Dimer of R, R-heptacyclic dimethyl acetal, and preparation and application thereof

Abstract

The invention discloses a dimer of R, R-heptacyclic dimethyl acetal in the following formula, namely [R, R-heptacyclic dimethyl acetal] 2, and discloses a preparation method of the dimer and application of the dimer as a thrombolytic agent in treatment of thrombotic diseases. Experiments prove that the [R, R-heptacyclic dimethyl acetal] 2 disclosed by the invention not only has a good thrombolysis effect, but also has the thrombolysis effect significantly stronger than that of the R, R-heptacyclic dimethyl acetal. Therefore, the invention provides an effective technical means for thrombolysis in treatment of thrombotic diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a dimer of R,R-heptacyclic dimethyl acetal, namely [R,R-heptacyclic dimethyl acetal]2, a preparation method thereof and application thereof as a thrombolytic agent in treating thrombotic diseases. The present invention belongs to the field of biomedicine. Background Art

[0002] The applicant has disclosed that the R,R-heptacyclic dimethyl acetal with the following structural formula is an intermediate of R,R-heptacyclic aldehyde; the applicant has disclosed that an oligopeptide can be introduced into the aldehyde group of R,R-heptacyclic aldehyde through an amination reduction reaction to prepare an antithrombotic R,R-heptacyclic ethyl oligopeptide; the applicant has disclosed that the R,R-heptacyclic dimethyl acetal with the following structural formula itself is a P-selectin inhibitor. At an oral dose of 1 μmol / kg / day, R,R-heptacyclic dimethyl acetal effectively inhibits tumor growth, at an oral dose of 1 μmol / kg, R,R-heptacyclic dimethyl acetal effectively inhibits arterial thrombosis, and at an oral dose of 1 μmol / kg, R,R-heptacyclic dimethyl acetal effectively inhibits venous thrombosis.

[0003]

[0004] The applicant knows that the aggregates of R,R-heptacyclic dimethyl acetal often show stronger biological activity. Therefore, the applicant further studied and prepared the dimer of R,R-heptacyclic dimethyl acetal, that is, prepared [R,R-heptacyclic dimethyl acetal] 2. The applicant screened the biological activities (including anti-arterial thrombotic activity, anti-venous thrombotic activity, thrombolytic activity, anti-tumor activity, anti-tumor metastasis activity and anti-inflammatory activity) that may be caused by [R,R-heptacyclic dimethyl acetal] 2 inhibiting P-selectin. Through further experiments, the applicant found that the dimer of R,R-heptacyclic dimethyl acetal showed excellent thrombolytic activity, and its thrombolytic activity was significantly stronger than the thrombolytic activity of R,R-heptacyclic dimethyl acetal.

[0005] Based on these findings, the applicant proposed the present invention. Summary of the invention

[0006] The technical problem to be solved by the present invention is to propose a dimer of R,R-heptacyclic dimethyl acetal, that is, to prepare [R,R-heptacyclic dimethyl acetal] 2. A method for preparing the dimer of R,R-heptacyclic dimethyl acetal [R,R-heptacyclic dimethyl acetal] 2 is established, and the thrombolytic activity of [R,R-heptacyclic dimethyl acetal] 2 is further evaluated. In order to achieve the above purpose, the present invention adopts the following technical means.

[0007] The first technical means proposes an R,R-heptacyclic dimethyl acetal dimer with the following structure, namely [R,R-heptacyclic dimethyl acetal] 2，

[0008]

[0009] The second technical means is to propose a method for preparing [R,R-heptacyclic dimethyl acetal] 2, which can be summarized into the following two steps:

[0010] 1. Prepare R,R-heptacyclic dimethyl acetal monomer according to the disclosed method;

[0011] 2Convert the R,R-heptacyclic dimethyl acetal monomer into the dimer [R,R-heptacyclic dimethyl acetal]2.

[0012] The third technical approach was to evaluate the thrombolytic activity of R,R-heptadyl dimethyl acetal monomer and dimer in the rat arteriovenous bypass catheter thrombolysis model, further confirming the effective application of [R,R-heptadyl dimethyl acetal]2 in the preparation of thrombolytic drugs.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention discloses a dimer composed of two R,R-heptacyclic dimethyl acetals, namely [R,R-heptacyclic dimethyl acetal]2. Experiments show that compared with the prior art, the [R,R-heptacyclic dimethyl acetal]2 of the present invention not only exhibits excellent thrombolytic activity, but also has significantly stronger thrombolytic activity than R,R-heptacyclic dimethyl acetal. Therefore, the present invention provides a more effective technical means for preparing thrombolytic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The synthetic route of the dimer of R,R-heptacyclic dimethyl acetal [R,R-heptacyclic dimethyl acetal] 2: i) 1,1,3,3-tetramethoxypropane and trifluoroacetic acid; ii) Pd / C, hydrogen; iii) 2-(7-oxybenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole; iv) vortex for 15 minutes, then ultrasonicate for 4 hours.

