A compound, composition and application thereof for Mycobacterium tuberculosis DprE1 enzyme inhibitors

Abstract

The present invention discloses a compound with structural formula I. The compound of the present invention exhibits strong inhibitory activity against Mycobacterium tuberculosis, has good antituberculosis effects, and has a significantly reduced MIC value compared with isoniazid, a first-line clinical drug. It has weak inhibitory effects on the proliferation of human hepatoma cells HepG2 and Chang cells and has good safety. In addition, in the experiment of Mycobacterium tuberculosis infection overexpressing DprE1, the antibacterial ability of the compound decreased, further verifying that the compound exerts its antibacterial effect by inhibiting the DprE1 enzyme, and it is a novel DprE1 inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical synthesis, and particularly relates to a compound for inhibiting Mycobacterium tuberculosis DprE1 enzyme and its application. Background Art

[0002] Tuberculosis is a multi-system disease caused by Mycobacterium tuberculosis, which can affect many parts of the body, with pulmonary tuberculosis being the most common. In the Global Tuberculosis Report 2019 released by the WHO, tuberculosis remains one of the top ten diseases related to death. In 2018, approximately 10 million people had tuberculosis, and 1.2 million people died from it. Approximately one-third of the global population are carriers of Mycobacterium tuberculosis, but only symptomatic tuberculosis patients are infectious. The treatment cycle of tuberculosis is long, generally requiring 6 - 9 months. Currently, the first-line drugs clinically mainly include isoniazid, rifampicin, ethambutol, and pyrazinamide, and these drugs can be used alone or in combination. However, the emergence of multi-drug resistant Mycobacterium tuberculosis has made the efficacy of these drugs worse. Second-line drugs such as aminoglycosides (amikacin, streptomycin, capreomycin, kanamycin) or fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin) are used to treat tuberculosis resistant to first-line drugs. However, with the emergence of multi-drug resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB), and totally drug-resistant tuberculosis (TDR-TB), there is an urgent need to develop anti-Mycobacterium tuberculosis drugs with a completely new mechanism for the treatment of tuberculosis.

[0003] Decaprenylphosphoryl-β-D-ribose oxidase (DprE1) is a flavoenzyme located on the cell wall of Mycobacterium tuberculosis and is one of the important targets for tuberculosis treatment discovered in recent years. It catalyzes the conversion of decaprenylphosphoryl-β-D-ribose (DPR) to decaprenylphosphoryl-2-keto-β-D-dehydrofuranose (DPX). Then DPX is converted to decaprenylphosphoryl-β-D-arabinofuranose (DPA) by DprE2. And DPA is the precursor of arabinogalactan and lipoarabinomannan in the mycobacterial cell wall. Therefore, DprE1 is crucial for the growth and ultimately the survival of the mycobacterial cell wall. In addition, since there is no cell wall in the human body, developing DprE1 selective inhibitors has relatively high safety for the long-term medication of patients infected with multi-drug resistant Mycobacterium tuberculosis.

[0004] Currently, four DprE1 inhibitors have entered clinical trials and can be mainly classified into covalent inhibitors and non-covalent inhibitors. Among them, covalent inhibitors include BTZ-043 and its water-soluble optimized derivative Macozinone. They mainly exert their inhibitory effects by forming a covalent bond with Cys387 on the DprE1 enzyme after the nitro group on the benzothiazinone nucleus is reduced to a nitroso group. Due to the electrophilic nature of BTZ-043 and related covalent inhibitors, they can react with nucleobases (adenine and guanine) present in the body. This reaction may trigger carcinogenic / mutagenic effects and genotoxicity. Non-covalent inhibition mainly includes the azaindole compound TBA-7371 and the benzopiperidine compound OPC-167832, both of which exhibit excellent activity against different Mycobacterium tuberculosis strains (MIC = 0.2 - 3.1 μM). However, the compound TBA-7371 has problems such as a short plasma half-life and moderate inhibitory activity against human PDE6 in the body; the compound OPC-167832 has a drug resistance problem due to two gene mutations (rv0678 and rv3790). Therefore, there is still an urgent need to develop novel scaffold DprE1 inhibitors. Summary of the Invention

[0005] The present invention relates to a pharmaceutically active compound and its pharmaceutically acceptable salt, which can be used for treating infections caused by Mycobacterium tuberculosis, such as pulmonary tuberculosis.

[0006] The present invention provides a pyrazolopyrimidinone derivative having a structure shown in General Formula I:

[0007]

[0008] Or an isomer of the structure shown in Formula I or I' or its pharmaceutically acceptable salt;

[0009] Wherein:

[0010] L is a chemical bond, -C(=O)(CH2)m- or C(=O). When L is a chemical bond, the corresponding N atom is directly connected to R3; m is 0, 1, 2, 3; the descriptions of the remaining substituents are as follows.

