CN116082460A - Cyclic peptide GG-8-6-Lys 1 Chemical preparation method and pharmaceutical application of (C) - Google Patents

Abstract

Cyclic peptide GG-8-6-Lys ¹ Chemical preparation method and pharmaceutical application of the same. The invention belongs to the technical field of chemistry and medicine, and relates to a novel cyclic polypeptide compound GG-8-6-Lys ¹ . The compound related to the invention is prepared by a polypeptide organic method, and the molecular formula of the compound is C ₅₂ H ₉₄ N ₁₀ O ₉ The molecular weight is 1002. The cyclic peptide is a cyclic peptide compound formed by connecting L-lysine, L-leucine, L-valine, L-isoleucine and L-proline residues through amide bonds according to a certain sequence, and has the structural formula of cyclo- (Lys) ¹ ‑Leu ² ‑Pro ³ ‑Ile ⁴ ‑Leu ⁵ ‑Leu ⁶ ‑Leu ⁷ ‑Val ⁸ ‑Leu ⁹ ). The in vitro anti-liver cancer cell activity screening shows that the anti-liver cancer cells HepG2 and HuH7 have slightly stronger activity than GG-8-6. The compound of the invention can be used as candidate compound of anti-liver cancer drugs.

Description

Cyclic peptide GG-8-6-Lys 1 Chemical preparation method and pharmaceutical application of (C)

Technical Field

The invention belongs to the technical field of chemistry and medicine, and relates to cyclopeptide GG-8-6-Lys ¹ Chemical preparation method and pharmaceutical application of the same. In particular the cyclic peptide GG-8-6-lys ¹ The synthesis method and the application in preparing the anti-liver cancer cell proliferation medicine.

Background

The data discloses that malignant tumors are a major cause of harm to human life and health. Investigation has shown that the cancer burden in China is continuously increasing due to aging of population, industrialization and progress of urbanization, changes in life and production modes, etc ^[1] . According to statistics, china is the country with highest cancer incidence rate, the liver cancer incidence rate of China currently accounts for 55% of the world, and the liver cancer death rate accounts for 45% of the world ^[2] . Liver cancer has created a serious threat to the health of people in China. Although clinical trials of drugs for treating advanced liver cancer are successful and more new drugs are marketed in batches, more patients with liver cancer are likely to survive for a long time; however, the toxicity of oncologic drugs and the major side effects on humans are the most important hurdles of chemotherapeutic drug treatment. In addition, the drug resistance of tumor cells to chemotherapeutic drugs has also been a hindrance to the chemotherapy of liver cancer ^[3] . Therefore, the development of the chemotherapeutic drug with novel structure, low toxicity and small side effect (strong specificity) is an important research content aiming at liver cancer treatment in the technical field.

It is common in the industry that natural products are one of the important channels for drug discovery. Cyclic peptides are a special natural product with a special structure, which form peptide bonds by connecting amino groups of amino acids with carboxyl groups end to end, the structure does not contain free amino groups and carboxyl groups, is insensitive to aminopeptidase and carboxypeptidase, and has relatively stable conformation in solution, so that compared with common linear peptides, the cyclic peptides have better in vivo stability and lipophilicity and higher tissue permeability ^[4,5] 。

The early-stage research of the research team of the application takes the geldanamycin B (Goniomycetin B) which is an active cyclic peptide of tumor cell toxicity and is separated from plants as a lead compound, chemically synthesizes series of cyclic peptide compounds GG-8-1 to GG-8-6 which have the same amino acid composition as the cyclic peptide geldanamycin B but different connection sequences, and applies for Chinese invention patents (201410050535. X and 201710716520.6) for the preparation method of the cyclic peptide compounds. Based on this, the research team further chemically synthesizes the structural analogue GG-8-6-Lys of the cyclic peptide GG-8-6 ¹ The in vitro anti-liver cancer cell proliferation activity screening finds that the anti-liver cancer cells HepG2 and HuH7 have activity stronger than GG-8-6, and the synthesis yield is more than four times that of GG-8-6. For this purpose, the inventors of the present application have sought to provide the cyclic peptide GG-8-6-Lys ¹ Chemical preparation method and application of the composition in preparing medicines for resisting liver cancer.

References relevant to the present invention are:

[1]Oberg K,Lamberts SW.Somatostatin analogues in acromegaly and gastroenteropancreatic neuroendo-crine tumors:past,present and future[J].Endocrine-Related Cancer,2016,23(12):551-566.

