Levodopa acyl oligopeptide compounds, synthesis and use thereof

Abstract

The application discloses levodopa acyl oligopeptide (levodopa acyl-AA1-AA2-Arg-Arg) with the following formula structure, wherein AA1-AA2 is a Leu residue, or AA1-AA2 is a Ser-Leu dipeptide residue, a preparation method of the levodopa acyl oligopeptide, and an application of the levodopa acyl oligopeptide in the preparation of an anti-arterial thrombosis drug are disclosed. The application provides a new candidate compound for the treatment of arterial thrombosis.

Description

Technical Field

[0001] This invention relates to a levodopayl-AA1-AA2-Arg-Arg compound, its preparation method, and its application in the preparation of anti-arterial thrombotic drugs. This invention belongs to the field of biomedicine. Background Technology

[0002] Levodopa, an open-ring derivative of tetrahydroisoquinoline alkaloids, has shown potential in antiplatelet aggregation, antioxidation, and neuroprotection. Arginine-rich peptides (such as Arg-Arg, Leu-Arg-Arg, and Ser-Leu-Arg-Arg) can bind to negatively charged regions on the surface of thrombi and are considered thrombus-targeting peptides. Based on this understanding, the inventors coupled levodopa with Arg-Arg, Leu-Arg-Arg, and Ser-Leu-Arg-Arg to prepare levodopayl-Arg-Arg, levodopayl-Leu-Arg-Arg, and levodopayl-Ser-Leu-Arg-Arg. Through extensive screening, levodopayl-Arg-Arg, levodopayl-Leu-Arg-Arg, and levodopayl-Ser-Leu-Arg-Arg were found to have excellent anti-arterial thrombotic effects. Based on the aforementioned understanding and discovery, the inventors proposed this invention. Summary of the Invention

[0003] The first aspect of this invention is to provide L-dopayl-Arg-Arg, L-dopayl-Leu-Arg-Arg, and L-dopayl-Ser-Leu-Arg-Arg having the following structures;

[0004] The second aspect of this invention is to provide a method for preparing the L-dopayl-Arg-Arg, L-dopayl-Leu-Arg-Arg, and L-dopayl-Ser-Leu-Arg-Arg structures; the method includes the following steps: 1. Arg(NO2)-Arg(NO2)-OBzl, Leu-Arg(NO2)-Arg(NO2)-OBzl and Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl were prepared by the DCC / HOBt method; 2. L-dopayl-Arg(NO2)-Arg(NO2)-OBzl, L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl and L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl were prepared by DCC / HOBt method; 3. The OBzl of L-dopayl-Arg(NO2)-Arg(NO2)-OBzl, L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl and L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl were removed by catalytic hydrogenation and reacted with NO2 to prepare L-dopayl-AA1-AA2-Arg-Arg with the structure described above.

[0005] The third aspect of this invention is to confirm the anti-arterial thrombotic activity of levodopam-Arg-Arg, levodopam-Leu-Arg-Arg, and levodopam-Ser-Leu-Arg-Arg and their application in the preparation of antithrombotic drugs. Attached Figure Description

[0006] Figure 1 shows the synthetic route of L-dopayl-Arg-Arg: i) DCC, HOBt, NMM, DMF, Boc-Arg(NO2), Arg(NO2)-OBzl; ii) Ethyl acetate solution of hydrogen chloride; iii) H2, Pd / C, L-dopayl-Arg(NO2)-Arg(NO2)-OBzl, MeOH.

[0007] Figure 2 shows the synthetic route of L-dopayl-Leu-Arg-Arg: i) DCC, HOBt, NMM, DMF, Boc-Leu, Arg(NO2)-Arg(NO2)-OBzl; ii) Ethyl acetate solution of hydrogen chloride; iii) H2, Pd / C, L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl, MeOH.