[0011] The present invention provides a compound having the structural formula II or II', its isomer or its pharmaceutically acceptable salt:

[0012]

[0013] Wherein:

[0014] R1 and R2 are each independently selected from H, C 1-3 alkyl or cycloalkyl;

[0015] R3 is selected from C 1-6alkyl, substituted or unsubstituted alicyclic, aromatic, aliphatic heterocyclic or aromatic heterocyclic rings;

[0016] selected from wherein W, X, Y, Z are each independently selected from C, N

[0017] R4, R5, R6, R7, R8, R9 are each independently selected from H, halogen, amino, C 1-6 alkoxy, C 1-6 alkyl;

[0018] R 10 selected from C 1-6 alkyl, C 1-6 cycloalkyl, said C 1-6 alkyl, C 1-6 cycloalkyl may optionally be substituted by 1 or more C 1-6 cycloalkyl, halogen, oxygen (i.e. =O), cyano.

[0019] In some embodiments of the present invention, the compound has the structural formula II-1 or II-1', its isomers or its pharmaceutically acceptable salts:

[0020]

[0021] wherein:

[0022] R1 is CH3, CH2CH3, CF3, cyclopropyl

[0023] R3 is CH3, CH2CH3, CF3, cyclopropyl, cyclopentyl, cyclohexyl, benzyl, wherein W, Q, T are each independently selected from C, N, and when one of them is selected from N, the other two are both C;

[0024] Ra, Rb, Rc, Rd, Re are each independently selected from H, halogen, nitro, amino, C 1-6 alkoxy, C 1-6 alkyl, trifluoromethyl.

[0025] As an embodiment, it has the structure shown by the general formula III or III':

[0026]

[0027] R5, R6 are each independently selected from H, Cl, F, C 1-3 alkyl, C 1-3 alkoxy;

[0028] R b 、R c are each independently selected from H, C 1-3 alkyl, trifluoromethyl.

[0029] In some embodiments of the present invention, the compound is characterized in that the A ring is selected from:

[0030]

[0031] In some embodiments of the present invention, the compound is characterized in that R3 is selected from:

[0032] CH3, CH2CH3, CF3,

[0033] In some embodiments of the present invention, the compound is characterized in that the compound is:

[0034]

[0035] In some embodiments of the present invention, there is provided a pharmaceutical composition comprising the compound according to any one of the above, its isomers, its pharmaceutically acceptable salts, solvates or prodrugs.

[0036] In some embodiments of the present invention, the use of the compound according to any one of the above, its isomers, its pharmaceutically acceptable salts, solvates or prodrugs, for preparing an inhibitor or a drug for inhibiting DprE1.

[0037] In some embodiments of the present invention, the use of the compound according to any one of the above, its isomers, its pharmaceutically acceptable salts, solvates or prodrugs, the drug is used for treating tuberculosis or mycobacterial infection.

[0038] Definitions and Explanations

[0039] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without a special definition, but should be understood in its ordinary meaning.

[0040] The term "pharmaceutically acceptable salt" refers to an inorganic acid salt or an organic acid salt of the compound, and the inorganic acid salts are selected from hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, carbonate, bicarbonate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate; the organic acid salts are selected from formate, acetate, octanoate, isobutyrate, oxalate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, succinate, fumarate, maleate, lactate, malate, citrate, tartrate, mesylate, esylate, benzenesulfonate, salicylate, picrate, glutamate, ascorbate, camphorate, camphorsulfonate, etc.

[0041] The term "isomer" refers to the geometric isomers and stereoisomers that the compounds of the present invention can exist as, such as cis-trans isomers, enantiomers, diastereoisomers, and their racemic mixtures and other mixtures, all of which mixtures are within the scope of the present invention.

[0042] The term "cis-trans isomer" refers to the configuration existing in a molecule where the double bond or the single bond of the ring-forming carbon atoms cannot rotate freely.

[0043] The term "enantiomer" refers to stereoisomers that are mirror images of each other.

[0044] The term "diastereoisomer" refers to stereoisomers that have two or more chiral centers and are not mirror images of each other between molecules.

[0045] "Optionally" or "optionally" means that the subsequently described event or condition may but does not necessarily occur, and this description includes the situation where the described event or condition occurs and the situation where the described event or condition does not occur.

[0046] The term "substituted" means that any one or more hydrogen atoms on a specific atom are replaced by substituents, which may include deuterium and variants of hydrogen, as long as the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the type and number of substituents can be arbitrary based on what is chemically achievable.

[0047] When any variable (such as R) appears more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted by 0 - 2 Rs, then the group may optionally be substituted by at most two Rs, and each R has an independent option in each case. In addition, combinations of substituents and / or their variants are only permitted if such combinations result in stable compounds.

[0048] Unless otherwise specified, the term "alkyl" is used to denote a straight-chain or branched-chain saturated hydrocarbon group, which can be mono-substituted (such as -CH2F) or multi-substituted (such as -CF3), and can be monovalent (such as methyl), divalent (such as methylene), or polyvalent (such as methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (such as n-propyl and isopropyl), butyl (such as n-butyl, isobutyl, s-butyl, t-butyl), pentyl (such as n-pentyl, isopentyl, neopentyl), etc.

[0049] Unless otherwise specified, cycloalkyl includes any stable cyclic or polycyclic hydrocarbon group, any carbon atom of which is saturated, and which may be mono- or polysubstituted and may be monovalent, divalent or polyvalent. Examples of such cycloalkyl groups include, but are not limited to, cyclopropyl, cyclopentane, cyclohexane, etc.