[2]Janin YL.Peptides with anticancer use or potential[J].Amino Acids,2003,25:1-40.

[3]Berger D,Ckarella R,Dutia M,Greenberger L,Hallett W,Paul R,Powell D.Novel multidrug resistance reversal agents[J].Journal of Medicinal Chemistry,1999,42(12):2145-2161.

[4]Rüegg C,HasmimM,Lejeune FJ,Alghisi GC.Antiangiogenic peptides and proteins:from experimental tools to clinical drugs[J].Biochimica et Biophysica Acta,2006,1765:155-177.

[5]Kaplan N,Morpurgo N,Linial M.Novel families of toxin-like peptides in insects and mammals:a computational approach[J].Journal of Molecular Biology,2007,369:553-566.。

disclosure of Invention

The invention aims to provide cyclopeptides GG-8-6-Lys based on the basis and the current state of the art ¹ Chemical preparation method and application of the composition in preparing medicines for resisting liver cancer. In particular the cyclic peptide GG-8-6-lys ¹ Is synthesized by (a)The method and the application in preparing the anti-liver cancer cell proliferation medicine.

The technical scheme of the invention comprises the following steps:

1. cyclic peptide GG-8-6-Lys ¹ Chemical synthesis of (a)

In the invention, the cyclic nonapeptide compound GG-8-6-Lys ¹ Is a cyclic peptide, which is formed by connecting nine alpha-L-amino acids end to end in sequence, and the sequence of peptide chains is as follows: cyclo- (Lys) ¹ -Leu ² -Pro ³ -Ile ⁴ -Leu ⁵ -Leu ⁶ -Leu ⁷ -Val ⁸ -Leu ⁹ ) (as shown in fig. 1); wherein the amino acid is lysine (Lys), valine (Val), leucine (Leu), proline (Pro), isoleucine (Ile); its structural molecular formula is C ₅₂ H ₉₄ N ₁₀ O ₉ The molecular weight is 1002.

The invention provides the cyclic nonapeptide compound GG-8-6-Lys ¹ The chemical synthesis method (shown in figure 2) comprises the steps of solid-phase synthesis of linear peptide, liquid-phase cyclization and side chain deprotection: the 2-CTC resin takes Fmoc protected amino acid as a raw material, the linear peptide is prepared by a solid-phase chemical method, the linear peptide is connected with a ring end to obtain cyclic peptide with a protecting group, and then a side chain is deprotected to obtain the target cyclic peptide.

More specifically, the method comprises the steps of,

the cyclic nonapeptide compound GG-8-6-Lys of the invention ¹ Is characterized in that it comprises the steps of:

(1) Connecting a first amino acid under DIEA (direct-coupled amplification) condition by taking 2-CTC (China-control computer) resin as a solid phase carrier, taking HATU/HOAt as a condensing agent, taking N-methylmorpholine as a base and taking N, N-dimethylformamide as a solvent, and sequentially realizing the construction of amide bonds according to the amino acid sequence to synthesize a linear peptide precursor; then taking HATU/HOAt as a condensing agent and N-methylmorpholine as alkali, and realizing liquid phase ring closure in a high-dilution solution taking acetonitrile as a solvent;

(2) The resultant ring-closing product GG-8-6-Lys ¹ Separating the crude product of the protected precursor by using a column chromatography technology under the following chromatographic conditions: 75% acetonitrile/water reversed phase column to obtain cyclic peptide protected precursor monomer, total yield 19.6%;

(3) Ring(s)Peptide GG-8-6-Lys ¹ Removing Boc protecting group of the protected precursor by using TFA/DCM (1:6) mixed solvent under ice bath condition to obtain the target cyclopeptide compound with the total yield of 14.2%;

(4) The cyclic peptide GG-8-6-Lys ¹ The purified product is detected by a high performance liquid chromatography instrument, and the elution conditions are as follows: acetonitrile: water 65:3, 94% product purity (fig. 3); the molecular weight of the product is 1002, and the molecular formula is: cyclo- (Lys) ¹ -Leu ² -Pro ³ -Ile ⁴ -Leu ⁵ -Leu ⁶ -Leu ⁷ -Val ⁸ -Leu ⁹ )。

(5) Para-cyclic nonapeptide compound GG-8-6-Lys ¹ Structural identification was performed and GG-8-6-Lys after purification ¹ Structural confirmation by HRMS, GG-8-6-Lys ¹ Molecular ion peaks in High Resolution Mass Spectrum (HRMS) are shown as [ M+Na ]] ⁺ 1025.7100 (calculated as 1025.7097) (shown in fig. 4).

