Coumarin derivatives and uses thereof and acly inhibitors and related diseases

Abstract

The application discloses 3-substituted coumarin derivatives 1a-14a and symmetrical biscoumarin derivatives 1b-25b and pharmaceutically acceptable salts, solvates or hydrates thereof. The compounds provided by the application are specific inhibitors of ATP citrate lyase (ACLY), and the half maximal inhibitory concentration (IC 50 ) of the former is 2-30 muM, and the IC 50 ) of the latter is 0.065-4 muM, so the compounds have potential therapeutic value for a plurality of diseases with abnormally high expression of ACLY, such as cancer, hyperlipidemia and arteriosclerotic cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug development, specifically relating to the application of 3-substituted coumarin derivatives and symmetrical dicoumarin derivatives in the preparation of ATP citrate lyase inhibitors and their use in related diseases. Background Technology

[0002] ATP citrate lyase (ACLY) is mainly located in the cytoplasm and is involved in the reaction of ATP and Mg. 2+ Under certain conditions, ACLY catalyzes the cleavage of citrate into acetyl-CoA and oxaloacetate. In this reaction, the substrate citrate originates from the tricarboxylic acid cycle, and the product acetyl-CoA is a precursor molecule for fatty acid and cholesterol biosynthesis (Wei XP, Schultz K, Bazilevsky GA, et al. Molecular basis for acetyl-CoA production by ATP-citratelyase[J]. Nature Structural & Molecular Biology, 2020, 27(1):33-41.). ACLY links the tricarboxylic acid cycle and lipid synthesis metabolism through enzymatic reactions and is considered a key enzyme in glucose and lipid metabolism (Chypre M, Zaidi N, Smans K. ATP-citrate lyase: A mini-review[J]. Biochemical and Biophysical Research Communications, 2012, 422(1):1-4.). ACLY is highly expressed in the liver, white adipose tissue, and pancreatic β-cells, and lowly expressed in the brain, heart, small intestine, and muscle tissue. ACLY is regulated at both the transcriptional and translational levels in vivo, mediating multiple signal transduction pathways and participating in various biological processes, including proliferation, apoptosis, homologous recombination, lipid synthesis, and energy metabolism (Granchi C. ATP citrate lyase (ACLY) inhibitors: An anti-cancer strategy at the crossroads of glucose and lipid metabolism[J]. European Journal of Medicinal Chemistry, 2018, 157: 1276-91).

[0003] ACLY's crucial role in the de novo lipid synthesis pathway makes it a natural target for lipid-lowering. Inhibiting ACLY activity suppresses acetyl-CoA production, which in turn inhibits fatty acid synthesis and the synthesis of triglycerides, which serve as precursors, thus achieving a lipid-lowering effect. Metabolic phenotypes are closely related to cancer development and progression. Disordered glucose metabolism and vigorous lipid synthesis, accompanied by abnormally high ACLY expression, have been observed in many cancers. ACLY is both a terminal enzyme in glycolysis and an initiating enzyme in lipid synthesis. Recent studies have shown that ACLY is a potential anti-cancer target. Therefore, the discovery of ACLY inhibitors not only has scientific significance in basic research but also has important application value in clinical practice for lipid-lowering and anti-cancer treatment.

[0004] Terminal carboxylic acid inhibitors and cycloamide inhibitors are the two main classes of synthetic ACLY inhibitors. These inhibitor molecules have played a crucial role in elucidating structure-activity relationships, exploring the mechanism of action of ACLY, and advancing related clinical research on ACLY. However, they suffer from problems such as low activity, limited variety, poor target specificity, and poor drug-likeness. Therefore, synthesizing ACLY inhibitors with novel skeletons, high activity, and good drug-likeness remains of significant practical importance.

[0005] Coumarin compounds have good medicinal value and are also an excellent source of material for synthesis. Many researchers have conducted research on the synthesis of coumarin compounds, and the technical routes are mature. This invention uses coumarin as the source material to synthesize a series of 3-substituted coumarin derivatives and dicoumarin derivatives, which are characterized as ACLY inhibitors. The application of these two types of coumarin derivatives in ACLY inhibitors has not been reported. Summary of the Invention

[0006] Coumarin derivatives, wherein the coumarin derivatives are one or more of 3-substituted coumarin derivatives and symmetrical dicoumarin derivatives, wherein the 3-substituted coumarin derivatives have one or more of the following general formulas I, II and III, and the symmetrical dicoumarin derivatives have one or more of the following general formula IV:

[0007]

[0008] in,

[0009] R1 is phenyl or 4-tert-butylbenzene;

[0010] R2 is selected from one of phenyl, p-chlorophenyl, p-fluorophenyl, p-phenolyl, 2,4-dichlorophenyl, p-methylphenyl, p-methoxyphenyl, cyclopropyl, cyclopentyl, cyclohexyl and 2-thienyl;

[0011] R3 is p-methoxyphenyl, and R4 is hydroxyl;

[0012] R5 is selected from one of the following: phenyl, p-phenol, p-fluorophenyl, p-cyanophenyl, p-methoxyphenyl, p-methylphenyl, 2-chlorophenol, 2-bromo-p-phenol, 2-fluoro-p-bromophenyl, 2-fluoro-p-fluorophenyl, phenyl, methyl, thiobutanol, n-pentyl, chlorobutyl, isopropyl, 2,2-dimethylbutyryl chloride, cyclopropyl, cyclobutyl, cyclopentyl, furanyl, 3-pyridyl, 4-pyridyl, and 2-pyridyl.