[0050] Unless otherwise specified, the term "halogen" by itself or as part of another substituent refers to a fluorine (F), chlorine (Cl), bromine (Br) or iodine (I) atom.

[0051] Unless otherwise specified, the term "alkoxy" means an alkyl group linked to the remainder of the molecule through an oxygen atom, where the alkyl group has the meaning as described in the present invention. Unless otherwise specified, C 1-6 alkoxy includes C1, C2, C3, C4, C5 and C6 alkoxy. Examples of alkoxy include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentyloxy and s-pentyloxy. The alkoxy group may optionally be substituted by one or more substituents described in the present invention.

[0052] Unless otherwise specified, the term "amino" means -NH2, -NH(alkyl) or -N(alkyl)(alkyl).

[0053] The compounds of the present invention exhibit strong inhibitory activity against Mycobacterium tuberculosis and have good antituberculosis effects. Compared with isoniazid, a first-line clinical drug, the MIC value is significantly reduced. The ability to inhibit the proliferation of human hepatoma cells HepG2 and Chang cells is relatively weak, and it has good safety. In addition, in the research on the mechanism, the antibacterial ability of the compound against the overexpressed DprE1 model bacteria is significantly decreased, which further verifies that the compound exerts its antibacterial effect by inhibiting the DprE1 enzyme and is a novel DprE1 inhibitor. Description of the Drawings

[0054] Figure 1 To utilize the Biolayer Interferometry (BLI) technology to determine the binding ability of compound 1 (ZJ-13) to the DprE1 protein. Detailed Embodiments

[0055] The present invention will be described in detail below by way of examples, but this does not mean any adverse limitation to the present invention. The present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. It will be obvious to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0056]

[0057] Preparation Example 1. 3-Methyl-N-(3-methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)benzamide (Compound 1) (ZJ-13)

[0058]

[0059] Step 1. 2-Chloroquinazolin-4(3H)-one (1-2)

[0060] Dissolve 2,4-dichloroquinazoline (2 g, 10.1 mmol) in tetrahydrofuran (10 ml), add 1N aqueous sodium hydroxide solution (10 ml), stir at room temperature for 4 h. After the reaction is completed, adjust the pH to 5 with 1N hydrochloric acid, extract with dichloromethane (3×50 ml), wash the organic phase with saturated brine (3×30 ml), dry over anhydrous sodium sulfate, remove the solvent to obtain a white solid (1-2); ESI-MS: m / z = 181 [M+H] + .

[0061] Step 2. 2-Hydrazinylidene-2,3-dihydroquinazolin-4(1H)-one (1-3)

[0062] Dissolve intermediate 1-2 (2 g, 11.0 mmol) in ethanol (20 mL), add hydrazine hydrate (2.77 g, 56.8 mmol), reflux and stir for 4 h. After the reaction is completed, filter by suction to obtain a white solid (1-3); ESI-MS: m / z = 177 [M+H] + .

[0063] Step 3. 2-(5-Amino-3-methyl-1H-pyrazol-1-yl)quinazolin-4(3H)-one (1-4)

[0064] Dissolve intermediate 1-3 (2 g, 11.4 mmol) in ethanol (20 ml), add 3-oxobutyronitrile (1.7 g, 20.4 mmol), reflux and stir for 4 h. After the reaction is completed, filter by suction to obtain a white solid (1-4); ESI-MS: m / z = 242 [M+H] + .

[0065] Step 4. 3-Methyl-N-(3-methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)benzamide (Compound 1) (ZJ-13)

[0066] Dissolve intermediate 1-4 (300 mg, 1.2 mmol) in dichloromethane (10 ml), successively add triethylamine (125 mg, 1.24 mmol) and 3-methylbenzoyl chloride, stir at room temperature for 30 min. After the reaction is completed, purify by silica gel column chromatography using DCM:MeOH (25:1) as the eluent to obtain the crude product, and then purify using a semi-preparative liquid chromatograph to obtain a white solid; 1 H NMR (400 MHz, CDCl3): δ 12.58 (s, 1H), 8.26 (dd, J = 7.9, 1.5 Hz, 1H), 7.85 (dt, J = 9.2, 1.9 Hz, 2H), 7.76 (ddd, J = 8.5, 7.2, 1.6 Hz, 1H), 7.58–7.54 (m, 1H), 7.50–7.42 (m, 3H), 6.88 (s, 1H), 2.50 (s, 3H), 2.31 (s, 3H). 13 C NMR (101 MHz, DMSO-D6) δ 162.91, 152.70, 141.45, 139.12, 135.19, 133.88, 133.11, 129.54, 127.94, 127.04, 126.36, 124.92, 120.01, 98.38, 21.40, 14.38. Retention time: 21.495 minutes, purity: 94.73%. ESI-MS: m / z = 360 [M + H] + .HRMS (ESI) (m / z): calcd for C20H17N5O2 [M + H] + = 360.1455, found 360.1451.

