CN115057807B

CN115057807B - 3- (2-isocyanobenzyl) indole derivative quorum sensing inhibitor and preparation method and application thereof

Info

Publication number: CN115057807B
Application number: CN202210545564.8A
Authority: CN
Inventors: 王江; 顾恭萍; 杨靓仪; 刘丹蕊; 贾爱群; 唐诗
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2023-06-23
Anticipated expiration: 2042-05-19
Also published as: CN115057807A

Abstract

The invention discloses a 3- (2-isocyanobenzyl) indole derivative quorum sensing inhibitor, a preparation method and application thereof, wherein the preparation method is efficient and convenient, the 3- (2-isocyanobenzyl) indole derivative can be used as a quorum sensing inhibitor for purple bacillus, pseudomonas aeruginosa and Serratia marcescens, wherein the inhibition rate of individual compounds on the envelope of the pseudomonas aeruginosa can be up to 75%, the inhibition rate of the individual compounds on the envelope of the Serratia marcescens can be up to 65%, and the destruction rate of the envelope of the humanized Serratia marcescens can be up to 62%.

Description

3- (2-isocyanobenzyl) indole derivative quorum sensing inhibitor and preparation method and application thereof

Technical Field

The invention relates to an indole derivative, a preparation method and application thereof, in particular to a 3- (2-isocyanobenzyl) indole derivative quorum sensing inhibitor, a preparation method and non-classical antibacterial application thereof.

Background

Pseudomonas aeruginosa (Pseudomonas aeruginosa) is a gram-negative bacterium which is commonly existing in air, water, soil, animal, plant and other tissues, the length of the bacterium is 1.5-5.0 mu m, the width is about 0.5-1.0 mu m, the bacterium is in a club shape or a linear shape, the length is different, the bacterium can grow in the range of 25-42 ℃, and the optimal growth temperature is 25-30 ℃. The bacterium is named because of its ability to secrete a copper green water-soluble pyocin (pyocianin) and a fluorescing water-soluble fluorescein (pyovindin) during growth. The thallus has flagella, terminal growth, even distribution, no capsule and spore. The pseudomonas aeruginosa has strong adaptability and can utilize various compounds as nutrient substances for self growth in various environments. Pseudomonas aeruginosa is one of the most main pathogenic bacteria causing nosocomial infection, belongs to conditional pathogenic bacteria, can infect through skin, oral cavity, mucous membrane and blood, can cause infection of various tissues and organs of human body, and seriously threatens human health. The pseudomonas aeruginosa is easy to infect patients with low immunity, such as burn patients, postoperative patients, patients with metabolic diseases and the like, can cause wound infection and abscess, and the pseudomonas aeruginosa can infect blood at an infection position and spread through the blood, thereby causing septicemia or bacteremia and having very high mortality. The quorum sensing system of Pseudomonas aeruginosa consists of las, rhl and pqs, the las system consists of LasI and LasR, and the rhl system consists of RhlI and RhlR. Of the three systems, las dominates, and the las system regulates rhl and pqs. The lasI gene can code and regulate the synthesis of a 3-oxoodecanonyl-homoserine lactone (3-oxo-C12-HSL) signal molecule, rhlI codes and synthesizes a signal molecule butryl-homoserine lactone (C4-HSL), PQS codes and synthesizes a signal molecule Pseudomonas Quinolone Signal (PQS), the synthesis of the PQS is regulated and controlled by genes pqsABDE and pqsH, and the synthesized PQS is combined with a receptor protein PqsR to activate the expression of a series of pathogenic genes. With the acceleration of bacterial growth, bacterial density gradually increases, and the signal molecule 3-oxo-C12-HSL synthesized by lasI gradually increases, and the signal molecule 3-oxo-C12-HSL is combined with the receptor protein LasR in cytoplasm to form a receptor complex, and the receptor complex is further combined with a target gene DNA receptor region, so that the expression of a series of virulence genes such as protease, elastase, hemolysin, hydrocyanic acid, pyocin and the like is activated. Meanwhile, the complex can also be combined with a lasI promoter to promote secretion of a signal molecule 3-oxo-C12-HSL, and the complex is circulated in this way to form a positive feedback regulation process. In the rhl system, rhlI encodes a synthetic signal molecule C4-HSL which binds to the cytoplasmic receptor protein RhlR to form a receptor complex which, on the one hand, binds to the target gene DNA receptor region, activates the expression of the associated virulence genes, such as rhamnolipids, alginates, biofilms, etc., and, on the other hand, binds to RhlI, promoting the synthesis of C4-HSL, and thus circulates, forming a positive feedback regulation process. The PQS system uses the PQS as a signal molecule, and can regulate and control the synthesis of pyocin and elastase lasB and can also regulate and control the rhl system.

Pseudomonas aeruginosa can secrete a variety of extracellular and intracellular virulence factors including proteases, pyocins, phosphatases, haemolysins, rhamnolipids, beta-lactamases, penicillin binding proteins, alginates, etc., which have an important role in the adhesion and infestation of Pseudomonas aeruginosa. The pyocin is one of the most important virulence factors of pseudomonas aeruginosa, belongs to phenazine compounds, and has hydrophilic and lipophilic dual properties, so that the pyocin can easily penetrate cell membranes to enter the inside of cells. On the one hand, the compound can inhibit the growth of other bacteria and reduce the competition between seeds, thereby being beneficial to the growth of the compound. On the other hand, pyocin readily penetrates the cell membrane into the host cell, thereby rendering it toxic to the host cell. In addition, the compound has oxidation-reduction dual properties, can serve as an electron carrier in the cell, and can increase the oxidation pressure of the host cell, so that the host cell is poisoned and dead. Studies have shown that the synthesis of pyocin is regulated by quorum sensing, the biosynthetic precursor is chorismate, and the precursor is converted into tricyclic compounds by 2 phzABCDEFG operon codes and regulation of the phzH, phzM, and phzS genes.

Since quorum sensing can regulate the production of virulence factors of pathogenic bacteria, and is closely related to the infection and pathogenicity of pathogenic bacteria, quorum sensing has become a new target of anti-infective drugs. Inhibiting quorum sensing of bacteria, namely inhibiting activity of related enzymes in quorum sensing systems or competitively inhibiting transmission of signal molecules under the condition of not affecting normal growth of bacteria, thereby achieving the effects of inhibiting quorum sensing, reducing production of virulence factors and reducing pathogenicity. Bacterial quorum sensing inhibitors (quorum sensing inhibitor, QSI) are broadly divided into two categories: small molecule compounds and degrading enzymes.

Serratia marcescens is a facultative anaerobe that is widely found in soil and water environments, the first species found in Serratia. Historically classification of Serratia bacteria has experienced a long period of confusion: the first edition of the handbook for identifying Burjie bacteria in 1923 defines Serratia as containing 23 species; eighth edition of Berger's Manual of bacteria identification in 1974 was incorporated into 1, serrtia marcescens; the ninth edition of the "Burjie's Manual of bacteria identification" in 2004 was redefined to be 11 species, with the addition of new species that were later isolated and identified, and currently there are 15 species of Serratia species. Serratia marcescens cells are spherical or short rod-shaped, and are one of the smallest bacteria. Serratia marcescens is classified into pigment-producing strains and pigment-non-producing strains. The colony of the pigment-producing strain is in a red round bulge shape, and the taste is slightly odorous; the non-pigment-producing strain is white or light yellow. Regarding its pathogenicity, serratia marcescens has been demonstrated to be able to infect humans, but it is only infectious to people with low immunity, most pathogenic Serratia marcescens with drug resistance are generally unable to produce prodigiosin.

