The invention relates to spiro [indoline-3, 3apos; -quinoline]-2, 2apos; design synthesis and application of-diketone piperidine carboxamide compound

Abstract

The invention discloses a preparation method and application of a spiro [indoline-3, 3 '-quinoline]-2, 2'-diketone piperidine formamide compound, and the structure of the compound is shown as a general formula 1: # imgabs0 # in the formula, R is various substituent groups such as halogen, methyl and the like. An enzyme inhibition activity experiment result shows that part of the compounds have a relatively strong inhibition effect on chitin synthetase and glucosamine-6-phosphate synthetase, and the compounds are double-target inhibitors of the chitin synthetase and the glucosamine-6-phosphate synthetase; meanwhile, the compound has a relatively strong inhibition effect on fungi such as candida albicans, aspergillus flavus, cryptococcus neoformans and aspergillus fumigatus. A drug resistance activity test result shows that part of the compounds have certain inhibitory activity on variant thalli such as fluconazole-resistant candida albicans, fluconazole-resistant cryptococcus neoformans and micafungin-resistant candida albicans, and have the potential of coping with the drug resistance problem. The compounds are simple and cheap in preparation raw materials and simple and convenient in synthesis method, and can be used as lead compounds for designing antifungal drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to spiro[indoline-3,3′-quinoline]-2,2′-dione derivatives, and specifically relates to the design and synthesis of spiro[indoline-3,3′-quinoline]-2,2′-diketopipectinamide compounds and their application in antimicrobial treatment. Background Art

[0002] Invasive fungal infections have become a global public health concern due to their high mortality and complex treatment, with an explosive growth rate particularly in immunocompromised populations. According to international epidemiological data, nearly 25% of the global population suffers from fungal infections of varying severity, resulting in 1.5 to 2 million deaths annually. Deep fungal infections account for a mortality rate of 30% to 90%. Pathogenic bacteria, primarily Candida, Aspergillus, and Cryptococcus, invade the bloodstream and organs, causing fatal infections such as candidemia and invasive aspergillosis, which have become common and critical clinical conditions. The rapid evolution of fungal resistance and the scarcity of available antifungal drugs present a significant challenge for clinical treatment. Notably, while superficial infections can be controlled with local therapies, deep fungal infections often involve the central nervous system (e.g., cryptococcal meningitis) or vital organs, making effective prognosis difficult to improve, even with potent drugs such as amphotericin B and echinocandins. This dire situation urgently requires the development of novel diagnostic and therapeutic strategies to address this global health threat.

[0003] The cell wall, a unique structure of fungi, and the biosynthesis of its core component, chitin, provide key targets for the development of highly selective antifungal drugs. Chitin synthase, a key enzyme regulating chitin synthesis, catalyzes the formation of β-1,4-glycosidic bonds from the substrate uridine diphosphate-N-acetylglucosamine. Because mammalian cells lack either CHS or a chitin synthesis pathway, inhibitors targeting this enzyme can precisely disrupt the integrity of the fungal cell wall, leading to osmotic imbalance and cell lysis while avoiding toxicity to human cells. This makes it an important target for the treatment of invasive fungal infections and the development of agricultural fungicides. Furthermore, glucosamine-6-phosphate synthase, the rate-limiting enzyme in hexosamine biosynthesis, indirectly influences chitin synthesis by regulating the supply of N-acetylglucosamine precursors. This enzymatic reaction marks the initiation and rate-limiting stages of chitin biosynthesis. The rapid turnover of human genes can mitigate the effects of its temporary inhibition, thus making glucosamine-6-phosphate synthase a promising antifungal target. It can be concluded that chitin synthase and glucosamine-6-phosphate synthase play an important role in the chitin biosynthesis pathway. Therefore, simultaneous inhibition of these two enzymes as targets can block the biosynthesis of chitin, thereby inhibiting the synthesis of fungal cell walls to achieve the purpose of sterilization.

[0004] 2-Indolinone structures exhibit diverse biodynamic activities; quinolinone derivatives, as N-heterocyclic carbonyl compounds, are widely found in pharmacologically active compounds; and spirocyclic structures are three-dimensional structures widely used in drug design. Based on the advantageous pharmacophore hybridization strategy, this study successfully constructed spiro[indoline-3,3′-quinoline]-2,2′-dione derivatives by assembling indolinone and quinolinone in three dimensions via spirocyclic rings. Furthermore, piperidinecarboxamides are widely used as linkers in the design of novel inhibitors of various enzymes, exhibiting promising biological activities. Research articles on this type of compounds can be found in: AkhaJa TN, Raval J P. 1,3-Dihydro-2H-indol-2-ones derivatives: design, synthesis, in vitro antibacterial, antifungal and antitubercular study[J]. European Journal of Medicinal Chemistry. 2011, 46(11): 5573-5579. Aly, AA; El-Sheref, EM; Mourad, A.-FE; Bakheet, MEM; S.4-Hydroxy-2-quinolones:syntheses,reactions and fused heterocycles[J].Molecular Diversity.2019,24(2),477-524.Dileep,KV;Sakai,N.;Ihara,K.;Kato-Murayama,M.;Nakata,A.;Ito,A.;Sivaraman,DM;Shin,JW;Yoshida,M.;Shirouzu,M.Piperidine-4-carboxamide as anew scaffold for designing secretory glutaminyl cyclase inhibitors[J].InternationalJournalof biological macromolecules.2021,170:415-23.None of these documents covers or includes the structure, synthesis method and use of the novel compounds involved in the present invention.