[0067] Preparation Example 2. N-(1-(6-chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 2)

[0068]

[0069] Step 1. 6-chloroquinazoline-2,4(1H,3H)-dione (2-2)

[0070] Reflux 2-amino-5-chlorobenzoic acid (2 g, 11.65 mmol) and urea (7 g, 116.5 mmol) for 5 h, cool the temperature to 100 °C, add water and stir for 10 min, filter by suction. Dissolve the filter cake in 0.2 N NaOH solution, stir at 100 °C for 10 min, adjust the pH to 3 with hydrochloric acid, and filter by suction to obtain a white solid; ESI-MS: m / z = 197 [M + 1] + .

[0071] Step 2. 2,4,6-Trichloroquinazoline (2-3)

[0072] Dissolve intermediate 2-2 (2 g, 10.2 mmol) in phosphorus oxychloride (20 ml), add dropwise N,N-dimethylaniline (1.23 g, 10.2 mmol), stir at 110 °C for 4 h. After the reaction is completed, pour it into ice water, extract with dichloromethane (3×50 ml). Wash the organic phase with saturated brine (3×30 ml), dry over anhydrous sodium sulfate, remove the solvent to obtain a white solid; ESI-MS: m / z = 233 [M+1] + 。

[0073] Step 3. 2,6-Dichloroquinazolin-4(3H)-one (2-4)

[0074] The synthesis step refers to Step 1 of Example 1, and use compound 2-3 instead of compound 1-1 to prepare compound 2-4; ESI-MS: m / z = 215 [M+1] + 。

[0075] Step 4. 6-Chloro-2-hydrazinylidene-2,3-dihydroquinazolin-4(1H)-one (2-5)

[0076] The synthesis step refers to Step 2 of Example 1, and use compound 2-4 instead of compound 1-2 to prepare compound 2-5; ESI-MS: m / z = 211 [M+1] + 。

[0077] Step 5. 2-(5-Amino-3-methyl-1H-pyrazol-1-yl)-6-chloroquinazolin-4(3H)-one (2-6)

[0078] The synthesis step refers to Step 3 of Example 1, and use 2-5 instead of 1-3 to prepare compound 2-6; ESI-MS: m / z = 276 [M+1] + 。

[0079] Step 6. N-(1-(6-Chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide

[0080] The synthesis step refers to Step 4 of Example 1, and use 2-6 instead of 1-4 to prepare compound 2; 11H NMR (400 MHz, CF3COOD) δ 11.48 (d, J = 3.3 Hz, 3H), 9.73 (s, 1H), 9.38 (d, J = 8.8 Hz, 1H), 9.30–9.20 (m, 3H), 9.02 (d, J = 6.8 Hz, 1H), 8.96 (dt, J = 7.3, 3.6 Hz, 1H), 4.01 (d, J = 2.8 Hz, 3H), 3.93 (d, J = 2.3 Hz, 3H). 13 13C NMR (101 MHz, CF3COOD) δ 171.57, 170.55, 156.21, 150.49, 148.62, 145.24, 143.10, 140.59, 138.26, 137.89, 133.62, 132.00, 130.57, 128.69, 127.79, 127.23, 118.97, 22.35, 13.75. Retention time: 24.787 minutes, purity: 95.01%. ESI-MS: m / z = 394 [M+H] + .

[0081] Preparation Example 3. N-(1-(6-Fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 3)

[0082]

[0083] The synthesis procedure refers to Example 2, and Compound 3 was prepared by using 2-amino-5-fluorobenzoic acid instead of Compound 2-1; 1 1H NMR (400 MHz, CF3COOD) δ 11.50 (s, 3H), 9.49 (dd, J = 7.7, 2.7 Hz, 1H), 9.44 (dd, J = 9.2, 4.4 Hz, 1H), 9.37–9.26 (m, 3H), 9.10 (d, J = 7.6 Hz, 1H), 9.04 (t, J = 7.6 Hz, 1H), 4.10 (s, 3H), 4.00 (s, 3H). 1313C NMR (101 MHz, CF3COOD) δ 170.73, 170.18, 164.63, 154.51, 149.29, 146.09, 144.35, 141.99, 137.16, 132.52, 130.86, 129.46, 128.79, 128.71, 128.43, 128.17, 126.08, 111.95, 21.15, 12.38. Retention time: 10.648 minutes, purity: 92.18%. ESI-MS: m / z = 378 [M+H] + .

[0084] Preparation Example 4. N-(1-(6-Methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 4)

[0085]

[0086] The synthesis procedure refers to Example 2, and 2-amino-5-methoxybenzoic acid is used instead of Compound 2-1 to prepare Compound 4; 1 1H NMR (400 MHz, CF3COOD) δ 11.50 (d, 2H), 9.46–9.30 (m, 3H), 9.25 (d, J = 10.8 Hz, 2H), 9.09 (dd, J = 22.4, 7.8 Hz, 1H), 8.98 (d, J = 7.7 Hz, 1H), 8.89–8.79 (m, 1H), 5.55 (s, 3H), 4.01 (s, 3H), 3.88 (s, 3H). 13 13C NMR (101 MHz, CF3COOD) δ 175.62, 144.59, 143.73, 141.81, 140.66, 137.06, 131.87, 130.68, 129.77, 129.42, 128.58, 126.59, 126.02, 106.76, 105.99, 56.83, 20.77. ESI-MS: m / z = 390 [M+H] + .