Prodigiosin is a family of natural red pigments having a tripyrrole ring structure with various biological activities such as anticancer, antimalarial, antibacterial, antifungal and antiprotozoal. Prodigiosin is mainly produced by serratia marcescens, pseudomonas, some species of actinomycetes and some marine bacteria. Prodigiosin is dark red, is a fat-soluble pigment, is almost insoluble in water, is soluble in methanol, is sensitive to p H, and is red under acidic conditions and yellow under alkaline conditions.

There is currently a debate on the relationship of quorum sensing to biofilm, however current studies indicate that quorum sensing can affect biofilm formation. In the initial attachment stage of bacteria, the quorum sensing system agr of staphylococcus aureus can inhibit the secretion of adhesins, the adhesins play an important role in the initial adhesion of the bacteria on the surface of an object, and compared with a wild strain, the agr mutant strain can secrete more adhesins, so that the adhesion on the surface of the object is facilitated. In Serratia liquefaciens, swrI regulates the synthesis of signal molecules, and after mutation of the genes, the formed coating is reduced in quantity and thin in thickness. The two genes, bsmA and bsmB, which regulate capsule formation are also regulated by quorum sensing systems. The outer surface of the envelope is covered with a thick polymer which is composed of polysaccharide, glycoprotein, lipoprotein, etc., and the synthesis process of these polymers is controlled by quorum sensing. The biofilm of the water vapor monad also needs to be regulated by a quorum sensing system in the maturation process, the biofilm biomass formed by the ahyI gene mutant strain is obviously lower than that of a wild strain, and the phenotype of the strain can be partially recovered after the signal molecule butyl-HSL is exogenously added. As early as 1998, a paper published in Science has introduced the relationship between the Pseudomonas aeruginosa las system and the envelope. After the lasI gene is mutated, the strain can not synthesize a 3-oxo-C12-HSL signal molecule, at the moment, the formed film has flat and loose surface and uniform shape, and the film formed by the wild strain has uneven structure, mushroom-shaped protrusions and compactness. The above studies indicate that there is indeed a certain link between quorum sensing systems and biofilms.

The QSI of plant source is found in marine red algae (Delisea pulchra) at the earliest time, the bromofuranone (halogenated furanones) which is extracted and separated from the red algae is a structural analogue of AHL signal molecule, and the compound has better inhibition activity on quorum sensing systems of various bacteria such as escherichia coli, vibrio freudenreichii, pseudomonas aeruginosa and the like.

Solenopsin A is an alkaloid isolated from an invading solenopsis invicta (Solenopsis invicta) body, and can competitively inhibit the combination of a signal molecule C4-HSL and a receptor protein RhlR, and inhibit the synthesis of pyocin. Two alkaloids are isolated from bryozoan extract, which are effective in inhibiting the transmission of pseudomonas aeruginosa signaling molecules. The compounds manoalide, manoalide monoacetate and secomboalide obtained from the body of sponge Luffariella variabilis are effective in inhibiting the expression of the lasB gene of the quorum sensing system of P.aeruginosa.

Penicillium is an important microbial resource, penicillin separated from penicillium saves lives of countless people and makes an important contribution to life health of the people. The penicillium restrictum is separated from stem of milk thistle herb, and the secondary metabolite omega-hydroxy rhein, emodin, (+) -2' S-isorhodoptillometrin and other active components of the penicillium restrictum have good inhibitory activity on quorum sensing systems of methicillin-resistant staphylococcus aureus, and the IC50 value of the penicillium restrictum is 8-120 mu M.

The quorum sensing inhibitor obtained from the natural world has the defects of low extraction rate, low yield, easy resource waste and the like, and some plant and animal resources are endangered resources and cannot be randomly utilized. Thus, researchers synthesize quorum sensing inhibitors by chemical synthesis methods, increasing the probability of obtaining positive compounds. By structural modification of signal molecules such as C4-HSL and 3-oxo-C12-HSL, a series of structural analogues of the signal molecules can be obtained, and the structural analogues can competitively bind with receptor proteins and block the binding of the signal molecules and the receptor proteins, so that quorum sensing systems are blocked, and the pathogenicity of bacteria is reduced. By changing the length, saturation and functional group of acyl side chain and the conformation of inner alicyclic ring, geske synthesizes a series of signal molecular structure analogues, and the research result shows that the structural analogues with LasR inhibitory activity are characterized in that the number of side chain atoms is 8 at most, and the side chain is straight chain or branched chain is aromatic ring; the end of the side chain is phenylacetic acid and the third carbon atom contains a lipophilic group. These compounds have varying degrees of inhibitory activity against the quorum sensing systems of Agrobacterium tumefaciens, pseudomonas aeruginosa and Vibrio freudenreichii. O' Loughlin, etc. simultaneously carry out structural modification on side chains and inner alicyclic rings, a series of signal molecular structure analogues are synthesized, wherein the activity of mBTL is best, the compound can obviously inhibit the generation of pseudomonas aeruginosa pyocin and biological envelope, the action targets are LasR and RhlR, and in vivo and in vitro experiments show that the compound can protect caenorhabditis elegans and human lung gland epithelial cells from being infected by pseudomonas aeruginosa.

In summary, the emergence of antibiotics has brought a good news to infected patients, but with the widespread use of antibiotics, more and more bacteria have developed resistance. Biofilms are an important mechanism for bacteria to develop resistance, and studies have shown that bacterial biofilms are closely related to quorum sensing. Therefore, finding novel quorum sensing inhibitors, blocking bacterial quorum sensing, inhibiting biofilm production is an important approach to the treatment of drug-resistant bacterial infections.

Disclosure of Invention

The invention aims to: the present invention aims to provide a 3- (2-isocyanobenzyl) indole derivative obtained by an organic synthesis means using an indole derivative and an anthranilic alcohol derivative; the invention also aims to provide a preparation method of the 3- (2-isocyanobenzyl) indole derivative, which is efficient and convenient; it is another object of the present invention to provide 3- (2-isocyanobenzyl) indole derivatives as inhibitors of quorum sensing and non-classical antibacterial applications of Serratia marcescens, pseudomonas aeruginosa and Violet bacilli.

The technical scheme is as follows: the 3- (2-isocyanobenzyl) indole derivative has the following structural general formula:

wherein the substituents R ¹ =at least one of H, me, OMe, F, cl, br, CN, R ² =at least one of H, me, OMe, F, cl, br, CN, ph, R ¹ Is substituent at any position of benzene ring, R ² Is substituent at any position on indole ring.

The preparation method of the 3- (2-isocyanobenzyl) indole derivative comprises the following steps:

(1) Weighing the compound 1, and reducing the compound by lithium aluminum hydride to obtain a product 2;

(2) Adding a compound 3 and a catalyst, and reacting to obtain a product 4;

(3) The product 4 reacts with formic acid and acetic anhydride continuously to obtain an intermediate product 5;

(4) Adding phosphorus oxychloride and a catalyst, carrying out dehydration reaction, and purifying by column chromatography to obtain a final product 6;

the reaction formula is as follows:

further, the reaction in the step (1) is anhydrous and anaerobic, the reaction solvent is tetrahydrofuran, the reaction temperature is 0-40 ℃, and the reaction time is 6-12 h.

Further, the reaction in the step (2) is anhydrous and anaerobic, the reaction solvent is 1, 2-dichloroethane, the catalyst is trifluoroacetic acid, the reaction temperature is 40-50 ℃, and the reaction time is 8-12 h.

Further, the solvent used in the reaction in the step (3) is tetrahydrofuran, the reaction temperature is 20-40 ℃, and the reaction time is 6-8 h.

Further, the solvent used in the reaction in the step (4) is dichloromethane, the catalyst is triethylamine, the reaction temperature is-10-0 ℃, and the reaction time is 3-5 hours.

