programs / apabetalone). Additionally, findings suggest that RVX-208 is effective in the advanced eradication of HIV- 1.4

[0008] Formula 1. Apabetalon (RVX-208)

[0009] Fabry disease is an inherited genetic disorder caused by mutations in the GLA gene which is responsible for the production of an enzyme called alpha-galactosidase A. These mutations render the enzyme either partially or completely non-functional. This leads to the accumulation of a lipid molecule called globotriaosylceramide (Gb3, also known as GL-3), resulting in cellular damage in tissues and consequently progressive, irreversible organ damage in the nervous, cardiac and renal systems. Due to the specific BRD inhibitory effect of RVX-208 (apabetalon),5Phase 1 / 2 studies for the treatment of Fabry disease are ongoing.

[0010] Since prehistoric times, people have used plants to treat various diseases. From the early 20th century, active compounds such as morphine, quinine, taxol and vincristine have been isolated from medicinal plants. These biologically active natural compounds obtained from plants have been pioneers in the design, synthesis and development of new drug molecules. Overtime, 60% of anticancer drugs and 75% of infectious drugs have been developed based on compounds of natural origin.

[0011] Flavones are benzo-gamma pyrone compounds (Formula 2) found in nature, responsible for the color and odor of vegetables and fruits. Approximately 9.000 different flavonoid derivatives have been isolated from plants, and for years, people have widely consumed flavone-rich plants for the treatment of various diseases.6

[0012] Formula 2. Flavone (benzo-y-piron)

[0013] Flavones are polyphenolic compounds abundantly found in the fruits and seeds of edible plants, teas prepared from plants and fruit juices. Flavonoids constitute an important part of the daily diet and are defined as dietary supplements that support health and prevent diseases. The daily intake of flavonoids from food is around 1.0 g-6

[0014] Studies have shown that flavones have anti-inflammatory, hypoglycemic and cardiovascular effects. Today, studies on the role of flavone-containing food consumption in cancer prevention continue intensively. The findings have shown that flavone compounds have important effects in cancer chemoprevention and chemotherapy. Proposed mechanisms include carcinogen inactivation, antiproliferation, cell cycle arrest, induction of apoptosis, differentiation, and inhibition of angiogenesis.6-13

[0015] Based on the "dihydroquinoxaline sulfonamide group" compounds studied for BRD inhibitor drug development, an effective BRD3 inhibitor compound was developed by combining the flavone ring (the core structure of compounds widely used for their therapeutic properties for thousands of years) with the quinoxaline group. The original compound developed with flavone-quinazoline structure demonstrates more selective effect by inhibiting BRD3, compared to apabetalone, which interacts with all of the BET proteins BRD2, BRD3 and BRD4. This selectivity gives our original molecule an advantage. The US Patent document US2021261539A1 in the prior art mentions a series of BET (bromodomain and extra-terminal domain) inhibitors with a novel structure, specifically targeting BRD4 (bromodomain containing protein 4), along with methods for preparing and using these inhibitors.

[0016] Upon examining existing studies in the field, there is a recognized need for the development of compounds with bromodomain3 (BRD3) inhibitory effects.

[0017] Objectives of the Invention

[0018] The objective of the invention is to develop an original drug candidate compound with bromodomain3 (BRD3) inhibitory activity.

[0019] Another objective of the invention is to create an original drug candidate compound with specific BRD3 inhibitory activity for the treatment of cardiovascular diseases in patients with atherosclerosis, type 2 diabetes, high-density lipoprotein (HDL).

[0020] A further objective of the invention is to develop an original drug candidate compound for the treatment of Fabry disease.

[0021] Detailed Description of the Invention

[0022] Within the scope of the invention, a compound with bromodamain3 (BRD3) inhibitory effect has been designed and synthesized.

[0023] The compound in question, designated as Formula I, consists of molecular groups formed by the combination of flavone and dihydroquinazoline / dihydroquinoxaline rings.

