Preparation method of sulfur-containing marine natural product Sunshiinamide

Abstract

The invention belongs to the technical field of chemical synthesis, and particularly relates to a novel preparation method of a sulfur-containing marine natural product Sunshiinamide. A series of Sunshiinamide analogues can be conveniently synthesized on the basis of a large number of experiments and by adjusting reagents used in related reactions, and compared with an existing method, the method has the remarkable difference that a chiral compound 5a does not need hydroxyl protection and chiral prosthetic group removal operation and then is linked with an amino acid fragment through subsequent condensation reaction, and the chiral compound 5a can be used for preparing the Sunshiinamide analogues. But can be directly used for ammonolysis reaction with amino acid fragments; the macrocyclization of the cyclic lipopeptide is not prepared through macrocyclic lactamization, but is prepared through macrocyclic lactonization; before the macrocyclization reaction of the key cyclic lipopeptide, an intramolecular disulfide bond is firstly constructed, an 8-membered ring intermediate is formed, and two Trt protecting groups are removed, so that the molecular weight of a macrocyclization precursor is reduced, and the molecular weight of a key macrocyclization product is not obviously reduced, thereby being beneficial to improving the efficiency. The invention is beneficial to the structure-function relationship or related biological research of the series of Sunshiinamide analogues.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis, and in particular relates to a new method for preparing a sulfur-containing marine natural product, Sunshinamide. Background Art

[0002] According to records, the ocean covers 70% of the earth's surface, and marine species account for about 50% of the total global biodiversity. At present, more than 1 million species of marine invertebrates and more than 25,000 species of fish have been discovered, and more than 30,000 natural active molecules have been isolated from marine organisms [Mar. Drugs 2018, 16, 41]. Because marine organisms live in a special environment of high salt, high pressure, low oxygen, low temperature and lack of sunlight, compared with natural products from land, marine natural products often show different structures or unique pharmacological activities. In recent years, more and more pharmacological studies have shown that secondary metabolites of marine organisms may have unique mechanisms of action in antibacterial, antiviral, antiparasitic, analgesic and anticancer activities, and it is expected that marine drugs with novel mechanisms of action will be developed, which has attracted more and more interest from scientists. As of 2021, about 49 marine natural products or their derivatives have been approved for marketing or entered clinical trials worldwide, and 11 marine drugs have been approved by European and American drug regulatory authorities, of which 4 are listed as anticancer drugs.

[0003] In 2018, Piel's research team discovered the marine spirochete Gynuella sunshinyii YC6258 from the Gram-negative bacteria of the halophilic plant moss rhizomes based on the identification method of genome sequencing. Through bioengineering methods, they produced and isolated multiple natural products, one of which is the disulfide-bonded cyclic dipeptide Sunshinamide (Angew. Chem. Int. Ed. 2018, 57, 14519–14523). Its structural characteristics are: it contains a 3-hydroxydecanoic acid side chain structural unit, a 13-membered cyclic peptide lipid structure, and a bridge ring molecule containing two adjacent cysteines bridged by a disulfide bond. This compound has strong cytotoxic activity against HeLa cells, IC 50 The value is 0.59μM, which is a novel anti-tumor lead.

[0004] In 2020, Goswami's research team reported its total synthesis method for the first time (Org.Lett.2020,22,1188-1192), determined its absolute stereoconfiguration, and evaluated its cytotoxicity on different human cancerous and non-cancerous cell lines. The results suggested that Sunshinamide is a new marine natural product with anti-tumor activity worthy of study.

[0005] In the synthetic method of Goswami's research team (Picture 2 ), firstly, octanal and chiral auxiliary (R)-1-(4-benzyl-2-thiothiazolidin-3-yl)ethanone were used to generate aldol diastereoisomers (dr=7:3) with 70% yield and stereoselectivity under TiCl4 / DIPEA low temperature conditions. The two diastereomers could be separated by silica gel column chromatography, and the absolute configurations of the two diastereomers (5a and 5b) were determined by the Mosher ester method, and then 5a with the desired configuration was further converted into the key intermediate 7a. Next, dipeptide compound 9 was obtained from L-cysteine ​​and D-phenylalanine using EDCI / HOBT / DIPEA as condensing agents with a yield of 86%. After removing the Fmoc protecting group of 9 with an organic base, it was condensed with the side chain 7a using HATU / HOAT / DIPEA as condensing agents to prepare tripeptide compound 10. Compound 10 was treated with TBAF to expose the alcoholic hydroxyl group (11). The hydroxyl group was then esterified with protected cysteine ​​and the product was prepared in the presence of MNBA (2-methyl-6-nitrobenzoic acid). Anhydride) / / DMAP / Et3N, it was esterified with protected cysteine ​​to obtain linear intermediate 12; 12 was deprotected from Boc and tert-butyl ester groups with TFA, followed by macrolactamization, using HATU / HOAt / DIPEA as condensing agents, to prepare macrolactam 13 with a yield of 72%; finally, 13 under I2 / MeOH-CH2Cl2 conditions under which intramolecular disulfide bonds were formed to obtain Sunshinamide with a yield of 90%.

