CN109810172B

CN109810172B - isoquinoline-3-formyl-PARGD (aa) aa, preparation thereof, anti-thrombus activity and application thereof

Info

Publication number: CN109810172B
Application number: CN201711161234.4A
Authority: CN
Inventors: 赵明; 彭师奇; 桂琳; 张筱宜
Original assignee: Capital Medical University
Current assignee: Capital Medical University
Priority date: 2017-11-21
Filing date: 2017-11-21
Publication date: 2021-01-01
Anticipated expiration: 2037-11-21
Also published as: CN109810172A

Abstract

The invention discloses isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa with the following formula, wherein aa is Phe, Ser or Val residue, and discloses a preparation method and anti-venous thrombosis activity thereof, so that the invention discloses application thereof in preparing anti-venous thrombosis medicaments.

Description

isoquinoline-3-formyl-PARGD (aa) aa, preparation thereof, anti-thrombus activity and application thereof

Technical Field

The invention relates to isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa, a preparation method thereof, and anti-venous thrombosis activity thereof, and thus the invention relates to application thereof in preparing anti-venous thrombosis medicaments. The invention belongs to the field of biological medicine.

Background

Thrombosis has become a disease with a high incidence and mortality. The number of venous thrombosis patients, including deep venous thrombosis and pulmonary embolism, exceeds the total number of myocardial infarction and apoplexy, and is higher than the total number of deaths caused by breast cancer and AIDS. The incidence of thrombosis increases exponentially with age, and the threat to the health of people in the aging countries in China is particularly serious. If the population base is counted, the negative influence of venous thrombosis on the Chinese county is particularly serious. The prevention and treatment of venous thrombosis has always been one of the major concerns in the medical field. Although warfarin was used clinically in 1941, its safety window was narrow. Low doses can lead to pulmonary embolism and high doses can lead to fatal bleeding. Although a large amount of heart blood is paid for inventing safe anti-vein thrombosis medicaments for more than 50 years, the effect is very little. In the research of antithrombotic drugs, the inventors have disclosed that intravenous injection of N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Ser) -Ser, N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Val) -Val and N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Phe) -Phe at a dose of 10nmol/kg is effective in inhibiting arterial thrombosis in rats and has no therapeutic effect on venous thrombosis. The inventors found isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Ser) -Ser, isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Val) -Val and isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Phe) -Phe in the blood of rats treated by them (see the following conversion formulae). In subsequent studies, the inventors further found that isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Ser) -Ser, isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Val) -Val and isoquinoline-3-formyl-Pro-Arg-Gly-Asp (Phe) -Phe have excellent anti-venous thrombosis activity. Thus, the inventors have proposed the present invention.

Disclosure of Invention

The first aspect of the present invention is the preparation of N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid esters according to known methods

Group ] -Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula aa is Ser, Val or Phe residue).

The second aspect of the present invention is to convert N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula, aa is Ser, Val or Phe residue) into isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula, aa is Ser, Val or Phe residue) by dehydrogenation and quantitative determination in rat serum.

The third aspect of the present invention is to evaluate the anti-thrombotic activity of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula, aa is Ser, Val or Phe residue) and observe bleeding side effects.

Drawings

FIG. 1 shows the synthetic route of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula, aa is Ser, Val or Phe residue).

Detailed description of the preferred embodiments

In order to further clarify the invention a series of examples are given below. It must be noted that these examples are purely illustrative. These examples are given for the purpose of fully clarifying the meaning and content of the present invention and are not to be construed as limiting the invention in any way.

EXAMPLE 1 preparation of N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (aa) -aa

N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (aa) -aa (in the formula, aa is Ser, Val or Phe residue) was prepared according to a known method.