[0013] Coumarin derivatives, characterized in that they contain one or more of the following compounds;

[0014] (1) 7,8-Dihydroxy-3-phenyl-2H-chromen-2-one

[0015] (2) 7,8-Dihydroxy-3-[4-(2-methylpropyl-2-yl)phenyl]-2H-chromen-2-one

[0016] (3) 3-Phenylacetyl-7,8-dihydroxy-2H-chromen-2-one

[0017] (4) 3-[(4-chlorophenyl)carbonyl]-7,8-dihydroxy-2H-chromen-2-one

[0018] (5) 3-[(4-fluorophenyl)carbonyl]-7,8-dihydroxy-2H-chromen-2-one

[0019] (6) 7,8-Dihydroxy-3-[(4-hydroxyphenyl)carbonyl]-2H-chromen-2-one

[0020] (7) 3-[(2,4-dichlorophenyl)carbonyl]-7,8-dihydroxy-2H-chromen-2-one

[0021] (8) 7,8-Dihydroxy-3-[(4-methylphenyl)carbonyl]-2H-chromen-2-one

[0022] (9) 7,8-Dihydroxy-3-[(4-methoxyphenyl)carbonyl]-2H-chromen-2-one

[0023] (10) 3-(cyclopropylcarbonyl)-7,8-dihydroxy-2H-chromen-2-one

[0024] (11) 3-(cyclopentylcarbonyl)-7,8-dihydroxy-2H-chromen-2-one

[0025] (12) 3-(cyclohexylcarbonyl)-7,8-dihydroxy-2H-chromen-2-one

[0026] (13) 7,8-Dihydroxy-3-(thien-2-ylcarbonyl)-2H-chromen-2-one

[0027] (14) 7,8-Dihydroxy-3-[hydroxy(4-methoxyphenyl)methyl]-2H-chromen-2-one

[0028] (15) 4,7,8-Trihydroxy-3-[phenyl(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one

[0029] (16) 4,7,8-Trihydroxy-3-[(4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one

[0030] (17) 3-[(4-fluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (18) 4-[bis(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]benzene-1-carboxylon

[0031] (19) 4,7,8-Trihydroxy-3-[(4-methoxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one

[0032] (20) 4,7,8-Trihydroxy-3-[(4-methylphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one

[0033] (21) 3-[(3-chloro-4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0034] (22) 3-[(3-bromo-4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0035] (23) 3-[(3-bromo-4-fluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0036] (24) 3-[(2,4-difluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0037] (25) 4,7,8-Trihydroxy-3-[2-phenyl-1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)ethyl]-2H-chromene-2-one

[0038] (26) 4,7,8-Trihydroxy-3-[(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one

[0039] (27) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)ethyl]-2H-chromene-2-one

[0040] (28) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxylidene-3-yl)pentyl]-2H-chromene-2-one

[0041] (29) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxylidene-3-yl)hexyl]-2H-chromen-2-one

[0042] (30) 3-[5-chloro-1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)pentyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0043] (31) 4,7,8-Trihydroxy-3-[2-methyl-1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)propyl]-2H-chromene-2-one

[0044] (32) 3-[3,3-dimethyl-1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)butyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0045] (33) 3-[cyclopropyl(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0046] (34) 3-[cyclobutyl(4,7,8-trihydroxy-2-oxoylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0047] (35) 3-[cyclohexyl(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one

[0048] (36) 4,7,8-Trihydroxy-3-[(tetrahydrofuran-3-yl)(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-2H-chromene-2-one

[0049] (37) 4,7,8-Trihydroxy-3-[(pyridin-3-yl)(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-2H-chromen-2-one (38) 4,7,8-Trihydroxy-3-[(pyridin-4-yl)(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-2H-chromen-2-one (39) 4,7,8-Trihydroxy-3-[(pyridin-2-yl)(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-2H-chromen-2-one

[0050] A coumarin derivative according to claim 1 or 2, characterized in that it further comprises one or more of the following pharmaceutically acceptable potassium salt, sodium salt, hydrochloride salt, phosphate salt, acetate salt, oxalate tartrate salt, or malonate salt of the coumarin derivative according to claim 1 or 2.

[0051] An ACLY inhibitor, characterized in that: the active ingredient comprises one or more of the 3-substituted coumarin derivatives or symmetrical dicoumarin derivatives as described in claim 1, 2 or 3 above.

[0052] A medicament for the prevention and / or treatment of ACLY-related diseases, characterized in that: the active ingredient comprises one or more of the 3-substituted coumarin derivatives or symmetrical dicoumarin derivatives as described in claim 1, 2 or 3 above.

[0053] The relevant diseases are characterized in that: the relevant diseases include one or more of cancer, hyperlipidemia, and atherosclerotic cardiovascular and cerebrovascular diseases; the cancer includes one or more of lung cancer, prostate cancer, and breast cancer.

[0054] The drug is characterized in that it contains pharmaceutically acceptable pharmaceutical excipients or carriers, and its dosage form may be tablets, pills, injections, capsules or granules.

[0055] The use of a coumarin derivative as described in claim 1, 2, or 3 in the preparation of an ATP citrate lyase inhibitor or in the prevention and / or treatment of ACLY-related diseases.

[0056] The compound provided by this invention is a specific inhibitor of ATP citrate lyase (ACLY), and the former has a half-maximal inhibitory concentration (IC50) of 1,000. 50 In the 2μM-30μM range, the latter's IC 50 These compounds, at concentrations of 0.065 μM to 4 μM, have potential therapeutic value for a variety of diseases with abnormally high ACLY expression, such as cancer, hyperlipidemia, and atherosclerotic cardiovascular diseases. Attached Figure Description

[0057] Figure 1The inhibitory activity of compound (12a) against ACLY

[0058] Figure 2 The inhibitory activity of compound (24b) against ACLY Detailed Implementation

[0059] The present invention will now be further illustrated with examples. These examples are merely illustrative and not intended to limit the scope of the invention.