[0006] In the first total synthesis of Sunshinamide, the macrocyclization seems to have a high yield, but the efficiency is not high for three reasons: first, the macrocyclic lactamization reaction needs to be carried out at an extremely dilute concentration, and the resulting macrocyclic lactamization still carries a large thiol protecting group Trt. When the protecting group is removed and a disulfide bond is formed, the molecular weight of the final product decreases sharply; second, the key intermediate 7a requires hydroxyl protection and de-chiralization of the chiral compound 5a, which is rather cumbersome; third, it is not convenient to synthesize Sunshinamide analogs in a diversified manner.

[0007] In addition, although Sunshinamide has a novel molecular structure and significant potential anti-tumor uses, the anti-tumor target of Sunshinamide has not yet been elucidated, and the structure-activity relationship research of its anti-tumor effect is still insufficient.

[0008] Based on the current status of the prior art, the inventors of the present application intend to provide a novel, concise, reliable and convenient new synthesis method for studying the structure-activity relationship of sunshinamide. Summary of the invention

[0009] The purpose of the present invention is to provide a novel, concise and reliable method for preparing Sunshinamide based on the current state of the art and the defects of the prior art synthetic routes. The present invention is based on a large number of experiments and by adjusting the reagents used in the relevant reactions, which can be conveniently synthesized into a series of Sunshinamide analogs, thereby facilitating their structure-activity relationship or related biological research. The synthetic route is as follows: Picture 3 shown.

[0010] Compared with the existing methods, the present invention is significantly different in that: 1. The chiral compound 5a does not need to be protected by hydroxyl groups, removed from the chiral auxiliary group, and then subjected to a subsequent condensation reaction with the amino acid fragment for linkage, but can be directly used for an aminolysis reaction with the amino acid fragment; 2. The macrocyclization of the cyclolipopeptide is not prepared by macrolactamization, but by macrolactonization; 3. Before the macrocyclization reaction of the key cyclolipopeptide, an intramolecular disulfide bond is first constructed to form an 8-membered ring intermediate, and two Trt protecting groups are removed, thereby reducing the molecular weight of the macrocyclization precursor, so that the molecular weight of the key macrocyclization product does not decrease significantly, thereby being beneficial to improving efficiency.

[0011] Specifically, the purpose of the present invention can be achieved by the above-mentioned synthetic route ( Picture 3 ). Using L-cysteine ​​as a starting material, in the first step, the thiol group of L-cysteine ​​is protected to obtain compound 15; subsequently, the amino group or carboxyl group thereof is protected to obtain compounds 16 and 17; in the condensation reaction of the fourth step, the two protected cysteines are connected through an amide bond to obtain compound 18; in the fifth step, compound 18 is deaminated and then condensed with D-amino-protected phenylalanine to obtain compound 19; in the sixth step, compound 19 is directly deaminated from the thiol protecting group and a disulfide bond is formed in the molecule to obtain compound 20; then, in the seventh step, compound 20 is deaminated from the protecting group to obtain free amine compound 21; in the eighth step, compound 21 is directly subjected to an aminolysis reaction with a chiral intermediate 5a to obtain compound 22; in the ninth step, the ester group of compound 22 is selectively hydrolyzed to obtain a carboxylic acid compound 23, and then compound 23 is macrolactonized to complete the preparation of Sunshinamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Picture 1 Molecular structure of sunshinamide.

[0013] Picture 2 The first total synthetic route to Sunshinamide.

[0014] Picture 3 The synthetic route of Sunshinamide in the present invention. DETAILED DESCRIPTION

[0015] The present invention is further illustrated by the following examples, but is not intended to be limiting of the present invention.

[0016] Example 1

[0017] Step 1: Preparation of compound 15

[0018]

[0019] Take a clean, dry 250 mL single-necked round-bottom flask, fill it with argon, add L-cysteine ​​14 (12.1 g, 100.0 mmol, 1.0 equiv) and triphenylmethanol (31.2 g, 120.0 mmol, 1.2 equiv), then add 100 mL of trifluoroacetic acid in an ice-salt bath at 0°C, and stir for 15 minutes. Warm to room temperature and continue stirring for 6 hours.

[0020] The reaction solution was concentrated under reduced pressure to remove most of the trifluoroacetic acid. Under 0°C ice-salt bath conditions, sodium hydroxide (4N) solution was slowly added dropwise to adjust the pH to 4-5, and a small amount of white solid was precipitated. After adding 100mL of ether to dissolve and dilute, the pH was adjusted to 5-6 with 10% sodium acetate solution, and the liquids were separated. The aqueous phase was extracted with ether (100mL×2), and the organic phases were combined, washed with 200mL of saturated brine, and dried over anhydrous sodium sulfate overnight. The anhydrous sodium sulfate was filtered, and the filtrate was taken and distilled under reduced pressure to obtain 31.4g of crude white solid powder, with a yield of 86%.

[0021] R f =0.40(PE / EA1 / 1); 1 HNMR(400MHz,DMSO-d6)δ7.32(m,15H),3.17(s,1H,C H NH2),2.55(m,1H,C H 2S),2.52–2.48(m,1H,C H 2S).