[0005] To research and develop novel antifungal inhibitors, the present invention designed and synthesized a class of spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide compounds. Their chitin synthase and glucosamine-6-phosphate synthase inhibitory activities, as well as their in vitro antifungal effects, were determined using polyoxin B and fluconazole as controls. Their antibacterial activity was also determined using chloramphenicol and levofloxacin as controls. To date, the novel compounds described in this invention have not been reported to demonstrate antimicrobial activity, but they may be used as novel antifungal agents. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a class of spiro[indoline-3,3′-quinoline]-2,2′-diketopipectinamide compounds; a second purpose of the present invention is to provide a method for preparing such compounds; and a third purpose of the present invention is to provide the use of the compounds in the preparation of anti-pathogenic microbial drugs.

[0007] To achieve the above-mentioned purpose, the present invention provides the following technical solution: The structure of the spiro[indoline-3,3′-quinoline]-2,2′-diketopipectinamide compound of the present invention is shown in Formula 1:

[0008]

[0009] Wherein R is various substituents such as halogen, methyl, etc. Specifically, the compound represented by general formula 1 is any one of the following compounds:

[0010]

[0011] The synthesis method of the above compound is shown in Scheme 1:

[0012]

[0013] Specifically, the above-mentioned Scheme 1 reaction conditions are as follows:

[0014] ab. 2-Nitrobenzyl bromide reacts with diethyl malonate to obtain compound 2, which is then reacted with 2,4-dinitrochlorobenzene to obtain compound 4; the solvent is tetrahydrofuran (dry), 2-nitrobenzyl bromide reacts with diethyl malonate and 2,

[0015] The molar ratio of 4-dinitrochlorobenzene is 1:1:1, and the reaction is carried out at room temperature for about 4 hours.

[0016] c. Compound 4 was subjected to nitro reduction to give compound 5. The solvent was ethanol-water solution (ethanol:water = 4:1), the molar ratio of compound 4 to reducing iron powder and ammonium chloride was 1:7.5:7.5, and the reaction was carried out at 80°C for about 5 hours.

[0017] df. 4-Piperidinecarboxylic acid was subjected to carboxyl protection (methanol), acid-amine condensation (various substituted acids), and ester hydrolysis to produce compounds 10a-10o. The solvents were methanol, dichloromethane, and a 1:1 solution of ethanol and water, respectively. The molar ratio of compound 7 to the various substituted acids was 1.1:1, and the reaction was allowed to proceed overnight at room temperature.

[0018] g. Compound 5 was condensed with compounds 10a-10o to produce compound 1. The solvent was N,N-dimethylformamide; the molar ratio of compound 5 to compounds 10a-10o was 1:1, and the reaction was carried out at room temperature overnight.

[0019] Anyone skilled in the art can prepare the corresponding compounds according to the above disclosed preparation methods.

[0020] The use of the above-mentioned spiro[indoline-3,3′-quinoline]-2,2′-diketopipectinamide compounds in the preparation of anti-pathogenic microbial drugs. The microorganisms are pathogenic bacteria or fungi, such as Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Proteus, Pseudomonas aeruginosa; Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus, etc. DETAILED DESCRIPTION

[0021] In order to deepen the understanding of the present invention, the present invention will be further described in detail with reference to the embodiments below. The embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention. However, some non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention are within the scope of protection of the present invention.

[0022] All reagents used were commercially purchased of analytical or chemical grade. Unless otherwise specified, no special treatment was performed. The reaction progress was monitored by TLC tracking, with color development at 254 nm under ultraviolet light, and partially with iodine and concentrated sulfuric acid. The melting point was determined using an X-4 micro melting point instrument. H-NMR and C-NMR spectra were determined using a Bruker Av-600 Fourier transform NMR spectrometer. The deuterated reagents used were CDCl3, DMSO, and the internal standard TMS. The chemical shift δ is in ppm, the coupling constant J is in Hz, and s, d, t, q, and m represent singlet, doublet, triplet, quartet, and multiplet, respectively. HRMS (ESI) was determined using Bruker impactⅡ.

[0023] Example 1: Diethyl 2-(2,4-dinitrobenzene)-2-(o-nitrobenzyl)malonate (4)