[0060] The main instruments, reagents, and consumables used in the experiment were ACLY and ADP-Glo. TM The Kinase Assay kit was purchased from Promega, Inc. (USA); the EnSight multi-plate reader and CulturPlate 384-well plates were purchased from PerkinElmer, Inc. (USA); and tris(hydroxymethyl)aminomethane, dithiothreitol, citric acid, coenzyme A, and dimethyl sulfoxide were purchased from Shanghai Yuanye Biotechnology Co., Ltd. All starting materials used in the preparation of the compounds of this invention are commercially available or can be prepared using existing methods.

[0061] Example 1: Synthetic route of the 3-substituted coumarin derivative of the present invention

[0062] (1) Synthesis of compounds 1a and 2a

[0063]

[0064] (a) CH3CO2K, (CH3CO)2O, 125℃, overnight. (b) methanol-HCl, 70℃, 3h.

[0065] 0.1 mol equivalents of trihydroxybenzaldehyde were dissolved in 10 mL of acetic anhydride, resulting in a concentration of 15 mg / mL. 0.1 mol equivalents of a phenylacetic acid derivative (structural formula R1C8H8O2) and 0.2 mol equivalents of potassium acetate were added, and the mixture was stirred and refluxed overnight. After TLC monitoring, the reaction was quenched and neutralized with saturated sodium bicarbonate solution. The mixture was extracted three times with ethyl acetate. The organic layer was washed with water and saturated brine, dried over anhydrous magnesium sulfate, and then rotary evaporated. The rotary evaporated product was purified by silica gel column chromatography, with the eluent being ethyl acetate-petroleum ether. The obtained intermediate product was hydrolyzed under acidic heating conditions (volume ratio, methanol:concentrated hydrochloric acid = 8:1, total volume 18 mL) for 3 h. After hydrolysis, water was added, followed by rotary evaporation and extraction with ethyl acetate. The organic layer was washed, dried, and rotary evaporated to obtain the final product. If the product was impure at this stage, it was further purified by silica gel column chromatography to obtain the pure product.

[0066] (2) Synthesis of compound 3a-13a

[0067]

[0068] (a)80℃,ethanol,piperidine,overnight

[0069] Dissolve 0.1 mol equivalents of tris(hydroxymethyl)benzaldehyde in 20 mL of ethanol to a concentration of 7.5 mg / mL. Add 0.12 mol equivalents of an active methylene ketoester derivative (general structural formula RCOCH2OCOCH2CH3) and 0.12 mol equivalents of piperidine, and stir under reflux overnight. After the reaction is complete, quench with dilute hydrochloric acid. If product precipitates, filter and purify the filter cake by silica gel column chromatography. If no product precipitates, extract with ethyl acetate and water by rotary evaporation. Wash and dry the organic layer, then purify by rotary evaporation and silica gel column chromatography to obtain the final product.

[0070] (3) Synthesis of compound 14a

[0071]

[0072] (a) NaBH4, 0℃, 30min.

[0073] 0.1 mol equivalents of 9a were dissolved in 20 mL of methanol to a concentration of 16 mg / mL. 0.3 mol equivalents of sodium borohydride were added, and the mixture was stirred and placed in an ice bath for 30 min. After the reaction was observed to be complete via TLC, water was added to quench the reaction, followed by rotary evaporation and extraction with ethyl acetate. The organic layer was washed, dried, and then purified by rotary evaporation and silica gel column chromatography to obtain the final product 14a.

[0074] The specific data for compounds 1a-14a are as follows:

[0075] (1) 7,8-Dihydroxy-3-phenyl-2H-chromen-2-one (1a): 30% yield as a yellow solid. Purity: 99%. LC-MS for C 15 H 10 O4: calculated, [M+H] + 255.06, found, 255.01.

[0076] (2) 7,8-Dihydroxy-3-[4-(2-methylpropyl-2-yl)phenyl]-2H-chromen-2-one (2a): 90% yield as a yellow solid. Purity: 99%. LC-MS for C 19 H 18 O4: calculated, [M+H] + 311.12, found, 311.07.

[0077] (3) 3-Phenylacetyl-7,8-dihydroxy-2H-chromen-2-one (3a): 98% yield as a yellow solid. Purity: 95%. LC-MS for C 16 H 10 O5: calculated, [M+H] + 283.05, found, 283.00.

[0078] (4) 3-[(4-chlorophenyl)carbonyl]-7,8-dihydroxy-2H-chromen-2-one (4a): 97% yield as a yellow solid. Purity: 99%. LC-MS for C 16 H9ClO5: calculated, [M+H] + 317.01, found, 316.58.

[0079] (5) 3-[(4-fluorophenyl)carbonyl]-7,8-dihydroxy-2H-chromene-2-one (5a): 15% yield as a yellow solid. 1 HNMR (400MHz, DMSO) δ8.48 (s, 1H), 7.68 (m, 2H), 7.33 (m, 3H), 6.90 (d, J = 8.3Hz, 1H). Purity: 93%. LC-MS for C 16 H9FO5: calculated, [M+H] + 301.04, found, 301.07.

[0080] (6) 7,8-Dihydroxy-3-[(4-hydroxyphenyl)carbonyl]-2H-chromene-2-one (6a): 21% yield as a yellow solid. 1 H NMR (400MHz, DMSO) δ8.16 (s, 1H), 7.75 (m, 2H), 7.16 (d, J = 8.5Hz, 1H), 6.85 (overlap, 2H). Purity: 95%. LC-MS for C 16 H 10 O6: calculated, [M+H] + 299.05, found, 299.07.