[0016] Figure 2 FT-ICR-MS and qCID spectra of the dimer of R,R-heptacyclic dimethyl acetal. DETAILED DESCRIPTION

[0017] In order 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.

[0018] Example 1 Preparation of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid benzyl ester (1)

[0019] A mixture of CH2Cl2 (150 mL), 1,1,3,3-tetramethoxypropane (5 mL) and trifluoroacetic acid (3.5 mL) was stirred at 0°C for 40 minutes. Then L-Trp-OBzl (17 mmol, 5 g) was gradually added to the mixture. The resulting reaction mixture was stirred at 60°C for 8 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that L-Trp-OBzl disappeared. The reaction mixture was concentrated under reduced pressure to obtain a yellow oil, which was washed with saturated NaHCO3 aqueous solution (30 mL × 3) and saturated NaCl aqueous solution (30 mL × 3) in turn. The ethyl acetate layer was dried over anhydrous Na2SO4 for 8 hours, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain 4.3 g (63%) of the title compound. FT-ICR-MS (m / e): 395.1971 [M+H] + .

[0020] Example 2 Preparation of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid (2)

[0021] 3.96 g (10.00 mmol) of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid benzyl ester (1) was dissolved in 100 mL of CH3OH. 400 mg of Pd / C was added to the obtained solution, and after stirring to form a uniform suspension, a three-way device was installed, and a hydrogen bag and a vacuum water pump were connected respectively, and the air in the reaction bottle was removed by hydrogen. Then, hydrogen was passed through the suspension for 18 hours while stirring. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that 1 disappeared. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.72 g (89%) of the title compound as a yellow solid. FT-ICR-MS (m / e): 303.1345 [MH] - .

[0022] Example 3 Preparation of R,R-heptacyclic dimethyl acetal (3R,R)

[0023] 1.32 g (4.35 mmol) of (3S)-1-(2,2-dimethoxyethyl)-2,3,4,9-tetrahydro-β-carlin-3-carboxylic acid (2) was dissolved in 50 mL of anhydrous DMF. 1.98 g (5.22 mmol) of 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and 0.58 g (4.35 mmol) of 1-hydroxybenzotriazole (HOBt) were added to the solution at 0°C with stirring. The pH of the reaction solution was then adjusted to 9 with collidine and stirred for 24 hours. TLC (petroleum ether / ethyl acetate = 1 / 1) showed that 2 disappeared. The reaction solution was concentrated under reduced pressure, and the obtained residue was dissolved in 100 mL of ethyl acetate. The obtained solution was washed with saturated NaHCO3 aqueous solution (30 mL × 3), saturated NaCl aqueous solution (30 mL × 3), 5% KHSO4 aqueous solution (30 mL × 3), saturated NaCl aqueous solution (30 mL × 3), 5% NaHCO3 aqueous solution (30 mL × 3), and saturated NaCl aqueous solution (30 mL × 3). The ethyl acetate layer was dried over anhydrous sodium sulfate for 8 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.25 g of dark yellow solid, which was separated by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) and then separated by silica gel column chromatography (petroleum ether / ethyl acetate = 2 / 1) to obtain 338 mg (35.8%) of R, R-heptacyclic dimethyl acetal (3R, R) as a light yellow solid. FT-ICR-MS (m / e): 573.2713 [M+H] + ;Mp:133-134℃; IR:3398.82,3300.26,2926.03,2831.12,2359.62,2341.99,1652.04,1451.45,1387.46,1328.93,1310 .92,1285.79,1231.27,1188.07,1120.46,1057.27,1006.96,970.05,838.08,739.70,704.22,668.01cm -1 ; 1HNMR (800MHz, DMSO-d6): δ / ppm=11.07(s,2H),7.39(d,J=7.8Hz,2H),7.34(d,J=8.2Hz,2H),7.08(t,J=7.6Hz,2H),6.97(t,J=7.4Hz,2H),5.93(dd,J1 =13.8Hz, J2=7.0Hz, 2H), 4.61(dd, J1=11.8Hz, J2=4.1Hz, 2H), 4.53(t, J=5. 4Hz,2H),3.34(s,6H),3.26(s,6H),3.22(m,2H),2.79(m,2H),2.25(m,4H); 13 C NMR (200 MHz, DMSO-d6): δ / ppm=165.4, 136.5, 133.4, 126.5, 121.8, 119.3, 118.4, 111.7, 106.1, 102.4, 53.6, 52.8, 52.7, 46.4, 36.8, 28.3; HPLC purity: 99.3% (Waters RPC18, 4.6×250 mm, 5 μm, CH3CN / H2O=50 / 50, flow rate=1 mL / min).

