CN117924431A - Natural cyclopeptide zelkovamycins simplified substance and preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of pharmaceutical technology, and in particular to a simplified natural cyclopeptide zelkovamycins, a preparation method and an application thereof. The simplified material has a compound of formula 1 or 2 or a pharmaceutically acceptable salt or prodrug thereof, and the simplified material is synthesized by a solid phase synthesis method of Fmoc chemistry, wherein the Fmoc protection is removed by a 20% piperidine DMF solution, the condensation reagent is HATU, and the base used for condensation is DIPEA. The cytotoxic activity of the simplified material on human liver cancer cell lines HepG2 and Huh7 was screened by the MTT method, and the simplified material has good anti-tumor activity and has certain application prospects in the development of corresponding drugs.

Description

A simplified natural cyclic peptide zelkovamycins and its preparation method and application

Technical Field

The invention relates to the technical field of pharmaceuticals, and in particular to a simplified natural cyclic peptide zelkovamycins, a preparation method and application thereof.

Background technique

Malignant tumors, also known as cancer, refer to the uncontrollable growth and spread of malignant cells and tissue infiltration that cause damage to normal body functions. After normal cells become cancer cells, the human body cannot restrain their spread and produces a series of symptoms. Cancer cells may also metastasize to various parts of the body, leading to more serious consequences or even death. Cancer has become the second leading cause of death among Chinese residents after cardiovascular and cerebrovascular diseases. Tumor treatment methods are mainly divided into five categories: surgical treatment, radiotherapy, chemotherapy, targeted therapy and immunotherapy. Seeking drugs with clear targets, good efficacy and few toxic side effects is the goal of drug research.

Cyclic peptide compounds have achieved great success in drug therapy. So far, there are more than 40 cyclic peptide drugs in clinical application, and more than 20 cyclic peptide drugs are in various stages of clinical research. On average, about one cyclic peptide drug enters the pharmaceutical market every year. Natural cyclic peptides are a class of cyclic compounds with special structure, wide biological activity and unique mechanism of action. They play a very important role in natural products. They are widely distributed in nature, and plants, animals, lower marine organisms, microorganisms, bacteria and pathogens all contain cyclic peptides. Many natural cyclic peptides have good biological activities, such as antibacterial, antiviral, antitumor and immunosuppression. Cyclic peptides can form a restricted conformation to enhance the binding performance with the target molecule and the selectivity for the receptor. They have the characteristics of low toxicity, easy penetration of cell membranes, and resistance to hydrolysis by exopeptidases and endopeptidases. However, natural products have the following disadvantages in drug research and development. First, the content of natural products in nature is too low, and it is difficult to obtain enough samples from nature for subsequent research and development; second, the structure of natural products is often too complex, and artificial preparation is not easy to achieve. However, due to the unique advantages of cyclopeptides, they provide valuable molecular models for drug design and have become a hot topic for chemists and pharmacists. The structure of zelkovamycins is shown below. It is a natural cyclopeptide containing thiazole residues and has certain antibacterial activity, but their content in microorganisms is low, and the natural cyclopeptide contains residues with chiral quaternary carbon, which is difficult to synthesize. Therefore, using zelkovamycins as a master template to obtain simplified zelkovamycins without quaternary carbon may quickly find new molecules with good biological activity, and this research has not yet been carried out.

Summary of the invention

The purpose of the present invention is to solve the above-mentioned problems. The present invention uses zelkovamycins as a model to provide a class of simplified natural cyclic peptide zelkovamycins with good anti-tumor activity, as well as a preparation method and application thereof.

In order to achieve the above object, the present invention adopts the following technical solutions:

A simplified natural cyclopeptide zelkovamycins, which has a compound of formula 1 or 2 or a pharmaceutically acceptable salt or prodrug thereof,

The present invention also provides a method for preparing a simplified natural cyclopeptide zelkovamycins with anti-tumor activity, which is synthesized by a solid phase synthesis method of Fmoc chemistry, comprising the following steps:

(1) Coupling the first amino acid to CTC resin:

DCM was added to CTC resin for soaking, and after the CTC resin was fully swollen for 0.5-1h, the filtrate was removed; the amino acid Fmoc-Sar-OH and collidine were dissolved in DCM to obtain a mixed solution, and the obtained mixed solution was added to the CTC resin soaked in DCM, shaken for 8-10h, the reaction solution was filtered out, and then washed three times with DCM and DMF alternately; then DCM/MeOH/DIPEA mixed solution was added to react for 30-50min, and after the reaction was completed, the resin was filtered and washed three times with DCM and DMF alternately to obtain Fmoc-Sar-OH-CTC resin; wherein the DCM/MeOH/DIPEA mixed solution was a solution of DCM, MeOH, and DIPEA mixed in a volume ratio of 17:2:1;