[0087] Preparation Example 5 3-Methyl-N-(3-methyl-1-(6-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)benzamide (Compound 5)

[0088]

[0089] The synthesis procedure refers to Example 2, and 2-amino-5-methylbenzoic acid is used instead of Compound 2-1 to prepare Compound 5;1 HNMR (400 MHz, CF3COOD) δ 11.69 (s, 3H), 9.83 (s, 1H), 9.60 (d, J = 8.6 Hz, 1H), 9.51 (d, J = 8.6 Hz, 1H), 9.45–9.39 (m, 2H), 9.21 (d, J = 7.6 Hz, 1H), 9.14 (t, J = 7.6 Hz, 1H), 4.23 (s, 3H), 4.17 (s, 3H), 4.12 (s, 3H). 13 C NMR (101 MHz, CF3COOD) δ 171.18, 170.67, 157.01, 148.93, 144.24, 143.52, 142.66, 141.85, 141.70, 136.91, 132.63, 130.73, 129.55, 126.60, 126.16, 123.62, 115.61, 21.25, 21.14, 13.01. ESI-MS: m / z = 374 [M+H] + .

[0090] Preparation Example 6 N-(1-(7-Chloro-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 6)

[0091]

[0092] The synthesis procedure refers to Example 2, and Compound 6 was prepared by using 2-amino-4-chlorobenzoic acid instead of Compound 2-1; 1 HNMR (400 MHz, CF3COOD) δ 11.50 (s, 3H), 9.92 (d, J = 8.2 Hz, 1H), 9.67 (d, J = 7.7 Hz, 1H), 9.44 (d, J = 6.1 Hz, 2H), 9.30–9.18 (m, 2H), 9.10 (t, J = 8.0 Hz, 1H), 4.26 (s, 3H), 4.08 (s, 3H). 13 C NMR (101 MHz, CF3COOD) δ 172.60, 170.47, 153.91, 149.61, 147.54, 144.66, 141.64, 138.33, 137.20, 132.68, 132.59, 130.63, 130.23, 130.09, 127.17, 125.98, 118.47, 21.24, 12.40. Retention time: 22.824 minutes, purity: 95.48%. ESI-MS: m / z = 394 [M+H] + .

[0093] Preparation Example 7 N-(1-(7-Fluoro-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 7)

[0094]

[0095] The synthesis procedure refers to Example 2, and Compound 7 was prepared by using 2-amino-4-fluorobenzoic acid instead of Compound 2-1; 1 HNMR(400MHz,CF3COOD)δ11.50(s,3H),9.91(dd,J=9.0,5.6Hz,1H),9.34–9.28(m,2H),9.09(d,J=7.6Hz,1H),9.06–9.00(m,2H),8.98–8.91(m,1H),4.06(s,3H),4.00(s,3H). 13 C NMR(101MHz,CF3COOD)δ171.51,170.66,170.08,168.91,155.32,150.35,144.23,142.00,137.14,132.56,130.89,130.69,130.58,129.37,126.11,119.95,119.70,113.39,21.17,12.58.Retention time:22.945minutes,purity:94.43%.ESI-MS:m / z=378[M+H] + .

[0096] Preparation Example 8 N-(1-(7-Methoxy-4-oxo-3,4-dihydroquinazolin-2-yl)-3-methyl-1H-pyrazol-5-yl)-3-methylbenzamide (Compound 8)

[0097]

[0098] The synthesis procedure refers to Example 2, and Compound 8 was prepared by using 2-amino-3-chlorobenzoic acid instead of Compound 2-1; 1 HNMR(400MHz,CF3COOD)δ11.68(s,3H),9.96(d,J=9.0Hz,1H),9.42–9.35(m,2H),9.20(d,J=7.6Hz,1H),9.12(t,J=7.7Hz,1H),9.07–9.02(m,2H),5.70(s,3H),4.14(s,3H),4.10(s,3H). 1313C NMR (101 MHz, CF3COOD) δ 171.35, 171.03, 170.56, 159.11, 150.20, 147.06, 143.28, 141.91, 136.79, 133.01, 130.61, 129.76, 129.53, 126.09, 121.81, 114.06, 108.22, 102.93, 57.11, 21.11, 13.37. Retention time: 17.713 minutes, purity: 95.64%. ESI-MS: m / z = 390 [M+H] + .

[0099] Preparation Example 9 N-(3-Methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)acetamide (Compound 9)

[0100]

[0101] The synthesis procedure refers to Example 1, and Compound 9 was prepared by using acetyl chloride instead of 3-methylbenzoyl chloride; ESI-MS: m / z = 284 [M+H] + 。

[0102] Preparation Example 10 N-(3-Methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)-3-(trifluoromethyl)benzamide (Compound 10)

[0103]

[0104] The synthesis procedure refers to Example 1, and Compound 10 was prepared by using 3-(trifluoromethyl)benzoyl chloride instead of 3-methylbenzoyl chloride; 1 1H NMR (400 MHz, CF3COOD) δ 11.68 (s, 3H), 10.00 (dd, J = 8.2, 1.4 Hz, 1H), 9.89 (d, J = 1.8 Hz, 1H), 9.88–9.83 (m, 1H), 9.69 (ddd, J = 8.5, 7.2, 1.4 Hz, 1H), 9.62 (d, J = 7.9 Hz, 1H), 9.56 (d, J = 8.4 Hz, 1H), 9.44–9.34 (m, 2H), 4.14 (s, 3H). 1313C NMR (101 MHz, CF3COOD) δ 171.26, 168.45, 156.89, 149.29, 146.60, 143.24, 139.73, 134.19, 133.83, 132.50, 132.23, 131.68, 130.53, 127.63, 126.22, 125.76, 124.42, 123.52, 116.11, 12.96. Retention time: 21.826 minutes, purity: 96.34%. ESI-MS: m / z = 414 [M+H] + .

