A PNPO inhibitor with anti-multiple myeloma effect and its preparation method and application

Abstract

The present invention relates to a PNPO inhibitor with anti-multiple myeloma effect, a preparation method and application thereof, and specifically to a compound of formula (I) or its stereoisomers, geometric isomers, tautomers, pharmaceutically acceptable salts or prodrugs. The PNPO small molecule inhibitor can effectively inhibit the expression of PNPO in MM cells and secreted exosomes, and further inhibit the proliferation and osteoclast differentiation of MM cells by affecting the stimulation of related signal pathways, thereby exerting an anti-multiple myeloma effect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a novel PNPO inhibitor, a preparation method thereof and an application thereof in preventing and treating multiple myeloma. Background Art

[0002] Multiple myeloma (MM) is a hematological malignancy characterized by the expansion of clonal plasma cells in the bone marrow, ranging from monoclonal gammopathy of undetermined significance (MGUS) to overt plasma cell leukemia and extramedullary myeloma [Kazandjian D: Multiple myeloma epidemiology and survival: A unique malignancy. Semin Oncol 2016, 43: 676-681.]. MM often causes end-organ damage, including anemia, renal impairment, osteolytic lesions [Mehdi SH, Nafees S, Mehdi SJ, Morris CA, Mashouri L, Yoon D: Animal Models of Multiple Myeloma Bone Disease. Front Genet 2021, 12: 640954.] and hypercalcemia [Mehdi SH, Nafees S, Mehdi SJ, Morris CA, Mashouri L, Yoon D: Animal Models of Multiple Myeloma Bone Disease. Front Genet 2021, 12: 640954.]. Since 1990, the global incidence has increased by 126% [JCowan A, Allen C, Barac A, Basaleem H, Bensenor I, Curado MP, Foreman K, Gupta R, Harvey J, Hosgood HD, et al: Global Burden of Multiple Myeloma: A Systematic Analysis for the Global Burden of Disease Study 2016. Jama Oncol 2018, 4: 1221-1227.], and the incidence of MM in China has been increasing year by year in recent years [Liu J, Liu W, Mi L, Zeng X, Cai C, MaJ, Wang L: Incidence and mortality of multiple myeloma in China, 2006-2016: an analysis of the Global Burden of Disease Study 2016. J Hematol Oncol 2019, 12: 136.]. The development of drugs for the treatment of MM has evolved from traditional chemotherapy and hormone drugs to the introduction of proteasome inhibitors (such as bortezomib, ixazomib and carfilzomib), immunomodulators (such as thalidomide, lenalidomide and pomalidomide), monoclonal antibodies targeting myeloma cell surface antigens (such as daratumab, eloizumab and isatuximab) [Frampton JE: Isatuximab: A Review of Its Use in Multiple Myeloma. Target Oncol 2021, 16: 675-686.] and autologous hematopoietic stem cell transplantation [Frampton JE: Isatuximab: A Review of Its Use in Multiple Myeloma. Target Oncol 2021, 16: 675-686.]. In the past 2 years, several new classes of therapies have been approved, including nuclear export inhibitors (selinexor), peptide-conjugated alkylates (melphalanflufenamide), and antibody-drug conjugates (belantamab mafodotin-blmf) [Richardson PG, Oriol A, Larocca A, Bladé J, Cavo M, Rodriguez-Otero P, Leleu X, Nadeem O, Hiemenz JW, Hassoun H, et al: Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma. J Clin Oncol 2021, 39: 757-767. Trudel S, Lendvai N, Popat R, Voorhees PM, Reeves B, Libby EN, Richardson PG, Anderson LJ, Sutherland HJ, Yong K, et al: Targeting B-cell maturation antigen with GSK2857916antibody-drug conjugate inrelapsed or refractory multiple myeloma(BMA117159):a dose escalation and expansion pHase 1trial.Lancet Oncol 2018,19:1641-1653.Lonial S,Lee HC,BadrosA,Trudel S,Nooka AK,Chari A,Abdallah AO,Callander N,Lendvai N,Sborov D,et al:Belantamab mafodotin for relapsed or refractory multiple myeloma(DREAMM-2):atwo-arm,randomised,open-label,pHase 2study.Lancet Oncol 2020,21:207-221. Ramasamy K, Gay F, Weisel K, Zweegman S, Mateos MV, Richardson P: Improving outcomes for patients with relapsed multiple myeloma: Challenges and considerations of current and emerging treatment options. Blood Rev 2021,49:100808.]. Idecaribene vicleucel (ide-cel) is a novel chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) and has recently been approved by the US FDA for relapsed and refractory multiple myeloma (RRMM) [Anderson LJ: Idecaribene vicleucel (ide-cel) CAR T-cell therapy for relapsed and refractory multiple myeloma. Future Oncol 2022, 18: 277-289. George LL, Deshpande SR, Cortese MJ, Kendall EK, Chattaraj A, Shah Z, Zhao J, Anwer F: Emerging Targets and Cellular Therapy for Relapsed Refractory Multiple Myeloma: A Systematic Review. Clin Lymp Homa Myeloma Leuk 2021, 21: 741-751.There are relatively few drugs available for MM patients in my country, mainly small molecule chemotherapy drugs and glucocorticoid drugs, as well as a few immunomodulatory drugs and proteasome inhibitors. Among targeted drugs, only plerixafor was launched in China in 2018. Although several new drugs for the treatment of multiple myeloma (MM) have been added in the past decade and the results have improved, there are still problems of drug resistance and relapse. Moreover, MM shows stronger invasiveness after relapse, which seriously affects the efficacy of new drugs and is still incurable [Anderson LJ: Idecabtagene vicleucel (ide-cel) CAR T-cell therapy for relapsed and refractory multiple myeloma. Future Oncol 2022, 18: 277-289. George LL, Deshpande SR, Cortese MJ, Kendall EK, Chattaraj A, Shah Z, Zhao J, Anwer F: Emerging Targets and Cellular Therapy for Relapsed Refractory Multiple Myeloma: A Systematic Review. Clin Lymp Homa Myeloma Leuk 2021, 21: 741-751.]. Therefore, researching and developing more effective and safe drugs is of great significance for the clinical treatment of MM. . Summary of the invention