(2) Synthesis of linear peptides

① The Fmoc-Sar-OH-CTC resin was shaken with a 20% piperidine/DMF solution for 2-10 min, and washed alternately with DCM and DMF for 3 times to obtain Sar-OH-CTC resin;

② Dissolve the next amino acid, HATU, and DIPEA in DMF to obtain a mixed solution, then add the obtained mixed solution to Sar-OH-CTC resin, shake for 2-4 hours, filter out the reaction solution, and wash with DCM and DMF alternately for 3 times to obtain CTC resin containing dipeptide;

③ Repeat the above steps ① and ② to sequentially couple the dipeptide-containing CTC resin with other amino acids in the order of the heptapeptide connection, and finally add a 20% piperidine/DMF solution to carry out a deprotection reaction, and wash it with DCM and DMF alternately to obtain a heptapeptide-containing CTC resin;

(3) Cleavage of linear peptides

A 0.5% TFA/DCM cutting solution was added to the CTC resin containing the heptapeptide, and after shaking for 20-40 minutes, the reaction solution was filtered and collected; the cutting solution was added again to react for 20-40 minutes, and the operation was repeated 3 times; the collected reaction solution was concentrated under reduced pressure to obtain a heptapeptide chain with the N-terminus protected by a Boc group;

(4) Removal of the Boc protecting group at the N-terminus of the chain precursor

The heptapeptide chain with the N-terminus protected by the Boc group was dissolved in DCM and placed in an ice bath for cooling and stirring, and TFA was slowly added, wherein the volume ratio of DCM to TFA was 1:1; after reacting for 30-50 minutes, TFA was blown away with nitrogen, 20-30 mL of DCM was added to the residue, and the solvent was evaporated under reduced pressure to obtain a crude heptapeptide chain product, which was directly used in the next step reaction;

(5) Synthesis of simplified natural cyclopeptide zelkovamycins

The crude product of the heptapeptide chain obtained in step (4) is dissolved in dry DCM, placed in an ice bath and stirred vigorously, and then a mixed solution obtained by dissolving HATU and HOAt in anhydrous DMF is added dropwise, and after cooling to 0-5°C, DIPEA is added, and then the temperature is raised to room temperature, and the reaction is carried out at room temperature for 48-60 hours; after the reaction is completed, the reaction is quenched with aqueous hydrochloric acid, and extracted with DCM, and the organic layer is washed with saturated _NaHCO3 solution and saturated brine respectively, and then dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude cyclic peptide, which is then purified by HPLC to obtain the target product;

The molar ratio of the crude heptapeptide chain to HATU and HOAt was 1:2:2, and the molar ratio of the crude heptapeptide chain to DIPEA was 1:4.5.

Preferably, in the above preparation method, in step (1), the molar ratio of the amino acid Fmoc-Sar-OH to collidine is 1:2.1.

Preferably, in the above preparation method, in step (2) ②, the molar ratio of amino acid, HATU and DIPEA is 1:1:3.2.

Preferably, in the above preparation method, in step (2) ②, the next amino acid is Fmoc-2-aminobutyric acid or Fmoc-Dhb-OH.

Preferably, in the above preparation method, in step (2) (3), amino acids are connected in the order of Fmoc-2-aminobutyric acid, Fmoc-glycine, Fmoc-D-2-amino-butyric acid, Fmoc-4-Methoxy-L-Tryptophan, and Boc-D-Thiazole-alanine; or amino acids are connected in the order of Fmoc-D-Abu-OH, Fmoc-Gly-OH, Fmoc-L-Thr-OH, Fmoc-L-Trp(4-OMe)-OH, and Fmoc-D-Ala(Thz)-OH.

Preferably, in the above preparation method, in step (5), the HPLC purification conditions are: YMC-Pack ODS-A semi-preparative column, 5 μm, 250×10 mm, and the eluent is a solution of acetonitrile and water in a volume ratio of 27:73 or a solution of methanol and water in a volume ratio of 30:70.

The invention also provides the use of the simplified natural cyclopeptide zelkovamycins in the preparation of anti-tumor drugs.

Preferably, the anti-tumor drug is a drug against HepG2 or Huh7 cancer cells.

In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

The present invention uses complex natural cycloheptapeptide zelkovamycins as a model to design and synthesize a class of simplified zelkovamycins analogs. The amino acid residues contained in the cycloheptapeptides are all commercial raw materials, and their rapid synthesis can be achieved through a solid phase synthesis method. Moreover, the simplified products have good anti-tumor effects on HepG2 and Huh7 cancer cells, and have certain application prospects in the preparation of corresponding anti-tumor drugs.