[0105] Preparation Example 11 4-Methyl-N-(3-methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)benzamide (Compound 11)

[0106]

[0107] The synthesis procedure refers to Example 1, and Compound 11 was prepared by using 4-methylbenzoyl chloride instead of 3-methylbenzoyl chloride; ESI-MS: m / z = 360 [M+H] + 。

[0108] Preparation Example 12 N-(3-Methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)nicotinamide (Compound 12)

[0109]

[0110] The synthesis procedure refers to Example 1, and Compound 12 was prepared by using nicotinoyl chloride instead of 3-methylbenzoyl chloride; 1 1H NMR (400 MHz, CF3COOD) δ 11.69 (s, 3H), 11.22 (d, J = 2.1 Hz, 1H), 10.84 (dd, J = 8.3, 1.8 Hz, 1H), 10.66 (d, J = 5.8 Hz, 1H), 10.00 (dd, J = 8.2, 1.6 Hz, 1H), 9.95 (ddd, J = 7.8, 5.9, 1.5 Hz, 1H), 9.74–9.66 (m, 1H), 9.54 (d, J = 8.5 Hz, 1H), 9.40–9.34 (m, 1H), 4.02 (d, J = 1.5 Hz, 3H). 13CNMR(101MHz,CF3COOD)δ173.75,161.37,160.06,150.08,148.16,145.77,143.35,141.85,141.37,140.53,134.52,130.69,129.84,128.30,120.66,115.37,13.70.Retention time:17.715minutes,purity:94.84%.ESI-MS:m / z=347[M+H] + .HRMS(ESI)(m / z):calcd forC18H14N6O2[M+H] + 347.1251,found347.1248.

[0111] Preparation Example 13 N-(3-Methyl-1-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-pyrazol-5-yl)-2-phenylacetamide (Compound 13)

[0112]

[0113] For the synthetic procedure, refer to Example 21 and prepare Compound 20 using 2-phenylacetic acid instead of 1-bromo-3-(trifluoromethyl)benzene; 1 HNMR(400MHz,CF3COOD)δ11.69(s,3H),9.92(d,J=8.2Hz,1H),9.72–9.65(m,1H),9.42–9.33(m,2H),9.15–9.01(m,5H),5.69(s,2H),4.01(s,3H). 13 C NMR(101MHz,CF3COOD)δ175.02,171.87,158.95,149.36,144.09,142.31,140.62,133.77,131.82,130.90,130.03,127.51,122.54,114.20,44.61,13.28.Retention time:18.683minutes,purity:94.65%.ESI-MS:m / z=360[M+H] + .HRMS(ESI)(m / z):calcd for C20H17N5O2[M+H] + 360.1455,found360.1450.

[0114] Preparation Example 14 2-(3-Methyl-5-(m-tolyl)-1H-pyrazol-1-yl)quinazolin-4(3H)-one (Compound 14)

[0115]

[0116] Dissolve intermediate 1-4 (100 mg, 0.41 mmol) in toluene, and successively add 1-bromo-3-methylbenzene (70 mg, 0.41 mmol), cesium carbonate (405 mg, 1.24 mmol), tris(dibenzylideneacetone)dipalladium(0) (38 mg, 0.04 mmol), and Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthene, 48 mg, 0.08 mmol); displace with nitrogen three times, reflux and stir for 12 h. After the reaction is completed, add water (50 ml) to the reaction solution, extract with dichloromethane (3 × 50 ml), wash the organic phase with saturated brine (3 × 30 ml), dry over anhydrous sodium sulfate, purify by silica gel column chromatography using DCM:MeOH (20:1) as the eluent to obtain the crude product, and purify using a semi-preparative liquid chromatograph to obtain compound 14; 1 H NMR (400 MHz, CF3CO2D) δ 11.69 (s, 3H), 9.99 (d, J = 7.8 Hz, 1H), 9.70–9.64 (m, 1H), 9.53 (d, J = 8.4 Hz, 1H), 9.36 (t, J = 7.7 Hz, 1H), 8.98 (s, 1H), 8.81 (ddd, J = 18.3, 15.5, 9.5 Hz, 2H), 4.09 (s, 3H), 3.99 (s, 3H). 13 C NMR (101 MHz, CF3CO2D) δ 170.46, 158.80, 155.94, 146.58, 146.22, 142.54, 139.63, 136.81, 131.05, 130.68, 129.39, 128.55, 127.86, 124.93, 117.88, 116.90, 20.86, 12.15. Retention time: 23.525 minutes, purity: 96.29%. ESI-MS: m / z = 332 [M + H] + . HRMS (ESI) (m / z): calcd for C19H17N5O [M + H] + 332.1506, found 332.1511.