[0024] Diethyl malonate (3.20 g, 20 mmol) dissolved in 150 mL of dry tetrahydrofuran was added to the reaction flask. Sodium hydride (0.96 g, 40 mmol) was added to the solution in an ice-water bath. Then, o-nitrobenzyl bromide (4.32 g, 20 mmol) dissolved in 25 mL of dry tetrahydrofuran was added dropwise using a constant pressure dropping funnel. The mixture was stirred at room temperature. The reaction progress was monitored by TLC, and the reaction was complete after approximately 5 h. The reaction mixture was quenched by adding 50 mL of dilute hydrochloric acid (0.1 M). Most of the tetrahydrofuran solvent was removed by rotary evaporation. 50 mL of water was added, and the mixture was extracted with ethyl acetate (3 × 70 mL). The organic phases were combined and washed sequentially with water (3 × 50 mL) and saturated sodium chloride solution (3 × 50 mL). The mixture was separated, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation to obtain a yellow oily liquid, compound 2, in an 85% yield. Compound 2 (5.90 g, 20 mmol) dissolved in 150 mL of dry tetrahydrofuran was added to the reaction flask. Sodium hydride (0.96 g, 40 mmol) was added to the solution under ice-water bath conditions. 1-Chloro-2,4-dinitrobenzene (4.05 g, 20 mmol) dissolved in 25 mL of dry tetrahydrofuran was then added dropwise using a constant pressure dropping funnel. The mixture was stirred at room temperature. The reaction progress was monitored by TLC, and the reaction was complete after approximately 5 h. 50 mL of dilute hydrochloric acid (0.1 M) was added to the reaction solution to quench the reaction. Most of the tetrahydrofuran solvent was removed by rotary evaporation. 50 mL of water was added thereto, and the mixture was extracted with ethyl acetate (3×70 mL). The organic phases were combined and the obtained organic phases were washed with water (3×50 mL) and saturated sodium chloride solution (3×50 mL) in sequence. The mixture was separated and dried over anhydrous Na2SO4. The solvent was removed by rotary evaporation to obtain a yellow oily liquid, which was dissolved in 20 mL of dichloromethane in a reaction flask. 200 mL of methanol was added thereto and stirred at room temperature to precipitate a light yellow solid. Stirring was continued for about 2 h, and the solid was filtered to obtain a crude compound 4. The dried crude compound 4 was slurried in 50 mL of methanol and filtered to obtain a white solid, which was compound 4 in a yield of 87%. 1 HNMR(600MHz,Chloroform-d)δ8.78(s,1H),8.12(s,1H),7.67(d,J=13.2Hz,1H),7.34(s,2H ),7.20(s,1H),7.02(d,J=8.8Hz,1H),4.42(s,2H),4.29–4.12(m,4H),1.21(t,J=7.1Hz,6H).

[0025] Example 2: 6-amino-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-2,2′-dione (5)

[0026] Reduced iron powder (5.58 g, 75 mmol) and ammonium chloride (5.35 g, 75 mmol) were weighed and added to a reaction flask. A mixture of 72 mL of ethanol and 18 mL of water was added to the reaction flask. After stirring at reflux for 30 min at 82°C, compound 4 (4.62 g, 10 mmol) was added and stirring and refluxing were continued for 5 h. The reaction progress was monitored by TLC. After completion of the reaction, the iron powder was removed by hot filtration to obtain a filtrate. The solvent was removed by rotary evaporation and the filtrate was purified by column chromatography using dichloromethane / methanol (1 / 20-1 / 30, V / V) to obtain a pale yellow solid, compound 5, in a yield of 52%. 1H NMR(600MHz,DMSO-d6)δ10.49(s,1H),10.37(s,1H),7.26–7.17(m,2H),7.00–6.94(m,2H),6.15(d,J=1.7Hz,1H),6 .02(d,J=8.1Hz,1H),5.88(dd,J=8.1,1.8Hz,1H),5.18(s,2H),3.39(d,J=15.7Hz,1H),2.82(d,J=15.8Hz,1H).13C NMR(151MHz,DMSO-d6)δ177.06,168.52,150.24,143.37,138.11,129.05,128.14,123 .32,122.98,121.56,117.19,115.26,106.77,96.61,54.33,34.83.HRMS(ESI):calcd for C16H13N3O2[M+H]+,280.1081,found,280.1082.

[0027] Example 3: 1-Benzoylpiperidine-4-carboxylic acid (10a)

[0028] To the reaction flask, piperidine-4-carboxylic acid (6.46 g, 50 mmol) dissolved in 30 mL of methanol was added dropwise to the reaction mixture using a constant pressure dropping funnel. After the addition was complete, the mixture was stirred and refluxed at 80°C for 2 h. After the reaction, the solvent was removed by rotary evaporation to yield compound 7 as a white solid in 80% yield. 1H NMR (600 MHz, Chloroform-d) δ 3.68 (s, 3H), 3.09 (dt, J = 12.4, 3.2 Hz, 2H), 2.64 (td, J = 12.4, 2.5 Hz, 2H), 2.43 (tt, J = 11.3, 3.9 Hz, 1H), 1.91–1.86 (m, 2H), 1.62 (qd, J = 11.5, 3.9 Hz, 2H).

[0029] Benzoic acid (1.22 g, 10 mmol) dissolved in 40 mL of dichloromethane was added to the reaction flask, EDCI (2.30 g, 12 mmol) and DMAP (1.22 g, 10 mmol) were weighed and added to the reaction flask. After stirring at room temperature for 30 min, compound 7 (1.58 g, 11 mmol) was weighed and added to the reaction flask. Stirring was continued at room temperature overnight. The reaction process was monitored by TLC. After the reaction was completed, the product was washed with 1 M dilute hydrochloric acid (3×80 mL), saturated sodium chloride solution (3×80 mL), saturated NaHCO3 solution (3×80 mL), and saturated sodium chloride solution (3×80 mL) in sequence, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The product was purified by column chromatography on petroleum ether / ethyl acetate (1 / 10 to 1 / 15, V / V) to give compound 9a as a colorless oily liquid with a yield of 55%. 1H NMR(600MHz,Chloroform-d)δ7.40(dtdd,J=8.0,6.0,3.8,2.1Hz,5H),4.54(s,1H),4.12(q,J=7.1Hz,1H),3.7 1(d,J=1.5Hz,3H),3.05(s,2H),2.59(td,J=10.7,5.4Hz,1H),2.04(s,1H),1.75(s,3H),1.26(t,J=7.1Hz,1H).