Detailed ways

In order to express the present invention more clearly, the present invention is further described below through specific examples.

The present invention provides a simplified natural cyclopeptide zelkovamycins, which has a compound of formula 1 or 2 or a pharmaceutically acceptable salt or prodrug thereof.

The following is a detailed description of the preparation method of the simplified natural cyclopeptide zelkovamycins by means of specific examples.

1. Preparation Example

Example 1: Preparation of Boc-D-Thiazole-alanine (7)

To synthesize the zelkovamycins simplified, the thiazole amino acid 7 must first be synthesized, and the synthetic route is as follows:

The specific synthetic route comprises the following steps:

(1) Synthesis of compound 3:

Boc-D-Ala-OH (3.00 g, 15.9 mmol) and HOBt (2.15 g, 15.9 mmol) were dissolved in DCM (90 mL), cooled to 0°C, and EDCI (3.8 g, 20.0 mmol) was added. After stirring at room temperature for 2 h, the reaction solution was cooled to 0°C again, and 79.5 mL of ammonia methanol solution (2 mol/L in MeOH) was slowly added dropwise to the reaction solution. The reaction was restored from 0°C to room temperature and stirred for 1 h. The solution was extracted with DCM and purified by column chromatography with the eluent being DCM:MeOH=25:1 to obtain 2.35 g of the product (compound 3) with a yield of 78.6%.

The structure of the product obtained in step (1) was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, chloroform-d, Jin Hz) δ (ppm): δ 6.42 (s, 1H), 5.92 (s, 1H), 5.22 (s, 1H), 4.20 (s, 1H), 1.43 (s, 8H), 1.36 (d, J = 7.11 Hz, 3H), 1.25 (d, J = 13.09 Hz, 1H).

(2) Synthesis of compound 4:

Compound 3 (2.00 g, 10.6 mmol) and Belleau reagent (3.35 g, 6.34 mmol) were dissolved in THF (85 mL) at 0°C, the reaction temperature was raised to room temperature, and the reaction was stirred for 2 h. The reaction mixture was then poured into a mixture of ice (80 g) and saturated NaHCO ₃ (80 mL), and extracted with EtOAc (3×100 mL). The organic layers were combined, washed with saturated saline solution (40 mL), dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography with an eluent of DCM:MeOH=5:1 gave 1.89 g of the product (compound 4) with a yield of 87.4%.

The structure of the product obtained in step (2) was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, Chloroform-d) δ 8.39 (s, 1H), 8.08 (s, 1H), 5.52 (s, 1H), 4.59 (s, 1H), 1.43 (d, J = 6.88 Hz, 3H), 1.40 (s, 9H).

(3) Synthesis of Compound 6:

Compound 4 (1.80 g, 8.82 mmol) was dissolved in DME (15 mL), cooled to -15 °C, KHCO ₃ (7.1 g, 71 mmol) was added, and after vigorous stirring at -15 °C for 5 min, ethyl bromopyruvate (3.3 mL) was added, and after the reaction solution was stirred evenly, TFAA (5 mL), 2,6-lutidine (8.8 mL) and DME (4.5 mL) were added in sequence. After stirring at -15 °C for 4 h, water (70 mL) was added to quench the reaction. Extracted with EtOAc (3×100 mL), the organic layers were combined, washed with saturated sodium bicarbonate (2×30 mL) and saturated brine (2×30 mL) in sequence, dried over anhydrous sodium sulfate, and concentrated. Purified by column chromatography, the eluent was DCM:MeOH=8:1, and the product (compound 6) 1.35 g was obtained, with a yield of 51.0%.

The structure of the product obtained in step (3) was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, Chloroform-d) δ 8.08 (s, 1H), 5.21 (s, 1H), 5.10 (s, 1H), 4.41 (q, J = 7.13 Hz, 2H), 1.62 (d, J = 6.82 Hz, 3H), 1.44 (s, 9H), 1.39 (t, J = 7.10 Hz, 3H).

(4) Synthesis of Compound 7:

Compound 6 (1.30 g, 4.33 mmol) was dissolved in 3 mL of methanol solution, cooled to 0°C, and THF (4.5 mL) and H ₂ O (3 mL) were added in the ratio of THF:MeOH:H ₂ O = 9:6:6 (v/v). LiOH (0.5 N, 18 mL) was then slowly added dropwise to the reaction solution, and stirred at room temperature for 6 h. Then, the pH of the reaction solution was adjusted to 2 with saturated KHSO ₄ at 0°C, extracted with EtOAc (3×100 mL), and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated. Purification by column chromatography with an eluent of DCM:MeOH = 10:1 gave 1.06 g of the product (Compound 7) with a yield of 89.0%.