[0081] (7) 3-[(2,4-dichlorophenyl)carbonyl]-7,8-dihydroxy-2H-chromene-2-one (7a): 20% yield as a gray solid. 1H NMR (400MHz, DMSO) δ8.62(s,1H),7.74(brs,1H),7.57(m,2H),7.36(m,1H),6.89(m,1H).Purity:95%.LC-MS for C 16 H8Cl2O5: calculated, [M+H] + 350.97, found, 350.80.

[0082] (8) 7,8-Dihydroxy-3-[(4-methylphenyl)carbonyl]-2H-chromene-2-one (8a): 72% yield as a yellow solid. 1 H NMR (400MHz, DMSO) δ8.05(s,1H),7.61(m,2H),7.25(m,2H),7.05(m,1H),6.46(m,1H),2.37(s,3H).Purity:99%.LC-MS for C 17 H 12 O5: calculated, [M+H] + 297.07, found, 297.22.

[0083] (9) 7,8-Dihydroxy-3-[(4-methoxyphenyl)carbonyl]-2H-chromen-2-one (9a): 99% yield as a yellow solid. Purity: 99%. LC-MS for C 17 H 12 O6: calculated, [M+H] + 313.06, found, 312.98.

[0084] (10) 3-(cyclopropylcarbonyl)-7,8-dihydroxy-2H-chromene-2-one (10a): 63% yield as a faint yellow solid. 1 H NMR (400MHz, DMSO) δ8.17(s,1H),7.05(d,J=8.5Hz,1H),6.37(d,J=8.6Hz,1H),3.33(M,1H),0.92~0.84(m,4H).Purity:99%.LC-MS for C 13 H 10 O5: calculated, [M+H] + 247.05, found, 247.05.

[0085] (11) 3-(cyclopentylcarbonyl)-7,8-dihydroxy-2H-chromene-2-one (11a): 25% yield as a faint yellow solid. 1 H NMR (400MHz, DMSO) δ8.53(s,1H),7.28(d,J=8.9Hz,1H),6.87(d,J=8.9Hz,1H)3.90(m,1H),1.83(m,2H),1.70(m,2H),1.56(m,4H).Purity:99%.LC-MS forC 16 H 16 O4: calculated, [M+H] + 275.08, found, 275.08.

[0086] (12) 3-(cyclohexylcarbonyl)-7,8-dihydroxy-2H-chromene-2-one (12a): 71% yield as a milkwhite solid. 1 H NMR(400MHz,DMSO)δ8.50(s,1H),7.28(d,J=8.9Hz,1H),6.87(d,J=8.9Hz,1H),3 .46(m,1H),1.82(m,2H),1.72(m,2H),1.36~1.15(m,5H).Purity:99%.LC-MSfor C 16 H 16 O5: calculated, [M+H] + 289.10, found, 289.11.

[0087] (13) 7,8-Dihydroxy-3-(thiophen-2-ylcarbonyl)-2H-chromene-2-one (13a): 58% yield as a gray solid. 1 HNMR(400MHz,DMSO)δ7.99(s,1H),7.87(dd,J=5.0,1.0Hz,1H),7.75(dd,J=3.8,1.0Hz,1H), 7.16(dd,J=5.0,3.8Hz,1H),6.97(d,J=8.7Hz,1H),6.23(d,J=8.7Hz,1H).Purity:99%.LC-MS for C 14 H8O5S: calculated, [M+H] + 289.01, found, 289.03.

[0088] (14) 7,8-Dihydroxy-3-[hydroxy(4-methoxyphenyl)methyl]-2H-chromen-2-one (14a): 19% yields a gray solid. Purity: 90%. LC-MS for C 17 H 14 O6: calculated, [M+H] + 315.08, found, 310.10.

[0089] Example 2: Determination of ACLY inhibitory activity of compound 12a

[0090] 1) Using compound 12a as the analyte, the stock solution concentration of compound 12a was 20 mM, and the solvent was dimethyl sulfoxide. 5 μL of the stock solution was added to 57.5 μL of aqueous buffer to obtain a 1.6 mM solution of compound 12a. The aqueous buffer solution consisted of: 40 mM tris(hydroxymethyl)aminomethane (Tris), 4 mM dithiothreitol, 0.01% lauryl alcohol polyoxyethylene ether, 10 mM MgCl2, 1% dimethyl sulfoxide, and pH 8.0.

[0091] 2) Dilute the ACLY stock solution (composition: 50 mM Tris, 400 mM NaCl, 10% glycerol) with aqueous buffer to a concentration of 30 nM ACLY. Place 2.5 μL of the diluted ACLY solution and 2.5 μL of the compound 12a solution into the same well of a 384-well plate. Gently tap the plate to mix the liquids thoroughly and incubate at room temperature for 15 min. Then add 5 μL of the substrate mixture to the well. The substrate mixture consists of 600 μM citrate, 150 μM adenosine triphosphate, 150 μM coenzyme A, and water as the solvent. Centrifuge at low speed (1200 rpm, 1 min, 20 °C) to ensure thorough mixing. The above preparation steps yield a sample solution with an initial concentration of 400 μM for compound 12a.

[0092] 3) The operation is the same as steps 1) and 2) above, except that the solution of compound 12a used is the same as the solution of compound 12a in step 1) which is serially diluted with aqueous buffer to obtain 8 concentrations of compound 12a: 400 μM, 80 μM, 16 μM, 3.2 μM, 0.64 μM, 0.13 μM, 0.03 μM and 0.01 μM.