The 3- (2-isocyanobenzyl) indole derivatives can be used as quorum sensing inhibitors of purple bacillus, pseudomonas aeruginosa and Serratia marcescens.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: a series of 3- (2-isocyanobenzyl) indole derivatives are synthesized; the 3- (2-isocyanobenzyl) indole derivative is used as a novel quorum sensing inhibitor and a non-classical antibacterial application of the rhodobacter martensii, serratia and Pseudomonas aeruginosa, the maximum inhibition rate of the 3- (2-isocyanobenzyl) indole derivative on the envelope of the Pseudomonas aeruginosa is 75%, the inhibition rate of the 3- (2-isocyanoberyl) indole derivative on the envelope of the Serratia marcescens is 65%, and the destruction rate of the 3- (2-isocyanoberyl) indole derivative on the envelope of the humanized Serratia marcescens is 62%.

Drawings

FIG. 1 is a primary screen of compounds for their induction activity against Pseudomonas aeruginosa populations;

FIG. 2 shows the results of preliminary screening of compounds with better activity for the induction activity of Pseudomonas aeruginosa population;

FIG. 3 is a preferred compound 1as formula;

FIG. 4 is a nuclear magnetic resonance hydrogen spectrum of compound 1 as;

FIG. 5 is a nuclear magnetic resonance spectrum of compound 1 as;

FIG. 6 is a graph of growth of compounds against Serratia marcescens;

FIG. 7 is a graph showing inhibition of compounds against Serratia marcescens by the envelope;

FIG. 8 is a cryo-scanning electron micrograph of a compound for inhibition of the envelope of Serratia marcescens;

FIG. 9 is a graph of the compound's membrane breakdown against Serratia marcescens;

FIG. 10 is a graph showing the results of toxicity factor experiments of compounds against Serratia marcescens;

FIG. 11 is a graph showing the results of a compound on Serratia marcescens;

FIG. 12 is a graph showing the results of a compound cytotoxicity test against Serratia marcescens;

FIG. 13 is a graph showing the results of a compound nematode experiment against Serratia marcescens;

FIG. 14 is a graph showing the growth of the compound human Serratia marcescens;

FIG. 15 is a graph showing inhibition of the envelope of the compound human Serratia marcescens;

FIG. 16 is a graph showing the breakdown of the envelope of the compound human Serratia marcescens;

FIG. 17 is a graph of virulence factors of the compound human Serratia marcescens;

FIG. 18 is a screen of compounds for quorum sensing activity against Serratia marcescens.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Example 1

A3- (2-isocyanobenzyl) indole derivative 1a has a structural formula shown in table 1.

The preparation method of the compound comprises the following steps:

(1) 2-aminobenzoic acid (10 mmol,1.0 equiv) was weighed into a Schlenk tube, purged with argon 3 times, treated anhydrous and anaerobic, and LiAlH was taken at 0deg.C ₄ (15 mmol,1.0 equiv) was dissolved in anhydrous THF (20 mL), mixed well, added dropwise to a Schlenk tube, reacted for 10 minutes, and the ice bath was removed. The mixture was then gradually warmed to room temperature. The progress of the reaction was monitored by thin layer chromatography. Distillation under reduced pressure, column chromatography (petroleum ether: etoac=2:1) purification gave the final product, 1.303g, 95% yield.

(2) 2-aminobenzyl alcohol (9.5 mmol,1.2 equiv) and 1H-indole (7.9 mmol,1.0 equiv) were weighed into a Schlenk tube, purged 3 times with argon, and 1, 2-dichloroethane (40 mL) solvent was added. Trifluoroacetic acid (2.85 mmol,0.3 equiv) was added dropwise, followed by slow heating and stirring overnight at 50 ℃. The reaction was monitored by thin layer chromatography and once the reaction was complete, saturated NaHCO was added ₃ The aqueous solution and aqueous phase were extracted three times with ethyl acetate (3×10 mL). Vacuum distilling, and purifying by column chromatographyThe final product. The mixture was purified by column chromatography on silica gel (petroleum ether: etoac=5:1) to give 1.347g of 2- (1H-indol-3-methyl) aniline in 60% yield.

(3) The reaction substrates formic acid (28.5 mmol,5.0 equiv) and acetic anhydride (11.4 mmol,2.0 equiv) were added to a 50mL flask. The reaction was stirred at room temperature for 1h. Then a solution of 2- (1H-indol-3-methyl) aniline dissolved in tetrahydrofuran is added. Stirring at room temperature for 1h, and monitoring the reaction by thin layer chromatography. After the completion of the reaction, the pH of the mixture was adjusted to neutrality with NaOH solution (1M), and extracted three times with ethyl acetate. Combined with organic layer, via anhydrous Na ₂ SO ₄ Drying, filtering and evaporating, and purifying the obtained mixture by silica gel column chromatography to obtain 1.22g of N- (-2- (1H-indole-3-methyl) benzamide with a yield of 81%.

(4) 20mL of anhydrous methylene chloride and N- (-2- (1H-indole-3-methyl) benzamide (4.6 mmol,1.0 eq.) were added to a 100mL flask, triethylamine (23 mmol,5.0 eq.) was added, the mixture was then cooled to 0deg.C, phosphorus oxychloride (9.2 mmol,2.0 eq.) was added to the solution and reacted under ice bath for 1 hour, and the reaction was monitored by thin layer chromatography ₃ The aqueous solution and aqueous phase were extracted three times with ethyl acetate (3X 10 mL). The organic phases were combined and extracted once more with saturated brine. Finally, the organic phase is treated with anhydrous Na ₂ SO ₄ And (5) drying. Vacuum distillation and purification by column chromatography to obtain the final product. The resulting mixture was purified by silica gel column chromatography (petroleum ether: etoac=5:1) to give the final product 1a:3- (2-isocyanobenzyl) -1-hydro-indole, 0.79g, 70% yield. Yellow solid, mp 65 ℃,75% yield; petroleum ether, etoac=5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.12(s,1H),7.57(d,J＝7.4Hz,1H),7.43(d,J＝8.1Hz,2H),7.34–7.30(m,2H),7.29–7.23(m,2H),7.19–7.10(m,2H),4.31(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.3,137.8,136.4,130.0,129.4,127.2,127.0,126.9,123.0,122.2,119.6,119.0,112.8,111.3,28.1.IR(KBr)3406,3055,2919,2122,1631,743cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₂ N ₂ [M-H] ^- 231.0928,found 231.0924。

Example 2

3- (2-isocyanobenzyl) indole derivative 1ac has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 4-methyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (4-methyl-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (4-methyl-1H-indole-3-methyl) benzamide in step (4), to give the product 1a as 1ac:3- (2-isocyanobenzyl) -4-methyl-1-hydro-indole. ¹ H NMR(400MHz,CDCl ₃ )δ8.23(s,1H),7.81–7.72(m,1H),7.53–7.47(m,1H),7.45–7.40(m,1H),7.37–7.27(m,3H),7.17(dd,J＝8.0,1.9Hz,1H),7.10(d,J＝2.5Hz,1H),4.40(s,2H),2.43(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ165.3,136.8,136.2,134.5,130.1,129.6,127.0,126.9,122.9,121.8,119.2,118.7,112.4,111.3,27.4,20.3.IR(KBr)3399,3054,2918,2851,2120,1577,744cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1079。

Example 3

3- (2-isocyanobenzyl) indole derivative 1ad has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1, except that 1H-indole was changed to 4-fluoro-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (4-fluoro-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (4-fluoro-1H-indole-3-methyl) benzamide in step (4), and 1a was changed to 1ad 3- (2-isocyanobenzyl) -4-fluoro-1-hydro-indole Yellow sol at mp 106℃ 66%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.89(s,1H),7.35(d,J＝8.0Hz,1H),7.20(d,J＝8.2Hz,1H),7.11–6.89(m,4H),6.87–6.74(m,2H),4.05(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.30,160.68,158.22,135.30,132.70(d,J＝3.9Hz),130.24(d,J＝8.6Hz),125.93,122.01,121.17,118.55,117.73,115.78(d,J＝20.9Hz),112.88(d,J＝25.4Hz),111.25,110.32(d,J＝1.8Hz),26.34. ¹⁹ F NMR(376MHz,CDCl ₃ )δ-114.17.IR(KBr)3447,3060,2921,2132,1588,1415,746cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ FN ₂ [M-H]-249.0834,found 249.0827。