[0003] The present study found that pyridoxine 5'-phosphate oxidase (PNPO) is highly expressed in multiple myeloma cells and secreted exosomes, promoting MM cell proliferation and affecting the bone marrow microenvironment through exosomes to promote osteoclast differentiation. PNPO small molecule inhibitors can effectively inhibit the expression of PNPO in MM cells and secreted exosomes, and further inhibit MM cell proliferation and osteoclast differentiation by affecting the stimulation of related signal pathways.

[0004] The present invention discovers for the first time that PNPO can be used as a new target for the development of drugs for treating multiple myeloma (MM), and opens up new mechanisms and methods for targeted treatment of MM through multiple pathways of action.

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a new target for treating MM, that is, to provide a PNPO inhibitor with anti-multiple myeloma effect.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the PNPO inhibitor so that it is easy to operate.

[0007] Another technical problem to be solved by the present invention is to provide the use of the PNPO inhibitor, namely, its use in the preparation of anti-multiple myeloma drugs.

[0008] In order to solve the above problems, the present invention adopts the following technical solutions:

[0009] A PNPO inhibitor, wherein the inhibitor is a compound represented by formula (I):

[0010]

[0011] or a stereoisomer, geometric isomer, tautomer, pharmaceutically acceptable salt or prodrug thereof, wherein:

[0012] R1 represents a single substituent or multiple substituents on the benzene ring, and R1 is selected from any one or more of a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkoxy group having 1 to 6 carbon atoms, a trifluoromethyl group, a halogen group, a nitro group, a cyano group, a hydroxyl group, a carboxyl group, an ester group, an amino group, a tetrazole group, a sulfonylamino group, and an aromatic ring or a heterocyclic ring condensed with a benzene ring;

[0013] R2 is selected from C1-C4 alkyl, ester, hydroxyl, amino, -(CH2)n-OH, -(CH2)n-NH2, n=1-2, a five-membered aromatic heterocycle containing 1-2 nitrogen atoms;

[0014] Ar is selected from an aromatic ring or an aromatic heterocyclic ring such as a carboxyl-substituted phenyl, furyl or thienyl group.

[0015] Preferably, the compound is selected from the following Table 1 compounds:

[0016] Table 1 Specific compounds

[0017]

[0018]

[0019]

[0020] Another aspect of the present invention relates to a method for preparing a compound of formula (I), comprising the following steps:

[0021]

[0022] a. Using 2-bromo-6-nitrophenol as the starting material, a nucleophilic substitution reaction is carried out with a benzyl halide under alkaline conditions to generate a derivative (2-2) for later use;

[0023] b. Take the derivative (2-2) obtained in step a, add carboxyl-substituted aryl (aromatic heterocyclic) boronic acid and alkaline solution in a reaction solvent, and carry out Suzuki coupling reaction under the catalysis of palladium catalyst to obtain derivative (2-3), which is set aside;

[0024] c. The derivative (2-3) obtained in step b is used as a raw material, hydrogenated and debenzylated in an ethyl acetate solution under palladium-carbon catalysis, and then salified with a hydrochloric acid solution to obtain an intermediate (2-4), which is set aside;

[0025] d. The intermediate (2-4) obtained in step c is first subjected to diazotization and then to coupling reaction with an acid, sodium nitrite, a commercially available similar compound (2-5) and a solvent at a certain temperature to obtain an intermediate (2-6) for later use;

[0026] e. The intermediate (2-6) obtained in step d is reacted with a commercially available similar compound (2-7) and an organic solvent under certain temperature conditions to undergo a condensation ring reaction to obtain the target compound (I).