[0093] 4) Incubate the 384-well plate at 37°C for 55 min, remove it, let it stand at room temperature for 5 min, and after it returns to room temperature, add ADP-Glo ​​to each well. TM10 μL of ADP-Glo ​​reagent from the Kinase Assay kit was added and reacted at room temperature for 40 min. Then, ADP-Glo ​​was added to each well. TM The kinase assay kit contains 20 μL of kinase detection reagent, which converts ADP to ATP and emits fluorescence over a period of 40 min. Fluorescence intensity is detected using a PE Ensight microplate reader. Data processing is performed using GraphPadPrism 8.0 to obtain the dose-response curve for compound 12a. Figure 1 The x-axis represents the logarithm of the molar concentration of compound 12a, and the y-axis represents the fluorescence intensity measured by the instrument. The inhibitory activity of compound 12a targeting ACLY is IC50. 50 =4.01±0.28μM, indicating that compound 12a is an inhibitor of ACLY.

[0094] The inhibitory activity against ACLY was determined using compounds 1a-14a as analytes, with the same procedures and conditions as in Example 2. The results are shown in Table 1.

[0095] Example 3: Synthetic route of the symmetrical dicoumarin derivative 1b-25b of the present invention

[0096]

[0097] (a)dichloroethane,MOMCl,DIPEA,0℃,RT.(b)methylbenzene,CO(OEt)2,NaH,80℃,2h.(c)ethanol,piperidine,

[0098] 30℃,overnight.(d)methanol,HCl,60℃,1h

[0099] Protection of the phenolic hydroxyl group: 0.1 mol equivalents of 2,3,4-trihydroxyacetophenone were dissolved in 20 mL of dichloroethane (mass concentration 8.4 mg / mL). 0.22 mol equivalents of diisopropylethylamine were added, and the mixture was pre-cooled in an ice bath. Then, 0.22 mol equivalents of chloromethyl methyl ether were slowly added dropwise, and the mixture was stirred at room temperature for 3 h. After the reaction was complete (monitored by TLC), water was added to quench the reaction, followed by extraction with dichloroethane. The organic layer was washed, dried, and rotary evaporated. The mixture was then purified by silica gel column chromatography. The eluent was dichloromethane-petroleum ether, yielding the protected intermediate.

[0100] Ring-closure reaction: 0.1 mol equivalent of the intermediate from the previous step was dissolved in 20 mL of anhydrous toluene and sonicated to promote dissolution; 0.2 mol equivalent of diethyl carbonate was added, and the mixture was pre-cooled in an ice bath; then 0.4 mol equivalent of sodium hydroxide was slowly added, and the mixture was stirred for 2 h. After the reaction was completed by TLC monitoring, water was added to quench the reaction, followed by acidification with a 10% (w / w) citric acid aqueous solution, and the product gradually precipitated. The intermediate was obtained by filtration.

[0101] Synthesis of dicumarol: 0.1 mol equivalent of the intermediate obtained from the previous cyclization reaction was dissolved in 20 mL of anhydrous ethanol. 0.12 mol equivalent of an aldehyde derivative (general structural formula RCHO) and a catalytic amount of piperidine were added. The reaction was stirred overnight at 30 °C. The reaction progress was monitored by TLC. If crystals precipitated after the reaction was complete, the intermediate was directly filtered; if no product precipitated, the crystals were quenched with water, rotary evaporated, extracted with ethyl acetate, and the organic layer was washed, dried, and rotary evaporated to obtain a symmetrical dicumarol intermediate with a protecting group.

[0102] Deprotection: The intermediate product obtained from the synthesis of dicumarol in the previous step was deprotected under acidic conditions at a volume ratio of methanol:concentrated hydrochloric acid = 5:1, with a total volume of 12 mL, at 60 °C for 1 h. After the reaction was completed, if crystals precipitated, the final product was obtained by direct filtration; otherwise, water was added and rotary evaporated, followed by extraction with ethyl acetate, washing, drying, rotary evaporation, and purification by silica gel column chromatography. The eluent was methanol-dichloroethane, yielding the final product.

[0103] The specific data for compounds 1b-25b are as follows:

[0104] (1) 4,7,8-Trihydroxy-3-[(4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (1b): 66% yielded as a gray solid. 1 H NMR (400MHz, DMSO) δ7.30(d,J=8.7Hz,2H),7.25(m,2H),7.17(m,1H),7.11(m,2H),6.84(d,J=8.7Hz,2H),6.34(s,1H).Purity:99%.LC-MS for C 25 H 16 O 10 :calculated,[M+H] + 477.07, found, 477.39.

[0105] (2) 4,7,8-Trihydroxy-3-[(4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (2b): 7% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.27(d,J=8.7Hz,2H),6.89(m,2H),6.81(d,J=8.7Hz,2H),6.64(m,2H),6.20(s,1H). 13 C NMR (101MHz, DMSO) δ165.50,165.31,155.27,149.26,142.55,131.83,127.52,114.94,113.98,112.16,110.00,101.62,34.64.Purity:98%.LC-MS for C 25 H 16 O 11 :calculated,[M+H] + 493.07, found, 493.39.

[0106] (3) 3-[(4-fluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (3b): 15% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.26 (d, J = 8.7Hz, 2H), 7.13 (m, 2H), 7.05 (m, 2H), 6.80 (d, J = 8.7Hz, 2H), 6.29 (br s, 2H). 13 C NMR (101MHz, DMSO) δ165.61,165.42,161.68,159.28,149.36,142.62,131.85,128.47 ,128.39,114.82,114.61,114.05,112.16,110.02,101.26,34.91.Purity:98%.LC-MS forC 25 H 15 FO 10 :calculated,[M+H] + 495.06, found, 495.38.