EXAMPLE 2 preparation of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp- (Ser) -Ser (10a)

50mg (0.04mmol) of N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl group]Pro-Ala-Arg-Gly-Asp (Ser) -Ser was dissolved in 1mL rat serum at 37 ℃ and the resulting solution was shaken at 37 ℃ for 4 hours, followed by TLC to monitor the disappearance of starting material. 2mL of methanol was added to the blood serum solution and shaken at 37 ℃ for 10 minutes, and the resulting mixed solution was centrifuged at 3000 rpm for 10 minutes. The residue obtained by centrifugation was sufficiently extracted with ultrapure water, and the extract was separated and centrifuged. The supernatant obtained by centrifugation was concentrated at 37 ℃ under reduced pressure, and the residue was sufficiently extracted with ultrapure water to separate the extract. The combined extracts were freeze-dried to yield 33mg (96%) of the title compound. ESI (-) -FT-MS:842.35219[ M-H ]]^-.Mp 118-119℃.[α]^D ₂₅＝-15.9(c＝1.3,CH₃OH).IR(cm^-1)3690,3454,3236,2932,2364,1640,1545,1455,1390,1175,1123,1036,643,512,426.¹H NMR(300MHz,DMSO)/ppm＝10.99(s,2H),9.22(s,1H),8.56(s,1H),8.54(s,1H),8.53(s,5H),8.04(m,6H),8.01(m,1H),7.48(m,1H),7.43(m,1H),5.10(m,5H),4.51(m,2H),4.33(d,J＝7.5Hz,2H),4.27(m,2H),4.03(d,J＝6Hz,1H),3.97(d,J＝2.4Hz,2H),3.92(d,J＝3.6Hz,1H),3.86(d,J＝7.5Hz,2H),3.83(d,J＝3Hz,3H),3.73-3.65(m,3H),3.58(m,2H),1.95(m,4H),1.414(m,2H),1.386(s,1H)。

EXAMPLE 3 preparation of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (Val) -Val (10b)

The procedure was followed as in example 1 from 50mg (0.04mmol) of N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl]Pro-Ala-Arg-Gly-Asp (Val) -Val gave 33mg (96%) of the title compound. ESI (-) -FT-MS:866.41722[ M-H ]]^-.Mp 142-143℃.[α]^D ₂₅＝-12.7(c＝1.6,CH₃OH).IR(cm^-1)3442,3253,2966,2380,1654,1542,1456,1390,1254,1171,1025,763,645,516,438.¹H NMR(300MHz,DMSO)/ppm＝10.98(s,2H),9.24(s,1H),8.55(s,1H),8.54(s,1H),8.52(s,1H),8.50(s,1H),8.07(m,1H),8.04(m,6H),7.49(m,1H),7.44(m,1H),4.52-4.46(m,2H),4.35-4.09(m,2H),4.07-4.03(m,2H),3.95-3.93(m,1H),3.52-3.44(m,1H),3.21-3.11(m,1H),2.81(d,J＝6.3Hz,1H),2.40-2.31(m,1H),2.03(m,4H),1.90(m,3H),1.65(m,2H),1.38(d,J＝7.2Hz,3H),0.87-0.77(m,12H)。

EXAMPLE 4 preparation of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (Phe) -Phe (10c)

The procedure was followed as in example 1 from 50mg (0.04mmol) of N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl]Pro-Ala-Arg-Gly-Asp (Phe) -Phe to obtain 38mg (97%) of the title compound. ESI (+) -FT-MS:964.18333[ M + H ]]⁺.Mp 135-137℃.[α]^D ₂₅＝-13.4(c＝1.1CH₃OH).IR(cm^-1)3445,2937,2363,1744,1645,1536,1452,1392,1273,1025,672,517,445.¹H NMR(300MHz,DMSO)/ppm＝10.99(s,2H),9.22(s,1H),8.54(s,1H),8.52(s,1H),8.06(m,5H),8.05(d,J＝7.5Hz,1H),7.50(m,1H),7.34-7.26(m,11H),4.81(m,2H),4.62(m,1H),4.51(m,1H),4.45(q,J＝8.0Hz,1H),4.28(m,3H),4.12(m,2H),3.15(m,3H),2.55(dd,J＝6.3Hz,J＝6.9Hz,1H),2.01(m,3H),1.97(m,1H),1.77(m,2H),1.55(m,2H),1.49(d,J＝7.8Hz,3H),1.22(d,J＝8.2Hz,3H)。