[0022] Step 2: Preparation of compound 16

[0023]

[0024] Take a clean and dry 250mL single-necked round-bottom flask, fill it with argon, add S-Trt-L-cysteine ​​15 (7.2g, 19.8mmol, 1.0equiv), add 50mL methanol and stir to dissolve. In an ice-salt bath at 0℃, slowly add dichlorothionyl (7.1mL, 99.0mmol, 5.0equiv) with a syringe, then slowly raise the temperature to reflux, and stir under reflux for 6h.

[0025] The reaction solution was concentrated under reduced pressure to remove most of the dichlorothionyl. 100 mL of ethyl acetate was added to dissolve the residue, and the organic phase was washed with saturated sodium bicarbonate (100 mL × 3), and then washed with saturated sodium chloride aqueous solution (100 mL), dried over anhydrous sodium sulfate overnight, filtered the anhydrous sodium sulfate, and the filtrate was distilled under reduced pressure to obtain 5.6 g of a white viscous liquid with a yield of 76%.

[0026] R f =0.45(PE / EA2 / 1); 1 H NMR (400MHz, CDC13) δ7.40 (d, J=7.6Hz, 5H), 7.29–7.12 (m, 10H), 3.62 (s, 3H, CH3), 3.18 (m, 1H, C H NH2),2.57(dd,J=12.4,4.6Hz,1H,C H 2S),2.45(dd,J=12.5,7.6Hz,1H,C H 2S).

[0027] Step 3: Preparation of compound 17

[0028]

[0029] Take a clean, dry 250 mL single-necked round-bottom flask, fill it with argon, add triphenylmethylthio-L-cysteine ​​15 (7.2 g, 19.8 mmol, 1.0 equiv), add 50 mL of 2M sodium hydroxide aqueous solution and 100 mL of 1,4-dioxane, stir and dissolve. In an ice-salt bath at 0 ° C, slowly add di-tert-butyl dicarbonate (5.4 mL, 23.7 mmol, 1.2 equiv) with a syringe, and stir at room temperature for 8 h.

[0030] The reaction solution was concentrated under reduced pressure to remove most of 1,4-dioxane, and 50 mL of ethyl acetate was added to dilute it. Sodium sulfite was slowly added dropwise to adjust the pH value to 2-3. The aqueous phase was extracted with ethyl acetate (100 mL×3). The organic phases were combined and washed with saturated brine (100 mL), dried over anhydrous sodium sulfate overnight, and the anhydrous sodium sulfate was filtered. The filtrate was taken and distilled under reduced pressure to obtain 7.7 g of a white viscous liquid with a yield of 84%.

[0031] R f =0.48(PE / EA 2 / 1); 1 H NMR (400MHz, CDC13): δl.46(s,9H),2.69(br,2H,C H 2S),4.21(s,1H,C H NH),4.97(s,1H,CHN H),7.20-7.44(m,15H),10.2(br,1H).

[0032] Step 4: Preparation of compound 18

[0033] [methylN-(N-(tert-butoxycarbonyl)-S-trityl-L-cysteinyl)-S-trityl-L-cysteinate]

[0034]

[0035] Take a clean and dry 250mL single-necked round-bottom flask, fill it with argon protection, add triphenylmethylthio-Boc-L-cysteine ​​17 (5.5g, 11.8mmol, 1.2equiv), condensation agent TBTU (3.8g, 11.8mmol, 1.2equiv) in sequence, add anhydrous dichloromethane (50mL), N,N-dimethylformamide (DMF) (5mL) and stir to suspend.

[0036] Take another 100mL single-necked round-bottom flask, add triphenylmethylthio-L-cysteine ​​methyl ester 16 (3.7g, 9.8mmol, 1.0equiv), and add anhydrous dichloromethane (50mL) to dissolve. In an ice-salt bath at 0℃, slowly add the solution to the above reaction suspension with a syringe, and continue stirring for 15 minutes. Subsequently, add triethylamine (2.0mL, 14.7mmol, 1.5equiv) to the reaction solution with a syringe, and stir for 4h after warming to room temperature.

[0037] 5 mL of saturated ammonium chloride was added dropwise to the reaction solution to quench the reaction. Water was added to wash the reaction solution (100 mL × 2), and the organic phase was continued to be washed with saturated brine (100 mL). Drying was done over anhydrous sodium sulfate overnight, and the anhydrous sodium sulfate was filtered. The filtrate was taken and distilled under reduced pressure to obtain a light yellow-brown viscous liquid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, PE:EA=8:1-5:1), and the eluent was concentrated under reduced pressure to obtain 6.37 g of a white foamy solid with a yield of 79%.

[0038] R f =0.48(PE / EA4 / 1); 1 H NMR (600MHz, CDC13) δ7.47(m,5H,ArH),7.40(m,5H,ArH),7.35–7.27(m,15H,ArH),7.23(m,5H,,ArH),6.65(brs,1H,N H CHCO2Me),4.84(brs,1H,N HBoc),4.50(dt,J=7.4,5.3Hz,1H,C H CO2Me),3.95(brs,1H,C H NHBoc),3.68(s,3H,OCH3),2.77(brs,1H,C H 2S),2.69(dd,J=12.1,5.0Hz,1H,C H 2S),2.63–2.53(m,2H,C H 2S),1.45(s,9H,OC Me 3). 13 C NMR (CDCl3, 150MHz): δ169.7( C O),169.5( C O), 154.6(NH C O2CMe3),143.6( C Ar ),128.9( C H Ar ),128.8( C H Ar ),127.4( C H Ar ),127.3( C H Ar ),126.3( C H Ar ),126.2( C H Ar ),79.7(O C Me3),66.9( C Ph3),66.5( C Ph3),52.8( C HNHCO2CMe3,rotamer),52.7( C HNHCO2CMe3,rotamer),51.9( C HNHCO),50.7(O Me ),33.2( C H2S),33.0( C H2S),27.6(OC Me 3).

