CN101906136A - Amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide and its synthesis method and application - Google Patents

Abstract

The invention discloses an amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide as well as its synthesis method and application. Its synthesis method includes: (1) synthesizing protected (3,4-dihydroxy-Phe)-Ser-Leu; (2) combining glycine, alanine, valine, leucine, isoleucine, benzene Alanine, tyrosine, tryptophan, serine, threonine, proline, glutamine, lysine, histidine, aspartic acid or glutamic acid were protected with protecting groups (3 , 4-dihydroxy-Phe)-Ser-Leu condensation, remove the protecting group, that is. In addition to good NO free radical scavenging activity, the pseudopeptide compound of the present invention also has the property of inhibiting vasodilation, and has excellent oral antithrombotic activity, and can be used as a drug with dual activities of free radical scavenging and antithrombotic.

Description

Amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide and its synthesis method and application

technical field

The present invention relates to pseudopeptides, in particular to pseudopeptides with dual activities of scavenging free radicals and antithrombotic activity obtained by condensation of amino acids with (3,4-dihydroxy-Phe)-Ser-Leu pseudotripeptide amino terminals. The present invention also relates to Their synthesis methods and their use as free radical scavenging antioxidants, vasodilator inhibitors and antithrombotic agents belong to the field of biomedicine.

Background technique

Compounds containing catechol structure have antioxidant capacity and can scavenge free radicals. Excessive accumulation of free radicals in the body can lead to various diseases, such as cardiovascular and cerebrovascular diseases, inflammation, aging, neurological diseases, etc. NO can dilate blood vessels, and when free radical scavengers exist, their vasodilator effects will be inhibited. Free radicals are also related to the aggregation of platelets, and superoxide free radicals can promote the aggregation of platelets. Flavonoids containing catechol structure can scavenge free radicals, thereby inhibiting the aggregation of platelets. Therefore, a series of pseudopeptides containing catechol structure were designed to scavenge free radicals and inhibit platelet aggregation.

Contents of the invention

One of the objectives of the present invention is to provide a class of pseudopeptides with good water solubility, long biological half-life, scavenging free radicals in the body and antithrombotic effect.

The second object of the present invention is to provide a method for synthesizing the above-mentioned pseudo-peptide;

The object of the invention is achieved through the following technical solutions:

Amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide, its structural formula is shown in general formula I:

通式I

Formula I

Wherein, AA is selected from hydrogen (H), glycine residue (Gly), alanine residue (Ala), valine residue (Val), leucine residue (Leu), isoleucine residue (Ile), phenylalanine residue (Phe), tyrosine residue (Tyr), tryptophan residue (Trp), serine residue (Ser), threonine residue (Thr), proline Acid residues (Pro), glutamine residues (Gln), lysine residues (Lys), histidine residues (His), aspartic acid residues (Asp) or glutamic acid residues (Glu) Residue; wherein, the amino acid or 3,4-dihydroxy-Phe are left-handed.

Another object of the present invention is to provide a method for synthesizing the above-mentioned amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide;

A method for synthesizing the above-mentioned amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide, comprising:

(1) Synthesis of protected (3,4-dihydroxy-Phe)-Ser-Leu;

(2) Glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine ( Tyr), tryptophan (Trp), serine (Ser), threonine (Thr), proline (Pro), glutamine (Gln), lysine (Lys), histidine (His), Aspartic acid (Asp) or glutamic acid (Glu) is condensed with protecting group-protected (3,4-dihydroxy-Phe)-Ser-Leu respectively, and the protecting group is removed to obtain the product.

The present invention combines the following understandings or completes the above technical solutions. Elevated levels of free radicals in the body can lead to diseases such as tumor diseases, autoimmune disorders, aging, rheumatoid arthritis, cardiovascular diseases, and neurodegenerative diseases, while antioxidants can remove free radicals and maintain normal physiological functions of the human body . Many natural compounds have a catechol structure and are excellent antioxidants. Flavonoids are a class of antioxidants containing catechol structure, which can effectively scavenge free radicals, and experiments have proved their anti-platelet aggregation activity. And 3,4-dihydroxy-Phe also contains a catechol structure, which has the ability to scavenge free radicals. In addition, its structure is similar to amino acids, and can be condensed into small molecular peptides as amino acid analogs. On the one hand, it can play its role in scavenging free radicals. On the other hand, the protection of its amino and carboxyl ends increases the water solubility of the entire molecule, making it difficult to metabolize the 3,4-dihydroxy-Phe part of the compound and improving the bioavailability.

Based on the above knowledge, the present inventor condensed sixteen kinds of amino acids with the amino terminal of the (3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide, so that the obtained molecules obtained four properties, that is, depending on the goodness of the peptide The water solubility depends on the catechol structure in the 3,4-dihydroxy-Phe structure, the antioxidant and free radical scavenging ability and the ability to resist platelet aggregation and depends on AA and Ser-Leu dipeptide pair 3, Protection of the 4-dihydroxy-Phe structure. In this way, the molecule obtained by condensing (3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide with sixteen kinds of amino acids becomes a good water-soluble, stable in vivo scavenging free radical and antithrombotic drug .