Example 4

3- (2-isocyanobenzyl) indole derivative 1ae has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1, except that 1H-indole was changed to 5-methyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (5-methyl-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (5-methyl-1H-indole-3-methyl) benzamide in step (4), and 1a was changed to 1ae:3- (2-isocyanobenzyl) -5-methyl-1-hydro-indole, 2a was changed to 2g in step (5), and 70%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.89(s,1H),7.34–7.29(m,1H),7.26(s,1H),7.21–7.16(m,3H),7.15–7.10(m,1H),6.99(dd,J＝8.3,1.6Hz,1H),6.88(d,J＝2.4Hz,1H),4.16(s,2H),2.39(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,138.0,134.8,130.1,129.5,128.9,127.6,127.0,126.9,126.0,123.9,123.4,118.6,112.1,111.1,28.1,21.7.IR(KBr)3293,3022,2923,2853,2140,1626,764cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1091。

Example 5

A3- (2-isocyanobenzyl) indole derivative 1af has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 5-methoxy-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (5-methoxy-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (5-methoxy-1H-indole-3-methyl) benzamide in step (4), and 1a was changed to 1af 3- (2-isocyanobenzyl) -5-methoxy-1-hydro-indole, 2a was changed to 2g in step (5), and 70%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.96(s,1H),7.42–7.36(m,1H),7.29–7.25(m,4H),7.24–7.18(m,1H),7.04(d,J＝2.4Hz,1H),6.94(d,J＝2.4Hz,1H),6.86(dd,J＝8.8,2.5Hz,1H),4.21(s,1H),3.82(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ154.1,137.7,131.5,130.0,129.4,127.7,127.0,126.9,123.8,112.6,112.3,112.0,100.9,55.9,28.0.IR(KBr)3396,3106,3055,2927,2832,2121,1211,727cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ O[M-H] ^- 261.1033,found261.1044。

Example 6

A3- (2-isocyanobenzyl) indole derivative 1ag has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1, except that 1H-indole was changed to 5-chloro-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (5-chloro-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (5-chloro-1H-indole-3-methyl) benzamide in step (4), and the product 1a was changed to 1ag 3- (2-isocyanobenzyl) -5-chloro-1-hydro-indole Brownsol, mp 112 ℃ 70%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.18(s,1H),7.44(s,1H),7.35(d,J＝6.9Hz,1H),7.27–7.19(m,4H),7.11(dd,J＝8.6,2.0Hz,1H),7.03(s,1H),4.16(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,137.3,134.8,129.9,129.6,128.4,127.2,127.0,126.0,125.3,124.6,122.6,118.4,112.5,112.4,28.0.IR(KBr)3281,3059,2924,2141,1625,1448,764cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ ClN ₂ [M-H] ^- 265.0538,found 265.0532。

Example 7

3- (2-isocyanobenzyl) indole derivative 1ah has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 5-bromo-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (5-bromo-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (5-bromo-1H-indole-3-methyl) benzamide in step (4), to give 1a as 1ah:3- (2-isocyanobenzyl) benzamide as a product) -5-bromo-1-hydro-indole. Brown solid, mp 111 ℃,60% yield; petroleum ether, etoac=5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.10(s,1H),7.50(d,J＝1.8Hz,1H),7.28–7.23(m,1H),7.18–7.06(m,5H),6.89(d,J＝2.4Hz,1H),4.04(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.2,137.3,135.1,129.9,129.6,129.0,127.3,127.0,126.0,125.1,124.4,121.5,112.9,112.4,28.0.IR(KBr)3395,3020,2922,2119,1663,1451,763cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ BrN ₂ [M-H] ^- 309.0033,found 309.0043。

Example 8

3- (2-isocyanobenzyl) indole derivative 1ai has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1, except that 1H-indole was changed to 6-methyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (6-methyl-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (6-methyl-1H-indole-3-methyl) benzamide in step (4), to give a product 1a as 1ai:3- (2-isocyanobenyl) -6-methyl-1-hydro-indole, brownsol, mp 97 ℃,65%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.08(s,1H),7.60(d,J＝8.1Hz,1H),7.50(dd,J＝7.5,1.7Hz,1H),7.42–7.27(m,3H),7.25(s,1H),7.14(d,J＝8.1Hz,1H),7.04(d,J＝2.4Hz,1H),4.39(s,2H),2.65(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.3,138.1,137.1,132.0,130.2,129.6,127.1,127.0,125.3,122.8,121.5,118.8,112.5,111.6,28.3,22.0.IR(KBr)3356,3057,2916,2854,2137,1626,769cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1085。

Example 9

3- (2-isocyanobenzyl) indole derivative 1aj has a structural formula shown in table 1.

The preparation of the compound is the same as in example 1 except that 1H-indole is changed to 6-chloro-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline is changed to 2- (6-chloro-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3) aniline in step (4)-methyl) benzamide to N- (-2- (6-chloro-1H-indole-3-methyl) benzamide, yielding product 1a to 1aj:3- (2-isocyanobenzyl) -6-chloro-1-hydro-indole, brown solid, mp 97 ℃,68% yield; petroleum ether, etoac=5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.13(s,1H),7.34(t,J＝8.6Hz,2H),7.28(d,J＝1.9Hz,1H),7.24–7.14(m,3H),7.02(dd,J＝8.4,1.8Hz,1H),6.96(d,J＝2.4Hz,1H),4.16(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,137.4,136.7,129.9,129.5,128.1,127.2,127.0,125.8,123.8,120.3,119.8,112.9,111.3.IR(KBr)3320,3070,2921,2141,1618,1408,771cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ ClN ₂ [M-H] ^- 265.0538,found 265.0532。

Example 10

A3- (2-isocyanobenzyl) indole derivative 1ak has a structural formula shown in table 1.

The preparation of the compound was carried out as in example 1, except that 1H-indole was changed to 7-methyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (7-methyl-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (7-methyl-1H-indole-3-methyl) benzamide in step (4), and the product 1a was changed to 1ak:3- (2-isocyanobenzyl) -7-methyl-1-hydro-indole, brownsol, mp 116 ℃,70%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.00(s,1H),7.36–7.31(m,2H),7.24–7.14(m,3H),7.04–6.96(m,3H),4.21(s,2H),2.44(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.1,137.9,136.0,130.1,129.5,127.0,126.9,126.8,122.9,122.8,120.6,119.9,116.7,113.2,28.2,16.7.IR(KBr)3318,3047,2924,2852,2143,1618,744cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1081。

Example 11

3- (2-isocyanobenzyl) indole derivative 1al has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1, and the preparation method of the compound is the same as in example 1, except that in the step (1), 2-aminobenzoic acidTo 3-chloro-2-aminobenzoic acid, 2-aminobenzyl alcohol in step (2) to 3-chloro-2-aminobenzyl alcohol, 2- (1H-indol-3-methyl) aniline in step (3) to 2- (1H-indol-3-methyl) -3-chloroaniline, and N- (-2- (1H-indol-3-methyl) benzamide in step (4) to N- (-2- (1H-indol-3-methyl) -3-chloro-benzamide to give the product 1a as 1al:3- (3-chloro-2-isocyanatobenzyl) -1-hydro-indole, brown sol, mp 61 ℃,60%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.02(s,1H),7.42(d,J＝8.0Hz,1H),7.24(d,J＝8.2Hz,1H),7.15–7.10(m,2H),7.06–7.02(m,1H),7.01–6.92(m,2H),6.88(d,J＝2.5Hz,1H),4.12(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ170.9,139.9,136.2,130.7,129.5,128.0,127.3,126.9,123.2,122.0,119.5,118.6,111.6,111.4,28.5.IR(KBr)3406,3058,2918,2119,1640,1353,747cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ ClN ₂ [M-H] ^- 265.0538,found 265.0537。