[0039] Step 5: Preparation of compound 19

[0040]

[0041] Take a clean and dry 100mL single-mouth round-bottom flask, add compound 18 (2.0g, 2.4mmol, 1.0equiv), dissolve it with 5% trifluoroacetic acid / dichloromethane solution, and stir at room temperature for 2h. The reaction solution was concentrated under reduced pressure to remove most of the trifluoroacetic acid, and 50mL of dichloromethane was added to dissolve it, and saturated sodium bicarbonate was added to adjust the pH to 7-8. After separation, the aqueous phase was extracted with dichloromethane (50mL×2). The organic phases were combined and washed with saturated sodium chloride (150mL). Drying with anhydrous sodium sulfate overnight, filtering the anhydrous sodium sulfate, and taking the filtrate under reduced pressure distillation to obtain a white foamy solid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, PE:EA=4:1-1:1), and the eluent was concentrated under reduced pressure to obtain a white foamy solid, that is, 6.37g of the free amine compound with a yield of 79%.

[0042] R f =0.68 (DCM / MeOH 20 / 1); 1 HNMR(400MHz,CDC13)δ7.59(d,J=8.1Hz,2H,N H 2),7.44(m,5H,ArH),7.34(m,5H,ArH),7.23(m,20H,,ArH),4.45(m,1H,C H CO2Me),4.12(m,1H,N H CHCO2Me),3.65(s,3H,OCH3),2.96(m,1H,,C H NH2),2.71(m,1H,C H 2S),2.54(m,3H,C H 2S). 13 CNMR (150MHz, CDC13) δ172.7( C O),170.7( C O),144.5( C Ar ),144.3( C Ar ),144.0( C Ar ),129.6( C H Ar ),129.4( C H Ar ),128.1( C H Ar ),128.0( C H Ar ),127.9( C H Ar ),127.0( C HAr ),126.8( C H Ar ),67.0( C Ph3),66.6( C Ph3),53.9( C HNH2),52.5( C HNHCO),50.87(O Me ),37.1( C H2S),33.8( C H2S).

[0043] Take a clean and dry 250mL single-necked round-bottom flask, fill it with argon protection, add Boc-D-phenylalanine (3.0g, 11.3mmol, 1.2equiv), condensation agent TBTU (3.6g, 11.3mmol, 1.2equiv) in sequence, add anhydrous dichloromethane (50mL), and N,N-dimethylformamide (DMF, 5mL) and stir to form a suspension.

[0044] Take another 100mL single-mouth round-bottom flask, add the free amine compound (6.7g, 9.3mmol, 1.0equiv), and add anhydrous dichloromethane (50mL) to dissolve. In an ice-salt bath at 0℃, slowly add the solution to the reaction suspension with a syringe and continue stirring for 15 minutes. Subsequently, add triethylamine (1.4mL, 10.0mmol, 1.5equiv) with a syringe, warm to room temperature and stir for 4h. Add 5mL of saturated ammonium chloride to the reaction solution to quench the reaction. Add water (100mL×2) to wash the reaction solution, and continue to wash the organic phase with saturated brine (100mL). Dry over anhydrous sodium sulfate overnight, filter the anhydrous sodium sulfate, and take the filtrate and distill under reduced pressure to obtain a light yellow-brown viscous liquid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, PE:EA=10:1-5:1), and the eluate was concentrated under reduced pressure to obtain 7.57 g of white foamy solid, namely compound 19, with a yield of 84%.

[0045] R f =0.40(PE / EA4 / 1); 1 HNMR(600MHz,CDCl3)δ7.35(m,5H,ArH),7.31(m,5H,ArH),7.22(m,15H,ArH),7.16-7.07(m,5H,ArH),6.39(brs,1H,N H CHCO2Me),6.18(brs,1H,N H CO),4.85(brs,1H,N H Boc),4.32(m,1H,C HCO2Me),4.18(m,1H,C H CO),3.86(m,1H,C H NHBoc,3.56(s,3H,OC H 3),3.04(dd,J=14.3,6.6Hz,1H,C H 2Ph),2.88(d,J=11.3Hz,1H,,C H 2Ph),2.58–2.49(m,3H,C H 2S),2.39(s,1H,C H 2S),1.28(s,9H,OC Me 3). 13 CNMR(CDCl3,150MHz):δ170.9( C O),170.1( C O),169.3( C O),155.2(NH C O2CMe3),144.3( C Ar ),144.2( C Ar ),136.5( C Ar ),129.6( C H Ar ),129.5( C H Ar ),129.2( C H Ar ),128.6( C H Ar ),128.1( C H Ar ),128.0( C H Ar ),126.9( C H Ar ),126.8( C H Ar ),80.2(O C Me3),67.1( C Ph3),66.9( C Ph3),55.9( C HNHCO2CMe3),52.4( C HNHCO),52.0( C HNHCO),51.5(O Me ),38.2( C H2S),33.4( C H2Ph),33.3(C H2S),28.2(OC Me 3).