[0024] Example 4 Preparation of R,R-heptacyclic dimethyl acetal 3R,R dimer [R,R-heptacyclic dimethyl acetal] 2

[0025] 100 mg of R,R-heptacyclic dimethyl acetal (3R,R) powder was dissolved in 5 mL of ultrapure water, and the obtained solution was vortexed for 15 minutes, and then placed in an ultrasonicator for 4 hours until the solution was completely colorless and transparent. The colorless and transparent solution was concentrated to dryness under reduced pressure to obtain a dimer of R,R-heptacyclic dimethyl acetal 3R,R, namely [R,R-heptacyclic dimethyl acetal] 2. The structure of [R,R-heptacyclic dimethyl acetal] 2 was confirmed by ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Figure 2The FT-ICR-MS spectrum of gives the assembly mode of 3-R,R in solution analyzed by qCID of FT-ICR-MS. The peak of two molecules of 3-R,R plus Na was seen at the mass number of 1167.50713 (the theoretical value is 1167.51674), and the theoretical value is consistent with the measured value. Under the conditions of FT-ICR-MS, the peak of two molecules of 3-R,R plus Na was bombarded, and the peak of one molecule of 3-R,R plus Na was seen at the mass number of 595.24867 (the theoretical value is 595.25325), and the theoretical value is consistent with the measured value. The peak of two molecules of 3-R,R plus Na and its qCID spectrum of 3-R,R plus Na under the conditions of FT-ICR-MS The peak of the cleavage of two molecules of 3-R,R plus Na and its cleavage of the peak of one molecule of 3-R,R plus Na indicate that the existence form of R,R-heptacyclic dimethyl acetal (3R,R) in solution is a dimer.

[0026] Example 5 Evaluation of the thrombolytic activity of [R,R-heptacyclic dimethyl acetal] 2

[0027] The present invention evaluates the thrombolytic activity of [R,R-heptacyclic dimethyl acetal] 2 on a rat model. During the evaluation, male SD rats (230±20g) were rested for 1 day and randomly divided into groups, with 10 rats in each group. The rats were anesthetized by intraperitoneal injection of 20% urethane saline solution. The anesthetized rats were fixed in a supine position, the right common carotid artery was separated, an artery clamp was clamped on the artery at the proximal end, surgical threads were inserted into the proximal end and the distal end, the surgical threads at the distal end were clamped on the fur with hemostats, a cannula was inserted at the distal end, the artery clamp was released, about 1mL of arterial blood was injected into a vertically fixed rubber tube (15mm long, 2.5mm inner diameter, 5.0mm outer diameter, and sealed with a rubber stopper at the bottom), and a stainless steel thrombus fixing bolt was quickly inserted into the tube. The thrombus fixing bolt is wound with a stainless steel wire with a diameter of 0.2 mm, the spiral part is 10 mm long, contains 15 spiral coils, the diameter of the spiral coil is 1.0 mm, and the handle is connected to the spiral, is 7.0 mm long, and is question mark shaped. After the blood coagulates for 40 minutes, the thrombus fixing bolt wrapped by the thrombus is carefully taken out from the rubber tube, and the initial weight of the thrombus fixing bolt is accurately weighed.

[0028] The bypass cannula consists of three sections. The middle section is a polyethylene tube with a length of 60 mm and an inner diameter of 3.5 mm. Both sides are polyethylene tubes with a length of 100 mm, an inner diameter of 1.0 mm, and an outer diameter of 2.0 mm. One end of the polyethylene tube is pulled into a 10 mm long and outer diameter of 1.0 mm sharp tip (for insertion into the rat carotid artery and vein), and the other end of the polyethylene tube is covered with a 7 mm long and outer diameter of 3.5 mm polyethylene tube (for connecting the middle section of polyethylene). The inner walls of the three sections are all silanized (1% silicone oil ether solution). The thrombus fixing bolt wrapped with a precisely weighed thrombus is placed in the middle section of polyethylene.