The evaluation on the rat thrombosis model shows that the free radical scavenger of the present invention has excellent oral antithrombotic activity, and can be used as an antithrombotic agent; The invented pseudopeptide has an excellent ability to scavenge free radicals, and can be used as a free radical scavenger; the experiment of inhibiting relaxation of rat thoracic aortic rings shows that the pseudopeptide of the present invention has excellent activity of inhibiting vasodilation.

Another object of the present invention is to provide a pharmaceutical composition containing the pseudopeptide compound of the present invention, which is composed of a therapeutically effective dose of the pseudopeptide compound of the present invention and pharmaceutically acceptable excipients or adjuvants ; That is: after the effective amount of the compound of the present invention is combined with a pharmaceutically acceptable carrier or diluent, it is prepared into any suitable pharmaceutical composition according to the conventional preparation method in this field. Usually the composition is suitable for oral administration and injection administration, and other administration methods are also suitable. The composition can be in the form of tablets, capsules, powders, granules, lozenges, suppositories, or liquid preparations such as oral liquids. According to different administration methods, the pharmaceutical composition of the present invention may contain 0.1%-99% by weight, preferably 10-60% by weight of the pseudopeptide compound of the present invention.

Description of drawings

Fig. 1 is a synthetic route diagram of the pseudopeptide of the present invention. i) DCC, HOBt, NMM; ii) hydrogen chloride/ethyl acetate solution (4N); iii) 10% palladium on carbon and hydrogen (0.02Mba). Where AA = glycine (Gly), alanine (Ala), valine Acid (Val), Leucine (Leu), Isoleucine (Ile), Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp), Serine (Ser), Threonine (Thr), proline (Pro), glutamine (Gln), lysine (Lys), histidine (His), aspartic acid (Asp), glutamic acid (Glu) residues.

Detailed ways

In order to further illustrate the present invention, a series of examples are given below. These examples are entirely illustrative, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

The preparation of embodiment 1 (3,4-dihydroxy-Phe)-Ser-Leu (4q)

1) Preparation of Boc-Ser-Leu-OBzl

Put 4.00g (10.18mmol) Leu-OBzl, 1.90g (9.25mmol) Boc-Ser and 1.44g (10.69mmol) HOBt in a 100mL eggplant bottle, add 50mLTHF, drop about 20 drops of N-methyl Morpholine, until the solution is clear, add dropwise a solution consisting of 2.28g (11.12mmol) of DCC and 15mL of anhydrous THF. Add about 20 drops of NMM to the obtained solution to adjust the pH value to 8-9. After the reaction mixture was stirred at 0° C. for 10 minutes, the ice bath was removed, and it was reacted at room temperature for 4 hours and then filtered with suction, and the filtrate was concentrated to dryness under reduced pressure. The obtained residue was dissolved in 70mL ethyl acetate, placed in a 100mL separatory funnel, washed successively with 5% aqueous sodium bicarbonate (20mL×3), saturated aqueous sodium chloride (20mL×3), 5% hydrogensulfate Potassium aqueous solution (20mL×3), saturated sodium chloride aqueous solution (20mL×3), 5% sodium bicarbonate aqueous solution (20mL×3), saturated sodium chloride aqueous solution (20mL×3). The ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain an oily substance soaked in petroleum ether for a period of time, then ground and washed to obtain 3.62 g (96.02%) of the title compound as a colorless solid. ESI ⁺ -MS(m/e)409[M+H] ⁺ .

2) Preparation of HCl Ser-Leu-OBzl

Put 1g of Boc-Ser-Leu-OBzl (2.45mmol) in a 50mL eggplant bottle, dissolve it with about 6mL of anhydrous ether, add about 5mL of 4N HCl/EtOAc dropwise under ice-bath conditions, and cover the drying tube after the dropwise addition. The ice bath was removed and the reaction was allowed to proceed at room temperature. After about ten minutes, it was observed that the reaction solution precipitated a white solid, that is, the reaction solution changed from clear to turbid, and the reaction ended after 1.5 hours. About 10 mL of anhydrous diethyl ether was added to the reaction liquid, and concentrated to dryness under reduced pressure. Then add anhydrous diethyl ether and stir evenly, let it stand, pour off the supernatant, repeat the operation 3 times, the precipitate is vacuum-dried to obtain 0.80 g (95.24%) of the title compound as a colorless solid. ESI ⁺ -MS(m/e)309[M+H] ⁺ 3) Preparation of Boc-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(1)

According to the preparation method of Boc-Ser-Leu-OBzl, 4.11g (93% ) the title compound as a pale yellow solid. Mp 130-132°C; ESI ⁺ -MS (m/e) 588[M+H] ⁺ ; [α] _D ²⁰ =-31.48 (c = 0.115, methanol).

4) Preparation of HCl H-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (2)

According to the preparation method of HCl·Ser-Leu-OBzl, 1.65 g (92.43%) of the title compound was obtained from 2 g (3.41 mmol) 1 as a colorless solid. ESI ⁺ -MS(m/e)488[M+H] ⁺ .