[0024] (Formula I) Here R; Ri: -H, -Cl, -CH3, -0CH3

[0025] R2: H, Cl, CH3, 0CH3JOH

[0026] R3: H, F, Cl, Br, 0CH3

[0027] R4: SO2 veya CH2 (Binding group)

[0028] Z: CH2, NH Y: C=O, NH, NCH3, NCH2CH3,

[0029] The open structure of the compound subject to the invention is specified in Formula II: [4-( ( 4-Chloro-3-(4-oxo-4H-chromen-2-yl)phenyl)sulfonyl)-6-fluoro-3,4- dihydro-quinoxalin-2( 1 H)-one]

[0030]

[0031] (Formula II)

[0032] At the stage of obtaining the inventive compound;

[0033] Synthesis of the original molecule: Synthesis of 2'-chloro-5'-flavone sulfonyl chloride:

[0034] Baker- Venkataraman synthesis method was used for flavone ring synthesis. Accordingly, the ester compound (I) formed by treatment of acetophenone with 2- chloro benzoyl chloride in the presence of pyridine was converted to diketone compound (II) when reacted in pyridine in the presence of potassium hydroxide. The diketone compound (II) gave the compound 2'-chloro flavone (III) when stirred in sulfuric acid at room temperature. The reaction of flavone (III) with chlorosulfonic acid gave flavone-SChCl (A) (Scheme 1).

[0035]

[0036] Scheme 1. Synthesis of flavone sulfonyl chloride (A)

[0037] Synthesis of 2-acetylphenyl 2-chlorobenzoate (I): 2-Chloro benzoyl chloride (0.071 mol) and o-hydroxyacetophenone (0.071 mol) in dry pyridine (0.055 mol) were heated at 100°C for 15 min. The reaction medium was cooled to room temperature, 40 ml of 15% HC1 was added and consumed with chloroform. The chloroform phase was washed 3 times with distilled water and dried over anhydrous Na2SO4 and concentrated in a rotary evaporator. The crude product was crystallized from ethanol. White crystalline material was obtained. Yield 66.19%, M.P.: 62°C. Synthesis of l-(2-chlorophenyl)-3-(2-hydroxyphenyl)propan-l, 3-dione (II): 2- Acetylphenyl 2-chlorobenzoate (I) (0.055 mol) was stirred in 60 ml pyridine in the presence of powdered potassium hydroxide (0.057 mol) at 60°C for 30 minutes. At the end of the time, 150 ml of distilled water was added to the reaction medium, cooled on ice and 6N HC1 was added until pH= 5. The yellow precipitate was filtered off and washed with 5% HC1, then with distilled water and dried. Yield 85.42%. M.P.: 95°C.

[0038] Synthesis of 2-(2'-chlorophenyl)-4J / -chromen-4-one (III): 1 -(2-Chlorophenyl)-3- (2-hydroxyphenyl)propane-l, 3-dione (II) (0.0465 mol) was stirred in 100 ml of concentrated H2SO4 for 15 min at room temperature. The reaction medium was poured onto ice. The precipitate was filtered off and washed twice with distilled water and dried. The crude product was crystallized from ethanol. Light cream coloured product was obtained. Yield 90.50%. M.P.: 190°C.

[0039] Synthesis of 4-chloro-3-(4-oxo-4J / -chromen-2-yl)benzenesulfonyl chloride (A): 25 ml of chlorosulfonic acid (0.376 mol) was cooled to 0-5°C and 0.01 mol of 22-(2'- chlorophenyl)-4J / -chromen-4-one (III) was added. The reaction medium was stirred at room temperature for 2 days. The mixture was poured dropwise onto 150 ml of crushed ice. The precipitate was filtered off and dried. 2.06 g of light cream coloured powder product was obtained. Yield 82.52% M.P.: 170-171°C (MS (ESI+) m / z (% relative intensity): (M+H, 100%): 356.1 (M+H, 100%).

[0040] Synthesis of 6-fluoro-3,4-dihydroquinoxalin-2(l B) (Scheme2):

[0041] Scheme 2. Synthesis of 6-fluoro-3.4-dihydroquinoxaline-2(l H)-one

[0042] 5-Fluoro-l,2-phenylenediamine (27.8 mmol) was dissolved in 30 ml DMF, to which triethylamine (TEA) (7.8 ml, 55.8 mmol) and ethyl 2-bromoacetate (30.5 mmol) were added. The reaction medium was stirred at room temperature for 16 hours, then heated at 80 °C for 3 hours. TEA and DMF were removed from the reaction medium by evaporation in vacuo. The residue was extracted 3 times with EtOAc by adding water. The EtOAc phase was washed with saturated NaHCOs solution and water, dried over Na2SO4 and evaporated in vacuo. The residue was dissolved in dichloromethane, precipitated by adding hexane (Dichloromethane- Hexane: 1 : 1) The precipitate was filtered off, dried. This process was repeated twice to purify the product. A light brown powder was obtained (3.0 g, yield: 75%). M.P. : 167°C. Synthesis of 4-((4-chloro-3-(4-oxo-4J / -chromen-2-yl)phenyl)sulfonyl)-6-fluoro-

[0043] 3,4-dihydro-quinoxaline-2(17i )one (Scheme3):