The structure of the product obtained in step (4) was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.84 (s, 1H), 7.76 (s, 1H), 4.96 (s, 1H), 1.36 (d, J=19.93 Hz, 10H), 1.27-1.17 (m, 3H); HRESITOFMS: m/z 295.0724 [M+Na] ⁺ (calcd for C ₁₁ H ₁₆ N ₂ NaO ₄ S, 295.0723).

Example 2: Preparation of Simplified Natural Cyclic Peptide Zelkovamycins 1

The synthetic route of the natural cyclopeptide zelkovamycins simplified 1 is as follows:

The specific preparation steps include:

(1) Coupling the first amino acid to CTC resin:

Weigh 300 mg of CTC resin and put it into a polypeptide solid phase synthesis sand core tube, add DCM to soak, make the CTC resin fully swell for 1 hour, and squeeze out the filtrate with an ear bulb; dissolve 1.06 mmol of amino acid Fmoc-Sar-OH and 2.27 mmol of collidine in DCM to obtain a mixed solution, add the obtained mixed solution to the CTC resin soaked in DCM, shake for 8 hours, filter out the reaction solution, and then wash it alternately with DCM and DMF three times; then add 10 mL of DCM/MeOH/DIPEA mixed solution to react to block unreacted sites, the reaction time is 50 minutes, after the reaction is completed, filter the resin and wash it alternately with DCM and DMF three times to obtain Fmoc-Sar-OH-CTC resin; wherein the DCM/MeOH/DIPEA mixed solution is a solution formed by mixing DCM, MeOH, and DIPEA in a volume ratio of 17:2:1;

(2) Synthesis of linear peptides

① Add 5 mL of 20% piperidine/DMF solution into the solid phase synthesis tube and shake for 10 min, then wash with DCM and DMF alternately for 3 times to obtain Sar-OH-CTC resin;

② Dissolve 1.06mmol Fmoc-2-aminobutyric acid, 1.06mmol HATU, and 3.4mmol DIPEA in 2mL DMF to obtain a mixed solution, then add the obtained mixed solution to Sar-OH-CTC resin, shake for 2h, filter out the reaction solution, and wash with DCM and DMF alternately for 3 times to obtain CTC resin containing dipeptide;

③ Repeat the above steps ① and ② to couple the dipeptide-containing CTC resin with Fmoc-2-aminobutyric acid, Fmoc-glycine, Fmoc-D-2-amino-butyric acid, Fmoc-4-Methoxy-L-Tryptophan, and Boc-D-Thiazole-alanine amino acids in sequence, and finally add a 20% piperidine/DMF solution to carry out a deprotection reaction, and wash it alternately with DCM and DMF to obtain a heptapeptide-containing CTC resin;

Wherein, the volume ratio of piperidine to DMF in the piperidine/DMF solution with a volume concentration of 20% is 1:4;

(3) Cleavage of linear peptides

5 mL of 0.5% TFA/DCM cleavage solution was added to the CTC resin containing the heptapeptide, and the mixture was shaken on a shaker for 20 min, and then the reaction solution was filtered and collected. The cleavage solution was added again and the mixture was reacted for 20 min, and the operation was repeated 3 times. The collected reaction solution was concentrated under reduced pressure to obtain a pale yellow solid crude product, i.e., a heptapeptide chain with a Boc group protected at the N-terminus. HRESITOFMS: m/z 892.3634 [M+Na] ⁺ (calcd for C ₄₀ H ₅₅ N ₉ NaO ₁₁ S, 892.3634).

Wherein, the volume ratio of TFA and DCM in the TFA/DCM cutting solution with a volume concentration of 0.5% is 5:95;

(4) Removal of the Boc protecting group at the N-terminus of the chain precursor

The N-terminal protecting group (Boc) was removed by using a dichloromethane-trifluoroacetic acid mixed system V _DCM : V _TFA = 1:1, and the Boc group was removed under ice bath conditions. The specific operation method is as follows: Take the heptapeptide chain (0.2mmol, 0.17g) protected by the Boc group at the N-terminus, dissolve it in 2mL DCM and place it in an ice bath for cooling and stirring, slowly add 2mL TFA, monitor the reaction by TLC, after 30min of reaction, blow away TFA with nitrogen, add 20mL DCM to the residue, and evaporate the solvent under reduced pressure to obtain a crude heptapeptide chain product, which is directly used in the next step reaction; HRESITOFMS: m/z 770.3294[M+H] ⁺ (calcd for C ₃₅ H ₄₈ N ₉ O ₉ S, 770.3290);