The invention carries out the cyclic peptide GG-8-6-Lys ¹ An in vitro anti-liver cancer activity test comprises,

(1) Preparation of GG-8-6-Lys ¹ A test solution;

(2) MTT assay for GG-8-6-Lys ¹ Inhibition of human hepatoma cells HuH7, hepG2, LM3 and 97H;

(3) Cell viability (%) = (OD dosing group-OD blank)/(OD control group-OD blank) ×100%, half inhibition concentration was calculated (half maximal inhibitory concentration, IC ₅₀ ) Evaluation of GG-8-6-Lys ¹ Inhibitory activity against human hepatoma cells HuH7, hepG2, LM3 and 97H;

(4) ATP method for detecting GG-8-6-Lys ¹ Inhibition of human hepatoma cells HuH7, hepG2, LM3 and 97H.

In the invention, the cell concentration of the in vitro anti-liver cancer activity experiment is 5 multiplied by 10 ⁴ cell/mL。

The invention proves that GG-8-6-Lys is proved by in vitro anti-liver cancer activity experiments ¹ Half Inhibition Concentration (IC) of human hepatoma cells HuH7 and HepG2 ₅₀ ) 8.59+ -0.97 μm and 7.34+ -0.33 μm, respectively, are significantly stronger than GG-8-6 (9.92+ -0.83 μm and 12.22+ -0.25 μm), and the activities of anti-liver cancer cells LM3 and 97H are slightly weaker than GG-8-6 (Table 1).

Table 1 shows the cyclic peptides GG-8-6 and GG-8-6-Lys ¹ Is compared with the in vitro anti-liver cancer cell proliferation activity.

Table 1.

The cyclopeptide GG-8-6-lys of the invention ¹ Can be used for preparing medicines for resisting liver cancer cell proliferation.

Drawings

FIG. 1 Cyclic peptide GG-8-6-Lys ¹ Is a chemical structure of (a).

FIG. 2 Cyclic peptide GG-8-6-Lys ¹ Is a synthetic route of (2).

FIG. 3 Cyclic peptide GG-8-6-Lys ¹ HPLC diagram of (2).

FIG. 4 cyclopeptide GG-8-6-Lys ¹ HRMS diagram of (2).

FIG. 5 shows the inhibition ratio of cyclic peptides detected by ATP method.

Detailed Description

The invention will now be further described with reference to examples, but the practice of the invention is not limited thereto.

Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.

Example 1, cyclopeptides GG-8-6-Lys ¹ Is synthesized by (a)

(1) First amino acid introduction resin:

2-CTC resin (1.0479 g,0.9mmol/g,100-200mesh,1% DVB) was taken in a solid phase synthesis reaction vessel, and 15mL of DCM was poured into the resin and swollen for 30min for use.

Fmoc-Val-OH (1.1058 g,3eq,0.27 mmol) was taken in a beaker, DMF 5mL was added for dissolution, poured into the swollen resin, DMF 5mL was added again, and the reaction was carried out for 3min, DIEA (800. Mu.L, 5eq,0.45 mmol) was added to the system and reacted for 2.5h.

After the reaction was completed, the resin was filtered, washed twice with DCM 10mL for 3min each and with DMF 10mL for 3min each.

10mL of the prepared blocking agent (MeOH/DCM/DIEA (2:17:1)) was poured into the washed resin and reacted for 30min. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL and twice with DMF 10 mL.

(2) Second amino acid introduction resin:

to the washed resin was added 10mL of 20% piperidine/DMF, reacted for 5min, filtered, poured into 10mL, reacted for 10min, and filtered. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL for 3min each and with DMF 10mL for 3min each. The modified Kaiser test was used to check if deprotection was successful: about 1mg of the resin is taken in a test tube, 4-5 drops of 5% ninhydrin/ethanol-pyridine-80% phenol ethanol (2:1:1) mixed solvent are added dropwise, and the mixture is heated for 1-2min to observe the color change. If blue, the deprotection is successful.

Fmoc-Leu-OH 1.1235g (3 eq,0.27 mmol), HATU 1.1334g (3 eq,0.27 mmol) and HOAt 0.3153g (3 eq,0.27 mmol) were taken in a beaker, 5mL of DMF was added to dissolve, poured into the resin, 5mL of DMF was added and reacted for 2-3min, 650. Mu.L (5 eq,0.45 mmol) of NMM was added to the system and reacted for 2h. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL and twice with DMF 10 mL.