Example 12

A3- (2-isocyanobenzyl) indole derivative 1am has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1, except that in step (1), 2-aminobenzoic acid is changed to 3-methyl-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol is changed to 3-methyl-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline is changed to 2- (1H-indole-3-methyl) -3-methylaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide is changed to N- (-2- (1H-indole-3-methyl) -3-methyl-benzamide, to give the product 1a 1am:3- (3-methyl-2-isocyano benzyl) -1-hydro-indole, brown solid, mp ℃,66%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.01(s,1H),7.50(d,J＝7.9Hz,1H),7.35–7.27(m,1H),7.20–7.13(m,1H),7.12–7.03(m,4H),6.99(d,J＝2.4Hz,1H),4.20(s,2H),2.41(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ168.2,137.8,136.4,135.2,128.9,128.1,127.4,127.3,123.2,122.2,119.6,119.1,112.9,111.4,28.5,19.1.IR(KBr)3301,3041,2919,2850,2141,1619,742cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1097。

Example 13

A3- (2-isocyanobenzyl) indole derivative 1an has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1, except that in step (1), 2-aminobenzoic acid is changed to 4-fluoro-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol is changed to 4-fluoro-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline is changed to 2- (1H-indole-3-methyl) -4-fluoroaniline, in step (4), N- (-2- (1H-indole-3-methyl) benzamide is changed to N- (-2- (1H-indole-3-methyl) -4-fluoro-benzamide, to obtain the product 1a is changed to 1an:3- (4-fluoro-2-isocyanatobenzyl) -1-hydro-indole, brown solid, mp123 ℃,65%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.89(s,1H),7.35(d,J＝7.9Hz,1H),7.20(d,J＝8.2Hz,1H),7.10–6.90(m,4H),6.84(d,J＝2.4Hz,1H),6.80(m,1H),4.05(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.3,160.7,158.2,135.3,132.7(d,J＝3.9Hz),130.2(d,J＝8.6Hz),125.9,122.0,121.2,118.6,117.7,115.8(d,J＝20.9Hz),112.9(d,J＝25.4Hz),111.2,110.3,26.3. ¹⁹ F NMR(376MHz,CDCl ₃ )δ-114.17.IR(KBr)3392,3058,2918,2128,1639,1092,746cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ FN ₂ [M-H] ^- 249.0834,found 249.0823。

Example 14

3- (2-isocyanobenzyl) indole derivative 1ao has a structural formula shown in table 1.

The preparation method of the compound was the same as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 4-methyl-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 4-methyl-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -4-methylaniline, and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (1H-indole-3-methyl) -4-methyl-benzamide, in step (4), to obtain a product 1a was changed to 1ao:3- (4-methyl-2-isocyanobenzyl) room-17 } 1-hydro-indole, brown solid, mp 138 ℃,68% yield; petroleum ether, etoac=5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.95(s,1H),7.30–7.24(m,1H),7.10–7.05(m,3H),6.96(dd,J＝8.2,7.1Hz,1H),6.93–6.88(m,1H),6.75–6.66(m,2H),4.26(s,2H),2.33(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ164.9,137.8,136.0,129.7,128.7,128.4,125.8,125.5,124.8,122.6,121.3,120.0,111.6,108.2,28.5,18.6.IR(KBr)3330,3051,2921,2853,2138,1548,756cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084,found 245.1080。

Example 15

A3- (2-isocyanobenzyl) indole derivative 1ap has a structural formula shown in Table 1.

The preparation method of the compound is the same as in example 1, except that in step (1), 2-aminobenzoic acid is changed to 5-methyl-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol is changed to 5-methyl-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline is changed to 2- (1H-indole-3-methyl) -5-methylaniline, in step (4), N- (-2- (1H-indole-3-methyl) benzamide is changed to N- (-2- (1H-indole-3-methyl) -5-methyl-benzamide, to obtain the product 1a is changed to 1ap 3- (5-methyl-2-isocyanatobenzyl) -1-hydro-indole, brown sol, mp 91 ℃,66%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.22(s,1H),7.78(d,J＝7.8Hz,1H),7.49–7.46(m,1H),7.43–7.37(m,2H),7.36–7.31(m,1H),7.25(s,1H),7.14–7.09(m,2H),4.39(s,2H),2.37(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ165.5,140.1,137.8,136.6(d,J＝1.4Hz),130.8,127.9,127.4,126.9,123.6,123.4,122.3,119.7,119.1,112.9,111.7(d,J＝1.9Hz),28.2,21.5.IR(KBr)3347,3053,2908,2843,2141,1616,738cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M+H] ⁺ 247.1230,found 247.1227。

Example 16

3- (2-isocyanobenzyl) indole derivative 1aq has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1The preparation of the compound was carried out as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 5-fluoro-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 5-fluoro-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -5-fluoroaniline, in step (4), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (1H-indole-3-methyl) -5-fluoro-benzamide, to give the product 1a as 1aq:3- (5-fluoro-2-isocyanatobenzyl) -1-hydro-indole, brown solid, mp79 ℃,75%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.12(s,1H),7.51–7.43(m,1H),7.39–7.30(m,2H),7.25–7.16(m,1H),7.13–7.03(m,2H),6.88(m,2H),4.20(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.4,163.8,161.3,141.0(d,J＝8.1Hz),136.6,128.7(d,J＝9.1Hz),127.1,123.5,122.4,119.9,118.9,117.0(d,J＝23.8Hz),114.3(d,J＝23.6Hz),111.7,111.6,28.2. ¹⁹ F NMR(376MHz,CDCl ₃ )δ-108.28.IR(KBr)3445,3059,2922,2132,1615,1338,745cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ FN ₂ [M-H] ^- 249.0834,found 249.0823。

Example 17

1ar of 3- (2-isocyanobenzyl) indole derivative has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1, except that in step (1), 2-aminobenzoic acid is changed to 5-chloro-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol is changed to 5-chloro-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline is changed to 2- (1H-indole-3-methyl) -5-chloroaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide is changed to N- (-2- (1H-indole-3-methyl) -5-chloro-benzamide, to obtain the product 1a is changed to 1ar 3- (5-chloro-2-isocyanatobenzyl) -1-hydro-indole, brown solid, mp 60 ℃,70%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ8.12(s,1H),7.47(d,J＝6.8Hz,1H),7.36–7.32(m,1H),7.26(d,J＝8.4Hz,1H),7.22–7.19(m,2H),7.16–7.08(m,2H),7.02(d,J＝2.4Hz,1H),4.18(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ167.8,139.8,136.5,135.4,130.1,128.0,127.3,127.0,123.2,122.4,122.0,119.8,118.8,111.8,111.4,28.0.IR(KBr)3408,3086,2919,2126,1634,1342,746cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ ClN ₂ [M-H] ^- 265.0538,found 265.0541。

Example 18

3- (2-isocyanobenzyl) indole derivative 1as has a structural formula shown in table 1.