5) Preparation of (3,4-dihydroxy-Phe)-Ser-Leu(4q)

Put 89 mg (17.0 μmol) 2 in a 25 mL eggplant bottle, dissolve it with 8 mL of methanol, add 10 mg of 10% palladium carbon, and after mixing evenly, connect the reaction bottle, vacuum pump, and hydrogen bag with a three-way connection, and first use a vacuum pump to pump Vacuum, then add H ₂ , repeat the operation 3 to 4 times, fill the reaction bottle with H ₂ , react at room temperature for about 8 hours, filter the reaction solution under reduced pressure, concentrate the filtrate under reduced pressure, soak in ether for a period of time, and precipitate a solid. The supernatant was decanted, and the ether was removed by precipitation in vacuo to obtain about 65 mg (96.31%) of the title compound as a colorless solid Mp 150-151°C; ESI ^- MS (m/e) 396 [MH] ^- ; [α] _D ²⁰ = -5.40 (c=0.115, methanol).

The preparation of embodiment 2 Gly-(3,4-dihydroxy-Phe)-Ser-Leu (4a)

1) Preparation of Boc-Gly-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3a)

According to the preparation method of Boc-Ser-Leu-OBzl, 1.73g (93.61%) of the title compound was obtained from 1.5g (2.865mmol) 2 and 0.55g (3.15mmol) Boc-Gly as a colorless solid Mp 107-108°C; ESI ⁺ -MS (m/e) 646[M+H] ⁺ , 668[M+Na] ⁺ ; [α] _D ²⁰ =-30.43 (c=0.100, methanol).

2) Preparation of Gly-(3,4-dihydroxy-Phe)-Ser-Leu (4a)

Put 100 mg (15.5 μmol) of 3a in a 25 mL eggplant bottle, dissolve it with 8 mL of methanol, add 10 mg of 10% palladium carbon, and after mixing evenly, vacuumize the reaction bottle with a tee, and then introduce H ₂ , repeat the operation 3 to 4 times, fill the reaction bottle with H ₂ , react at room temperature for about 8 hours, filter the reaction solution under reduced pressure, concentrate the filtrate under reduced pressure, soak in ether for a period of time, a solid precipitates, pour off the supernatant, precipitate and remove the ether in a vacuum A colorless solid was obtained. Under the condition of ice bath, 4 mL of 4N HCl/ethyl acetate was slowly added dropwise to the solid powder, the drying tube was covered, the ice bath was removed, and the reaction was suspended at room temperature for 2 hours. After the reaction, add 8 mL of anhydrous ether, concentrate under reduced pressure, obtain a solid and then soak it in anhydrous ether, pour off the supernatant, repeat the operation 3 times, remove the ether from the obtained solid in vacuo to obtain about 67 mg (94.97%) of the title compound, as Colorless solid Mp 162-164°C; ESI ^- MS (m/e) 454[MH] ^- ; [α] _D ²⁰ =-12.09 (c = 0.115, methanol).

The preparation of embodiment 3Ala-(3,4-dihydroxy-Phe)-Ser-Leu (4b)

1) Preparation of Boc-Ala-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3b)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.70 g (90.17%) of the title compound was obtained as a colorless solid from 0.60 g (3.15 mmol) of Boc-Ala. Mp 107-108°C; ESI ⁺ -MS (m/e) 658[M+H] ⁺ ; [α] _D ²⁰ =-31.48 (c=0.115, methanol).

2) Preparation of Ala-(3,4-dihydroxy-Phe)-Ser-Leu (4b)

Following the preparation method of 4a, 68 mg (95.61%) of the title compound were obtained from 100 mg (15.2 μmol) of 3b as a colorless solid. Mp 163-165°C; ESI ^- -MS(m/e) 467[MH] ^- ; [α] _D ²⁰ =-4.25 (c=0.105, methanol).

The preparation of embodiment 4Val-(3,4-dihydroxy-Phe)-Ser-Leu (4c)

1) Preparation of Boc-Val-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3c)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.78 g (90.56%) of the title compound were obtained from 0.68 g (3.15 mmol) of Boc-Val as a colorless solid. Mp 84-86°C; ESI ⁺ -MS (m/e) 687[M+H] ⁺ , 709[M+Na] ⁺ , 725[M+K] ⁺ ; [α] _D ²⁰ =-41.44°(c =0.130, methanol).

2) Preparation of Val-(3,4-dihydroxy-Phe)-Ser-Leu(4c)

66 mg (91.28%) of the title compound was obtained as a colorless solid from 100 mg (14.9 μmol) of 3c following the preparation method of 4a. Mp 155-157°C; ESI ^- -MS(m/e) 495[MH] ^- ; [α] _D ²⁰ =17.20 (c=0.125, methanol).

The preparation of embodiment 5Leu-(3,4-dihydroxy-Phe)-Ser-Leu (4d)

1) Preparation of Boc-Leu-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3d)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.82 g (90.74%) of the title compound were obtained as a colorless solid from 0.73 g (3.15 mmol) of Boc-Leu. Mp 132-134°C; ESI ⁺ -MS (m/e) 701 [M+H] ⁺ ; [α] _D ²⁰ =-42.77 (c = 0.135, methanol).