[0046] Step 6: Preparation of compound 20

[0047]

[0048] Take a clean, dry 500mL single-necked round-bottom flask, add iodine (I2) (2.5g, 10.0mmol, 5.0equiv), and dissolve it with dichloromethane / methanol (850mL / 50mL). Dissolve compound 19 (2.0g, 2.0mmol, 1.0equiv) in 50mL dichloromethane, and slowly add it dropwise to the above iodine solution using a constant pressure drip funnel (5min), and stir at room temperature for 1h.

[0049] 50 mL of 5% sodium thiosulfate was added to the reaction solution to quench the reaction, and the reaction solution changed from dark brown to white. After separation, the aqueous phase was extracted with dichloromethane (50 mL × 2), and the organic phase was washed with saturated sodium bicarbonate (300 mL × 2) and saturated brine (500 mL) in sequence. Drying with anhydrous sodium sulfate overnight, filtering anhydrous sodium sulfate, and taking the filtrate under reduced pressure distillation to obtain a white powdery solid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel DCM: EA: MeOH = 40: 10: 1), and the eluent was concentrated under reduced pressure to obtain 492 mg of a white powdery solid, with a yield of 54%.

[0050] R f =0.40(DCM / EA / MeOH 15 / 5 / 1); 1 H NMR(600MHz,CDCl3)δ7.79(brs,1H,N H COCHBn), 7.38(m,1H,MeOOCCHN H CO),7.24–7.10(m,5H,ArH),5.31(m,1H,N H Boc), 5.05–4.82(m, 3H, 3*C H NHCO,overlap),3.78(s,3H,OC H 3),3.41(d,J=13.5Hz,1H,(CO2Me)CHC H 2S),3.05–2.95(m,1H,C H 2Ph),2.95–2.85(m,1H,C H 2Ph),2.80(d,J=14.2Hz,1H,C H 2S),2.64(m,2H,C H 2S,(CO2Me)CHCH 2S),1.34(s,9H,OC Me 3). 13 C NMR (150MHz, CDCl3): δ172.2( C ONHCO2Me),171.0(NH C OCH(N)Bn),170.0(CONH C O2Me),155.1(NH C O2CMe2),136.5( Cq Ar ),129.3( C H Ar ),128.3( C H Ar ),126.7( C H Ar ),79.8(O C Me3),54.7(NHCO C H(N)Bn),52.8(O Me ),52.1(CONH C HCO2Me),51.8[ C HNHCOCH(N)Bn],42.1( C H2S),41.6

[0051] [(CO2Me)NHCH C H2S],39.8( C H2Ph),28.1(OC Me 3).

[0052] Step 7: Preparation of compound 21

[0053]

[0054] Take a clean and dry 100 mL single-necked round-bottom flask, add compound 20 (1.0 g, 2.0 mmol, 1.0 equiv), dissolve it in 5% trifluoroacetic acid dichloromethane solution, and stir at room temperature for 2 h.

[0055] The reaction solution was concentrated under reduced pressure to remove most of the trifluoroacetic acid, 50 mL of dichloromethane was added to dissolve, and saturated sodium bicarbonate was added to adjust the pH to 7-8. After separation, the aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, washed with saturated sodium chloride (150 mL), dried over anhydrous sodium sulfate overnight, anhydrous sodium sulfate was filtered, and the filtrate was distilled under reduced pressure to obtain a white foamy solid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, DCM: EA: MeOH = 20: 10: 1-10: 5: 1), and the eluent was concentrated under reduced pressure to obtain 551 mg of a white foamy solid, with a yield of 72%.

[0056] 1 H NMR(600MHz,CDCl3)δ8.18(s,1H,N H COCHBn),7.38–7.16(m,5H,ArH),6.17(m,1H,MeOOCCHN H CO),4.98(m,1H,C H NHCO),4.88(d,1H,C H NHCO), 3.84(d, J=2.2Hz, 3H, OC H 3),3.59(dd,J=9.8,4.3Hz,1H,C H NH2),3.42(m,1H,C H 2S),3.25(m,1H,C H 2S),3.03–2.81(m,2H,C H 2Ph),2.67–2.56(m,2H,C H 2S). 13 C NMR (150MHz, CDCl3): δ173.7( C O),171.9( C O),170.3( C O),137.7( Cq Ar ),129.2( C H Ar ),128.7( C H Ar ),126.9( C H Ar ),56.7(NHCO C H(N)Bn),53.1( OMe ),52.1( C HNHCO),50.0( C HNH2),42.1( C H2S),42.1( CH2S),41.0( C H2Ph).

[0057] Step 8: Preparation of compound 22

[0058]

[0059] Take a clean and dry 100 mL single-necked round-bottom flask, fill it with argon protection, add compound 21 (500 mg, 1.3 mmol, 1.0 equiv) and DMAP (398 mg, 3.2 mmol, 2.5 equiv) in sequence, and dissolve them in 50 mL of anhydrous dichloromethane.