[0030] Compound 9a (0.74 g, 3 mmol), sodium hydroxide (0.24 g, 6 mmol), and 20 mL of water dissolved in 20 mL of ethanol were added to a reaction flask and stirred at reflux for 4 h at 80°C. The reaction was monitored by TLC. After completion of the reaction, most of the ethanol solvent was removed by rotary evaporation. 20 mL of water was added, and some impurities were removed by extraction with ethyl acetate (3 × 20 mL). The pH of the aqueous phase was adjusted to approximately 3 with 1 M dilute hydrochloric acid, and then extracted with ethyl acetate (3 × 70 mL). The organic phases were combined, washed with saturated sodium chloride solution (3 × 50 mL), separated, and dried over anhydrous Na2SO4. The solvent was removed by rotary evaporation to obtain a colorless oily liquid. Recrystallization from ethyl acetate / petroleum ether gave compound 10a as a white solid in a 60% yield. 1H NMR (600MHz, DMSO-d6) δ7.44(dd,J=4.9,1.6Hz,3H),7.37(dd,J=6.5,3.1Hz,2H),4.32(s,1H),3.53(s ,1H),3.01(d,J=79.0Hz,2H),2.54(ddt,J=11.2,7.2,4.0Hz,1H),1.84(d,J=78.0Hz,2H),1.50(s,2H).

[0031] According to the method of Example 3, compounds 10b-10o can be prepared.

[0032] Example 4: 1-Benzoyl-N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide (1a)

[0033] Compound 10a (0.47 g, 2 mmol) dissolved in 20 mL of N,N-dimethylformamide was added to a reaction flask. EDCI (0.50 g, 2.6 mmol), HOBT (0.32 g, 2.4 mmol), and N-methylmorpholine (0.77 g, 7.6 mmol) were then added sequentially in an ice-water bath. After stirring for 30 minutes, compound 5 (0.56 g, 2 mmol) was added to the flask and stirred overnight at room temperature. The reaction progress was monitored by TLC. After completion, 100 mL of water was added and the mixture was extracted with ethyl acetate (3 × 70 mL). The combined organic phases were washed sequentially with 0.1 M dilute hydrochloric acid (3 × 70 mL) and saturated NaCl solution (3 × 70 mL), dried over anhydrous Na2SO4, and the solvent removed by rotary evaporation. The mixture was purified by column chromatography using dichloromethane / methanol (1:15-1:20, V / V) to obtain compound 11a as a white solid in a 34% yield. NMR(600MHz,DMSO-d6)δ7.48–7.41(m,4H),7.38(dd,J=6.5,2.9Hz,2H),7.26(t,J=7.7Hz,1H),7 .18(dd,J=20.7,7.3Hz,2H),7.03–6.97(m,2H),6.90(dd,J=17.4,7.7Hz,1H),6.82(d,J=9.8Hz, 1H),6.34(d,J=8.2Hz,1H),4.49(s,1H),3.62(s,1H),3.42(d,J=15.8Hz,1H),3.10–2.96(m,1H) ,2.93(d,J=16.0Hz,1H),2.85(d,J=16.2Hz,1H),2.59(t,J=11.4Hz,1H),1.90–1.44(m,4H).13C NMR(151MHz,DMSO-d6)δ176.54,173.35,169.49,167.73,143.03,140.45,137.98,136.70,129.84,129.05,128.90,128.30,12 7.14,124.83,123.16,123.10,121.15,115.42,115.14,112.28,101.93,54.68,47.08,43.14,34.52,34.09.HRMS(ESI):calcd for C29H26N4O4[M+Na]+,517.1846,found,517.1846.

[0034] The following compounds 1b-1o were prepared by the same method as in Example 4.

[0035] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-((2-methylbenzoyl)piperidine-4-carboxamide (1b)

[0036] Yield 33.2%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.44 (s, 1H), 7.27 (dq, J = 23.1, 7.6 Hz, 4H), 7.21–7.10 (m, 3H), 7.03–6.96 (m, 2H), 6.90 (dd, J = 17.5, 7.7 Hz, 1H), 6.82 (d, J = 7.7 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4.57 (d, J = 12.9 Hz, 1H), 3. 42(d,J=15.9Hz,1H),3.01(t,J=11.9Hz,1H),2.93(d,J=15.9Hz,1H),2.85(d,J=16.2Hz,1H),2.58(s,1H ),2.21(d,J=35.1Hz,3H),1.89(d,J=12.6Hz,1H),1.68(dd,J=23.7,11.6Hz,1H),1.60–1.37(m,2H).13C NMR(151MHz,DMSO-d6)δ176.55,173.31,168.90,167.72,143.03,140.43,137.98,137.87,137.07,129.04,128.61,128.30,127.8 1,124.84,123.16,122.64,121.73,121.14,115.42,115.14,101.92,54.68,47.09,34.52,34.09,28.68,18.99.HRMS(ESI):calcd for C30H28N4O4[M+Na]+,531.2003,found,531.2001.

[0037] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(3-methylbenzoyl)piperidine-4-carboxamide (1c)

[0038] Yield 35.0%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.45 (s, 1H), 7.32 (t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.8 Hz, 2H), 7.22–7.14 (m, 3H), 7.00 (dd, J = 13.8, 7.5 Hz, 2H), 6.82 (d, J = 7.3 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4. 49(s,1H),3.63(d,J=17.3Hz,1H),3.42(d,J=15.8Hz,1H),3.06(s,1H),2.93(d,J=16.0Hz ,1H),2.85(d,J=16.2Hz,1H),2.59(t,J=11.4Hz,1H),2.34(s,3H),1.91–1.48(m,4H).13C NMR(151MHz,DMSO-d6)δ176.54,173.36,169.59,167.73,143.03,140.45,138.28,137.98,136.70,130.42,129.05,128.75,12 8.30,127.59,124.83,124.13,123.16,123.10,121.15,115.42,112.28,101.92,54.68,43.16,34.52,21.36.HRMS(ESI): calcd for C30H28N4O4[M+Na]+,531.2003,found,531.2001.