EXAMPLE 5 evaluation of anti-thrombotic Activity of 10a-c

Male SD rats (250 + -20 g) were acclimatized and fasted for one day, anesthetized with a 20% urethane solution intraperitoneally 2min prior to surgery, and fixed on a plate. 2mL of blood was taken from the carotid artery and used for the measurement of blood related indices. The abdomen of the rat was prepared, disinfected, and the abdominal cavity was opened along the white line (down to the coagulated gland and up to the corner of the liver). The organs such as small intestine in the abdominal cavity were removed and wrapped with gauze soaked with normal saline. The perivascular connective tissue was isolated blunt, exposing the inferior vena cava and its branches. The abdominal aorta and inferior vena cava were peeled off below the renal veins, and the inferior vena cava was ligated with saline-soaked sutures at the junction of the inferior vena cava and left renal vein. Moving organs such as intestines back to the abdominal cavity according to the anatomical position, and suturing the abdominal cavity layer by using a suture line. The physiological saline solution of 10a-c was injected from the tail vein at a dose of 1nmol/kg, the positive control warfarin at a dose of 4.87 μmol/kg, and the negative control was physiological saline. After circulating for 4 hours at 25-28 ℃, the abdominal cavity of the rat is opened, the branches are ligated one by one, and the 2cm inferior vena cava is removed from the ligation site at the junction of the inferior vena cava and the left renal vein, from which the thrombus is removed. The thrombus was weighed and the results were counted using the t-test. The operation was performed in two alternating groups of 12 each. The thrombus was weighed in table 1. The results show that 10a-c is effective in inhibiting venous thrombosis at a dose of 1nmol/kg, and the unexpected technical effects of the present invention are compared with N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Ser) -Ser, N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Val) -Val and N- [ (3S) -1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Arg-Gly-Asp (Phe) -Phe which have no therapeutic effect on venous thrombosis at a dose of 10 nmol/kg. In addition, the effective dose of 10a-c is 1/4870 for warfarin without the bleeding side effects of warfarin. The present invention has an unexpected technical effect.

Anti-thrombotic Activity of tables 110 a-c

Compound (I)	Dosage form	Thrombus wet weight (mean + -SD mg)
			Physiological saline	3ml/kg	25.35±1.41
Warfarin	4.87μmol/kg	13.13±3.71
			10a	1nmol/kg	13.00±2.89^a
10b	1nmol/kg	12.87±2.53^a
			10c	1nmol/kg	13.12±2.98^a

a) P <0.01 to saline and p >0.05 to warfarin; n is 12.

Claims

1. isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp of the formula (aa) -aa, wherein aa is a Ser, Val or Phe residue,

2. a process for the preparation of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-asp (aa) -aa of claim 1, comprising the steps of:

1) preparing N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Ser) -Ser according to the disclosed method;

2) preparing N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Val) -Val according to the disclosed method;

3) preparation of N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Phe) -Phe according to published procedures;

4) converting N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Ser) -Ser in rat serum to isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (Ser) -Ser;

5) converting N- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Val) -Val to isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (Val) -Val in rat serum;

6) n- [ (3S) -N-1,2,3, 4-tetrahydroisoquinoline-3-formyl ] -Pro-Ala-Arg-Gly-Asp (Phe) -Phe was converted in rat serum to isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (Phe) -Phe.

3. Use of isoquinoline-3-formyl-Pro-Ala-Arg-Gly-Asp (aa) -aa of claim 1 in the preparation of an anti-venous thrombosis medicament.