[0008] Figure 3 shows the synthetic route of L-dopayl-Ser-Leu-Arg-Arg: i) DCC, HOBt, NMM, DMF, Boc-Ser, Leu-Arg(NO2)-Arg(NO2)-OBzl; ii) Ethyl acetate solution of hydrogen chloride; iii) H2, Pd / C, L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl, MeOH. Detailed Implementation

[0009] To further illustrate the present invention, a series of embodiments are given below. These embodiments are purely illustrative and are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

[0010] Example 1: Preparation of Boc-Arg(NO2)-Arg(NO2)-OBzl Using a conventional liquid-phase synthesis method in the presence of DCC and HOBt, 833 mg (58%) Boc-Arg(NO2)-Arg(NO2)-OBzl was obtained from 757 mg (2.37 mmol) Boc-Arg(NO2) and 1102 mg (2.37 mmol) Arg(NO2)-OBzl, as a colorless solid. ESI-MS (m / e): 611 [M+H] + .

[0011] Example 2: Preparation of HCl·Arg(NO2)-Arg(NO2)-OBzl 824 mg (1.35 mmol) of Boc-Arg(NO2)-Arg(NO2)-OBzl was thoroughly mixed with 20 mL of an ethyl acetate solution of hydrogen chloride (4 M). The mixture was stirred for 30 minutes to obtain a clear solution. This clear solution was then concentrated under reduced pressure. The residue was dissolved in 50 mL of anhydrous ethyl acetate, and the resulting ethyl acetate solution was further concentrated under reduced pressure. The residue was then dissolved again in 50 mL of anhydrous ethyl acetate, and the resulting ethyl acetate solution was further concentrated under reduced pressure to remove free hydrogen chloride, yielding 638 mg (87%) HCl·Arg(NO2)-Arg(NO2)-OBzl as a colorless solid. ESI-MS (m / e): 511 [M+H] + .

[0012] Example 3 Preparation of L-dopayl-Arg(NO2)-Arg(NO2)-OBzl Following the procedure in Example 1, 243 mg (36.20%) of levodopa-Arg(NO2)-Arg(NO2)-OBzl was obtained from 168 mg (0.85 mmol) of levodopa and 434 mg (0.85 mmol) of HCl·Arg(NO2)-Arg(NO2)-OBzl, as a colorless solid. ESI-MS (m / e): 690 [M+H] + ; 1 HNMR (300 MHz, DMSO- d 6): δ / ppm = 8.67 (d, J =3.1 Hz, 2H), 8.45 (d, J = 7.2 Hz, 1H), 7.94(m, 2H), 7.35 (s, 5H), 6.61 (m, 2H), 5.11 (s, 2H), 4.30 (d, J= 11.2 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 3.68 (d, J = 3.1Hz, 2H), 3.14 (s, 4H), 2.77 (d, J = 11.2 Hz, 2H), 1.99 (s, 2H), 1.77 (s, 1H), 1.64 (s, 1H), 1.52 (m, 4H), 1.19 (m, 4H).