[0027] Further, the solvent used in step a includes esters, dichloromethane, tetrahydrofuran, toluene and acetonitrile, etc., preferably acetonitrile. The base used can be sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide, etc., preferably sodium carbonate or potassium carbonate. The molar ratio of 2-bromo-6-nitrophenol to halobenzyl and base is 1:1-1.5:1-1.5, the molar volume ratio to the solvent is 1 mol / 300mL-600mL, the reaction temperature is reflux, and the reaction time is 2-8 hours.

[0028] Further, in step b, the palladium catalyst used for Suzuki coupling can be palladium acetate, [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride, tetrakis(triphenylphosphine)palladium or other suitable catalysts; preferably tetrakis(triphenylphosphine)palladium. The solvent used includes a mixed solvent of toluene, tetrahydrofuran, ethylene glycol dimethyl ether, 1,4-dioxane and water, etc.; preferably a mixed solvent of 1,4-dioxane and water. The base used includes sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide and potassium hydroxide, etc., preferably sodium carbonate or potassium carbonate.

[0029] The molar ratio of the derivative (2-2) to the carboxyl-substituted aryl (aromatic heterocyclic) boronic acid, the catalyst and the base in step b is 1:1-1.5:0.02-0.2:2-5, the molar volume ratio of the derivative (2-2) to water and the organic solvent is 1 mol / 200mL-600mL, nitrogen protection, the reaction temperature is reflux, and the reaction time is 2-8 hours.

[0030] The carboxyl-substituted aryl (aromatic heterocyclic) boronic acid is preferably 3-carboxyphenylboronic acid.

[0031] Furthermore, in step c, the molar ratio of the derivative (2-3) to palladium carbon is 1:0.1-0.5, the molar ratio of (2-3) to solvent ethyl acetate is 1 mol / 200mL-600mL, hydrogen is used for catalysis, the reaction temperature is 40°C-60°C, and the reaction time is 20-40 hours.

[0032] Further, in the diazotization reaction in step d, the acid is at least one of hydrochloric acid and sulfuric acid, and more preferably the acid is hydrochloric acid; the molar ratio of the intermediate (2-4) in the diazotization reaction to the 20% aqueous solution of sodium nitrite is 1:1 to 1.5; more preferably, the molar ratio of the intermediate (2-4) in the diazotization reaction to the 20% aqueous solution of sodium nitrite is 1:1; the reaction temperature in the diazotization reaction is -10°C to 10°C, and more preferably, the reaction temperature in the diazotization reaction is 0°C to 5°C; the organic solvent in the coupling reaction is at least one of water / methanol and water / ethanol, and more preferably, the organic solvent in the coupling reaction is Water / ethanol, mass ratio is 1:1; the mass ratio of the intermediate (2-4) in the coupling reaction to the organic solvent is 1:40-80, more preferably, the mass ratio of the intermediate (2-4) in the coupling reaction to the organic solvent is 1:60; the molar ratio of the intermediate (2-4) in the coupling reaction to the commercially available similar compound (2-5) is 1:0.9-1.5; more preferably, the molar ratio of the intermediate (2-4) in the coupling reaction to the commercially available similar compound (2-5) is 1:1; the reaction temperature in the coupling reaction is 10°C-35°C, more preferably, the reaction temperature in the diazotization reaction is 20°C-30°C.

[0033] Furthermore, the organic solvent for the condensation ring-closing reaction in step e is at least one of toluene, ethanol, acetonitrile, acetic acid, and tetrahydrofuran, and the mass ratio of the intermediate (2-6) to the organic solvent is 1:6 to 10. The molar ratio of the intermediate (2-6) to the analogous compound (2-7) is 1:0.8 to 1.5, and the reaction temperature is 60°C to 130°C.

[0034] Another aspect of the present invention relates to the use of PNPO inhibitors in the preparation of drugs for preventing and treating multiple myeloma.

[0035] In one embodiment, the PNPO inhibitor (I) exhibits a certain acidity, and the PNPO inhibitor (I) can be reacted with an alkali metal, an alkaline earth metal hydroxide, an alkali metal carbonate or an alkaline earth metal carbonate, an alkali metal bicarbonate or an alkaline earth metal bicarbonate to form a salt.