(5) Synthesis of simplified natural cyclopeptide zelkovamycins

The crude product of the heptapeptide chain obtained in step (4) (0.12 mmol, 0.09 g) was dissolved in dry 78 mL DCM, stirred vigorously under an ice bath, and then a mixed solution obtained by dissolving HATU (0.24 mmol, 90 mg) and HOAt (0.24 mmol, 32 mg) in anhydrous DMF (2 mL) was added dropwise. After cooling to 0°C, DIPEA (4.5 eq, 0.54 mmol, 100 μL) was added, and then the temperature was raised to room temperature and reacted at room temperature for 48 h. After the reaction was completed, the reaction was quenched with 0.1 M HCl (12 mL), and extracted with DCM (2×75 mL). The organic layer was washed with saturated NaHCO ₃ solution (2×15 mL) and saturated brine (2×25 mL), respectively, and then dried over anhydrous sodium sulfate, filtered and concentrated to obtain a light yellow solid, i.e., a crude cyclopeptide, which was prepared and purified using Agilent 1260 Infinity liquid chromatography. The HPLC purification conditions were: YMC-Pack ODS-A semi-preparative column, 5 μm, 250×10 mm, eluent: V _acetonitrile : V _water = 27:73, yield 22.5%.

The structure of the target product obtained in this example was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, CDCl3, Jin Hz) δ (ppm): 10.73 (s, 1H), 9.38 (s, 1H), 8.91 (d, J = 8.52 Hz, 1H), 8.59 (d, J = 7.40 Hz ,1H),8.03(s,1H),7.52(t,J＝14.87Hz,1H),7.11-6.95(m,3H),6.83(d,J＝8.12Hz,1H),6.52(d,J＝ 7.78Hz,1H),5.59-5.43(m,2H),5.28-5.13(m,2H),5.00(d,J＝16.93Hz,1H),4.17(d, J＝15.30Hz,1H),4.12-4.02(m,1H),3.99(s,3H),3.75(dd,J＝17.48,7.92Hz,1H),3.58(dd,J＝15.50,3.40Hz,1H ),3.51-3.37(m,2H),3.10(s,3H),2.27-2.09(m,1H),2.07-1.95(m,1H),1.89(d,J＝7.08Hz,3H),1.72( d, J = 7.09 Hz, 3H), 1.63 (s, 3H), 0.95-0.86 (m, 6H);

¹³ C NMR (101 MHz, CDCl ₃ ) δ 171.93, 171.74, 171.43, 169.52, 169.47, 169.15, 167.30, 160.15, 153.58, 150.46, 136.85, 130.71, 125.08, 122.75, 122.72, 116.51, 110.72, 106.52, 105.98, 100.08, 57.87, 55.66, 54.64, 52.66, 51.24, 45.55, 42.01, 37.58, 28.49, 21.57, 21.28, 20.95, 11.50, 11.45, 10.52.

HRESITOFMS: m/z 774.3012 [M+Na] ⁺ (calcd for C ₃₅ H ₄₅ N ₉ NaO ₈ S, 774.3004).

Example 3: Preparation of Simplified Natural Cyclic Peptide Zelkovamycins 2

The synthetic route of the natural cyclopeptide zelkovamycins simplified product 2 is as follows:

The specific preparation steps include:

(1) Coupling the first amino acid to CTC resin:

Weigh 300 mg of CTC resin and put it into a polypeptide solid phase synthesis sand core tube, add DCM to soak, make the CTC resin fully swell for 0.5 hours, and squeeze out the filtrate with an ear bulb; dissolve 1.06 mmol of amino acid Fmoc-Sar-OH and 2.27 mmol of collidine in DCM to obtain a mixed solution, add the obtained mixed solution to the CTC resin soaked in DCM, shake for 10 hours, filter out the reaction solution, and then wash it alternately with DCM and DMF three times; then add 10 mL of DCM/MeOH/DIPEA mixed solution to react to block unreacted sites, the reaction time is 30 minutes, after the reaction is completed, filter the resin and wash it alternately with DCM and DMF three times to obtain Fmoc-Sar-OH-CTC resin; wherein the DCM/MeOH/DIPEA mixed solution is a solution formed by mixing DCM, MeOH, and DIPEA in a volume ratio of 17:2:1;

(2) Synthesis of linear peptides

① Add 3 mL of 20% piperidine/DMF solution into the solid phase synthesis tube and shake for 2 minutes, then wash with DCM and DMF alternately for 3 times to obtain Sar-OH-CTC resin;