(3) Third amino acid introduction resin:

to the washed resin was added 10mL of 20% piperidine/DMF, reacted for 5min and filtered. After the reaction was completed, the resin was filtered, washed twice with 10mL of DCM for 3min each and twice with 10mL of DMF.

Fmoc-Leu-OH 1.1248g (3 eq,0.27 mmol), HATU 1.1312g (3 eq,0.27 mmol) and HOAt 0.3167g (3 eq,0.27 mmol) were taken in a beaker, 5mL of DMF was added to dissolve, poured into the resin, 5mL of DMF was added and reacted for 2-3min, 650. Mu.L (5 eq,0.45 mmol) of NMM was added to the system and reacted for 2h. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL and twice with DMF 10 mL.

(4) The other amino acids were introduced into the resin in sequence:

to the washed resin was added 10mL of 20% piperidine/DMF, reacted for 5min, filtered, poured into 10mL, reacted for 10min, filtered and Fmoc protecting group removed. After the reaction was completed, the resin was filtered, washed twice with 10mL of DCM for 3min each and twice with 10mL of DMF. And detecting whether the deprotection is successful.

Fmoc-AA-OH (3 eq,0.27 mmol), HATU (3 eq,0.27 mmol) and HOAt (3 eq,0.27 mmol) were taken in a beaker, dissolved by adding 5mL of DMF, poured into the resin, and reacted for 2-3min, 650. Mu.L (5 eq,0.45 mmol) of NMM was added to the system and reacted for 2h. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL and twice with DMF 10 mL.

(5) Release of target linear peptide product:

to the washed resin was added 10mL of 20% piperidine/DMF, reacted for 5min, filtered, poured into 10mL, reacted for 10min, filtered and Fmoc protecting group removed. After the reaction was completed, the resin was filtered, washed twice with DCM 10mL and twice with DMF 10 mL.

To the washed resin was added 15mL of 1% TFA/DCM, stirred for 15min, filtered, the filtrate was collected, the resin was washed 3 times with 15mL of MeOH/DCM (5:95) for 3min each and the filtrate was collected each time. This operation was repeated 3 times and all filtrates were combined. The modified Kaiser test was used to check whether the cleavage was complete (no blue change indicates complete cleavage). All the filtrates were evaporated to dryness after cleavage to give 0.84g of the target linear peptide with a total yield of 86.0%.

(6) Linear peptide-related loops:

dissolving 0.84g of linear peptide in 20mL MeCN in a beaker, pouring into a reaction flask, adding MeCN into the reaction flask, and diluting the linear peptide to 10 ^-3 mmol/L, and stirring for standby. HATU 1.2653g (3 eq,0.27 mmol) and HOAt 0.4756g (3 eq,0.27 mmol) were weighed into a beaker and dissolved in MeCN, poured into the reaction flask and stirred until clear. The reaction system was placed in ice bath conditions and stirred for 30min. Another small beaker was poured into 15mL MeCN, 1300. Mu.L (10 eq,0.9 mmol) of NMM was added and added dropwise to the reaction system under ice-bath conditions. After the dripping is finished, the reaction is continued for 30min under the ice bath, the ice bath is removed, and the reaction is carried out for 48h at room temperature. After the reaction is finished, filtering and spin-drying the filtrate to obtain the target cyclopeptide GG-8-6-Lys ¹ Protecting the crude precursor.

Cyclic peptide GG-8-6-Lys ¹ Purification of crude protected precursor: the product is subjected to reverse phase C ₁₈ Column chromatography 75% MeCN/H ₂ O is eluted and purified to obtain GG-8-6-Lys ¹ 194.8mg of protected precursor in 22.8% yield, 19.6% overall yield.

(7) Removing Boc protecting groups

The protected precursor 194.8mg was taken in a reaction flask, 7mL of TFA/DCM (1:6) was added and stirred for 30min, the solvent was spun dry, and the appropriate amount of DCM and 5% Na were added ₂ CO ₃ Oscillating, detecting by pH test paper, regulating pH to 8-9, drying DCM layer, repeating for three times, and spin drying to obtain cyclopeptide GG-8-6-Lys ¹ 126.6mg of monomer was found in 65.0% yield, 14.2% overall yield.

(8) Structure validation

Subjecting said GG-8-6-Lys ¹ Dissolving with methanol, and detecting by High Resolution Mass Spectrum (HRMS), wherein [ M+Na ]] ⁺ Peak abundance was 1002.7100, consistent with the target cyclic peptide.