2) Preparation of Leu-(3,4-dihydroxy-Phe)-Ser-Leu(4d)

Following the preparation method of 4a, 67 mg (91.96%) of the title compound was obtained from 100 mg (14.3 μmol) of 3d as a colorless solid. Mp 145-147°C; ESI ^- -MS(m/e) 509[MH] ^- ; [α] _D ²⁰ =-16.38°(c=0.105, methanol).

The preparation of embodiment 6Ile-(3,4-dihydroxy-Phe)-Ser-Leu (4e)

1) Preparation of Boc-Ile-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3e)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.78 g (88.75%) of the title compound were obtained from 0.73 g (3.15 mmol) of Boc-Ile as a colorless solid. Mp 68-70°C; ESI ⁺ -MS (m/e) 701[M+H] ⁺ , 723[M+Na] ⁺ , 739[M+K] ⁺ ; [α] _D ²⁰ ＝-38.06(c＝ 0.120, methanol).

2) Preparation of Ile-(3,4-dihydroxy-Phe)-Ser-Leu(4e)

Following the preparation method of 4a, 64 mg (87.84%) of the title compound were obtained from 100 mg (14.3 μmol) of 3e as a colorless solid. Mp 148-149°C; ESI ^- -MS(m/e) 509[MH] ^- ; [α] _D ²⁰ =7.30°(c=0.115, methanol).

The preparation of embodiment 7Phe-(3,4-dihydroxy-Phe)-Ser-Leu (4f)

1) Preparation of Boc-Phe-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3f)

Following the preparation of Boc-Ser-Leu-OBzl, 1.92 g (91.29%) of the title compound were obtained as a colorless solid from 0.84 g (3.15 mmol) of Boc-Phe. Mp 173-175°C; ESI ⁺ -MS (m/e) 735 [M+H] ⁺ ; [α] _D ²⁰ = -35.91 (c = 0.110, methanol)

2) Preparation of Phe-(3,4-dihydroxy-Phe)-Ser-Leu(4f)

According to the preparation method of 4a, 70 mg (94.45%) of the title compound was obtained from 100 mg (15.2 μmol) of 3f as a colorless solid. Mp 143-145°C; ESI ^- -MS(m/e) 543[MH] ^- ; [α] _D ²⁰ =-9.57 (c=0.115, methanol).

Preparation of Example 8 Tyr-(3,4-dihydroxy-Phe)-Ser-Leu (4g)

1) Preparation of Boc-Tyr-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3g)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.95 g (90.74%) of the title compound was obtained as a colorless solid from 0.89 g (3.15 mmol) of Boc-Tyr. Mp 153-154°C; ESI ⁺ -MS (m/e) 751[M+H] ⁺ , 773[M+Na] ⁺ ; [α] _D ²⁰ =-35.19 (c=0.115, methanol).

2) Preparation of Tyr-(3,4-dihydroxy-Phe)-Ser-Leu (4g)

Following the preparation method of 4a, 69 mg (92.41%) of the title compound was obtained from 100 mg (13.3 μmol) 3 g as a colorless solid. Mp 151-153°C; ESI ^- -MS(m/e) 559[MH] ^- ; [α] _D ²⁰ =-23.01 (c=0.125, methanol).

The preparation of embodiment 9Trp-(3,4-dihydroxy-Phe)-Ser-Leu (4h)

1) Preparation of Boc-Trp-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3h)

Following the preparation of Boc-Ser-Leu-OBzl, 2.00 g (90.30%) of the title compound were obtained as a colorless solid from 0.96 g (3.15 mmol) of Boc-Trp. Mp 160-162°C; ESI ⁺ -MS (m/e) 774[M+H] ⁺ ; [α] _D ²⁰ =-37.07 (c = 0.100, methanol).

2) Preparation of Trp-(3,4-dihydroxy-Phe)-Ser-Leu(4h)

Following the preparation method of 4a, 70 mg (92.81%) of the title compound was obtained from 100 mg (12.9 μmol) in 3 h as a colorless solid. Mp 169-171°C; ESI ^- -MS(m/e) 582[MH] ^- ; [α] _D ²⁰ =-10.43 (c=0.100, methanol).

Embodiment 10 Preparation of Ser-(3,4-dihydroxy-Phe)-Ser-Leu (4i)

1) Preparation of Boc-Ser-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3i)

According to the preparation method of Boc-Ser-Leu-OBzl, 1.80 g (93.20%) of the title compound were obtained from 0.65 g (3.15 mmol) of Boc-Ser as a colorless solid. Mp 93-95°C; ESI ⁺ -MS (m/e) 675[M+H] ⁺ ; [α] _D ²⁰ =-37.16 (c=0.115, methanol).

2) Preparation of Ser-(3,4-dihydroxy-Phe)-Ser-Leu(4i)

Following the preparation method of 4a, 66 mg (91.91%) of the title compound were obtained from 100 mg (14.8 μmol) of 3i as a colorless solid. Mp 149-151°C; ESI ^- -MS(m/e) 483[MH] ^- ; [α] _D ²⁰ =-16.97 (c=0.110, methanol).