② Dissolve 1.06mmol Fmoc-Dhb-OH, 1.06mmol HATU, and 3.4mmol DIPEA in 2mL DMF to obtain a mixed solution, then add the obtained mixed solution to Sar-OH-CTC resin, shake for 4 hours, filter out the reaction solution, and wash with DCM and DMF alternately for 3 times to obtain CTC resin containing dipeptide;

③ Repeat the above steps ① and ② to couple the dipeptide-containing CTC resin with Fmoc-D-Abu-OH, Fmoc-Gly-OH, Fmoc-L-Thr-OH, Fmoc-L-Trp(4-OMe)-OH, and Fmoc-D-Ala(Thz)-OH amino acids in sequence, and finally add a 20% piperidine/DMF solution to carry out a deprotection reaction, and wash it alternately with DCM and DMF to obtain a heptapeptide-containing CTC resin;

Wherein, the volume ratio of piperidine to DMF in the piperidine/DMF solution with a volume concentration of 20% is 1:4;

(3) Cleavage of linear peptides

5 mL of 0.5% TFA/DCM cleavage solution was added to the CTC resin containing the heptapeptide, and the mixture was shaken on a shaker for 40 min, and then the reaction solution was filtered and collected; the cleavage solution was added again and reacted for 40 min, and the operation was repeated 3 times; the collected reaction solution was concentrated under reduced pressure to obtain a pale yellow crude product, i.e., a heptapeptide chain with a Boc group protected at the N-terminus; HRESITOFMS: m/z 908.3580 [M+Na] ⁺ (calcd for C ₄₀ H ₅₅ N ₉ NaO ₁₂ S, 908.3583);

Wherein, the volume ratio of TFA and DCM in the TFA/DCM cutting solution with a volume concentration of 0.5% is 5:95;

(4) Removal of the Boc protecting group at the N-terminus of the chain precursor

The N-terminal protecting group (Boc) was removed by using a dichloromethane-trifluoroacetic acid mixed system V _DCM : V _TFA = 1:1, and the Boc group was removed under ice bath conditions. The specific operation method is as follows: Take the heptapeptide chain (0.2mmol, 0.17g) protected by the Boc group at the N-terminus, dissolve it in 2mL DCM and place it in an ice bath for cooling and stirring, slowly add 2mL TFA, monitor the reaction by TLC, after 30min of reaction, blow away TFA with nitrogen, add 30mL DCM to the residue, and concentrate under reduced pressure to remove the solvent to obtain a crude heptapeptide chain product, which is directly used in the next step reaction; HRESITOFMS: m/z 786.3237[M+H]+(calcd for C ₃₅ H ₄₈ N ₉ O ₁₀ S, 786.3239).

(5) Synthesis of simplified natural cyclopeptide zelkovamycins

The crude product of the heptapeptide chain obtained in step (4) (0.12 mmol, 0.09 g) was dissolved in dry 78 mL DCM, placed in an ice bath and stirred vigorously, and then a mixed solution obtained by dissolving HATU (0.24 mmol, 90 mg) and HOAt (0.24 mmol, 32 mg) in anhydrous DMF (2 mL) was added dropwise. After cooling to 5°C, DIPEA (4.5 eq, 0.54 mmol, 100 μL) was added, and then the temperature was raised to room temperature and reacted at room temperature for 60 h. After the reaction was completed, the reaction was quenched with 0.1 M HCl (12 mL), and extracted with DCM (2×75 mL). The organic layer was washed with saturated NaHCO ₃ solution (2×15 mL) and saturated brine (2×25 mL), respectively, and then dried over anhydrous sodium sulfate, filtered and concentrated to obtain a light yellow solid, i.e., a crude cyclopeptide, which was prepared and purified using Agilent 1260 Infinity liquid chromatography. The HPLC purification conditions were: YMC-Pack ODS-A semi-preparative column, 5 μm, 250×10 mm, eluent: V _methanol : V _water = 30:70, yield 26.5%.

The structure of the target product obtained in this example was characterized by nuclear magnetic resonance, and the results are as follows.

¹ H NMR (400 MHz, CDCl ₃ , Jin Hz)δ(ppm):10.7393(s,1H),9.3476(s,1H),8.8551(t,J＝8.15Hz,2H),8.1096(s,1H),7.5815(s,1H),7.1244-6.9684(m,2H),6.7928(dd,J＝11.77,7.31Hz,2H),6.4630(d,J＝7.76Hz,1H),5.6218(dd,J＝8.10,5.02Hz,1H),5.5532-5.4333(m,1H),5.3588-5.2625(m,1H),5.1972(q,J＝6.99Hz,1H),4.9857(d,J＝16.95Hz,1 H), 4.3943-4.2390 (m, 2H), 4.182-3.9702 (m, 2H), 3.9125 (d, J = 6.38 Hz, 3H), 3.7542 (dd, J = 17.50, 8.19 Hz, 2H), 3.6590 (dd, J = 15.81, 3.29 Hz, 1H), 3.4146 (d, J = 16.96 Hz, 1H), 3.1079 (s, 3H), 2.0987-1.9303 (m, 2H), 1.8966 (d, J = 7.03 Hz, 3H), 1.7450 (d, J = 7.09 Hz, 3H), 1.3497 (d, J = 6.46 Hz, 3H), 0.8967 (t, J = 7.34 Hz, 3H);