[0117] Biological experiments

[0118] Example 15 Minimum inhibitory concentration test for Mycobacterium tuberculosis

[0119] Detection principle: In this study, a luminescent strain of Mycobacterium tuberculosis H37Ra expressing the luxCDABE gene was constructed, and rapid counting of bacteria was achieved by measuring the relative light units (RLU). Therefore, by detecting the RLU, the growth status of bacteria can be determined, and thus whether the compound has an inhibitory effect on bacterial growth can be evaluated.

[0120] Detection steps: Mycobacterium tuberculosis H37Ra was grown in 7H9 liquid medium supplemented with OADCZ enrichment broth, 0.2% glycerol, and 0.05% Tween 80 to aid in bacterial dispersion. The initial RLU value of the test bacteria was measured and diluted to 10 - 15 μL using the medium. 196 μL of the diluted bacterial suspension was added to a 1.5 mL centrifuge tube containing 4 μL of the test compound. The centrifuge tube was incubated at 37 °C, and the RLU values were measured on the 3rd and 7th days. Isoniazid was used as the positive control and dimethyl sulfoxide as the negative control. The minimum inhibitory concentration (MIC) based on RLU was defined as the minimum compound concentration that could reduce >90% of the RLU compared to the negative control.

[0121] Detection results: As shown in Table 1, the in vitro inhibitory activity (MIC) of most compounds against Mycobacterium tuberculosis H37Ra was less than 10 μM.

[0122] Table 1. In vitro inhibitory activity of the compounds of the present invention against Mycobacterium tuberculosis H37Ra strain. A indicates MIC < 1 μM; B indicates IC 50 1 - 10 μM; C indicates IC 50 10 - 100 μM.

[0123]

[0124]

[0125] As can be seen from Table 1, the compounds of the present invention have excellent in vitro inhibitory activity against Mycobacterium tuberculosis H37Ra strain.

[0126] Example 16 HepG2 cell proliferation inhibition experiment

[0127] Detection principle: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous (4,5 - dimethylthiazol - 2 - yl) - 2,5 - diphenyl - 2H - tetrazolium bromide (MTT) to water - insoluble blue - violet formazan crystals and deposit them in the cells, while dead cells do not have this function. Within a certain range of cell numbers, the amount of formazan crystals formed is proportional to the number of cells. Therefore, this method can be used to quantify the number of living cells and thus evaluate the cytotoxicity of the compound to HepG2.

[0128] Detection step: Inoculate HepG2 cells into a 96-well plate at a cell density of 3×10 4 cells / well and place them in a 5% CO2 cell incubator for 24 hours (37 °C). After the cells adhere to the wall, add the test compound with gradient dilution and continue to culture for 72 hours. Add 20 μL of MTT solution (5 mg / mL) to each well and incubate for 4 hours. Subsequently, add 100 μL of triple buffer to each well and continue to incubate for 24 hours. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance value of each well at 570 nm and convert it to the survival rate. The survival rate calculation formula is: Survival rate (%) = (experimental group - blank group) / (control group - blank group) × 100%, and use GraphPad Prism 8.0 software to analyze the data and fit the IC 50 value.

[0129] Detection results: As shown in Table 2, the in vitro inhibitory activities (IC 50 values) of the compounds against HepG2 cells are all greater than 100 μM.

[0130] Example 17 Chang cell proliferation inhibition experiment

[0131] Detection principle: Use the MTT method (see Example 16) to detect the proliferation inhibition of the test compound against Chang cells.

[0132] Detection step: Chang (Human liver cell) cells are cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum. Inoculate the cells into a 96-well plate at a density of 3000 cells / well and place them in a 5% CO2 incubator for 24 hours. After the cells adhere to the wall, treat the cells with the serially diluted test compound and incubate for 72 hours. Subsequently, add 20 μL of MTT solution (5 mg / mL) to each well and incubate for 4 hours. Then, add 100 μL of SDS-HCl-PBS triple buffer to each well and incubate overnight at 37 °C. Use a spectrophotometer to measure the absorbance at 570 nm with a reference wavelength of 650 nm. The survival rate calculation formula is: Survival rate (%) = (experimental group - blank group) / (control group - blank group) × 100%, and use GraphPad Prism 8.0 software to analyze the data and fit the IC 50 value.

[0133] Detection results: As shown in Table 2, the in vitro inhibitory activities (IC 50 values) of the compounds against Chang cells are all greater than 100 μM.

[0134] Table 2. In vitro inhibitory activities of the compounds of the present invention against HepG2 cells and Chang cells, A represents IC 50< 10 μM; B represents IC 50 10 - 100 μM; C represents IC 50 > 100 μM.

[0135]

[0136] As can be seen from the above table, the compounds of the present invention have relatively weak inhibitory effects on the proliferation of HepG2 cells and Chang cells. Combining with the results of Example 15, it is suggested that while the compounds of the present invention have good antibacterial activity against Mycobacterium tuberculosis H37Ra strain, they have relatively low toxicity to human cells and good safety.