[0039] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(4-methylbenzoyl)piperidine-4-carboxamide (1d)

[0040] Yield 38.9%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.47–7.43 (m, 1H), 7.29–7.23 (m, 5H), 7.19 (d, J = 7.4 Hz, 1H), 7.04–6.96 (m, 2H), 6.91 (dd, J = 16.9, 7.8 Hz, 1H), 6.84–6.80 (m, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4.47 (s, 1H), 3.67(s,1H),3.42(d,J=15.8Hz,1H),3.04(s,1H),2.93(d,J=16.0Hz,1H),2.85(d,J=16.2Hz,1 H),2.58(t,J=11.4Hz,1H),2.33(s,3H),1.78(dd,J=64.2,12.0Hz,2H),1.60–1.49(m,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.38,169.63,167.73,143.03,140.45,139.50,137.98,133.74,129.35,129.04,12 8.30,127.29,124.83,123.16,123.10,121.15,115.42,112.28,101.92,54.68,43.18,34.52,21.36.HRMS(ESI):calcd for C30H28N4O4[M+H]+,509.2183,found,509.2183.

[0041] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(4-ethylbenzoyl)piperidine-4-carboxamide (1e)

[0042] Yield 33.7%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.45 (d, J = 1.4 Hz, 1H), 7.32–7.24 (m, 5H), 7.18 (dd, J = 20.5, 7.4 Hz, 1H), 7.03–6.96 (m, 2H), 6.82 (dd, J = 8.2, 1.7 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4.48 (s, 1H), 3 .67(s,1H),3.42(d,J=15.8Hz,1H),3.06(s,1H),2.93(d,J=16.0Hz,1H),2.85(d,J=16.2H z,1H),2.68–2.56(m,3H),1.92–1.67(m,2H),1.61–1.49(m,2H),1.19(t,J=7.6Hz,3H).13C NMR(151MHz,DMSO-d6)δ176.54,173.38,169.63,167.73,145.68,143.03,140.45,137.98,134.00,129.04,128.30,128.1 9,127.36,124.83,123.16,123.10,121.15,115.42,112.28,101.92,54.68,43.19,34.53,28.44,15.84.HRMS(ESI):calcd for C31H30N4O4[M+Na]+,545.2159,found,545.2158.

[0043] 1-(2,5-Dimethylbenzoyl)-N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide (1f)

[0044] Yield 33.9%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.44 (s, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.1 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 7.00 (dd, J = 12.8, 7.3 Hz, 2H), 6.91 (dd, J = 16.2, 7.2 Hz, 1H), 6.82 (s, 1H), 6.34 (d, J = 8.1 Hz, 1H), 4.56 (d, J = 1 2.5Hz,1H),3.42(d,J=15.8Hz,1H),3.24(d,J=16.2Hz,1H),3.00(t,J=12.4Hz,1H),2.93(d,J=15.9Hz,1H),2.87–2.77 (m,1H),2.58(s,1H),2.31–2.10(m,6H),1.88(d,J=11.1Hz,1H),1.68(dd,J=20.4,12.0Hz,1H),1.60–1.34(m,2H).13C NMR(151MHz,DMSO-d6)δ176.55,173.32,169.04,169.01,167.73,143.03,140.44,137.98,136.99,129.04,128.61,128.30,127.8 1,124.85,123.16,122.64,121.73,121.14,115.42,115.14,112.29,101.92,54.68,47.09,34.52,28.69,20.90.HRMS(ESI):calcd forC31H30N4O4[M+Na]+,545.2159,found,545.2156.

[0045] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(2-methoxybenzoyl)piperidine-4-carboxamide (1 g)

[0046] Yield 38.3%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.49–7.42 (m, 1H), 7.38 (q, J = 6.6 Hz, 1H), 7.26 (t, J = 7.4 Hz, 1H), 7.21–7.13 (m, 3H), 7.07 (d, J = 8.4 Hz, 1H), 7.02–6.97 (m, 3H), 6.91 (dd, J = 17.6, 7.9 Hz, 1H), 6.82 (d, J = 6.4 Hz, 1H), 6.33 (d, J = 6.7 Hz, 1H), 4.5 3(d,J=11.9Hz,1H),3.79(d,J=4.4Hz,3H),3.42(d,J=15.6Hz,1H),3.05–2.73(m,4H),2.56(dt,J=12.0,7.4Hz,1H) ,1.85(d,J=8.3Hz,1H),1.67(dd,J=22.5,12.7Hz,1H),1.55(ddd,J=17.5,12.9,8.3Hz,2H),1.44–1.36(m,1H).13C NMR(151MHz,DMSO-d6)δ176.55,169.05,167.73,143.03,137.98,137.87,129.04,128.61,128.30,127.81,12 3.16,123.10,122.64,121.73,121.15,115.41,115.14,55.89,54.68,47.09,34.52,34.08.HRMS(ESI):calcd for C30H28N4O5[M+Na]+,547.1952,found,547.1950.