The preparation of embodiment 11T-(3,4-dihydroxy-Phe)-Ser-Leu (4j)

1) Preparation of Boc-Thr-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3j)

Following the preparation of Boc-Ser-Leu-OBzl, 1.83 g (92.83%) of the title compound were obtained from 0.69 g (3.15 mmol) of Boc-Thr as a colorless solid. Mp 123-125°C; ESI ⁺ -MS (m/e) 689[M+H] ⁺ ; [α] _D ²⁰ =-37.90 (c = 0.100, methanol).

2) Preparation of Thr-(3,4-dihydroxy-Phe)-Ser-Leu(4j)

Following the preparation of 4a, 67 mg (92.56%) of the title compound was obtained from 100 mg (14.5 μmol) of 3j as a colorless solid. Mp 148-150°C; ESI ^- -MS(m/e) 497[MH] ^- ; [α] _D ²⁰ =-10.18 (c=0.110, methanol).

The preparation of embodiment 12P-(3,4-dihydroxy-Phe)-Ser-Leu (4k)

1) Preparation of Boc-Pro-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3k)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.78 g (90.82%) of the title compound were obtained from 0.68 g (3.15 mmol) of Boc-Pro as a colorless solid. Mp 81-83°C; ESI ⁺ -MS (m/e) 685[M+H] ⁺ ; [α] _D ²⁰ =-27.96 (c=0.095, methanol).

2) Preparation of Pro-(3,4-dihydroxy-Phe)-Ser-Leu(4k)

Following the preparation of 4a, 69 mg (95.54%) of the title compound was obtained from 100 mg (14.6 μmol) of 3k as a colorless solid. Mp 135-137°C; ESI ^- -MS(m/e) 493[MH] ^- ; [α] _D ²⁰ =-44.09 (c=0.110, methanol).

The preparation of embodiment 13Gln-(3,4-dihydroxy-Phe)-Ser-Leu (4l)

1) Preparation of Boc-Gln-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl (3l)

Following the preparation method of Boc-Ser-Leu-OBzl, 1.87 g (91.28%) of the title compound were obtained as a colorless solid from 0.78 g (3.15 mmol) of Boc-Gln. Mp 168-169°C; ESI ⁺ -MS (m/e) 716[M+H] ⁺ ; [α] _D ²⁰ =-37.68 (c = 0.105, methanol).

2) Preparation of Gln-(3,4-dihydroxy-Phe)-Ser-Leu(4l)

Following the preparation method of 4a, 67 mg (91.25%) of the title compound was obtained from 100 mg (14.00 μmol) of 3l as a colorless solid. Mp 150-152°C; ESI ^- -MS(m/e) 524[MH] ^- ; [α] _D ²⁰ =-1.26 (c=0.095, methanol).

The preparation of embodiment 14Lys-(3,4-dihydroxy-Phe)-Ser-Leu (4m)

1) Preparation of Boc-Lys(Boc)-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3m)

Following the preparation method of Boc-Ser-Leu-OBzl, 2.07 g (88.64%) of the title compound were obtained as a colorless solid from 1.09 g (3.15 mmol) of Boc-Lys (Boc). Mp 106-108°C; ESI ⁺ -MS (m/e) 816[M+H] ⁺ ; [α] _D ²⁰ =-31.48 (c = 0.115, methanol).

2) Preparation of Lys-(3,4-dihydroxy-Phe)-Ser-Leu

Following the preparation method of 4a, 60 mg (93.14%) of the title compound were obtained from 100 mg (12.3 μmol) of 3m as a colorless solid. Mp 162-163°C; ESI ^- -MS(m/e) 524[MH] ^- ; [α] _D ²⁰ =-21.24 (c=0.110, methanol).

The preparation of embodiment 15His-(3,4-dihydroxy-Phe)-Ser-Leu(4n)

1) Preparation of Boc-His(Boc)-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3n)

Following the preparation method of Boc-Ser-Leu-OBzl, 2.18 g (92.33%) of the title compound were obtained as a colorless solid from 1.12 g (3.15 mmol) of Boc-His (Boc). Mp 115-117°C; ESI ⁺ -MS (m/e) 825[M+H] ⁺ ; [α] _D ²⁰ =-26.79 (c=0.105, methanol).

2) Preparation of His-(3,4-dihydroxy-Phe)-Ser-Leu(4n)

Following the preparation method of 4a, 59 mg (91.04%) of the title compound were obtained from 100 mg (12.1 μmol) of 3n as a colorless solid. Mp 151-153°C; ESI ^- -MS(m/e) 533[MH] ^- ; [α] _D ²⁰ =-16.23 (c=0.115, methanol).

The preparation of embodiment 16Asp-(3,4-dihydroxy-Phe)-Ser-Leu(4o)

1) Preparation of Boc-Asp(OBzl)-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3o)

Following the preparation method of Boc-Ser-Leu-OBzl, 2.06 g (89.20%) of the title compound were obtained as a colorless solid from 1.06 g (3.15 mmol) of Boc-Asp(OBzl). Mp 136-138°C; ESI ⁺ -MS (m/e) 807[M+H] ⁺ , 829[M+Na] ⁺ ; [α] _D ²⁰ =-35.23 (c=0.095, methanol).