[0013] Example 4: Preparation of L-dopayl-Arg-Arg 248 mg (0.36 mmol) of L-dopayl-Arg(NO2)-Arg(NO2)-OBzl was dissolved in 40 mL of MeOH, and 40 mg of Pd / C was added. The suspension was purged with hydrogen gas for 4 hours, and TLC (Cl2CH2 / MeOH = 30 / 1) showed that L-dopayl-Arg(NO2)-Arg(NO2)-OBzl disappeared. The reaction mixture was filtered to remove Pd / C, and the filtrate was concentrated under reduced pressure to give 174 mg (95%) of L-dopayl-Arg-Arg, a colorless solid. ESI-MS (m / e): 510 [M+H] + ; 1 HNMR (300 MHz, DMSO- d 6): δ / ppm = 8.83 (s, 1H), 8.51 (s, 1H), 8.38 (d, J = 7.5 Hz, 1H), 8.05 (d, J =12.7 Hz, 4H), 6.70 (q, J = 5.4 Hz, 1H), 6.66 (d, J = 3.8 Hz, 1H), 6.53 (dd, J 1 = 8.1 Hz J 2 = 2.1 Hz, 1H), 4.36 (t, J = 7.5 Hz, 1H), 4.17 (q, J = 5.4 Hz, 1H), 4.02-3.93 (m, 1H), 3.91 (d, J = 3.8 Hz, 1H), 3.84-3.65 (m, 2H), 3.05 (dd, J 1 = 8.1 Hz J2= ​​2.1 Hz, 2H), 2.97 (m, 1H), 2.67 (m, 2H), 2.66 (m, 1H), 1.45 (m, 6H); 13 CNMR (75 MHz, D2O) δ / ppm = 176.85, 172.04, 168.79, 156.75, 156.71, 144.29, 143.66, 126.02, 121.78, 117.08, 116.49, 54.13, 53.85, 53.28, 40.64, 40.58, 36.11, 28.48, 28.22, 24.57, 24.12; HPLC purity was 98.94%.

[0014] Example 5: Preparation of compound Boc-Leu-Arg(NO2)-Arg(NO2)-OBzl Following the procedure in Example 1, 1.83 g (74%) of Boc-Leu-Arg(NO2)-Arg(NO2)-OBzl was obtained from 791 mg (3.42 mmol) of Boc-Leu and 1.75 g (3.42 mmol) of HCl·Arg(NO2)-Arg(NO2)-OBzl, as a colorless solid. ESI-MS (m / e): 724 [M+H] + .

[0015] Example 6: Preparation of HCl·Leu-Arg(NO2)-Arg(NO2)-OBzl Following the procedure in Example 2, 613 mg (87%) HCl·Leu-Arg(NO2)-Arg(NO2)-OBzl was obtained from 819 mg (1.13 mmol) Boc-Leu-Arg(NO2)-Arg(NO2)-OBzl, as a colorless solid. ESI-MS (m / e): 624 [M+H] + .

[0016] Example 7 Preparation of L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl Following the procedure in Example 1, 484 mg (71%) of levodopa-Leu-Arg(NO2)-Arg(NO2)-OBzl was obtained from 168 mg (0.85 mmol) of levodopa and 530 mg (0.85 mmol) of HCl·Leu-Arg(NO2)-Arg(NO2)-OBzl. The levodopa-Leu-Arg(NO2)-Arg(NO2)-OBzl was a colorless solid. ESI-MS (m / e): 803 [M+H] + ; 1HNMR (300 MHz, DMSO- d 6): δ / ppm = 9.19 (s,1H), 8.98 (s, 1H), 8.57 (s, 1H), 8.11 (s, 2H), 7.34 (d, J = 6.5 Hz, 5H), 6.65(s, 1H), 6.53 (s, 1H), 5.11 (s, 2H), 4.28 (s, 2H), 4.02 (dd, J = 6.5, 5.6 Hz,3H), 3.81 (s, 2H), 1.61 (s, 9H), 1.50 (s, 10H), 0.88 (d, J = 5.6 Hz, 6H).

[0017] Example 8: Preparation of L-dopayl-Leu-Arg-Arg (d-lrr) Following the procedure in Example 2, 351 mg (94%) of levodopayl-Leu-Arg-Arg was obtained from 484 mg (0.60 mmol) of levodopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl, as a colorless solid. ESI-MS (m / e): 623 [M+H] + ; 1 HNMR (300 MHz, DMSO- d 6): δ / ppm = 9.10 (s, 1H), 8.90 (s, 1H), 8.35 (s, 1H),8.18 (d, J = 7.6 Hz, 1H), 7.96 (m, 2H), 7.25 (s, 7H), 6.64 (s, 1H), 6.47 (s,1H), 4.40 (q, J = 7.6 Hz, 1H), 4.28 (m, 1H), 4.06 (m, 2H), 3.09 (t, J = 5.8 Hz, 4H), 2.91 (s, 1H), 2.79 (s, 1H), 1.71 (s, 2H), 1.54 (m, 12H), 1.41 (s, 3H), 0.89 (t, J = 5.8 Hz, 6H); 13CNMR (75 MHz, D2O): δ / ppm = 177.59, 173.85, 172.20, 170.16, 156.69, 143.75, 143.42, 129.64, 122.33, 115.64, 112.23, 58.22, 54.34, 53.48, 52.54, 51.70, 40.61, 40.52, 39.89, 28.89, 28.64, 27.52, 26.94, 24.55, 24.22, 23.82, 21.57; HPLC purity was 98.9%.