[0060] Take a clean and dry 50mL single-mouth round-bottom flask, add the chiral side chain 5a (591mg, 1.5mmol, 1.2equiv), dissolve it with 10mL of anhydrous dichloromethane, and slowly add the solution to the reaction solution with a syringe under 0℃ ice-salt bath conditions, warm to room temperature, and stir the reaction overnight. Add 5mL of saturated ammonium chloride solution to quench the reaction, wash with water (50mL×3), wash with saturated brine (50mL), add anhydrous sodium sulfate to dry overnight, filter the anhydrous sodium sulfate, and take the filtrate to distill under reduced pressure to obtain a yellow oily liquid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, PE:DCM:EA:MeOH=40:20:10:1-20:10:5:1), and the eluate was concentrated under reduced pressure to obtain 373mg of colorless solid with a yield of 52%.

[0061] 1 H NMR (600MHz, CDCl3) δ7.87 (t, J=7.5Hz, 1H, CHN H CO),7.35(m,1H,CHN H CO),7.24–7.10(m,5H,ArH),6.76–6.68(m,1H,C H OH),5.25(dd,J=12.8,6.8Hz,1H,CHN H CO),4.94(td,J=11.4,3.5Hz,1H,NHC H CO), 4.85(d, J=10.1Hz,1H,NHC H CO), 3.88(d, J=10.0Hz,1H,NHC H CO),3.78(s,3H,OCH3),3.40(dd,J=13.6,3.7Hz,1H,C H 2S),3.05(dd,J=14.2,6.9Hz,1H,C H2Ph),2.95(dd,J=13.7,7.9Hz,1H,C H 2Ph),2.79(d,J=17.0Hz,1H,C H 2S),2.68(dt,J=19.0,11.8Hz,2H,C H 2S),2.31–2.28(m,1H,COC H 2),2.22–2.17(m,1H,COC H 2),1.46–1.23(m,12H,6*CH2),0.88(t,J=4.0Hz,3H,C H 3). 13 C NMR (150MHz, CDCl3) δ171.3,171.2,169.7,169.1,135.9,128.3,128.1,127.5,125.9,67.6,52.5,52.1,51.2,51 .20,4.11,41.5,40.6,38.1,37.6,36.0,30.9,30.4,30.4,29.1,28.6,28.6,28.4,28.4,28.2,24.5,21.6,13.0.

[0062] Step 9: Preparation of Sunshinamide

[0063]

[0064] Take a clean and dry 100mL single-mouth round-bottom flask, fill it with argon, add compound 22 (200mg, 0.36mmol, 1.0equiv), trimethyltin hydroxide (163mg, 0.90mmol, 2.5equiv) in sequence, add 25mL of anhydrous tetrahydrofuran with a syringe to dissolve, and stir at room temperature for 4h. After the reaction solution is concentrated under reduced pressure to remove tetrahydrofuran, 50mL of ethyl acetate is added to dissolve, and then washed with 1M hydrochloric acid aqueous solution (50mL×2), the aqueous phase is extracted with ethyl acetate (50mL), the organic phases are combined, and washed with saturated brine (50mL). Add anhydrous sodium sulfate to dry overnight, filter the anhydrous sodium sulfate, and take the filtrate and distill it under reduced pressure to obtain a rotten egg-smelling solid, that is, intermediate 23.

[0065] Take a clean and dry 500mL single-mouth round-bottom flask, fill it with argon, add 2-methyl-6-nitrobenzoic anhydride (MNBA, 309mg, 0.90mmol, 2.5equiv), DMAP (9mg, 0.07mmol, 0.2equiv) in sequence, add 450mL of anhydrous tetrahydrofuran and stir to dissolve, then add triethylamine (0.1mL, 0.72mmol, 2.0equiv) with a syringe and continue stirring. Dissolve the crude compound 23 in 50mL of anhydrous tetrahydrofuran, slowly add the solution dropwise to the above reaction solution (2h), and after the addition, stir at room temperature for 16h. Add 5mL of saturated ammonium chloride to quench the reaction, concentrate the reaction solution under reduced pressure, add 50mL of ethyl acetate, wash with water (50mL×2), wash with saturated brine (50mL), add anhydrous sodium sulfate to dry overnight, filter the anhydrous sodium sulfate, and take the filtrate to distill under reduced pressure to obtain a colorless solid. The crude product was purified by silica gel column chromatography (elution conditions: 300-400 mesh silica gel, PE:EA=8:1-4:1), and the eluate was concentrated under reduced pressure to obtain 41 mg of a colorless solid, i.e., the final product Sunshinamide, with a yield of 22%.

[0066] R f =0.46(PE / EA 2 / 1); 1 H NMR (600MHz, DMSO-d6) δ9.24(d,J=5.1Hz,1H),8.70(d,J=8.4Hz,1H),7.27(d,J=2.6Hz,5H),7.01(d,J=10.9Hz,1H),4.98–

[0067] 4.89(m,1H),4.85(td,J=10.6,4.8Hz,1H),4.70(td,J=8.6,6.4Hz,1H),4.07( d,J=4.2Hz,1H),3.53(dd,J=15.1,4.0Hz,1H),3.43(dd,J=15.1,4.8Hz,1H),3 .07(dt,J=15.1,2.2Hz,1H),2.99(dd,J=15.0,10.4Hz,1H),2.88(t,J=6.7Hz, 2H), 2.50 (t, J = 2.0Hz, 2H), 1.57 (s, 2H), 1.24 (s, 10H), 0.85 (t, J = 6.6Hz, 3H).