[0044] Scheme 3. Synthesis of 4-((4-chloro-3-(4-oxo-4J / -chromen-2-yl)phenyl)sulfonyl)- 6-fluoro-3,4-dihydro-quinoxalin-2(177)-one (Formula II)

[0045] 6-Fluoro-3,4-dihydroquinoxaline-2(U7)-one (B) (0.7 mmol) was dissolved in 5 ml of tetrahydrofuran, 2 ml of pyridine was added and 2'-chloro-5'-flavone sulfonyl chloride (A) (1.01 mmol) was added, stirred at room temperature for 24 hours. The reaction medium was evaporated on a rotary evaporator and water was added. The precipitate formed on addition of water was filtered off. When no precipitate formed with the addition of water, it was extracted 3 times with chloroform. The chloroform phase was dryed with anhydrous sodium sulfate and evaporated in a rotary evaporator. The crude product was purified by column chromatography using a chloroform:methanol:ammonia solution (10: 1 :0.5) solvent system. Silica gel 60 (230-400 mesh ASTM) was used for this. 60 mg of pure product was obtained. Yield 20.17%, M.P. 255-257°C, MS (ESI+) m / z (% relative intensity): 486.08 (M+H, 100%), 488.02 (M+2+H, 34%).

[0046] ’H-NMR (CDCh, 400 MHz, 6, ppm): 4.39 (s, 2H, NCH2), 6.58 (s, 1H, 3-H), 6.61 (dd, 1H, . / o=9,6Hz, Jm=2.4Hz,QN-H), 6.99 (td, 1H, Jo=8.8Hz, Jm=2.4Hz, QN-H), 7.47(dd, 1H, . / o=8,8Hz, Jm=2.0Hz,QN-H), 7.53-7.59 (m, 2H, 6,3’-H), 7.65 (d, 1H, Jo=8.8Hz, 8-H), 7.77 (yayvan s, 1H, 6’-H), 7.84-7.86 (m, 2H, 7,4’-H), 8.10 (d, 1H, Jo=8.0Hz, 5-H), 10.49 (s,lH, N-H).

[0047] 13C NMR (DMSO, 100 MHz, 6, ppm): 176.53, 164.51, 161.37(d, JCF=243.1 HZ). 160.39, 155.89, 137.68, 135.39(d, JCF=11.4HZ), 134.82, 134.74, 132.33, 131.69, 130.08, 129.26, 129.16, 125.94, 124.95, 123.12, 119.59, 118.39, 112.92, 109.52(d, JCF=22.9HZ), 103.01 (d, JCF=25.9HZ), 48.81.

[0048] Elementary Analysis: C23H14CIFN2O5S; calculated: C, 56.92; H, 2.91; N, 5.78; S, 6.61; found: C, 56.52; H, 3.09; N, 5.68; S, 6.59.

[0049] Investigation of the effect of bromodomain inhibition:

[0050] In this context, BRD2, BRD3 and BRD4 inhibitory effects of the synthesized substances were investigated using Bromodomain inhibitor assay kits.

[0051] Analyses were performed using 348 reaction plates. Before starting the experiment, all kit components were diluted and prepared as recommended by the manufacturer. First, 5 pl of Tb (terbium)-labeled donor and 5 pl of dye-labeled acceptor were applied to all wells. Then 2 pl of each sample at varying concentrations (100, 50, 25, 10, 1 pM) was added to the wells reserved for the samples and 2 pl of 10% DMSO was added to the wells reserved for the positive and negative control groups. 5 pl of BET bromodomain ligand was added to the wells containing the positive control and samples and 5 pl of non-acetylated ligand 1 was added to the wells containing the negative control group. Finally, 3 pl of BRD2 / BRD3 / BRD4 protein (6 ng / pl) was added to all wells to start the reaction. The plate was incubated at room temperature for 2 hours and fluorescence intensity was measured using a plate reader. Donor emission was measured at a wavelength of 620 nm and acceptor emission at a wavelength of 665 nm. Data analysis was performed using the ratio of these two measurements. The inventive compound tested (4-((4-Chloro-3-(4-oxo-4J / -chromen-2- yl)phenyl)sulfonyl)-6-fluoro-3,4-dihydroquinoxaline-2(U7)-one (C)) was found to inhibit BRD3 with an IC50 value of 0.33 pM.