[0036] Furthermore, the PNPO inhibitor (I) or its salt and a pharmaceutically acceptable carrier are prepared into a drug in the form of tablets, capsules, granules, sprays, injections, microcapsules, ointments or transdermal controlled-release patches.

[0037] In one embodiment, when the PNPO inhibitor (I) or its salt provided by the present invention is made into tablets, the PNPO inhibitor (I) salt and the carrier lactose or corn starch, and the lubricant magnesium stearate are added as needed, mixed evenly, and then compressed into tablets.

[0038] In one embodiment, when the PNPO inhibitor (I) or its salt provided by the present invention is made into capsules, the PNPO inhibitor (I) salt and the carrier lactose or corn starch are evenly mixed, granulated, and then filled into capsules to make capsules.

[0039] In one embodiment, when the PNPO inhibitor (I) or its salt provided by the present invention is made into granules, the composition and the diluent lactose or corn starch are evenly mixed, granulated, and dried to make granules.

[0040] In one embodiment, when the PNPO inhibitor (I) or its salt provided by the present invention is prepared into an injection, the PNPO inhibitor (I) salt is added with a solubilizer, stirred evenly, heated at 80°C for 30 minutes, filtered, the pH value is adjusted, filtered with a fritted glass funnel or other filter until clear, filled, and sterilized at 100 to 115°C for 30 minutes to prepare an injection.

[0041] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0042] 1. The series of PNPO inhibitors (I) provided by the present invention can effectively inhibit the proliferation of MM cells and solve the defect of traditional targeted drugs with large toxic and side effects.

[0043] 2. The preparation method of the series of PNPO inhibitors (I) provided by the present invention uses 2-bromo-6-nitrophenol as a raw material, and sequentially undergoes nucleophilic substitution, Suzuki coupling, palladium carbon reduction, diazotization followed by coupling, condensation and cyclization to obtain the target compound. The reaction conditions of each step are mild, the operability is strong, the production efficiency is high, the cost is low, and the finished product yield is high and the purity is high.

[0044] The beneficial technical effects of the present invention are detailed in the following test examples.

[0045] definition:

[0046] The term "halogen" or the prefix "halo" refers to F, Cl, Br or I.

[0047] "Alkoxy" refers to the group -OR, where R is a substituted or unsubstituted alkyl group.

[0048] As described in the present invention, the substituent is connected to the central ring by a bond to form a ring system (as shown in Formula I) and R1 represents a single substituent or multiple substituents on the benzene ring, which can be substituted at any substitutable position on the ring.

[0049] The term "pharmaceutically acceptable" as used herein refers to any substance that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered.

[0050] The term "prodrug" used in the present invention represents a compound that is converted into the compound shown in Formula I in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug compound of the present invention can be an ester. In the existing invention, the esters that can be used as prodrugs include phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound in the present invention contains a hydroxyl group, which can be acylated to obtain a compound in the form of a prodrug. Other prodrug forms include phosphate esters, such as these phosphate ester compounds that are obtained by phosphorylation of the hydroxyl group on the parent.

[0051] The term "prodrug" used in the present invention represents a compound that is converted into the compound shown in Formula I in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug compound of the present invention can be an ester. In the existing invention, the esters that can be used as prodrugs include phenyl esters, aliphatic (C1-24) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound in the present invention contains a hydroxyl group, which can be acylated to obtain a compound in the form of a prodrug. Other prodrug forms include phosphate esters, such as these phosphate ester compounds that are obtained by phosphorylation of the hydroxyl group on the parent.

[0052] The compounds of the present invention may contain asymmetric centers or chiral centers, and therefore exist in different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to, diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane polarized light. When describing optically active compounds, the prefix D, L or R, S is used to indicate the absolute configuration of the chiral center of the molecule. The prefix d, l or (+), (-) is used to name the sign of rotation of the plane polarized light of the compound, the prefix (-) or l means that the compound is left-handed, and the prefix (+) or d means that the compound is right-handed. The chemical structures of these stereoisomers are the same, but their stereostructures are different. Specific stereoisomers can be enantiomers, and mixtures of isomers are usually called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in no stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers, devoid of optical activity.

[0053] The pharmaceutically acceptable carrier used in the present invention refers to a non-toxic carrier, adjuvant or vehicle that does not destroy the pharmacological activity of the compound formulated together. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 1 is the general structural formula diagram of the compound of the present invention.

[0055] Figure 2 The synthetic reaction flow chart of the compound of the present invention is shown in FIG.

[0056] Figure 3 This is a graph showing the test results of the affinity test between the compound I-1 of the present invention and the PNPO protein.