The preparation method of the compound is the same as in example 1, except that in step (1), 2-aminobenzoic acid is changed to 6-methyl-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol is changed to 6-methyl-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline is changed to 2- (1H-indole-3-methyl) -6-methylaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide is changed to N- (-2- (1H-indole-3-methyl) -6-methyl-benzamide, to give the product 1a 1as:3- (6-methyl-2-isocyano benzyl) -1-hydro-indole, brown solid, mp 116 ℃,67%yield;petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.88(s,1H),7.61(d,J＝7.7Hz,1H),7.28–7.22(m,2H),7.18–7.08(m,4H),6.52–6.46(m,1H),4.20(s,2H),2.25(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ165.4,138.9,136.4,136.1,131.5,127.3,127.0,125.0,122.2,122.0,119.5,118.8,112.8,111.3,25.5,19.9.IR(KBr)3397,3059,2923,2868,2124,1616,744cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M-H] ^- 245.1084, found245.1090. The structural formula and nuclear magnetic hydrogen spectrum and carbon spectrogram of 1as are shown in figures 3, 4 and 5 respectively.

Example 19

3- (2-isocyanobenzyl) indole derivative 1at has a structural formula shown in table 1.

The procedure for the preparation of this compound was as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 6-chloro-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 6-chloro-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -6-chloroaniline, and in step (4), N- (-2- (1H-indole-3-)Methyl) benzamide becomes N- (-2- (1H-indole-3-methyl) -6-chloro-benzamide, yielding product 1a becomes 1at:3- (6-chloro-2-isocyanobenzyl) -1-hydro-indole, brown solid, mp101 ℃,70%yield,petroleum ether:EtOAc =5:1. ¹ H NMR(400MHz,CDCl ₃ )δ7.86(s,1H),7.73–7.64(m,1H),7.31(dd,J＝8.1,1.3Hz,1H),7.18–7.12(m,2H),7.12–7.06(m,2H),7.00(t,J＝8.0Hz,1H),6.71–6.68(m,1H),4.30(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ167.6,136.3,136.3,135.7,131.1,128.0,127.8,127.2,126.1,123.0,122.2,119.7,119.2,111.7,111.5,26.4.IR(KBr)3417,3075,2923,2119,1638,1384,740cm ^-1 ；HRMS(ESI):calcd for C ₁₆ H ₁₁ ClN ₂ [M-H] ^- 265.0538,found 265.0536。

Example 20

A3- (2-isocyanobenzyl) indole derivative 1ab has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 4-methoxy-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (4-methoxy-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (4-methoxy-1H-indole-3-methyl) benzamide in step (4), to give a product 1a changed to 1ab:3- (2-isocyanobenzyl) -4-methoxy-1-hydro-indole, ¹ H NMR(400MHz,CDCl ₃ )δ8.23(s,1H),7.81–7.72(m,1H),7.53–7.47(m,1H),7.45–7.40(m,1H),7.37–7.27(m,3H),7.17(dd,J＝8.0,1.9Hz,1H),7.10(d,J＝2.5Hz,1H),4.40(s,2H),3.3(s,3H)。

Example 21

A3- (2-isocyanobenzyl) indole derivative 1au has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 6-cyano-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (5-cyano-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (5-cyano-1H-indole-3-methyl) benzamide in step (4), and 1a was changed to 1 au-3- (2-isocyanobenzyl) -5-cyano-1-hydro-indole as a product ¹ H NMR(400MHz,CDCl ₃ )δ8.12(s,1H),7.57(d,J＝7.4Hz,1H),7.43(d,J＝8.1Hz,2H),7.34–7.30(m,2H),7.29–7.23(m,2H),7.19–7.10(m,2H),4.31(s,2H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.3,137.8,136.4,130.0,129.4,127.2,127.0,126.9,123.0,122.2,119.6,119.0,112.8,111.3,28.1。

Example 22

3- (2-isocyanobenzyl) indole derivative 1av has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 7-fluoro-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (7-fluoro-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (7-fluoro-1H-indole-3-methyl) benzamide in step (4), to give 1a as 1ac:3- (2-isocyanobenzyl) -7-fluoro-1-hydro-indole. ¹ H NMR(400MHz,CDCl ₃ )δ7.80(s,1H),7.35(d,J＝8.0Hz,1H),7.20(d,J＝8.2Hz,1H),7.11–6.89(m,4H),6.87–6.74(m,2H),3.82(s,2H)。

Example 23

3- (2-isocyanobenzyl) indole derivative 1aw has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 6-bromo-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (6-bromo-1H-indole-3-methyl) aniline in step (3), and N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (6-bromo-1H-indole-3-methyl) benzamide in step (4), to give 1a as 1aw:3- (2-isocyanobenzyl) -6-bromo-1-hydro-indole. ¹ H NMR(400MHz,CDCl ₃ )δ8.06(s,1H),7.53(d,J＝1.7Hz,1H),7.36(dd,J＝13.0,7.7Hz,2H),7.29(dd,J＝8.1,1.4Hz,1H),7.25–7.16(m,3H),7.08–7.00(m,1H),4.21(s,2H)。

Example 24

A3- (2-isocyanobenzyl) indole derivative 1ax has a structural formula shown in table 1.

The preparation of the compound is the same as in example 1 except that 1H-indole is changed to 6-methoxy-1H-indole in step (2) and 2- (1H-indole-3-methyl) aniline is changed to 2- (6-methoxy) aniline in step (3)The N- (-2- (1H-indol-3-methyl) benzamide in step (4) was changed to N- (-2- (6-methoxy-1H-indol-3-methyl) benzamide to give the product 1a as 1ax 3- (2-isocyanobenzyl) -6-methoxy-1-hydro-indole). ¹ H NMR(400MHz,CDCl ₃ δ7.98(s,1H),7.36(d,J＝7.7Hz,1H),7.20(dd,J＝3.0,7.5,1.7Hz,1H),7.09(t,J＝8.0Hz,1H),6.97(d,J＝8.2Hz,1H),6.89(d,J＝2.4Hz,1H),6.46(d,J＝7.8Hz,2H),4.40(s,1H),3.82(s,3H)。

Example 25

3- (2-isocyanobenzyl) indole derivative 1ay has a structural formula shown in table 1.

The procedure for the preparation of this compound was as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 5-methoxy-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 5-methoxy-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -5-methoxyaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (1H-indole-3-methyl) -5-methoxy-benzamide to give product 1a as 1ay:3- (5-methoxy-2-isocyanatobenzyl) -1-hydro-indole ¹ H NMR(400MHz,CDCl ₃ )δ8.22(s,1H),7.78(d,J＝7.8Hz,1H),7.49–7.46(m,1H),7.43–7.37(m,2H),7.36–7.31(m,1H),7.25(s,1H),7.14–7.09(m,2H),4.39(s,2H),3.34(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ165.5,140.1,137.8,136.6(d,J＝1.4Hz),130.8,127.9,127.4,126.9,123.6,123.4,122.3,119.7,119.1,112.9,111.7,50.3,28.2。

Example 26

3- (2-isocyanobenzyl) indole derivative 1az has a structural formula shown in table 1.

The procedure for the preparation of this compound was as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 6-fluoro-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 6-fluoro-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -6-fluoroaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (1H-indole-3-methyl) benzamidePhenyl) -6-fluoro-benzamide, giving product 1a as 1az:3- (6-fluoro-2-isocyanobenzyl) -1-hydro-indole ¹ H NMR(400MHz,CDCl ₃ δ8.01(s,1H),7.74(d,J＝7.8Hz,1H),7.34(dt,J＝8.0,1.0Hz,1H),7.25–6.98(m,6H),4.26(dd,J＝1.9,0.9Hz,2H)。

Example 27

A3- (2-isocyanobenzyl) indole derivative 1aaa has a structural formula shown in table 1.