[0018] Example 9 Preparation of L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl Following the procedure in Example 1, 584 mg (75%) of levodopa-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl was obtained from 168 mg (0.85 mmol) of levodopa and 711 mg (0.85 mmol) of HCl·Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl. The levodopa-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl was a colorless solid. ESI-MS (m / e): 890 [M+H] + ; 1 HNMR (300 MHz, DMSO- d 6): δ / ppm= 9.19 (s, 1H), 9.01 (s, 1H), 8.98 (s, 1H), 8.57 (s, 1H), 8.11 (s, 2H), 7.34(d, J = 6.5 Hz, 5H), 6.65 (s, 1H), 6.53 (s, 1H), 5.11 (s, 2H), 4.94 (s, 1H), 4.28 (s, 2H), 4.16 (d, J = 6.6 Hz, 2H), 4.02 (dd, J 1 = 6.5 Hz J 2= ​​5.6 Hz, 3H),3.81 (s, 2H), 3.51 (t, J = 6.6 Hz, 1H), 1.61 (s, 9H), 1.50 (s, 10H), 0.88 (d, J =5.6 Hz, 6H).

[0019] Example 10: Preparation of L-dopayl-Ser-Leu-Arg-Arg (d-slrr) Following the procedure in Example 2, 351 mg (94%) of levodopayl-Ser-Leu-Arg-Arg was obtained from 584 mg (0.65 mmol) of levodopayl-Ser-Leu-Arg-Arg, as a colorless solid. ESI-MS (m / e): 710 [M+H] + ; 1 HNMR (300 MHz, DMSO- d 6): δ / ppm = 8.75 (s, 2H), 8.41 (s, 4H), 7.46 (s, 9H), 6.76 (s, 1H), 6.61 (d, J = 7.4 Hz, 2H), 6.47 (d, J = 7.4 Hz, 2H), 4.38(s, 1H), 4.20 (s, 3H), 3.97 (s, 1H), 3.74 (s, 2H), 3.03 (s, 5H), 2.67 (s,1H), 1.59 (s, 11H), 0.84 (m, 6H); 13 CNMR (75 MHz, D2O): δ / ppm = 178.20, 173.36, 172.36, 171.40, 156.67, 156.64, 144.18, 143.35, 129.42, 121.66, 117.03, 116.31, 54.68, 54.53, 53.35, 52.14, 40.68, 40.55, 40.02, 37.25, 28.89, 27.93, 24.49, 24.33, 24.20, 21.82; HPLC purity was 98.60%.

[0020] Example 11: Evaluation of the antithrombotic activity of levodopam-AA1-AA2-Arg-Arg 1) Pull the polyethylene tube into a thin tube with one end at an angle, with a fixed length of 10.0 cm, and insert it into the right jugular vein (larger diameter) and the left carotid artery (smaller diameter); the middle section of the polyethylene tube is fixed to a length of 8.0 cm, with the thrombus line pressed against the direction of carotid artery cannulation, and the tube must be filled with heparin before cannulation.