[0057] Figure 4 This is a graph showing the experimental results of the effect of the positive control drug on MM cell proliferation.

[0058] Figure 5 This is a graph showing the test results of the effect of the compound I-1 of the present invention on the proliferation of MM cells.

[0059] Figure 6 This is a graph showing the test results of the effect of the compound I-2 of the present invention on the proliferation of MM cells.

[0060] Figure 7 This is a graph showing the test results of the effect of the compound I-3 of the present invention on the proliferation of MM cells.

[0061] Figure 8 This is a graph showing the test results of the effect of the compound I-4 of the present invention on the proliferation of MM cells.

[0062] Fig. 9 This is a graph showing the test results of the effect of the compound I-5 of the present invention on the proliferation of MM cells.

[0063] Fig.10 This is a graph showing the test results of the effect of the compound I-6 of the present invention on the proliferation of MM cells.

[0064] Fig.11 This is a graph showing the test results of the effect of the compound I-7 of the present invention on the proliferation of MM cells.

[0065] Fig.12 This is a graph showing the test results of the effect of the compound I-8 of the present invention on the proliferation of MM cells.

[0066] Fig.13 It is the H-NMR chart of compound I-1 of the present invention.

[0067] Fig.14 It is the H-NMR chart of compound I-2 of the present invention.

[0068] Fig.15This is the H-NMR chart of compound I-3 of the present invention.

[0069] Fig.16 It is the H-NMR chart of compound I-4 of the present invention.

[0070] Fig.17 This is the H-NMR chart of compound I-5 of the present invention.

[0071] Fig.18 This is the H-NMR chart of compound I-6 of the present invention.

[0072] Fig.19 It is the H-NMR chart of compound I-7 of the present invention.

[0073] Fig. 20 It is the H-NMR chart of compound I-8 of the present invention. DETAILED DESCRIPTION

[0074] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0075] In the present invention, the structure of the compound is determined by mass spectrometry (MS) and / or nuclear magnetic resonance (1HNMR) equipment. The H-NMR diagrams of compounds I-1 to I-8 are as follows: Figures 13 to 20 .

[0076] The compounds of the present invention can be prepared according to conventional methods in the art, and using suitable reagents, raw materials and purification methods known to those skilled in the art. The preparation method of the compounds of the present invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such a combination can be easily carried out by a technician in the field to which the present invention belongs.

[0077] Example 1 Synthesis of 2-benzyloxy-3-bromonitrobenzene (2-2)

[0078]

[0079] 2.18 g (10 mmol) of nitro-6-bromophenol, 1.88 g (11 mmol) of benzyl bromide and 1.52 g (11 mmol) of potassium carbonate were mixed in acetonitrile (30 ml) and refluxed for 6 hours until the reaction was complete. The mixture was cooled to room temperature, evaporated under reduced pressure to remove the solvent, and concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the eluent to obtain 2-benzyloxy-3-bromonitrobenzene (2-2) (3.03 g, 98.39%) as a white solid. 1H NMR(500MHz,DMSO)δ8.07(dd,J=8.1,1.4Hz,1H),8.00(dd,J=8.2,1.4Hz,1H), 7.49(d,J=7.3Hz,2H),7.46–7.39(m,3H),7.38(d,J=8.2Hz,1H),5.13(s,2H).

[0080] Example 2 Preparation of 3'-nitro-2'-benzyloxy-[1,1'-biphenyl]-3-carboxylic acid

[0081]

[0082] 2.93 g (9.50 mmol) of compound 2-2 obtained in Example 1, 1.74 g (10.45 mmol) of 3-carboxyphenylboronic acid, 2.01 g (19 mmol) of sodium carbonate, and 329.46 mg (0.28 mmol) of tetrakistriphenylphosphine palladium were mixed in 1,4-dioxane (50 ml) and water (10 ml), and the mixture was refluxed at 95° C. for 8 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, water (20 ml) was added, and 1 The pH was adjusted to acidic with 10 mol / L dilute hydrochloric acid, solid precipitated, extracted with ethyl acetate, evaporated under reduced pressure to remove the solvent, concentrated to dryness to obtain a concentrate, and the crude product was separated and purified by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-nitro-2'-benzyloxy-[1,1'-biphenyl]-3-carboxylic acid (2-3) (2.87 g, 86.65%) as a light yellow solid.