[0052] References:

[0053] 1. Picaud, S., Wells, C., Felletar, I., Brotherton, D., Martin, S., Savitsky, P., Diez-Dacal, B., Philpott, M., Bountra, C., Lingard, H., Fedorov, O., Muller, S., Brennan, P.E., Knapp, S., Filippakopoulos, P. 2013a. “RVX-208, an inhibitor of BET transcriptional regulators with selectivity for the second bromodomain”, Proc Natl Acad Sci U S A. 110, 19754-19759. Ghosh vd. 2017

[0054] 2. Ghosh, G.C., Bhadra, R., Ghosh, R.K., Banerjee, K., Gupta, A. 2017. “RVX 208: A novel BET protein inhibitor, role as an inducer of apo A-I / HDL and beyond”, Cardiovascular Therapeutics, 35, el2265, 1-10.

[0055] 3. Wasiak, S., Dzobo, K.E., Rakai, B.D., Kaiser, Y., Versloot, M., Bahjat, M., Stotz, S.C., Fu, L., Sweeney, M., Johansson, J.O., Wong, N.C.W., Stroes, E.S.G., Kroon, J., Kulikowski, E. 2020. “BET protein inhibitor apabetalone (RVX-208) suppresses pro-infammatory hyper-activation of monocytes from patients with cardiovascular disease and type 2 diabetes”, Clin Epigenet, 12(166), 1-19.

[0056] 4. Zhang, X.X., Lin, J., Liang, T.Z., Duan, H., Tan, X.H., Xi, B.M., Li, L., Liu,

[0057] S.W. 2019. “The BET bromodomain inhibitor apabetalone induces apoptosis of latent HIV-1 reservoir cells following viral reactivation”, Acta Pharmacologica Sinica, 40,98-110.

[0058] 5. Morgado-Pascual, J.L., Rayego-Mateos, S., Tejedor, L., Suarez-Alvarez, B., Ruiz-Ortega, M. 2019. “Bromodomain and Extraterminal Proteins as Novel Epigenetic Targets for Renal Diseases”, Front Pharmacol., 10, 1315, 1-15.

[0059] 6. Kittakoop, P. 2015. “Anticancer Drugs and Potential Anticancer Leads Inspired by Natural Products”, Studies in Natural Products Chemistry, 44,251-307. Bontempo, P., Mita, L., Miceli, M., Doto, A., Nebbioso, A., De Bellis, F., Conte, M., Minichiello, A., Manzo, F., Carafa, V., Basile, A., Rigano, D., Sorbo, S., Cobianchi, R.C., Schiavone, E.M., Ferrara, F., De Simone, M., Vietri, M.T., Cioffi, M., Sica, V., Bresciani, F., De Lera, A.R., Altucci, L., Molinari, AM. 2007. “Feijoa sellowiana derived natural Flavone exerts anti-cancer action displaying HDAC inhibitory activities”, The International Journal of Biochemistry & Cell Biology, 39, 1902-1914. Ramos, S. 2008. “Cancer Chemoprevention And Chemotherapy: Dietary Polyphenols And Signalling Pathways”, Mol Nutr Food Res, 52,507-526. Anso', E., Zuazo, A., Irigoyen, M., Urdaci, M. C., Rouzaut, A., Martinez- Irujo, J. J. 2010. “Flavonoids inhibit hypoxia-induced vascular endothelial growth factor expression by a HIF-1 independent mechanism”, Biochemical Pharmacology, 79, 1600-1609. Attoub, S., Hassan, A.H., Vanhoecke, B., Iratni, R., Takahashi, T., Gaben, A.-M., Bracke, M., Awad, S., John, A., Kamalboor, H.A., Al Sultan, M.A., Arafat, K., Gespach, C., Petroianu, G. 2011.” Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells”, European Journal of Pharmacology, 651,18-25. Romagnolo, D.F., Selmin, O. I., 2012. “Flavonoids and Cancer Prevention: A Review of the Evidence”, Journal of Nutrition in Gerontology and Geriatrics, 31,206-238. Singh, P., Tomar, R.S., Rath, S.K. 2015. “Anticancer potential of the histone deacetylase inhibitor-like, effects of flavones, a subclass of polyphenolic compounds: a review”. Mol Biol Rep, 42,1515-1531. Miceli, M., Bontempo, P., Nebbioso, A., Altucci, L. 2014. “Natural compounds in epigenetics: A current view”, Food and Chemical Toxicology, 73, 71-83.

Claims

CLAIMS1. The invention is a compound of formula I having a bromodomain3 (BRD3) inhibitory effect,(Formula I) characterized in thatRi is -H, -Cl, -CH3or -OCH3R2is H, Cl, CH3, OCH3or OHR3is H, F, Cl, Br or OCH3R4is; SO2 or CH2.

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