¹³ C NMR (101 MHz, CDCl ₃ )δ173.11,171.47,171.38,169.83,169.31,169.21,167.34,160.51,153.77,150.03,136.71,130.55,124.86,123.19,122.88,116.34,110.91,106.55,105.66,99.71,77.48,77.16,76.84,66.88,62.29,55.48,54.74,52.89,51.29,45.51,41.62,37.65,28.31,21.32,21.17,20.73,11.49,10.66.

HRESITOFMS: m/z 790.2959 [M+H] ⁺ (calcd for C ₃₅ H ₄₅ N ₉ NaO ₉ S, 790.2953).

2. Anti-tumor activity test

1. Cell type: The cells used in this experiment are human liver cancer cell lines HepG2 and Huh7, both of which are from Shanghai Cell Bank.

2. Experimental reagents and instruments, as shown in Table 1 and Table 2:

Table 1 Main raw materials and sources

Table 2 Main experimental instruments

3. Anti-tumor experimental methods

(1) Cell inoculation and culture: When inoculating, cells are digested with an appropriate amount of 0.25% trypsin and cultured at 37°C with 5% CO ₂ . The culture medium is usually replaced every 2 to 3 days. Cells can be subcultured every 6 to 7 days. After 3 to 4 subcultures, cells in the logarithmic growth phase are usually used for experiments.

(2) MTT assay to determine anti-tumor cell activity

① Cell seeding plate: Take tumor cells in the logarithmic growth phase and add them to 1 mL of culture medium to make a cell suspension. Digest the cells with an appropriate amount of 0.25% trypsin, inoculate the cells in a 96-well plate at 4,000 cells per well, 100 μL per well, and culture in a 37°C, 5% CO ₂ incubator overnight.

② Cell administration: fully dissolve the target compound and positive control drug in DMSO, prepare the solution with 1% DMEM, add different concentrations of the test solution to the culture wells, set up 3 replicates for each concentration, and add 100 μL of sample solution to each well. The blank control group has 100 μL of 1% DMEM culture medium per well.

③ ELISA detection: before the end of the culture, the supernatant in each well was discarded, and 10 μL of freshly prepared MTT serum-free culture medium (concentration 5 mg/mL) was added to each well. After incubation at 37°C, 5% CO ₂ for 4 hours, the supernatant was aspirated, 150 μL DMSO was added to each well, and the reaction was shaken for 5 minutes to fully dissolve the formazan precipitate in the cells. The optical density (OD) was measured with an ELISA reader at a reference wavelength of 450 nm and a detection wavelength of 570 nm. The cell proliferation inhibition rate at the corresponding concentration was calculated according to the inhibition rate formula, and the IC ₅₀ value was calculated. All experiments were repeated 3 times and the average value was taken.

④Cell growth inhibition rate (%) = (1-average OD value of the drug-treated group/average OD value of the control group) × 100%.

(3) Anti-tumor activity data, as shown in Table 3:

Table 3 Antitumor activity data of simplified natural cyclopeptide zelkovamycins 1 and 2

As shown in Table 3, the simplified natural cyclopeptide zelkovamycins synthesized in Example 1-2 has good anti-tumor activity against human liver cancer cell lines HepG2 and Huh7. It can be seen that the simplified natural cyclopeptide zelkovamycins synthesized in the present invention has certain application prospects in the development of corresponding drugs.

The above description is a detailed description of the preferred feasible embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modified changes completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims (9)