[0083] Example 3 Preparation of 3'-amino-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid

[0084]

[0085] 2.87 g (8.25 mmol) of compound 2-3 obtained in Example 2 was dissolved in ethyl acetate (100 ml), 430.5 mg of 15% palladium carbon was added, and hydrogen was passed through the mixture under pressure at 50°C overnight until the reaction was complete. The mixture was cooled to room temperature, filtered, concentrated, and a small amount of ethanol was added to dissolve the mixture. 4N HCl was added, stirred, and concentrated to dryness to obtain 3'-amino-2'-hydroxy-[1,1'-biphenyl]-3-carboxylic acid hydrochloride (2-4) (1.14 g, 52.2%) as a purple solid.

[0086] Example 4 Preparation of ethyl 2-[3'-(2'-hydroxy-3-carboxylic acid biphenyl)hydrazono]-3-oxobutanoate

[0087]

[0088] 1.14 g (4.3 mmol) of compound 2-4 obtained in Example 3 was dissolved in 100 mL of 1N hydrochloric acid, and a 20% aqueous solution of 296.7 mg (4.3 mmol) of sodium nitrite was added dropwise under ice bath. After the addition, the mixture was reacted under ice bath for 1 h. 559 mg (4.3 mmol) of ethyl acetoacetate was added under stirring, and the reaction was continued for 45 min. The pH was adjusted to 6-8 with triethylamine, 30 mL of ethanol and 30 mL of water were added, and the mixture was warmed to room temperature and the reaction was continued for 1 hour. The pH was adjusted to 2-3 with 1N HCl, and the mixture was extracted with ethyl acetate. The solvent was removed by evaporation under reduced pressure, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 6:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 2-[3'-(2'-hydroxy-3-carboxylic acid biphenyl) hydrazono]-3-oxobutanoic acid ethyl ester (2-6) (910.7 mg, 57.2%) as a brown solid. 1 H NMR (500MHz, DMSO) δ14.73(s,1H),12.63(s,1H),9.45(s,1H),8.10(s,1H),7.94(d,J=7.7Hz,1H),7.78(d,J=7 .6Hz,1H),7.66-7.54(m,2H),7.18-7.02(m,2H),4.28(td,J=14.0,6.8Hz,2H),2.43(s,3H),1.28-1.21(m,3H).

[0089] Example 5 Preparation of 3'-[2-[(2Z)-1-(4-cyano)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-1)

[0090]

[0091] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 75.5 mg (0.204 mmol) of 4-cyanophenylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid, and refluxed at 130°C with stirring for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(4-cyano)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-1) (41 mg, 46.65%) as an orange-yellow solid. 1 H NMR (500MHz, DMSO) δ13.59(s,1H),13.05(s,1H),9.77(s,1H),8.15–8.08(m,3H),7.96(d,J=7.8Hz,1H),7.88(d,J=8 .8Hz,2H),7.79(d,J=7.8Hz,1H),7.68(dd,J=7.8,1.6Hz,1H),7.61(t,J=7.7Hz,1H),7.18–7.10(m,2H),2.33(s,3H).

[0092] Example 6 3'-[2-[(2Z)-1-(4-fluoro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene

[0093] Preparation of [2-[4-[(2-hydroxy-3-biphenyl)-1-yl]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-2)

[0094]

[0095] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 75.6 mg (0.204 mmol) of 4-fluorophenylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid, and refluxed at 130°C with stirring for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(4-fluoro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-2) (31 mg, 35.85%) as an orange solid. 1H NMR (500MHz, DMSO) δ13.69(s,1H),13.04(s,1H),9.71(s,1H),8.13(s,1H),7.96(d,J=7.8Hz,1H),7.94–7.90(m,2H),7.79( d,J=7.8Hz,1H),7.70(dd,J=7.4,1.8Hz,1H),7.61(t,J=7.7Hz,1H),7.29(t,J=8.8Hz,2H),7.18–7.11(m,2H),2.32(s,3H).

[0096] Example 7 Preparation of 3'-[2-[(2Z)-1-(4-trifluoromethyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-3)

[0097]

[0098] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 63.8 mg (0.3 mmol) of 4-trifluoromethylphenylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid, and refluxed at 130°C with stirring for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(4-trifluoromethyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-3) (35 mg, 36.28%) as a yellow solid. 1 H NMR (500MHz, DMSO) δ13.67(s,1H),13.04(s,1H),9.77(s,1H),8.16(d,J=8.4Hz,2H),8.13(s,1H),7.96(d,J=7 .7Hz,1H),7.81(t,J=8.0Hz,3H),7.71(d,J=7.2Hz,1H),7.61(t,J=7.7Hz,1H),7.19–7.12(m,2H),2.35(s,3H).