[0047] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(3-methoxybenzoyl)piperidine-4-carboxamide (1h)

[0048] Yield 36.9%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.45 (s, 1H), 7.35 (t, J = 7.9 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.18 (dd, J = 20.5, 7.3 Hz, 1H), 7.02–6.97 (m, 3H), 6.94–6.89 (m, 2H), 6.85–6.79 (m, 1H), 6.34 (d, J = 8.2 Hz, 1H ),4.49(s,1H),3.78(s,3H),3.63(s,1H),3.42(d,J=15.8Hz,1H),3.06(s,1H),2.93(d,J=16.0H z,1H),2.85(d,J=16.2Hz,1H),2.59(t,J=11.4Hz,1H),1.79(d,J=76.8Hz,2H),1.56(s,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.35,169.15,167.73,159.58,143.03,140.45,138.10,137.98,130.13,129.05 ,128.30,124.83,123.17,122.64,121.15,119.08,115.50,112.45,101.92,55.69,54.68,43.14.HRMS(ESI):calcd for C30H28N4O5[M+H]+,525.2133,found,525.2130.

[0049] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(4-methoxybenzoyl)piperidine-4-carboxamide (1i)

[0050] Yield 37.6%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.46 (s, 1H), 7.36 (d, J = 8.6 Hz, 2H), 7.26 (t, J = 7.6 Hz, 1H), 7.18 (dd, J = 20.6, 7.3 Hz, 2H), 6.99 (dd, J = 17.3, 8.1 Hz, 4H), 6.91 (dd, J = 17.4, 7.7 Hz, 1H), 6.82 (d, J = 8.2 Hz ,1H),6.34(d,J=8.2Hz,1H),4.44(s,1H),3.79(d,J=5.0Hz,3H),3.42(d,J=15.8Hz,1H),2.89( dd,J=52.9,16.1Hz,3H),2.58(t,J=11.7Hz,1H),1.79(s,2H),1.55(q,J=11.1,10.1Hz,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.40,169.49,167.73,160.56,143.03,140.46,137.98,129.27,129.05,128.58 ,128.30,124.83,123.17,121.15,115.42,114.11,112.28,101.92,55.70,54.68,43.22,34.52.HRMS(ESI):calcd for C30H28N4O5[M+Na]+,547.1952,found,547.1950.

[0051] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(2-nitrobenzoyl)piperidine-4-carboxamide (1J)

[0052] Yield 25.3%; yellow solid; 1H NMR (600 MHz, DMSO-d6) δ 8.19 (d, J = 8.2 Hz, 1H), 7.85 (t, J = 7.4 Hz, 1H), 7.70 (t, J = 7.8 Hz, 1H), 7.56–7.46 (m, 1H), 7.26 (t, J = 7.7 Hz, 1H), 7.20–7.15 (m, 1H), 7.04–6.97 (m, 2H), 6.94–6.87 (m, 1H), 6.82(t,J=6.2Hz,1H),6.34(d,J=8.2Hz,1H),4.57–4.46(m,1H),3.42(t,J=12.9Hz,2H),3.08(s ,1H),2.95–2.83(m,2H),2.61(t,J=11.2Hz,1H),1.91(d,J=11.2Hz,1H),1.72–1.51(m,3H).13C NMR(151MHz,DMSO-d6)δ176.55,173.25,169.05,167.73,143.04,140.45,137.98,133.08,130.64,129.05,128.40,127.81,125.1 8,124.83,123.16,122.64,121.73,121.15,115.42,115.14,112.28,101.91,54.69,47.09,42.94,34.52,34.09.HRMS(ESI): calcd for C29H25N5O6[M+Na]+,562.1697,found,562.1696.

[0053] 1-(2-Chlorobenzoyl)-N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide (1k)

[0054] Yield 33.5%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.59–7.45 (m, 1H), 7.42 (dt, J = 14.6, 4.7 Hz, 3H), 7.26 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.03–6.97 (m, 2H), 6.81 (dd, J = 9.1, 4.4 Hz, 1H) ,6.34(d,J=8.2Hz,1H),4.60–4.49(m,1H),3.42(d,J=15.8Hz,1H),3.31(d,J=13.9 Hz,1H),3.10–2.98(m,1H),2.93(d,J=15.9Hz,1H),2.85(t,J=12.5Hz,1H),2.59(q ,J=13.2,11.4Hz,1H),1.89(d,J=12.6Hz,1H),1.75–1.44(m,3H).13CNMR(151MHz, DMSO-d6)δ176.55,173.27,167.72,165.92,143.03,140.42,137.98,136.56,130. 91,130.80,129.94,129.58,129.05,128.30,128.27,128.18,124.85,123.17,121 .14,115.42,112.26,101.92,54.68,46.46,45.85,42.98,34.52.HRMS(ESI):calcd forC29H25ClN4O4[M+Na]+,551.1457,found,551.1459.

[0055] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(4-fluorobenzoyl)piperidine-4-carboxamide (1l)

[0056] Yield 32.3%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.46 (dd, J = 8.4, 5.6 Hz, 3H), 7.30–7.24 (m, 3H), 7.20 (d, J = 7.4 Hz, 1H), 7.02–6.97 (m, 2H), 6.82 (dd, J = 8.2, 1.5 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4.47 (s, 1 H),3.62(s,1H),3.42(d,J=15.7Hz,1H),3.08(s,1H),2.93(d,J=15.9Hz,1H),2.83(s,1 H),2.59(ddd,J=14.9,11.4,3.6Hz,1H),1.80(d,J=65.0Hz,2H),1.62–1.50(m,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.34,168.62,167.73,163.73,162.10,143.04,140.45,137.98,133.06,133.03,129.82,129.77,12 9.05,128.30,124.84,123.17,123.10,121.15,115.92,115.78,115.42,112.28,101.92,54.75,54.68,43.10,34.52.HRMS(ESI):calcd for C29H25FN4O4[M+Na]+,535.1752,found,535.1748.