[0137] Example 18 Minimum Inhibitory Concentration Test of Mycobacterium tuberculosis Model Bacteria with Overexpressed DprE1

[0138] Detection principle: In this study, a Mycobacterium tuberculosis H37Ra luminescent strain expressing the luxCDABE gene and overexpressing DprE1 was constructed, and rapid counting of bacteria was achieved by measuring the relative light unit (RLU). Therefore, by detecting the RLU, the growth of bacteria can be judged, and thus whether the compound has an inhibitory effect on bacterial growth can be evaluated.

[0139] Detection steps: Mycobacterium tuberculosis H37Ra expressing the luxCDABE gene and overexpressing DprE1 grows in 7H9 liquid medium, supplemented with OADCZ enrichment solution, 0.2% glycerol and 0.05% Tween 80 to help disperse the bacteria. The initial RLU value of the test bacteria is measured and diluted to 10 - 15 μL using the medium. 196 μL of the diluted bacterial suspension is added to a 1.5 mL centrifuge tube containing 4 μL of the test compound. The centrifuge tube is incubated at 37 °C, and the RLU value is measured on the 3rd and 7th days. Isoniazid is used as the positive control and dimethyl sulfoxide as the negative control. The minimum inhibitory concentration (Minimum inhibitory concentration, MIC) based on RLU is defined as the minimum compound concentration that can reduce > 90% RLU compared to the negative control.

[0140] Detection results: As shown in Table 3, compound 1 (ZJ - 13) has strong antibacterial ability against the normal H37Ra strain, but its antibacterial ability decreases sharply in the H37Ra strain with overexpressed DprE1, and the sensitivity decreases by about 128 - fold. However, the antibacterial activities of PBTZ169 and Rifampin do not change significantly, suggesting the specific targeting and dependence of compound 1 (ZJ - 13) on the DprE1 enzyme.

[0141] Table 3. In vitro inhibitory activities of the compounds of the present invention against Mycobacterium tuberculosis H37Ra strain and Mycobacterium tuberculosis H37Ra strain with overexpressed DprE1 enzyme

[0142]

[0143] Example 19 Experiment for Measuring the Binding Affinity between a Compound and a Protein by Biolayer Interferometry (BLI)

[0144] Detection principle: The DprE1 protein forms a biolayer at the end of the biosensor. When binding to a compound, it causes a change in the molecular weight at the end of the sensor, thereby leading to a change in the thickness of the biolayer. After light passes through the biolayer of the sensor, transmission and reflection occur to form interference light waves. The change in the thickness of the biolayer causes a relative displacement of the interference light waves. The interference light waves before and after biomolecular binding are detected by a spectrometer to form an interference spectrum, which is displayed as the real-time displacement (nm) of the interference pattern. Finally, the molecule to be detected is analyzed based on the changes in the pattern before and after molecular binding.

[0145] Detection steps: Use Octet RED96e (ForteBio) to measure the dose-dependent binding affinity of a compound to DprE1 by biolayer interferometry. After pretreatment with HBS-P buffer containing 0.01 M HEPES, 0.15 M NaCl, 0.05% v / v surfactant P20, and 1% bovine serum albumin (BSA), use the second-generation amide reaction (AR2G) biosensor probe (ForteBio) to immobilize the DprE1 protein with reactive amino groups. All analyses were performed using a standard protocol at 30 °C in a 96-well black plate, with a total volume of 200 μL per well. All data were analyzed using Octet Data Analysis software. The association rate and dissociation rate were fitted using a 1:1 binding kinetic model, and the equilibrium dissociation constant (Kd) value was calculated from the ratio of K off (dissociation rate constant) to K on (association rate constant).

[0146] Detection results: As Figure 1 shown, compounds 1 (ZJ-13) at different concentrations (2.058 μM - 166.7 μM) were passed over the immobilized DprE1 protein. The results of the interference spectrum showed that the binding mode between compound 1 and the DprE1 protein was fast binding and slow dissociation. After software calculation, the affinity between the two was 1.35 μM. Using the same experimental protocol, we also tested the binding ability of the positive compound PBTZ169 and the spectral antibacterial drug rifampicin to the DprE1 protein. The results are shown in Table 3. Compound 1 exhibited an affinity comparable to that of PBTZ169, and both were significantly superior to rifampicin, indicating that compound 1 has good targeting properties.

Claims

1. A pyrazolopyrimidinone derivative, characterized in that, It has the structure shown in General Formula I: or a pharmaceutically acceptable salt thereof; wherein: L is a chemical bond, -CO-, -CO-CH2-, and when L is a chemical bond, the corresponding N atom is directly connected to R3; R1 is methyl; R2 is H; R3 is: CH3, Ring A is selected from:

2. The compound according to claim 1, wherein: It has the structures shown in General Formulas II and II'; The substituents are defined in the same way as in claim 1.

3. The compound according to claim 1, wherein The compound is selected from:

4. A pharmaceutical composition, characterized in that, It includes the compound described in any one of Claims 1-3 and its pharmaceutically acceptable salt.

5. Use of the compound according to any one of claims 1-3 and its pharmaceutically acceptable salts, characterized in that, It is used for preparing an inhibitor or a drug that inhibits DprE1.

6. The use according to claim 5, characterized in that, The drug is a drug for treating tuberculosis or mycobacterial infection.

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