The procedure for the preparation of this compound was as in example 1, except that in step (1), 2-aminobenzoic acid was changed to 5-bromo-2-aminobenzoic acid, in step (2), 2-aminobenzyl alcohol was changed to 5-bromo-2-aminobenzyl alcohol, in step (3), 2- (1H-indole-3-methyl) aniline was changed to 2- (1H-indole-3-methyl) -5-bromoaniline, and in step (4), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (1H-indole-3-methyl) -bromo-benzamide, to give product 1a as 1ay:3- (5-bromo-2-isocyanobenzyl) -1-hydro-indole ¹ H NMR(400MHz,CDCl ₃ δ8.09(s,1H),7.49(dd,J＝7.9,1.1Hz,1H),7.41–7.33(m,3H),7.25–7.20(m,2H),7.15–7.07(m,2H),4.22(s,2H)。

Example 28

A3- (2-isocyanobenzyl) indole derivative 1aab has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1 except that 1H-indole was changed to 2-phenyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (2-phenyl-1H-indole-3-methyl) aniline in step (3), N- (-2- (1H-indole-3-methyl) benzamide was changed to N- (-2- (2-phenyl-1H-indole-3-methyl) benzamide in step (4) to give a product 1a was changed to 1aab 3- (2-isocyanobenzyl) -2-phenyl-1-hydro-indole, ¹ H NMR(400MHz,CDCl ₃ δ8.28(d,J＝8.4Hz,1H),7.40–7.33(m,5H),7.30–7.24(m,3H),7.23(dd,J＝1.6,0.7Hz,1H),7.21–7.16(m,3H),7.04–6.98(m,1H),4.03(s,2H)。

example 29

3- (2-isocyanobenzyl) indole derivative 1aac has a structural formula shown in table 1.

The preparation of the compound was the same as in example 1, except for the step (2) In (3) the 2- (1H-indole-3-methyl) aniline is changed into 2- (2-methyl-1H-indole-3-methyl) aniline, in (4) the N- (-2- (1H-indole-3-methyl) benzamide is changed into N- (-2- (2-methyl-1H-indole-3-methyl) benzamide to obtain the product 1a which is changed into 1aac 3- (2-isocyano benzyl) -2-methyl-1-hydrogen-indole, ¹ H NMR(400MHz,CDCl ₃ δ7.88(s,1H),7.41–7.36(m,1H),7.33–7.28(m,2H),7.22–7.16(m,2H),7.12(dd,J＝7.8,7.1,1.2Hz,1H),7.07–6.98(m,2H),4.18(s,2H),2.40(s,3H)。

example 30

3- (2-isocyanobenzyl) indole derivative 1aad has a structural formula shown in table 1.

The preparation of the compound was carried out in the same manner as in example 1, except that 1H-indole was changed to N-methyl-1H-indole in step (2), 2- (1H-indole-3-methyl) aniline was changed to 2- (N-methyl-1H-indole-3-methyl) aniline in step (3), and N- (N- (1H-indole-3-methyl) benzamide was changed to N- (-2- (2-methyl-1H-indole-3-methyl) benzamide in step (4), to give a 1aad:3- (2-isocyanobenzyl) -2-methyl-1-hydro-indole, yellow sol, mp 130 ℃,60%yield;petroleum ether:EtOAc =5:1 as the product. ¹ H NMR(400MHz,CDCl ₃ )δ7.48(d,J＝7.9Hz,1H),7.31(d,J＝7.6Hz,1H),7.27–7.16(m,4H),7.13(m,1H),7.09–7.03(m,1H),6.85(s,1H),4.18(s,2H),3.66(s,3H). ¹³ C NMR(100MHz,CDCl ₃ )δ166.6,138.1,137.3,130.1,129.5,127.8,127.8,127.0,126.9,121.9,119.2,119.2,111.3,109.5,32.8,28.0.IR(KBr)3052,2922,2851,2119,1602,1491,740cm ^-1 ；HRMS(ESI):calcd for C ₁₇ H ₁₄ N ₂ [M+H] ⁺ 247.1230,found247.1222。

Table 1 3- (2-isocyanobenzyl) indole derivatives of formula

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Application examples

Experimental material and equipment

Screening for Activity of Violet bacillus CV026 (report indicating Strain)

Picking CV026 single colony, culturing in LB liquid medium at 28deg.C and 180rpm for 17 hr, and regulating bacterial liquid OD ₆₂₀ =0.1. 100mL of LB solid medium was poured into a petri dish uniformly as a bottom plate. 1mL of the culture broth was inoculated into 100mL of LB solid medium (cooled to 50 ℃ C.) at an inoculum size of 1%, followed by addition of kanamycin (20. Mu.g/mL) and signal molecule C6-HSL (5. Mu.M), and after mixing, the mixture was poured onto a bottom plate uniformly to serve as an upper plate. After the medium had solidified, 5. Mu.L of the compound was spotted on the upper plate, and incubated at 28℃for 24 hours with methanol as a negative control, and the change in the color of the medium was observed. Each set of experiments was repeated 3 times.

(II) preliminary screening for quorum sensing inhibition activity of pseudomonas aeruginosa

The inhibitory effect of 40 isonitrile compounds on pyocin was quantitatively determined by inoculating 0.1% overnight of a culture of pseudomonas aeruginosa in LB broth with barley malt base as positive control and dimethyl sulfoxide (DMSO) as negative control. After incubation at 37℃for 24 hours, the change in colour of the pyocin was observed.

Determination of Minimum Inhibitory Concentration (MIC)

MIC determination of isonitrile compound against pseudomonas aeruginosa was performed according to the method set forth by the standard of clinical laboratory council (CLSI, 2015). Pseudomonas aeruginosa single colonies were picked and inoculated into 5mL of Luria-Bertani broth (Sangon Biotech, shanghai, china) for activation for 17h as a mother liquor. Adding appropriate amount of mother solution into LB culture medium, and adjusting OD ₆₂₀ =0.05, at which time the bacterial concentration in the medium was about 1.5×10 ^{^5} CFU/mL. Adding isonitrile compound, and adopting double dilution method to make gradient dilution, and DMSO is used as negative control. 200 mu L of each concentration is added into a 96-well plate, cultured for 24 hours at 37 ℃ and 150rpm, and OD is measured by an enzyme-labeled instrument ₆₂₀ . Three replicates were performed in the experiment.

Determination of pyocin

Picking single bacterial colony of pseudomonas aeruginosa PAO1 to 5mL LB culture mediumIs activated for 17 hours and used as seed liquid. Seed solution was inoculated into new LB medium at 0.1% inoculum size, different concentrations of isonitrile compound were added, DMSO as negative control, and barley malt alkali as positive control. The cells were incubated at 37℃for 17 hours at 180 rpm. The culture broth was filtered through a 0.22 μm sterile filter to give a sterile filtrate for use. Determination of the content of pyocin: extracting the supernatant with chloroform (5/3, v/v), standing for layering, collecting lower organic phase, adding 1mL of 0.2M HCl, mixing, centrifuging at 4deg.C and 10000rpm for 10min, collecting 200 μL of upper solution, and measuring OD by enzyme-labeling instrument ₅₂₀ 。

Inhibition by coating

Reference is made to the method of Damiano et al and modifications are made. And (3) picking a single bacterial colony of pseudomonas aeruginosa, and activating the single bacterial colony in 5mL of LB culture medium for 17 hours to obtain seed liquid. 50. Mu.L of seed solution was added to 50mL of TSB medium (containing 1% glycerol) at a ratio of 1:1000, and different concentrations of isonitrile compound and isonitrile compound were added, DMSO as a negative control. 200. Mu.L of the mixed culture solution was added to a 96-well plate, and the mixture was allowed to stand at 37℃for 24 hours. The upper culture solution was aspirated, washed 3 times with PBS, the planktonic bacteria were washed off, dried in an oven at 60℃for moisture, and fixed with 200. Mu.L of methanol for 15min. The methanol was sucked off and dried in an oven at 60 ℃. mu.L of 0.05% crystal violet was added for 15min. Absorbing crystal violet, washing with PBS for 3 times, removing excessive unattached crystal violet dye, and drying in a 60 ℃ oven. After drying, 200. Mu.L of 95% ethanol was added and decolorized in a shaker at 37℃and 180rpm for 15min. Taking 150 mu L of decolorized solution, and measuring OD by using an enzyme-labeled instrument ₅₇₀ 。

(III) initial screening of serratia marcescens quorum sensing activity:

picking NJ01 single colony into LB liquid culture medium, culturing at 28deg.C and 180rpm for 17 hr, and regulating bacterial liquid OD ₆₂₀ =0.1. 100mL of LB solid medium was poured into a petri dish uniformly as a bottom plate. 1mL of the culture solution was inoculated into 100mL of LB solid medium (cooled to 50 ℃ C.) at an inoculum size of 1%, and the mixture was uniformly poured onto the bottom plate as the upper plate. After the culture medium was solidified, 5. Mu.L of the isonitrile compound organism was spotted on an upper plate, DMSO was used as a negative control, and the culture medium was incubated at 28℃for 24 hours to observe the change in color of the culture medium. Each set of experiments was repeated 3 times.