Claims

1. A method for preparing sunshinamide, a sulfur-containing marine natural product, characterized in that: According to the following synthetic route: Using L-cysteine ​​as a starting material, in the first step, the thiol group of L-cysteine ​​is protected to obtain compound 15; subsequently, the amino group or carboxyl group thereof is protected to obtain compounds 16 and 17; in the condensation reaction of the fourth step, two protected cysteines are connected through an amide bond to obtain compound 18; in the fifth step, compound 18 is deaminated and then condensed with D-amino-protected phenylalanine to obtain compound 19; in the sixth step, compound 19 undergoes thiol protection and simultaneously forms an intramolecular disulfide bond to obtain compound 20; then, in the seventh step, compound 20 is deaminated to obtain free amine compound 21; in the eighth step, compound 21 directly undergoes aminolysis reaction with chiral intermediate 5a to obtain compound 22; in the ninth step, the ester group of compound 22 is selectively hydrolyzed to obtain carboxylic acid compound 23, and then compound 23 undergoes macrocyclic lactonization reaction to complete the preparation of Sunshinamide; In the above synthetic route, the thiol protecting group is P 1 , the carboxyl protecting group is P 2 , the aminolysis protecting group is P 3 ; Among them, P 1 Selected from CPh3 (ie, Trt) or t-Bu; P 2 is selected from Me, Et, i-Pr, or t-Bu; P 3 Selected from Boc, Cbz or Bn.

2. The preparation method according to claim 1, characterized in that: The synthetic route of Sunshinamide is as follows: These include: Using L-cysteine ​​as a starting material, in the first step, the thiol group of L-cysteine ​​is protected to obtain compound 15; subsequently, the amino group or carboxyl group thereof is protected to obtain compounds 16 and 17; in the condensation reaction of the fourth step, two protected cysteines are connected through an amide bond to obtain compound 18; in the fifth step, compound 18 is deaminated and then condensed with D-amino-protected phenylalanine to obtain compound 19; in the sixth step, compound 19 undergoes thiol protection and simultaneously forms an intramolecular disulfide bond to obtain compound 20; then, in the seventh step, compound 20 is deaminated to obtain free amine compound 21; in the eighth step, compound 21 directly undergoes aminolysis reaction with chiral intermediate 5a to obtain compound 22; in the ninth step, the ester group of compound 22 is selectively hydrolyzed to obtain carboxylic acid compound 23, and then compound 23 undergoes macrocyclic lactonization reaction to complete the preparation of Sunshinamide; In the above synthetic route, the thiol protecting group is P 1 , the carboxyl protecting group is P 2 , the aminolysis protecting group is P 3 ; Among them, P 1 is CPh3 (i.e. Trt); P 2 for Me;P 3 For Boc.

3. The preparation method according to claim 2, characterized in that: The synthetic route of Sunshinamide is as follows: in Using L-cysteine ​​as a starting material, in the first step, the thiol group of L-cysteine ​​is protected to obtain compound 15; subsequently, the amino group or carboxyl group thereof is protected to obtain compounds 16 and 17; in the condensation reaction of the fourth step, two protected cysteines are connected through an amide bond to obtain compound 18; in the fifth step, compound 18 is deaminated and then condensed with D-amino-protected phenylalanine to obtain compound 19; in the sixth step, compound 19 undergoes thiol protection and simultaneously forms an intramolecular disulfide bond to obtain compound 20; then, in the seventh step, compound 20 is deaminated to obtain free amine compound 21; in the eighth step, compound 21 directly undergoes aminolysis reaction with chiral intermediate 5a to obtain compound 22; in the ninth step, the ester group of compound 22 is selectively hydrolyzed to obtain carboxylic acid compound 23, and then compound 23 undergoes macrocyclic lactonization reaction to complete the preparation of Sunshinamide; In the above synthetic route, in the fourth step, compounds 16 and 17 undergo a condensation reaction in the presence of a condensing agent, under a suitable solvent and temperature to obtain compound 18; wherein the condensing agent is selected from DMAP, Et3N, DIPEA, EDCI, HATU, HOAt, HOBt, TBTU or a combination thereof; The solvent is selected from toluene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, 1,4-dioxane, N,N-dimethylformamide, acetonitrile or a combination thereof; the temperature is 0-80°C; In the step 5, the amino protecting group Boc of compound 18 is removed in the presence of a suitable acid, under a suitable solvent and temperature to obtain a free amine, and the obtained free amine is condensed with D-amino-protected phenylalanine (D-BocNH-Phe-OH) under a suitable solvent and temperature to obtain compound 19; wherein the acid is selected from trifluoroacetic acid or HCl; the condensing agent is selected from DMAP, Et3N, DIPEA, EDCI, HATU, HOAt, HOBt, TBTU or a combination thereof; the solvent is selected from toluene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, acetonitrile or a combination thereof; and the temperature is 0-80°C; In the reaction of step 6, compound 19 undergoes desulfhydrylation in the presence of a suitable reagent, in a suitable solvent and at a suitable temperature, and simultaneously forms a disulfide bond within the molecule to obtain compound 20; wherein the reagent is I2; the solvent is selected from methanol, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, acetonitrile or a combination thereof; and the temperature is 0-80°C; In the reaction of step 7, compound 20 is subjected to the removal of the amino protecting group Boc in the presence of a suitable acid, under a suitable solvent and temperature to obtain compound 21; wherein the acid is selected from trifluoroacetic acid or HCl; the solvent is selected from toluene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, acetonitrile or a combination thereof; and the temperature is 0-80°C; In the reaction of step 8, compound 21 is condensed with compound 5a under a suitable base, solvent and temperature to obtain compound 22; wherein the base is selected from DMAP, Et3N or DIPEA; the solvent is selected from toluene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, acetonitrile or a combination thereof; and the temperature is 0-80°C; In the reaction of step 9, compound 22 undergoes methyl ester hydrolysis under a suitable base, solvent and temperature to obtain free acid 23, and the obtained free acid 23 undergoes macrocyclic lactonization under a suitable condensing agent, solvent and temperature to obtain the final product Sunshinamide; wherein the base is selected from LiOH, NaOH, KOH, Bu3SnOH or Me3SnOH; the condensing agent is selected from DMAP, Et3N, DIPEA, 2,4,6-trichlorobenzoyl chloride, 2-methyl-6-nitrobenzoic anhydride (MNBA) or a combination thereof; the solvent is selected from toluene, tetrahydrofuran, dichloromethane, 1,2-dichloroethane, N,N-dimethylformamide, acetonitrile or a combination thereof; and the temperature is 0-80°C.