[0099] Example 8 Preparation of 3'-[2-[(2Z)-1-(2-methyl-4-nitro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-4)

[0100]

[0101] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 61.1 mg (0.3 mmol) of 2-methyl-4-nitrophenylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid, and refluxed at 130°C with stirring for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(2-methyl-4-nitro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-4) (43 mg, 45.41%) as a yellow solid. 1 H NMR (500MHz, DMSO) δ13.59(s,1H),9.74(s,1H),8.27(s,1H),8.16(d,J=8.0Hz,1H),8.11(s,1H),7.95(d,J=7.5Hz,1H),7.78(d, J=7.5Hz,1H),7.72(d,J=6.2Hz,1H),7.65(d,J=8.8Hz,1H),7.60(t,J=7.7Hz,1H),7.19–7.11(m,2H),2.42(s,3H),2.33(s,3H).

[0102] Example 9 Preparation of 3'-[2-[(2Z)-1-(naphthalen-1-yl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-5)

[0103]

[0104] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 58.4 mg (0.3 mmol) of 1-naphthylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid, and refluxed at 130° C. with stirring for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(naphthalen-1-yl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-5) (40 mg, 43.06%) as a yellow solid. 1H NMR (500MHz, DMSO) δ13.68(s,1H),12.98(s,1H),9.64(s,1H),8.11(s,1H),8.06–8.01(m,2H),7.94(d,J=7.8Hz,1 H),7.88–7.84(m,1H),7.80–7.73(m,2H),7.60(ddd,J=10.0,7.6,4.0Hz,5H),7.16(d,J=4.4Hz,2H),2.36(s,3H).

[0105] Example 10 Preparation of 3'-[2-[(2Z)-1-(4-nitro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-6)

[0106]

[0107] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 56.9 mg (0.3 mmol) of p-nitrophenylhydrazine hydrochloride and dissolved in 5 mL of glacial acetic acid, and the mixture was refluxed and stirred at 130°C for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(4-nitro)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazine]-2'-hydroxy-3-biphenylcarboxylic acid (I-6) (39 mg, 42.44%) as a yellow solid. 1 H NMR (500MHz, DMSO) δ13.58(s,1H),13.00(s,1H),9.77(s,1H),8.29(d,J=9.1Hz,2H),8.17(d,J=9.1Hz,2H),8.13(s,1H),7.97(d,J=7 .8Hz,1H),7.79(d,J=7.7Hz,1H),7.66(d,J=7.8Hz,1H),7.61(t,J=7.7Hz,1H),7.18–7.14(m,1H),7.11(t,J=7.8Hz,1H),2.33(s,3H).

[0108] Example 11 Preparation of 3'-[2-[(2Z)-1-(4-chloro-3-hydroxy)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-7)

[0109]

[0110] 74.1 mg (0.2 mmol) of compound 2-6 obtained in Example 4 was added to 64.21 mg (0.3 mmol) of 2,4-dichlorophenylhydrazine hydrochloride, dissolved in 5 mL of glacial acetic acid solution, refluxed and stirred at 130°C for 6 hours under nitrogen protection. After the reaction was completed, it was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the concentrate was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent, and 1% acetic acid was added to prevent silica gel column adsorption to obtain 3'-{N'-[3-methyl-5-oxo-1-(2,4-dichlorophenyl)-1,5-dihydropyrazole-4-ylidene]hydrazine}-2'-hydroxybiphenyl-3-carboxylic acid (I-7) (80.13 mg, 82.90%), the product was an orange solid. 1H NMR (500MHz, DMSO-d6) δ (ppm): 13.56 (s, 1H), 12.99 (s, 1H), 9.69 (s, 1H), 8.11 (s, 1H), 7.95 (d, J = 7.8Hz, 1H), 7.8 6(s,1H),7.78(d,J=7.8Hz,1H),7.72(dd,J=7.2,2.4Hz,1H),7.62-7.58(m,3H),7.18-7.13(m,2H),2.31(s,3H).

[0111] Example 12 Preparation of 3'-[2-[(2Z)-1-(4-carboxyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-8)

[0112]

[0113] 74.1 mg (0.2 mmol) of the compound obtained in Example 4 was added to 45.64 mg (0.3 mmol) of 4-hydrazinobenzoic acid and dissolved in 5 mL of glacial acetic acid, and the mixture was refluxed and stirred at 130°C for 6 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, petroleum ether was added and evaporated under reduced pressure to remove the solvent, and the mixture was concentrated to dryness to obtain a concentrate. The crude product was separated and purified by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 8:1 as the eluent and 1% acetic acid to prevent silica gel column adsorption to obtain 3'-[2-[(2Z)-1-(4-carboxyl)-3-methyl-5-oxo-1,5-dihydro-4H-pyrazole-4-ylidene]hydrazino]-2'-hydroxy-3-biphenylcarboxylic acid (I-8) (31.40 mg, 34.90%) as a yellow solid. 1H NMR(500MHz,DMSO-d6)δ13.84–13.56(m,1H),12.91(s,2H),9.75(s,1H),8 .14(d,J=1.9Hz,1H),8.09(d,J=8.7Hz,2H),8.03(d,J=8.7Hz,2H),7.97(d, J=7.7Hz,1H),7.84–7.79(m,1H),7.73(dd,J=7.7,2.0Hz,1H),7.62(t,J=7 .7Hz, 1H), 7.19 (dd, J = 7.6, 2.1Hz, 1H), 7.16 (t, J = 7.7Hz, 1H), 2.36 (s, 3H).