[0029] Continue to separate the trachea of ​​the anesthetized rat and perform endotracheal intubation. Separate the left external jugular vein of the rat, insert surgical thread into both the proximal and distal ends, and carefully cut an oblique cut on the exposed left external jugular vein. Insert the oblique end of the prepared bypass tube into the proximal end of the opening of the left external jugular vein, and fix the handle of the thrombus spiral in the middle section away from the bypass tube. Use a syringe to push an accurate amount of heparin sodium saline solution (50IU / kg) through the sharp tube at the other end. At this time, do not remove the syringe from the polyethylene tube. Clamp the hose between the syringe and the polyethylene tube with a hemostatic forceps. Use an artery clamp to stop bleeding at the proximal end of the right common carotid artery, and carefully cut an oblique cut on the right common carotid artery not far from the artery clamp. Pull out the syringe from the tip of the polyethylene tube and insert the tip of the polyethylene tube into the proximal end of the arterial oblique cut. Use No. 4 surgical sutures to fix the artery and vein at both ends of the bypass tube.

[0030] The saline solution (dose of 3 mL / kg) or the saline solution of R,R-heptacyclic dimethyl acetal (dose of 1 μmol / kg) or the saline solution of [R,R-heptacyclic dimethyl acetal]2 (dose of 0.1 μmol / kg) in the syringe was slowly injected into the stomach of the rat through the esophagus, or the saline solution of urokinase (dose of 20000 IU / kg) in the scalp needle was slowly injected into the proximal vein away from the thrombus fixation spiral in the middle section of the bypass tube so that it can act on the thrombus in the order of vein-heart-artery through the blood circulation. The arterial clamp was opened to allow blood to flow from the artery to the vein through the bypass tube. The timing was started from the time of the injection. After 1 hour, the thrombus fixation bolt was removed from the middle section of the bypass tube and the final weight of the thrombus fixation bolt was accurately weighed. The initial weight of the thrombus fixation bolt minus the final weight of the thrombus fixation bolt was obtained to obtain the thrombus weight loss.

[0031] The thrombus weight reduction in Table 1 shows that the thrombus weight reduction in rats treated with an oral dose of 0.1 μmol / kg of [R,R-heptacyclic dimethyl acetal]2 was significantly greater than that in rats treated with saline (P<0.01 compared with saline). The thrombus weight reduction in Table 1 also shows that the thrombus weight reduction in rats treated with an oral dose of 0.1 μmol / kg of [R,R-heptacyclic dimethyl acetal]2 was significantly greater than that in rats treated with urokinase at an intravenous dose of 20000 IU / kg (P<0.01 compared with urokinase). The 0.1 μmol / kg [R,R-heptacyclic dimethyl acetal]2 treatment had an outstanding technical effect.

[0032] Table 1 Thrombolytic activity of [R,R-heptacyclic dimethyl acetal] 2

[0033] Therapeutic agents dose Thrombus weight loss, mean ± SD mg Normal saline 0.1mL / 10g 23.31±1.55 <![CDATA[[R,R-1,7-Dioxaspiro[5.5]undecane]]> 0.1 μmol / kg <![CDATA[34.11±2.24 a ]]> R,R-Heptacyclic dimethyl acetal 1μmol / kg <![CDATA[28.69±2.01 b ]]> Urokinase 20000IU / kg 29.10±2.02

[0034] a) Compared with normal saline, R,R-heptacyclic dimethyl acetal and urokinase, P<0.01; b) Compared with normal saline, P<0.01, compared with urokinase, P>0.05; n=10.

Claims

1. A dimer of R,R-heptacyclic dimethyl acetal, characterized in that: The dimer is of the following structure: [R,R-heptacyclic dimethyl acetal]2, 2. A method for preparing the dimer of R,R-heptacyclic dimethyl acetal according to claim 1, characterized in that: The method described comprises the following steps: 1) preparing R,R-heptacyclic dimethyl acetal; 2) Preparation of [R,R-heptacyclic dimethyl acetal] 2.

3. Use of the dimer of R,R-heptacyclic dimethyl acetal according to claim 1 in the preparation of a thrombolytic agent.

Images