4. The preparation method according to claim 3, characterized in that: The synthetic route of Sunshinamide is as follows: in: Using L-cysteine ​​as a starting material, in the first step, the thiol group of L-cysteine ​​is protected to obtain compound 15; subsequently, the amino group or carboxyl group thereof is protected to obtain compounds 16 and 17; in the condensation reaction of the fourth step, two protected cysteines are connected through an amide bond to obtain compound 18; in the fifth step, compound 18 is deaminated and then condensed with D-amino-protected phenylalanine to obtain compound 19; in the sixth step, compound 19 undergoes thiol protection and simultaneously forms an intramolecular disulfide bond to obtain compound 20; then, in the seventh step, compound 20 is deaminated to obtain free amine compound 21; in the eighth step, compound 21 directly undergoes aminolysis reaction with chiral intermediate 5a to obtain compound 22; in the ninth step, the ester group of compound 22 is selectively hydrolyzed to obtain carboxylic acid compound 23, and then compound 23 undergoes macrocyclic lactonization reaction to complete the preparation of Sunshinamide; In the above synthetic route, in the fourth step, compounds 16 and 17 undergo condensation reaction in the presence of a condensing agent, in a suitable solvent and at a temperature to obtain compound 18; wherein the condensing agent is selected from DMAP, Et3N, DIPEA, TBTU or a combination thereof; the solvent is selected from dichloromethane, N,N-dimethylformamide or a combination thereof; the temperature is 0-40°C; In the fifth step, the amino protecting group Boc of compound 18 is removed in the presence of a suitable acid, under a suitable solvent and temperature to obtain a free amine, and the obtained free amine is condensed with D-amino-protected phenylalanine (D-BocNH-Phe-OH) under a suitable solvent and temperature to obtain compound 19; wherein the acid is selected from trifluoroacetic acid; the condensing agent is selected from DMAP, Et3N, DIPEA, TBTU or a combination thereof; the solvent is selected from dichloromethane, N,N-dimethylformamide or a combination thereof; and the temperature is 0-40°C; In the sixth step, compound 19 undergoes desulfhydrylation in the presence of a suitable reagent, under a suitable solvent and temperature, and simultaneously forms a disulfide bond within the molecule to obtain compound 20; The reagent is I2; the solvent is selected from methanol, dichloromethane or a combination thereof; the temperature is 0-40°C; In the reaction of step 7, compound 20 is subjected to removal of the amino protecting group Boc in the presence of a suitable acid, under a suitable solvent and temperature to obtain compound 21; wherein the acid is selected from trifluoroacetic acid; the solvent is selected from dichloromethane; and the temperature is 0-40°C; In the eighth step, compound 21 is condensed with compound 5a under a suitable base, solvent and temperature to obtain compound 22; wherein the base is selected from DMAP; the solvent is selected from tetrahydrofuran, dichloromethane, N,N-dimethylformamide or a combination thereof; and the temperature is 0-40°C; In the reaction of step 9, compound 22 undergoes methyl ester hydrolysis under a suitable base, solvent and temperature to obtain free acid 23, and the obtained free acid 23 undergoes macrocyclic lactonization under a suitable condensing agent, solvent and temperature to obtain the final product Sunshinamide; wherein the base is selected from Bu3SnOH or Me3SnOH; the condensing agent is selected from DMAP, Et3N, DIPEA, 2-methyl-6-nitrobenzoic anhydride (MNBA) or a combination thereof; the solvent is selected from tetrahydrofuran; and the temperature is 0-40°C.

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