[0057] N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)-1-(3-fluorobenzoyl)piperidine-4-carboxamide (1m)

[0058] Yield 35.9%; white solid; 1H NMR (600 MHz, DMSO-d6) δ 7.52–7.43 (m, 2H), 7.32–7.18 (m, 5H), 7.03–6.97 (m, 2H), 6.82 (dd, J = 8.2, 1.6 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4.48 (s, 1H), 3.58 (s, 1H), 3.42 (d, J = 15.8 Hz, 1H), 3.09 (s, 1H), 2.94 (d, J = 16.0 Hz, 1H), 2.85 (d, J = 15.8 Hz, 1H), 2.59 (t, J = 11.4 Hz, 1H), 1.80 (d, J = 88.6 Hz, 2H), 1.57 (s, 2H). 13C NMR(151MHz,DMSO-d6)δ176.54,173.31,167.73,163.12,161.50,143.03,140.44,139.02,137.98,131.21,129.04,128.30,124 .84,123.16,123.10,121.15,116.79,116.65,115.42,114.27,114.12,112.29,101.92,54.68,43.05,34.52.HRMS(ESI): calcd for C29H25FN4O4[M+H]+,513.1933,found,513.1932.

[0059] 1-(4-Bromobenzoyl)-N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide (1n)

[0060] Yield 35.6%; pale yellow solid; 1H NMR (600 MHz, DMSO-d6) δ 7.65 (d, J = 8.3 Hz, 2H), 7.45 (s, 1H), 7.35 (d, J = 8.3 Hz, 2H), 7.29–7.16 (m, 2H), 7.03–6.95 (m, 2H), 6.82 (d, J = 9.4 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1H), 4. 47(s,1H),3.60(d,J=18.8Hz,1H),3.42(d,J=15.8Hz,1H),3.08(s,1H),2.93(d,J=1 6.0Hz,1H),2.83(s,1H),2.59(t,J=11.3Hz,1H),1.91–1.68(m,2H),1.56(s,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.31,168.48,167.73,143.03,140.44,137.98,135.81,131.91,131.85,130.29,129.43,1 29.05,128.30,124.84,123.20,123.17,123.11,121.15,115.42,112.29,101.92,54.68,43.06,34.52.HRMS(ESI):calcdfor C29H25BrN4O4[M+H]+,573.1132,found,573.1132.

[0061] 1-(3-Bromobenzoyl)-N-(2,2′-dioxo-1′,4′-dihydro-2′H-spiro[indoline-3,3′-quinoline]-6-yl)piperidine-4-carboxamide (1o)

[0062] Yield 32.6%; pale yellow solid; 1H NMR (600 MHz, DMSO-d6) δ 7.68–7.54 (m, 2H), 7.51–7.37 (m, 3H), 7.26 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 7.3 Hz, 1H), 7.04–6.95 (m, 2H), 6.82 (d, J = 7.9 Hz, 1H), 6.34 (d, J = 8.2 Hz, 1 H),4.47(s,1H),3.57(s,1H),3.42(d,J=15.7Hz,1H),3.09(s,1H),2.94(d,J=15.9H z,1H),2.84(s,1H),2.59(t,J=11.2Hz,1H),1.80(d,J=83.3Hz,2H),1.57(s,2H).13C NMR(151MHz,DMSO-d6)δ176.54,173.30,167.73,143.04,140.44,139.01,137.98,132.67,131.18,129.80,129.05,128.30,1 27.36,126.10,124.84,123.17,123.11,122.17,121.15,115.42,112.29,101.93,54.68,43.05,34.52.HRMS(ESI):calcdfor C29H25BrN4O4[M+Na]+,595.0952,found,595.0944.

[0063] Example 5: Enzyme inhibition activity test of spiro[indoline-3,3′-quinoline]-2,2′-diketopipetidinecarboxamide compounds

[0064] Chitin synthase (CHS) extracted from tropical yeast (ATCC 750) can specifically bind to the substrate UDP-GlcNAc on a support and incubate to produce chitin. The resulting chitin binds to wheat germ agglutinin (WGA) pre-coated on a 96-well plate and becomes immobilized. After washing away excess reagent with a buffer solution, horseradish peroxidase-wheat germ agglutinin complex (WGA-HRP) is added to bind to the immobilized chitin. Subsequently, the excess WGA-HRP is washed away. The activity of horseradish peroxidase (WGA) is tested using a 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution. The reaction is terminated with a 2M H2SO4 solution, and the OD value of each well is measured at 450 nm. The CHS inhibition rate (IP) of each drug can be calculated. Each group was tested in triplicate. The results are shown in Table 1.

[0065] Glucosamine-6-phosphate synthase (GFA) was isolated by culturing Escherichia coli (E. coli) carrying the gene for glucosamine-6-phosphate synthase. This enzyme catalyzes the release of p-nitroaniline from L-γ-glutamyl-p-nitroaniline (GLUPA) in a reaction system using the substrate. GFA activity and the GFA inhibition rate (IP) of each drug were determined by spectrophotometric measurement of absorbance at 420 nm. Each group was tested in triplicate. The results are shown in Table 2. The reaction system consisted of (final concentrations): 1 mmol / L substrate (GLUPA), 1 mmol / L ethylenediaminetetraacetic acid (EDTA), 1 mmol / L dithiothreitol (DTT), and 0.5 mmol / L phenylmethylsulfonyl chloride (PMSF) dissolved in 20 mmol / L 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, pH 7.5) buffer.