[0021] 2) Male SD rats weighing 200±20g were acclimatized to their environment and fasted for one day before surgery. They were randomly divided into a saline group (blank control, oral dose of 0.3mL / 100g, 12 rats), an aspirin group (positive control, oral dose of 167μmol / kg, 12 rats), and a levodopam-AA1-AA2-Arg-Arg group (oral dose of 0.1μmol / kg, 12 rats). Thirty minutes after oral administration, the rats were anesthetized by intraperitoneal injection of 20% urethane solution (7 mL / kg), and surgery began 2 minutes later. During the surgery, the rat was placed supine on a fixation board. The skin of the neck was cut open, and the right common carotid artery and left jugular vein were separated. A precisely weighed suture was placed under the blood vessel and ligated at the distal end. A small incision was made at the distal end of the vein, and a cannula was inserted into the vein. Heparin was injected, and then the syringe used for injecting heparin was removed. The suture was then secured, and the proximal end of the artery was clamped with an arterial clamp. A small incision was made at the distal end of the artery, and the arterial end was ligated. After securing the suture, the arterial clamp was released, establishing an extracorporeal circulation bypass. After 15 minutes of circulation, the vein was cut to observe whether blood circulation was normal. If blood circulation was normal, the suture with thrombus was removed from the arterial end. Uncoagulated blood was absorbed with filter paper, and the weight of the suture with thrombus was accurately weighed. The weight of the thrombus was obtained by subtracting the weight of the suture from the weight of the thrombus. The data are listed in Table 1. The thrombus weights in the table indicate that levodopam-Arg-Arg and levodopam-Leu-Arg-Arg exhibit excellent anti-arterial thrombotic effects at an oral dose of 0.1 μmol / kg (compared to normal saline and aspirin). p <0.01). It is evident that the anti-arterial thrombotic effects of levodopam-Arg-Arg and levodopam-Leu-Arg-Arg are 1670 times stronger than those of aspirin. The thrombus weights in the table further demonstrate that, at an oral dose of 0.1 μmol / kg, the anti-arterial thrombotic activity of levodopam-Ser-Leu-Arg-Arg is significantly stronger than that of physiological saline. p <0.01). However, at an oral dose of 0.1 μmol / kg, the anti-arterial thrombotic activity of levodopayl-Ser-Leu-Arg-Arg was significantly weaker than that of aspirin at an oral dose of 167 μmol / kg. p <0.05).

[0022] Table 1. Effects of levodopamyl-AA1-AA2-Arg-Arg on arterial thrombosis in rats. a) Compared with normal saline, P<0.01; compared with aspirin, P>0.05; b) Compared with normal saline, P<0.01; compared with aspirin, P<0.05; n=12.

Claims

1. L-dopayl-AA1-AA2-Arg-Arg with the following structure, In the formula, AA1-AA2 are Leu residues, or AA1-AA2 are Ser-Leu dipeptide residues.

2. The method for preparing the L-dopayl-AA1-AA2-Arg-Arg according to claim 1, characterized in that, The method includes the following steps: 1) Arg(NO2)-Arg(NO2)-OBzl, Leu-Arg(NO2)-Arg(NO2)-OBzl and Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl were prepared using the DCC / HOBt method; 2) L-dopayl-Arg(NO2)-Arg(NO2)-OBzl, L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl and L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl were prepared by DCC / HOBt method; 3) The L-dopayl-AA1-AA2-Arg-Arg described in claim 1 is prepared by catalytic hydrogenation to remove OBzl from L-dopayl-Arg(NO2)-Arg(NO2)-OBzl, L-dopayl-Leu-Arg(NO2)-Arg(NO2)-OBzl and L-dopayl-Ser-Leu-Arg(NO2)-Arg(NO2)-OBzl and NO2.

3. The use of the levodopayl-AA1-AA2-Arg-Arg as described in claim 1 in the preparation of antithrombotic drugs.

4. The application according to claim 3, characterized in that, The application of the aforementioned levodopayl-AA1-AA2-Arg-Arg in the preparation of anti-arterial thrombotic drugs.

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