[0107] (4) 4-[bis(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]benzene-1-carboxylonitrile (4b): 20% yields a white solid.1 H NMR (400MHz, DMSO) δ7.69 (m, 2H), 7.33 (m, 2H), 7.27 (d, J = 8.7Hz, 2H), 6.80 (d, J = 8.7Hz, 2H), 6.37 (br s, 1H). 113 C NMR(101MHz,DMSO)δ165.97,165.27,149.44,147.00,142.72,132.05,131.90,12 7.85,119.13,114.13,112.16,110.09,108.34,100.63,36.09.Purity:98%.LC-MS for C 26 H 15 NO 10 :calculated,[M+H] + 502.07, found, 502.40.

[0108] (5) 4,7,8-Trihydroxy-3-[(4-methoxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (5b): 23% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.27 (d, J = 8.7Hz, 2H), 7.01 (m, 2H), 6.81 (ovelap, 4H), 6.25 (br s, 1H). 13 C NMR(101MHz,DMSO)δ165.52,165.17,157.36,149.40,142.55,131.88,127.6 2,113.99,113.57,112.25,109.79,101.54,54.98,34.65.Purity:99%.LC-MS for C 26 H 18 O 11 :calculated,[M+H] + 507.08, found, 507.42.

[0109] (6) 4,7,8-Trihydroxy-3-[(4-methylphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (6b): 35% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.30~6.83 (overlap, 8H), 6.30 (s, 1H), 2.26 (s, 3H).13 C NMR(101MHz,DMSO)δ166.07,165.49,149.98,143.01,136.61,135.12,132.40,12 9.28,126.93,114.47,112.82,110.05,101.93,35.45,20.97.Purity:92%.LC-MS forC 26 H 18 O 10 :calculated,[M+H] + 491.09, found, 491.42.

[0110] (7) 3-[(3-chloro-4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (7b): 13% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.13(d,J=8.4Hz,2H),6.91(br s,1H),6.82(d,J=8.4Hz,1H),6.75(d,J=8.4Hz,1H),6.67(d,J=8.4Hz,2H),6.09(s,1H). 13 C NMR (101MHz, DMSO) δ168.69,164.99,150.11,148.08,142.93,135.23,131.34,127.66 ,126.38,118.69,116.05,114.17,112.91,111.01,100.62,35.15.Purity:90%.LC-MS for C 25 H 15 ClO 11 :calculated,[M+H] + 527.03, found, 527.83.

[0111] (8) 3-[(3-bromo-4-hydroxyphenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (8b): 2% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.26 (d, J = 8.5 Hz, 2H), 7.11 (br s, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.81, 6.80 (overlap, 3H), 6.20 (ds, 1H).13 C NMR(101MHz,DMSO)δ165.78,165.36,151.90,149.31,142.65,131.85,130.56,12 7.12,116.16,114.09,112.13,110.22,108.97,101.17,34.53.Purity:90%.LC-MS forC 25 H 15 BrO 11 :calculated,[M+H] + 570.98,found,570.98.

[0112] (9) 3-[(3-bromo-4-fluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (9b): 9% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.38~7.17 (overlap, 5H), 6.81 (d, J = 8.8Hz, 2H), 6.29 (s, 1H). 13 C NMR (101MHz, DMSO) δ165.71,165.27,157.74,155.33,149.49,142.70,138.59,131.93,131.25,12 8.14,116.38,116.17,114.11,112.22,109.99,107.67,107.46,100.93,34.96.Purity:92%.LC-MS forC 25 H 14 BrFO 10 :calculated,[M+H] + 572.98,found,572.98.

[0113] (10) 3-[(2,4-difluorophenyl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (10b): 4% yield as a white solid. Purity: 85%. LC-MS for C 25 H 14 F2O 10 :calculated,[M+H] + 513.06, found, 513.37.

[0114] (11) 4,7,8-Trihydroxy-3-[2-phenyl-1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)ethyl]-2H-chromene-2-one (11b): 5% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.18 (d, J = 8.7Hz, 2H), 7.13 ~ 7.06 (m, 4H), 6.99 (m, 1H), 6.63 (d, J = 8.7Hz, 2H), 5.15 (t, J = 8.3Hz, 2H), 3.40 (overlap, 2H). 13 C NMR(101MHz,DMSO)δ168.37,164.51,163.05,147.67,142.73,141.85,131.07,128.5 5,127.53,125.17,114.00,113.11,110.77,101.17,35.86,33.25.Purity:98%.LC-MS forC 26 H 18 O 10 :calculated,[M+H] + 491.09, found, 491.42.

[0115] (12) 4,7,8-Trihydroxy-3-[(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (12b): 40% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.50 (d, J = 8.9 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H). 13 C NMR (101MHz, DMSO) δ166.83,163.50,150.55,143.31,132.19,114.86,112.36,108.30,91.12,22.44.Purity:99%.LC-MS for C 19 H 12 O 10 :calculated,[M+H] + 401.04, found, 401.30.

[0116] (13) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxomylidene-3-yl)ethyl]-2H-chromene-2-one (13b): 5% yield as a white solid. Purity: 96%. LC-MS for C 20 H 14 O 10 :calculated,[M+H] + 415.06, found, 415.32.

[0117] (14) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)pentyl]-2H-chromene-2-one (14b): 5% yield as a white solid. Purity: 85%. LC-MS for C 23 H 20 O 10 :calculated,[M+H] + 457.11, found, 457.40.