2) Preparation of Asp-(3,4-dihydroxy-Phe)-Ser-Leu(4o)

Following the preparation method of 4a, 62 mg (95.91%) of the title compound was obtained from 100 mg (12.6 μmol) of 3o as a colorless solid. Mp 147-149°C; ESI ^- -MS(m/e) 511[MH] ^- ; [α] _D ²⁰ =21.68 (c=0.125, methanol).

Embodiment 17 Preparation of Glu-(3,4-dihydroxy-Phe)-Ser-Leu(4p)

1) Preparation of Boc-Glu(OBzl)-(3,4-dihydroxy-Phe)-Ser-Leu-OBzl(3p)

Following the preparation method of Boc-Ser-Leu-OBzl, 2.11 g (92.98%) of the title compound were obtained as a colorless solid from 1.02 g (3.15 mmol) of Boc-Glu (OBzl). Mp 67-68°C; ESI ⁺ -MS (m/e) 793[M+H] ⁺ ; [α] _D ²⁰ =-38.06 (c=0.120, methanol).

2) Preparation of Glu-(3,4-dihydroxy-Phe)-Ser-Leu(4p)

Following the preparation method of 4a, 63 mg (96.54%) of the title compound were obtained from 100 mg (12.4 μmol) of 3p as a colorless solid. Mp 133-135°C; ESI ^- -MS(m/e) 525[MH] ^- ; [α] _D ²⁰ =-5.64 (c=0.115, methanol).

Test example 1 Antithrombotic activity test of compound of the present invention administered orally

1) Rat surgery and instruments

SD rats (male, 220-230 g) were orally administered the compound of the present invention at a dose of 10 nmol·kg ^-1 , and were anesthetized by intraperitoneal injection of urethane solution at a dose of 1200 mg-kg ^-1 30 min later. Anesthetized rats were fixed in a supine position, the right common carotid artery was separated, and the arterial clamp was clamped at the proximal end, and the proximal and distal ends were respectively threaded into the surgical thread, and the surgical thread at the distal end was clamped on the fur with a hemostat, ready to be placed in the Intubation at the distal end.

2) Intubation

The intubation tube is a silanized polyethylene hose, which is divided into three sections. The middle section is a polyethylene hose with a length of 60.0 mm and an inner diameter of 3.5 mm; both ends are the same polyethylene tube with a length of 100.0 mm, an inner diameter of 1.0 mm, and an outer diameter of 2.0 mm. mm One end of the tube is drawn into a tip tube (for insertion into a rat carotid artery or vein) with an outer diameter of 1.0 mm. Put 6cm-long black surgical thread into the numbered clean penicillin vials and weigh them; then take out the silk thread and put it into the thicker middle section of the prepared cannula according to the number.

Open the right arterial clamp of the rat, use a syringe to fill the tube with heparin saline solution (50IU·kg ^-1 ) through the tip of the tube, and then insert the arterial end of the cannula into the right common carotid artery of the rat. The heparin was slowly injected into the rats.

3) Dosing solution

Drugs: physiological saline solution (3ml·kg ^-1 , administered orally), aspirin (a dose of 30 mg/kg, given intravenously according to the method of Test Example 1), a physiological saline solution of the compound of the present invention (a dose of 10 nmol/kg, Oral administration) of physiological saline solution.

4) Thrombus weighing

，并做t检验。15 minutes after the start of timing, cut off the arteriovenous cannula, stop the circulation, carefully take out the silk thread with ophthalmic forceps, gently dip the blood drop on the filter paper, put it into the pre-weighed penicillin vial, accurately weigh and record. Calculate the wet weight of the thrombus. Each drug was administered 11 times. Statistical thrombus wet weight in each group

, and do a t-test.

5) Results

After oral administration, the compounds of the present invention have good antithrombotic activity. The results are listed in Table 2.

Table 1. Antithrombotic activity of compounds of the present invention administered orally

n=10; NS=normal saline; ASP=aspirin; a. Compared with normal saline, P<0.01.

The dose-effect relationship of oral administration of test example 2 compound 4i of the present invention

1) Rat surgery and instruments

SD rats (male, 220-230g) were orally administered 4i at doses of 10nmol·kg ^-1 , 1nmol·kg ^-1 and 0.1nmol·kg ^-1 , and were anesthetized by intraperitoneal injection of urethane solution at a dose of 1200mg-kg ^-1 30min later. Anesthetized rats were fixed in a supine position, the right common carotid artery was separated, and the arterial clamp was clamped at the proximal end, and the proximal and distal ends were respectively threaded into the surgical thread, and the surgical thread at the distal end was clamped on the fur with a hemostat, ready to be placed in the Intubation at the distal end.

2) Intubation

The intubation tube is a silanized polyethylene hose, which is divided into three sections. The middle section is a polyethylene hose with a length of 60.0 mm and an inner diameter of 3.5 mm; both ends are the same polyethylene tube with a length of 100.0 mm, an inner diameter of 1.0 mm, and an outer diameter of 2.0 mm. mm One end of the tube is drawn into a tip tube (for insertion into a rat carotid artery or vein) with an outer diameter of 1.0 mm. Put 6cm-long black surgical thread into the numbered clean penicillin vials and weigh them; then take out the silk thread and put it into the thicker middle section of the prepared cannula according to the number.