[0066] Table 1 CHS inhibition rate of compounds 1a-1o at a concentration of 300 μg / mL

[0067]

[0068] Table 2 GFA inhibition rate of compounds 1a-1o at a concentration of 300 μg / mL

[0069]

[0070] Example 6: In vitro antimicrobial activity of spiro[indoline-3,3′-quinoline]-2,2′-diketopipectinamides

[0071] Antimicrobial activity was determined in strict accordance with the Clinical and Laboratory Standards Institute (CLSI) antimicrobial susceptibility testing protocol using the half-dilution method in 96-well plates. The antifungal activity of the target compounds was determined by the MIC (minimum inhibitory concentration). Three Gram-positive bacteria, Candida albicans (ATCC 76615), Aspergillus fumigatus (GIMCC 3.19), Candida fumigatus (ATCC 16870), and Cryptococcus neoformans (ATCC 208821), methicillin-resistant Staphylococcus aureus (MRSAN315), Staphylococcus aureus (ATCC 25923), and Bacillus subtilis (ATCC 6633), and three Gram-negative bacteria, Escherichia coli (JM 109), Pseudomonas aeruginosa (ATCC 9027), and Proteus (ATCC 8427), were selected as test strains. Positive controls included fluconazole and polyoxin B; chloramphenicol and levofloxacin. The test and control drugs were dissolved in dimethyl sulfoxide (DMSO) and prepared using sterile water using a half-dilution method to a concentration gradient of 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg / mL. 100 μL of the strain solution and 100 μL of the test compound solution were placed in a 96-well plate and incubated with shaking at 37°C for 24 hours. MIC values were determined by comparing the clarity of the culture medium in each well with that of the positive wells and blank wells. Each experiment was performed in triplicate. The results are shown in Tables 3 and 4.

[0072] Table 3 MIC values of compounds 1a-1o against fungi in vitro (μg / mL)

[0073]

[0074] Note a: In the blank control and negative control (containing 0.5% DMSO solution), the colonies grew normally and the solution was turbid.

[0075] Table 4 MIC values of compounds 1a-1o against bacteria in vitro (μg / mL)

[0076]

[0077]

[0078] Note a: In the blank control and negative control (containing 0.5% DMSO solution), the colonies grew normally and the solution was turbid.

[0079] As can be seen from Tables 3 and 4, compounds 1a-1o prepared in the present invention have weak inhibitory activity against bacteria, but exhibit certain inhibitory effects against all four fungi. In particular, compounds 1c, 1d, 1j, 1n, and 1o exhibit moderate inhibitory activity against Candida albicans (MIC = 16 μg / mL), comparable to polyoxin B. Compounds 1f, 1j, 1k, and 1n also exhibit moderate inhibitory activity against Aspergillus fumigatus (MIC = 16 μg / mL), comparable to polyoxin B. For Cryptococcus neoformans, compounds 1j, 1k, 1l, and 1o exhibit moderate inhibitory activity (MIC = 16 μg / mL), comparable to polyoxin B. For Aspergillus flavus, compounds 1j, 1k, 1m, and 1o exhibit inhibitory activity (MIC = 16 μg / mL) comparable to polyoxin B. Based on the above data, it can be concluded that some of the synthesized compounds have specific inhibitory activity against fungi.

[0080] Example 7: In vitro anti-drug-resistant fungal activity test of spiro[indoline-3,3′-quinoline]-2,2′-diketopipetidinecarboxamide compounds

[0081] Candida albicans (ATCC 76615), Cryptococcus neoformans (ATCC 208821), Aspergillus fumigatus (GIMCC3.19), and Candida fumigatus (ATCC 16870) were selected as experimental strains for inducing drug-resistant fungi. Micafungin and fluconazole were used as the incubation agents. The MIC values of the bioactive compounds 1j, 1k, 1n, and 1o against the drug-resistant strains were determined (using procedures similar to those used for antifungal activity assays). The experimental results are shown in Table 5.

[0082] Table 5 MIC values (μg / mL) of compounds 1j, 1k, 1n and 1o against drug-resistant fungi

[0083]

[0084] Note: a: This experiment was not performed; b: In the negative control group (containing 0.5% DMSO solution), the colonies grew normally and the solution was turbid.

Claims

1. The spiro[indoline-3,3'-quinoline]-2,2'-diketopipectinamide compounds of the present invention have the following characteristic structures: As shown in general formula 1: Wherein R is various substituents such as halogen, hydrocarbon group, etc.

2. As claimed in claim 1, the specific structure of the spiro[indoline-3,3'-quinoline]-2,2'-diketopipectinamide compound is as follows:

3. Use of the compound according to claim 1 in the preparation of an anti-pathogenic microbial drug. The microorganism is a pathogenic bacterium or pathogenic fungus, including Escherichia coli, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Proteus, Pseudomonas aeruginosa; Candida albicans, Cryptococcus neoformans, Aspergillus flavus, Aspergillus fumigatus; fluconazole-resistant Candida albicans, fluconazole-resistant Aspergillus fumigatus, fluconazole-resistant Cryptococcus neoformans, fluconazole-resistant Aspergillus flavus, and micafungin-resistant Candida albicans.

4. Use of the compound according to claim 1 as a dual-target inhibitor of chitin synthase and glucosamine-6-phosphate synthase in medicine.

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