[0118] (15) 4,7,8-Trihydroxy-3-[1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)hexyl]-2H-chromene-2-one (15b): 8% yielded as a white solid. 1 H NMR(400MHz,DMSO)δ7.31(d,J=8.7Hz,2H),6.85(d,J=8.7Hz,2H),4.80(t,J=8.2Hz,1H), 2.10(m,2H),1.21(ovelap,2H),1.13(ovelap,2H),0.82(ovelap,2H),0.76(ovelap,3H). 13 C NMR(101MHz,DMSO)δ166.71,164.73,149.99,142.73,132.42,114.24,113.05, 109.72,103.00,31.61,31.53,29.55,27.90,22.41,14.28.Purity:90%.LC-MS forC 24 H 22 O 10 :calculated,[M+H] + 471.12, found, 471.43.

[0119] (16) 3-[5-chloro-1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)pentyl]-4,7,8-trihydroxy-2H-chromene-2-one (16b): 17% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.16(d,J=8.4Hz,2H),6.65(d,J=8.4Hz,2H),4.77(t,J=8.2Hz,1H),3.54(d,J=6.7Hz,2H),2.01(m,2H),1.67(m,2H),1.23(m,2H). 13 C NMR (101MHz, DMSO) δ168.3,165.0,147.8,142.8,131.3,113.9,113.1,111.0,101.4,45.6,32.2,31.3,29.4,25.3.Purity:98%.LC-MS forC 23 H 19 ClO 10 :calculated,[M+H] + 491.07, found, 491.85.

[0120] (17) 4,7,8-Trihydroxy-3-[2-methyl-1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)propyl]-2H-chromene-2-one (17b): 4% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.14 (d, J = 8.6 Hz, 2H), 6.63 (d, J = 8.6 Hz, 2H) 4.38 (d, J = 11.0 Hz, 1H), 3.00 (m, 1H), 0.76 (d, J = 6.3 Hz, 6H). 13 CNMR(101MHz,DMSO)δ168.00,165.28,147.84,142.89,131.31,113.89,112.99,110.96,101.36,29.03,25.81,21.74.Purity:98%.LC-MS for C 22 H 18 O 10 :calculated,[M+H] + 443.09, found, 443.38.

[0121] (18) 3-[3,3-dimethyl-1-(4,7,8-trihydroxy-2-oxoylidene-3-yl)butyl]-4,7,8-trihydroxy-2H-chromene-2-one (18b): 74% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.15(d,J=8.2Hz,2H),6.72(d,J=8.2Hz,2H),4.86(t,J=6.3Hz,1H),2.01(d,J=6.3Hz,2H),0.80(s,9H).Purity:96%.LC-MS for C 24 H 22 O 10 :calculated,[M+H] + 471.12, found, 471.43.

[0122] (19) 3-[cyclopropyl(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (19b): 8% yield as a white solid. Purity: 97%. LC-MS for C 22 H 16 O 10 :calculated,[M+H] + 441.07, found, 441.36.

[0123] (20) 3-[cyclobutyl(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (20b): 8% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.13(d,J=8.6Hz,2H),6.63(d,J=8.6Hz,2H),4.80(d,J=11.8Hz,1H),3.64(m,1H),1.74(m,2H),1.71(m,2H),1.51(m,2H). 13 C NMR (101MHz, DMSO) δ168.16,165.47,147.85,142.87,131.31,113.88,113.00,110.97,99.79,38.63,34.38,27.10,16.86.Purity:90%.LC-MS for C 23 H 18 O 10 :calculated,[M+H]+ 455.09, found, 455.39.

[0124] (21) 3-[cyclohexyl(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-4,7,8-trihydroxy-2H-chromene-2-one (21b): 30% yield as a white solid. 1 H NMR (400MHz, DMSO) δ7.30 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.6 Hz, 2H) 4.51 (d, J = 11.8 Hz, 1H), 2.63 (m, 1H), 1.61 (m, 4H), 1.15 (m, 4H), 0.86 (m, 2H). 13 C NMR (101MHz, DMSO) δ170.34,142.29,131.91,113.83,101.47,37.62,34.77,31.25,25.97,25.71.Purity:98%.LC-MS for C 25 H 22 O 10 :calculated,[M+H] + 483.12, found, 483.44.

[0125] (22) 4,7,8-Trihydroxy-3-[(tetrahydrofuran-3-yl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (22b): 17% yielded as a white solid. 1 H NMR (400MHz, DMSO) δ7.31(d,J=8.6Hz,2H),6.82(d,J=8.6Hz,2H),4.72(d,J=1 1.5Hz,1H),3.78(m,1H),3.64(m,3H),3.25(m,1H),1.86(m,1H),1.47(m,1H). 13 C NMR (101MHz, DMSO) δ170.39,149.49,142.44,131.91,113.94,112.41,102.03,101.94,71.68,67.37,38.15,34.94,31.03.Purity:97%.LC-MS for C 23 H 18 O 11 :calculated,[M+H] + 471.08, found, 471.39.

[0126] (23) 4,7,8-Trihydroxy-3-[(pyridin-3-yl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (23b): 31% yield as a white solid. Purity: 90%. LC-MS for C 24 H 15 NO 10 :calculated,[M+H] + 478.07, found, 478.38.