Open the right arterial clamp of the rat, use a syringe to fill the tube with heparin saline solution (50IU·kg ^-1 ) through the tip of the tube, and then insert the arterial end of the cannula into the right common carotid artery of the rat. The heparin was slowly injected into the rats.

3) Dosing solution

Drugs: 4i is formulated into physiological saline solution at dosages of 10nmol·kg ^-1 , 1nmol·kg ^-1 and 0.1nmol·kg ^-1 for oral administration.

4) Thrombus weighing

，并做t检验。15 minutes after the start of timing, cut off the arteriovenous cannula, stop the circulation, carefully take out the silk thread with ophthalmic forceps, gently dip the blood drop on the filter paper, put it into the pre-weighed penicillin vial, accurately weigh and record. Calculate the wet weight of the thrombus. Each drug was administered 11 times. Statistical thrombus wet weight in each group

, and do a t-test.

5) Results

After oral administration, at the doses of 10nmol·kg ^-1 , 1nmol·kg ^-1 , 0.1nmol·kg ^-1 , 4i exerts antithrombotic effect dependently. The results are listed in Table 3.

Table 2 The dose-effect relationship of oral administration of 4i

Test example 3 The compound of the present invention detects scavenging NO free radical with ESR method

a. Solution preparation:

①Preparation of MGD solution: Dissolve 7.325mg of MGD in 1mL of pure water to obtain a 25mM MGD solution;

②Preparation of FeSO ₄ solution: Dissolve 3.475 mg of FeSO ₄ 7H ₂ O in 1 mL of pure water to obtain a FeSO ₄ solution with a concentration of 12.5 mM;

③ Preparation of SNAP solution: Dissolve 25 mg of SNAP in 1 mL of purified water to obtain a mother solution (green) with a concentration of 110 μM, take 10 μL of the mother solution and dilute to 1 mL to obtain a SNAP solution with a concentration of 1 μM;

④ Preparation of sample solution: Dissolve about 1 mg of the sample to be tested in 1 mL of pure water to obtain a sample solution with a concentration of 1 mM.

b.ESR parameter setting:

Table 3ESR parameter setting

c. Data determination:

(1) Mix ① and ② uniformly at a volume ratio of 1:1 to prepare brown [(MGD) ₂ -Fe ²⁺ ] complex ⑤;

(2) Mix ⑤, ④, and ③ in equal proportions in turn, and the blank group replaces ④ with the same volume of pure water;

(3) After incubating the mixed solution at 37° C. for 30 min, each mixed solution was sucked up with a 2 mm quartz tube, and placed in the resonant cavity of an ESR spectrometer for measurement.

d. Calculation of the scavenging percentage of NO free radicals:

Record the pH value (peak intensity) of the first peak in the three main peaks in the spectrogram, the blank group (pure water group) is recorded as PH ^BLANK , and the test compound group is PH ^Drug , according to the formula "the scavenging percentage of free radicals=( PH ^BLANK - PH ^Drug )/PH ^BLANK × 100%" to calculate the scavenging percentage of the test compound to the NO radical (n=3).

e. Experimental results: the compounds of the present invention have a good ability to scavenge NO free radicals

Table 4ESR measures the percentage of scavenging of NO free radicals by compounds

Wherein, the concentration of the compound is 10 ^-3 mol/L (n=3); statistical method: t test method

^a means that the compound’s scavenging percentage of NO free radicals is compared with 3,4-dihydroxy-Phe, P<0.05; ^b means the compound’s scavenging percentage of NO free radicals is compared with 3,4-dihydroxy-Phe, P<0.05 0.01; ^c represents the percentage of scavenging NO radicals of the compound compared with 4q, P<0.05; ^d represents the percentage of scavenging of NO radicals of the compound compared with 4q, P<0.01.

Test Example 4 Compounds of the present invention inhibit rat thoracic aorta ring relaxation experiment

1. Experiment preparation:

a. Surgical instruments: ophthalmic scissors (straight), ophthalmic forceps (straight), ophthalmic forceps (curved), 16cm surgical scissors, watch glass

b. Instrument: LMS-2B two-channel physiological recorder (Chengdu Instrument Factory);

Muscle tension transducer (Beijing Xinhang Xingye Science and Trade Co., Ltd.);

THS-15 CNC super constant temperature bath (Ningbo Tianheng Instrument Factory).

c. Reagents: NE, Ach (Shanghai No.3 Reagent Factory)

NaCl, CaCl ₂ , NaHCO ₃ , NaH ₂ PO ₄ , MgCl ₂ 6H ₂ O, EDTA-2Na (Beijing Chemical Plant)

KCl (Beijing Yili Fine Chemicals Co., Ltd.)

Anhydrous glucose (Sinopharm Chemical Reagent Co., Ltd.)

a. Experimental animals: healthy SD male rats (Department of Experimental Animals, Capital Medical University, about 200g)

2. Experimental steps:

a. Preparation of Krebs-Henseleit (K-H) solution:

①Salt stock solution: NaCl 95.5g+KCl 5.5g+MgCl ₂ ·6H ₂ O 3.3g+CaCl ₂ 3.7g mixed with 1000mL H ₂ O, ultrasonically dissolved.