Example 2, cyclopeptides GG-8-6-Lys ¹ In vitro anti-hepatoma cell proliferation activity experiment

The cytotoxic effect of the compounds was determined using the MTT method. Cells in the logarithmic growth phase were prepared into cell suspensions in DMEM medium containing 10% fetal bovine serum, inoculated in 96-well plates, and 3 duplicate wells were set for each concentration of compound. Mu.l of cells (containing 5000 tumor cells) per well were placed in a 5% CO2 incubator at 37 ℃. After a certain period of compound action, 20. Mu.l MTT solution was added to each well and incubated at 37℃for 4 hours. After that, the supernatant was discarded, 150. Mu.L of DMSO was added, and the OD value was measured at 490nm using an ELISA reader, and the experimental results were repeated three times.

Cell viability (%) = (OD dosing group-OD blank)/(OD control group-OD blank) ×100%.

The results showed that the cyclic peptide GG-8-6-Lys ¹ Half Inhibition Concentrations (IC) of human hepatoma cells HuH7, hepG2, LM3 and 97H ₅₀ ) 8.59.+ -. 0.97. Mu.M, 7.34.+ -. 0.33. Mu.M, 17.29.+ -. 1.82. Mu.M and 27.49.+ -. 2.99. Mu.M, respectively (FIG. 2).

The cytotoxic effect of the compounds was verified using the ATP method. A synergistic ATP detection kit (Beyotime Biotechnology, shanghai, china) was used. The same tumor cells were detected using the MTT method and incubated with the same concentration of compound. After 24 hours of continuous exposure, 200. Mu.L of ATP lysate was added to each well to extract ATP. After a short incubation time, the buffer was centrifuged at 15000g for 5min at 4 ℃. Collecting supernatant, and refrigerating. 20. Mu.L of supernatant was taken, mixed with 100. Mu.L of ATP working solution (ATP assay solution: ATP assay diluent=1:4) per well, and transferred to a black six-well plate. The amount of luminescence was measured immediately during a counting integration time of 1 second using a luminometer (Bio-Tek, USA). Chemiluminescent counts are proportional to ATP content. Inhibition = (solvent-treated ATP level-compound-treated ATP level)/solvent-treated ATP level x 100%.

Claims (5)

1. Cyclic peptide compound GG-8-6-Lys of the formula ¹ ' the cyclic peptide is a cyclic peptide compound formed by connecting L-lysine, L-leucine, L-valine, L-isoleucine and L-proline residues through amide bonds according to a certain sequence, and the structural formula of the cyclic peptide compound is cyclo- (Lys) ¹ -Leu ² -Pro ³ -Ile ⁴ -Leu ⁵ -Leu ⁶ -Leu ⁷ -Val ⁸ -Leu ⁹ ) The molecular weight is 1002.

2. GG-8-6-Lys according to claim 1 ¹ ' is characterized in that the preparation method comprises the following steps,

the preparation method comprises adopting 2-CTC resin as solid phase carrier, constructing amide bond sequentially according to amino acid sequence, synthesizing linear peptide by solid phase method,

which comprises the steps of the method,

(1) The cyclization process is realized in a highly diluted solution with HATU/HOAt as condensing agent and N-methylmorpholine as alkali and acetonitrile as solvent, and the side chain is deprotected to obtain cyclic peptide GG-8-6-Lys ¹ The purity is more than 93 percent.

(2) The cyclic peptide compound contains a lysine (Lys) residue with a naked amino group.

3. GG-8-6-Lys according to claim 2 ¹ ' characterized in that the cyclic peptide GG-8-6-Lys ¹ Protection of the precursor side chain deprotection was performed under TFA/DCM (1:6) conditions.

4. The cyclic peptide compound GG-8-6-Lys according to claim 1 ¹ The application in preparing anti-liver cancer drugs;

the GG-8-6-Lys ¹ Has the activity of resisting liver cancer cell proliferation in vitro,

ATP method for detecting GG-8-6-Lys ¹ Has inhibitory effect on human hepatoma cells, and has half Inhibitory Concentration (IC) ₅₀ ) 8.59.+ -. 0.97 (7.62-9.56) μM, 7.34.+ -. 0.33 (7.01-7.67) μM, 17.29.+ -. 1.82 (15.47-19.11) μM and 27.49.+ -. 2.99 (24.50-30.48) μM, respectively.

5. The method of claim 4, wherein the human hepatoma cells are hepatoma cells HuH7, hepG2, LM3 and 97H.

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