[0127] (24) 4,7,8-Trihydroxy-3-[(pyridin-4-yl)(4,7,8-trihydroxy-2-oxomylidene-3-yl)methyl]-2H-chromene-2-one (24b): 16% yield as a white solid. 1 H NMR (400MHz, DMSO) δ8.68(m,2H),7.78(m,2H),7.21(d,J=8.6Hz,2H),6.76(d,J=8.6Hz,2H),6.39(br s,1H). 13 C NMR (101MHz, DMSO) δ167.60,165.01,164.57,149.18,143.03,140.63,131.86,125.45,114.31,111.81,111.17,99.20,37.75.Purity:99%.LC-MS for C 24 H 15 NO 10 :calculated,[M+H] + 478.07, found, 478.38.

[0128] (25) 4,7,8-Trihydroxy-3-[(pyridin-2-yl)(4,7,8-trihydroxy-2-oxylidene-3-yl)methyl]-2H-chromene-2-one (25b): 5% yield as a white solid. 1 H NMR (400MHz, DMSO) δ8.63 (dd, J=6.0, 1.6Hz, 1H), 8.45 (td, J=8.0, 1.6Hz, 1H), 7.86 (ovelap, 1H),7.83(ovelap,1H),7.18(d,J=8.6Hz,2H),6.74(d,J=8.6Hz,2H),6.42(d,J=1.0Hz,1H). 13C NMR(101MHz,DMSO)δ168.22,164.02,158.26,149.01,146.24,143.09,141.3 9,131.77,125.93,124.30,114.24,111.51,97.97,36.72.Purity:99%.LC-MS for C 24 H 15 NO 10 :calculated,[M+H] + 478.07, found, 478.38.

[0129] Example 4: Determination of ACLY inhibitory activity of compound 24b

[0130] 1) Using compound 24b as the analyte, the stock solution concentration of compound 24b was 20 mM, and the solvent was dimethyl sulfoxide. 1 μL of the stock solution was added to 61.5 μL of aqueous buffer to obtain a 0.32 mM solution of compound 24b. The aqueous buffer solution consisted of: 40 mM tris(hydroxymethyl)aminomethane (Tris), 4 mM dithiothreitol, 0.01% lauryl alcohol polyoxyethylene ether, 10 mM MgCl2, 1% dimethyl sulfoxide, and pH 8.0.

[0131] 2) Dilute the ACLY stock solution (composition: 50 mM Tris, 400 mM NaCl, 10% glycerol) to 30 nM ACLY with aqueous buffer. Place 2.5 μL of the diluted ACLY solution and 2.5 μL of the compound 24b solution into the same well of a 384-well plate. Gently tap the plate to mix the liquids thoroughly and incubate at room temperature for 15 min. Then add 5 μL of the substrate mixture to the well. The substrate mixture consists of 600 μM citrate, 150 μM adenosine triphosphate, 150 μM coenzyme A, and water as the solvent. Centrifuge at low speed (1200 rpm, 1 min, 20 °C) to ensure thorough mixing. The above preparation steps yield a sample solution with an initial concentration of 80 μM for compound 24b.

[0132] 3) The operation is the same as steps 1) and 2) above, except that the solution of compound 24b used is the same as the solution of compound 24b in step 1) which is serially diluted with aqueous buffer to obtain sample solutions of compound 24b at concentrations of 80 μM, 16 μM, 3.2 μM, 0.64 μM, 0.13 μM, 0.03 μM, 0.005 μM and 0.001 μM.

[0133] 4) Incubate the 384-well plate at 37°C for 55 min, remove it, let it stand at room temperature for 5 min, and after it returns to room temperature, add ADP-Glo ​​to each well. TM 10 μL of ADP-Glo ​​reagent from the Kinase Assay kit was added and reacted at room temperature for 40 min. Then, ADP-Glo ​​was added to each well. TM The kinase assay kit contains 20 μL of kinase detection reagent, which converts ADP to ATP and emits fluorescence over a period of 40 min. Fluorescence intensity is detected using a PE Ensight microplate reader. Data processing is performed using GraphPadPrism 8.0 to obtain the dose-response curve for compound 24b. Figure 2 The x-axis represents the logarithm of the molar concentration of compound 24b, and the y-axis represents the fluorescence intensity measured by the instrument. The inhibitory activity of compound 24b targeting ACLY is IC50. 50 =0.065±0.020μM, indicating that compound 24b is an inhibitor of ACLY.

[0134] The inhibitory activity against ACLY was determined using compounds 1b-25b as analytes, with the same procedures and conditions as in Example 3. The results are shown in Table 2.

[0135] Table 1. Inhibitory activity of compounds 1a-14a against ACLY

[0136]

[0137]

[0138] Table 2. Inhibitory activity of compounds 1b-25b against ACLY

[0139]

[0140]

Claims

1. A coumarin derivative, wherein the coumarin derivative has the following structural formula: ;in, R5 in general formula IV is 4-pyridyl.

2. A coumarin derivative according to claim 1, characterized in that: It also includes one or more of the pharmaceutically acceptable potassium salt, sodium salt, hydrochloride salt, phosphate salt, acetate salt, oxalate tartrate salt, or malonate salt of the coumarin derivative of claim 1.

3. An ACLY inhibitor, characterized in that: The active ingredient comprises the coumarin derivative as described in claim 1 or 2 above.

4. A medicine for the prevention and / or treatment of ACLY-related diseases, characterized in that: The active ingredient comprises the coumarin derivative as described in claim 1 or 2 above.

5. The drug according to claim 4, characterized in that: The relevant diseases include one or more of cancer, hyperlipidemia, and atherosclerotic cardiovascular and cerebrovascular diseases; the cancers include one or more of lung cancer, prostate cancer, and breast cancer.

6. The drug according to claim 4, characterized in that: It contains pharmaceutically acceptable excipients or carriers, and its dosage form may be tablets, pills, injections, capsules or granules.

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