[0114] Example 13 Experimental study on the affinity of the PNPO inhibitor (I) prepared by the present invention to the PNPO protein

[0115] The affinity test of the compound for PNPO was conducted using surface plasmon resonance technology (SPR), and the instrument was a Reichert4SPR molecular interaction instrument. First, the PNPO protein was fixed on the channel of the dextran sensor chip, and the adjacent channel was used as a reference for kinetic detection. The concentration gradient injection of the I-8 small molecule compound was set. After the program was completed, the SPR curve was fitted by the analysis software to calculate the Kd value of PNPO and the small molecule compound.

[0116] Results (see Figure 3 ) is as follows: The affinity Kd value of compound I-8 for PNPO is 1.88 μM, and it has a strong binding force with PNPO. The experimental results provide a basis for subsequent research on binding sites and compound structure-activity relationships. Therefore, the present invention provides a new mechanism for developing targeted treatment of MM.

[0117] Example 14 Effect of the PNPO inhibitor (I) of the present invention on MM cell proliferation

[0118] The wild-type cells of MM cell line ARP1 and PNPO OE cells of ARP1 were collected, proteins were extracted, and Western blot was used to detect the expression of PNPO protein in each cell line. We found that after PNPO cDNA was transfected into cells, the expression of PNPO protein was significantly higher than that of the control group, indicating that the MM cell line with high expression of PNPO was successfully constructed. The succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and its absorption value is measured at a wavelength of 570nm using an enzyme reader. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. The number of living cells can be judged based on the absorbance value (OD) measured. The larger the OD, the stronger the cell activity. Specific steps: 1) ARP1 wild-type, ARP1 overexpressing and ARP1 knockdown cell lines in the logarithmic growth phase were inoculated in a sterile 96-well plate, with 3 replicate wells in each group, and 6 concentration gradients of drugs were added, and incubated in an incubator at 37°C, 5% CO2 for 48 hours; 2) Freshly prepared MTT reagent was added to each well, and incubated in an incubator at 37°C, 5% CO2 for 4 hours; 3) Centrifuged in a centrifuge, and 150 μL DMSO was added to each well; 4) The OD of each well was measured at a wavelength of 570 nm using an ELISA reader, and the growth inhibition rate of the drug on myeloma cells was calculated according to the following formula: inhibition rate = [1-(experimental group OD-blank group OD) / (control group OD-blank group OD)]×100%; a curve was drawn, and IC50 was solved.

[0119] The results are as follows (see Tables 2-10 for details, and Figure 4-Figure 12 ): Bortezomib (BTZ) was used as a positive drug to test the effects of the above 8 compounds on MM cell proliferation. The above synthesized compounds have good anti-proliferative activity against MM cells, and can target and bind to PNPO to achieve the purpose of inhibiting MM cell proliferation, and have good development prospects in the treatment of multiple myeloma.

[0120] Table 2 BTZ inhibition rate of MM cells

[0121]

[0122] Table 3 Inhibitory effect of I-1 on MM cells

[0123]

[0124] Table 4 Inhibition rate of I-2 on MM cells

[0125]

[0126] Table 5 Inhibition rate of I-3 on MM cells

[0127]

[0128] Table 6 Inhibition rate of I-4 on MM cells

[0129]

[0130] Table 7 Inhibition rate of I-5 on MM cells

[0131]

[0132] Table 8 Inhibition rate of I-6 on MM cells

[0133]

[0134] Table 9 Inhibition rate of I-7 on MM cells

[0135]

[0136] Table 10 Inhibition rate of I-8 on MM cells

[0137]

[0138] And by comparing and analyzing the compounds I-1 to I-8 synthesized by the present invention, the IC50 value of compound I-8 on MM cells was 2.88 μm, showing the best activity.

[0139] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A PNPO inhibitor, characterized in that The compound is selected from I-1 to I-8: 。 2. Use of the PNPO inhibitor according to claim 1 in the preparation of a drug for treating multiple myeloma.

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