②Sodium dihydrogen phosphate solution: mix 1.56g NaH ₂ PO ₄ with 100mL H ₂ O, and dissolve it by ultrasonic.

③Ethylenediaminetetraacetic acid disodium solution: EDTA-2Na 1.12g is mixed with 100mL H ₂ O, and dissolved by ultrasonic.

④K-H solution: NaHCO ₃ 5.04g+ anhydrous glucose 6.00g+1500mL H ₂ O+36mL② solution+3mL③ solution+225mL① solution+1236mL H ₂ O

Among them, H ₂ O is distilled water, and the solutions ①②③ should be kept away from light; ④ must be prepared in order, each step must ensure that the solids are completely dissolved, and the solution must be prepared and used on the same day. Each solution must be completely dissolved and clear.

b. Preparation of rat thoracic aortic rings:

About 200g male SD rats were killed by neck dislocation, the sternum was cut open, the heart was exposed, the heart was picked up with forceps, and the thoracic aorta was removed along the thoracic vertebrae. Immediately isolate the thoracic aorta from other tissues in K-H buffer, leaving the endothelium intact. Cut the thoracic aorta into 2mm-wide vascular rings in K-H buffer solution for use.

Hang a 1g weight on the muscle tension transducer (range 5g), and record the number of grids the pointer moves. Suspend the thoracic aortic ring on the muscle tension transducer, immerse it in a bath bath with continuous oxygenation (95% O ₂ +5% CO ₂ ), adjust the tension of the thoracic aortic ring so that the moving grid of the pointer is the same as the hanging 1g weight The code moves the same number of grids.

c. Determination of the target substance 4a-q inhibiting Ach relaxation activity of blood vessel strips

After the 1g pretensioned baseline leveled off, add NE (0.2mg/mL, 10μL, final concentration 0.1μmol/L) to the bath, and the blood vessel strips contracted. After stabilization, add the aqueous solution of the target compound 4a-q (1mg/mL , 15 μL, final concentration 1 μmol/L) and Ach (1 mg/mL, 15 μL, final concentration 5×10 ^-5 mol/L) were recorded after the relaxation was stable. Wash the aortic ring 3-5 times with KH buffer. Repeat the experiment.

3. Calculate the percentage of inhibition of relaxation:

The number of relaxation grids L ^Ach caused by Ach, the number of relaxation grids L ^Drug caused by adding the compound to be tested and Ach, the number of contraction grids L NE caused by ^NE . According to the formula "inhibition of relaxation percentage = (L ^Ach - L ^Drug )/L ^NE × 100%" to calculate the inhibition percentage of the test compound on Ach-induced relaxation of blood vessel strips (n=3).

4. Experimental results: The experimental results show that all the compounds of the present invention have good activity of inhibiting vasodilation.

Table 5 The compound of the present invention is to the inhibitory percentage of the vasodilation caused by Ach

Wherein, the final concentration of the compound is 10 ^-6 mol/L (n=3); statistical method: t test method

^a represents the inhibition percentage of the vasodilation caused by the compound to Ach compared with 3,4-dihydroxy-Phe, P＜0.01; ^b represents the inhibition percentage of the compound to the vasodilation caused by Ach and (3,4-dihydroxy-Phe )-Ser-Leu, P<0.01.

Claims (8)

1. Amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide, its structural formula is shown in general formula I:

Formula I Wherein, AA is selected from hydrogen (H), glycine residue (Gly), alanine residue (Ala), valine residue (Val), leucine residue (Leu), isoleucine residue (Ile), phenylalanine residue (Phe), tyrosine residue (Tyr), tryptophan residue (Trp), serine residue (Ser), threonine residue (Thr), proline Acid residues (Pro), glutamine residues (Gln), lysine residues (Lys), histidine residues (His), aspartic acid residues (Asp) or glutamic acid residues (Glu) Residues. 2. a method for synthesizing amino acid-(3,4-dihydroxyl-Phe)-Ser-Leu pseudopeptide according to claim 1, comprising the following steps: (1) Synthesis of protected (3,4-dihydroxy-Phe)-Ser-Leu; (2) Glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, proline, glutamine, Lysine, histidine, aspartic acid or glutamic acid are respectively condensed with (3,4-dihydroxy-Phe)-Ser-Leu protected by the protecting group, and the protecting group is removed to obtain the product. 3. A pharmaceutical composition for scavenging free radicals, characterized in that: the amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide and the pharmaceutically effective amount according to claim 1 It consists of acceptable carriers or excipients. 4. An anti-vasodilation pharmaceutical composition, characterized in that: the amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide and the pharmaceutically effective amount of amino acid-(3,4-dihydroxy-Phe)-Ser-Leu according to claim 1 It consists of acceptable carriers or excipients. 5. An antithrombotic pharmaceutical composition, characterized in that: amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide and pharmaceutically acceptable Composed of acceptable carriers or excipients. 6. The use of the amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide according to claim 1 in the preparation of free radical scavenging drugs. 7. The use of the amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide according to claim 1 in the preparation of anti-vasodilation drugs. 8. The use of the amino acid-(3,4-dihydroxy-Phe)-Ser-Leu pseudopeptide according to claim 1 in the preparation of antithrombotic drugs.