Determination of Minimum Inhibitory Concentration (MIC), method operation is referred to above.

Determination of prodigiosin

Inoculating S.marcescens NJ01 single colony into fresh LB culture medium, 28 ℃,180rpm, overnight, regulating OD of bacterial liquid ₆₀₀ =0.4, added to 2mL of LB medium at a ratio of 1%, to which 50 μg/mL of isonitrile compound was added, blank control; DMSO is used as a negative control, and 50 mug/mL of isonitrile compound is used as a positive control. Culturing at 28deg.C and 180rpm for 16-24 hr, sucking 1mL of each group of bacterial liquid into centrifuge tube, centrifuging at 10000rpm for 10min, collecting supernatant for use (or filtering with 0.22 μm sterile filter head to obtain sterile filtrate), adding 1mL of acidified ethanol (4% ethanol solution containing 1M HCl) into centrifuge tube, centrifuging at 10000rpm for 10min, transferring supernatant into 96-well plate, and measuring OD ₅₃₄ 。

Inhibition and destruction of the coating, the procedure is described above.

And (5) performing cytotoxicity experiments.

(IV) results and statistical analysis

Data are expressed as mean ± standard deviation. Prior to analysis, the variance homogeneity test of Levene was used to evaluate the equivalence of all variables. Statistical differences were determined by the level test, analysis of variance (ANOVA) and Tukey-Kramer test. Statistical analysis was performed using Graphpad Prism. p is less than or equal to 0.05, and has statistical significance.

Results of the inhibition of Pseudomonas aeruginosa PAO1 by the compounds of Table 2

Note that: the concentration of the compound is unified as follows: 0.78 μg/mL.

1. The MIC of all compounds was around 50. Mu.g/mL. The primary screening result of the induction activity of the compound on the pseudomonas aeruginosa population is shown in figure 1 and figure 2. The inductive activity of the whole population is obvious.

2 from the data in the table, the inhibition of the film of the compound 1as on Pseudomonas aeruginosa is as high as 75%. Is the best compound. The active compounds are preferably present in relatively large amounts. The following are provided:

3. a series of quorum sensing phenotypic characterization is performed with the optimal compound, which fully demonstrates that the compound has good quorum sensing activity.

Further study of 4 quorum sensing showed that: phenotypic characterization of compounds for response to Serratia marcescens populations demonstrated: FIG. 18 shows the results of a preliminary screening of Serratia marcescens for a series of compounds. Preliminary results indicate that compound 1as is superior in activity.

(1) Growth curve

The growth curve of Serratia marcescens is shown in figure 6. The growth curve of the compound humanized Serratia marcescens is shown in FIG. 14. It was demonstrated that at compound concentrations below 1.56ug/mL, DMSO was the control group, without affecting bacterial growth.

(2) The film inhibition chart shows that the film inhibition rate is as high as 65% when the concentration of the compound is 1.56 mug/mL, and the effect is obvious. Wherein RSV is a positive control and DMSO is a negative control.

(3) The figure of the inhibition of the compound to the serratia marcescens is shown in figure 7, and the figure of the frozen scanning electron microscope of the inhibition of the compound to the serratia marcescens is shown in figure 8. FIG. 9 is a graph showing the damage of the compound to Serratia marcescens envelope. The SEM (cryoscanning electron microscope) with suppressed film damage effect is good. At a compound concentration of 1.56 μg/mL, the membrane destruction rate was as high as 40%, where RSV (vanillin) was the positive control and DMSO was the negative control.

(4) Toxicity factor experiment: FIG. 10 is a graph showing the results of toxicity factor experiments of compounds against Serratia marcescens. Catalase, protease, extracellular polysaccharide and mouse Li Tangzhi, and has good inhibition effect.

(5) Electrophoresis experiments, and FIG. 11 is a diagram showing the results of electrophoresis experiments of compounds against Serratia marcescens. The results demonstrate that the gradient and effect are very pronounced.

(6) Cytotoxicity experiments the results of the cytotoxicity experiments of the compounds against Serratia marcescens are shown in FIG. 12.IC (integrated circuit) ₅₀ Compound toxicity was relatively low =47.21 μm,

(7) The results of the compound nematode experiments on Serratia marcescens are shown in FIG. 13. Nematode experiments further illustrate the toxicity problem of compounds.

Phenotypic characterization of compounds induced to human Serratia marcescens populations demonstrated:

growth curve graph of human Serratia marcescens

(1) Growth curves, demonstrating that at compound concentrations below 1.56ug/mL, bacterial growth was not affected, with DMSO being the control.

(2) The inhibition of human Serratia marcescens by the compounds is shown in FIG. 15. The membrane disruption pattern of human Serratia marcescens is shown in FIG. 16. The inhibition effect of the coating is obvious and reaches 50%, and the effect is high. The film failure chart shows that the film failure rate is as high as 62%, which is obvious.

(3) Virulence factor graph: FIG. 17 is a graph showing virulence factors of the humanized Serratia marcescens. The extracellular polysaccharide, mouse Li Tangzhi and protease are measured with good effect.

Claims

1. A 3- (2-isocyanobenzyl) indole derivative, characterized by the following structural formula:

wherein the substituents R ¹ =one of Me, OMe, F, cl, br, CN, R ² =one of H, OMe, F, cl, br, CN, ph, R ¹ Is substituent at any position of benzene ring, R ² Is substituent at any position on indole ring.

2. A process for the preparation of a 3- (2-isocyanobenzyl) indole derivative according to claim 1, comprising the steps of:

(2) Adding a compound 3 and a catalyst, and reacting to obtain a product 4;

the reaction formula is as follows:

3. the method for preparing 3- (2-isocyanobenzyl) indole derivatives according to claim 2, wherein the reaction in the step (1) is anhydrous and anaerobic, the reaction solvent is tetrahydrofuran, the reaction temperature is 0-40 ℃, and the reaction time is 6-12 h.

4. The method for preparing 3- (2-isocyanobenzyl) indole derivatives according to claim 2, wherein the reaction in the step (2) is an anhydrous and anaerobic reaction, the reaction solvent is 1, 2-dichloroethane, the catalyst is trifluoroacetic acid, the reaction temperature is 40-50 ℃, and the reaction time is 8-12 hours.

5. The process for producing 3- (2-isocyanobenzyl) indole derivatives according to claim 2, wherein the solvent used in the reaction in step (3) is tetrahydrofuran, the reaction temperature is 20 to 40 ℃ and the reaction time is 6 to 8 hours.

6. The process for preparing 3- (2-isocyanobenzyl) indole derivatives according to claim 2, wherein the solvent used in the reaction in the step (4) is methylene dichloride, the catalyst is triethylamine, the reaction temperature is-10 to 0 ℃, and the reaction time is 3 to 5 hours.

7. Use of a 3- (2-isocyanobenzyl) indole derivative according to claim 1 for the preparation of quorum sensing inhibitors of pseudomonas aeruginosa, serratia marcescens and violacein.