1. A simplified natural cyclopeptide zelkovamycins, characterized in that the simplified product has a compound of formula 1 or 2 or a pharmaceutically acceptable salt or prodrug thereof. 2. The method for preparing a simplified natural cyclopeptide zelkovamycins according to claim 1, characterized in that it comprises the following steps: (1) Coupling the first amino acid to CTC resin: Add DCM to CTC resin and soak it, so that the CTC resin is fully swollen for 0.5-1h, and then remove the filtrate; dissolve the amino acid Fmoc-Sar-OH and collidine in DCM to obtain a mixed solution, add the obtained mixed solution to the CTC resin soaked in DCM, shake for 8-10h, filter out the reaction solution, and then wash it alternately with DCM and DMF three times; then add DCM/MeOH/DIPEA mixed solution to react for 30-50min, after the reaction is completed, filter the resin and wash it alternately with DCM and DMF three times to obtain Fmoc-Sar-OH-CTC resin; wherein the DCM/MeOH/DIPEA mixed solution is a solution formed by mixing DCM, MeOH, and DIPEA in a volume ratio of 17:2:1; (2) Synthesis of linear peptides ① The Fmoc-Sar-OH-CTC resin was shaken with a 20% piperidine/DMF solution for 2-10 min, and washed alternately with DCM and DMF for 3 times to obtain Sar-OH-CTC resin; ② Dissolve the next amino acid, HATU, and DIPEA in DMF to obtain a mixed solution, then add the obtained mixed solution to Sar-OH-CTC resin, shake for 2-4 hours, filter out the reaction solution, and wash with DCM and DMF alternately for 3 times to obtain CTC resin containing dipeptide; ③ Repeat the above steps ① and ② to sequentially couple the dipeptide-containing CTC resin with other amino acids in the order of the heptapeptide connection, and finally add a 20% piperidine/DMF solution to carry out a deprotection reaction, and wash it alternately with DCM and DMF to obtain a heptapeptide-containing CTC resin; (3) Cleavage of linear peptides A 0.5% TFA/DCM cutting solution was added to the CTC resin containing the heptapeptide, and after shaking for 20-40 minutes, the reaction solution was filtered and collected; the cutting solution was added again to react for 20-40 minutes, and the operation was repeated 3 times; the collected reaction solution was concentrated under reduced pressure to obtain a heptapeptide chain with the N-terminus protected by a Boc group; (4) Removal of the Boc protecting group at the N-terminus of the chain precursor The heptapeptide chain with the N-terminus protected by the Boc group was dissolved in DCM and placed in an ice bath for cooling and stirring, and TFA was slowly added, wherein the volume ratio of DCM to TFA was 1:1; after reacting for 30-50 minutes, TFA was blown away with nitrogen, 20-30 mL of DCM was added to the residue, and the solvent was evaporated under reduced pressure to obtain a crude heptapeptide chain product, which was directly used in the next step reaction; (5) Synthesis of simplified natural cyclopeptide zelkovamycins The crude product of the heptapeptide chain obtained in step (4) is dissolved in dry DCM, placed in an ice bath and stirred vigorously, and then a mixed solution obtained by dissolving HATU and HOAt in anhydrous DMF is added dropwise, and after cooling to 0-5°C, DIPEA is added, and then the temperature is raised to room temperature, and the reaction is carried out at room temperature for 48-60 hours; after the reaction is completed, the reaction is quenched with aqueous hydrochloric acid, and extracted with DCM, and the organic layer is washed with saturated _NaHCO3 solution and saturated brine respectively, and then dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude cyclic peptide, which is then purified by HPLC to obtain the target product; The molar ratio of the crude heptapeptide chain to HATU and HOAt was 1:2:2, and the molar ratio of the crude heptapeptide chain to DIPEA was 1:4.5. 3. The preparation method according to claim 2, characterized in that, in step (1), the molar ratio of amino acid Fmoc-Sar-OH to collidine is 1:2.1. 4. The preparation method according to claim 2, characterized in that in step (2) ②, the molar ratio of amino acid, HATU and DIPEA is 1:1:3.2. 5. The preparation method according to claim 2, characterized in that in step (2) ②, the next amino acid is Fmoc-2-aminobutyric acid or Fmoc-Dhb-OH. 6. The preparation method according to claim 2, characterized in that in step (2) (3), amino acids are connected in the order of Fmoc-2-aminobutyric acid, Fmoc-glycine, Fmoc-D-2-amino-butyric acid, Fmoc-4-Methoxy-L-Tryptophan, and Boc-D-Thiazole-alanine; or amino acids are connected in the order of Fmoc-D-Abu-OH, Fmoc-Gly-OH, Fmoc-L-Thr-OH, Fmoc-L-Trp(4-OMe)-OH, and Fmoc-D-Ala(Thz)-OH. 7. The preparation method according to claim 2, characterized in that in step (5), the HPLC purification conditions are: YMC-Pack ODS-A semi-preparative column, 5 μm, 250×10 mm, and the eluent is a solution composed of acetonitrile and water in a volume ratio of 27:73 or a solution composed of methanol and water in a volume ratio of 30:70. 8. Use of the simplified natural cyclopeptide zelkovamycins according to claim 1 in the preparation of anti-tumor drugs. 9 . The use according to claim 8 , characterized in that the anti-tumor drug is a drug against HepG2 or Huh7 cancer cells.