CN106543032B - Tetracyclic diterpene compound and preparation and application - Google Patents

Abstract

The present invention belongs to the fields of medicinal chemistry, pharmacology and preparation, and specifically relates to tetracyclic diterpene compounds, preparation, pharmacological action mechanism research, application and preparation, and relates to the treatment of tetracyclic diterpene compounds for anticoagulation, cerebral ischemia, cerebral apoplexy, cerebral palsy, etc. Uses for edema, cerebral infarction, and neurological impairment.

Description

Tetracyclic diterpene compound and its preparation and application

technical field

The present invention belongs to the fields of medicinal chemistry, pharmacology and preparation, and specifically relates to tetracyclic diterpene compounds, preparation, pharmacological action mechanism research, application and preparation, and relates to the treatment of tetracyclic diterpene compounds for anticoagulation, cerebral ischemia, cerebral apoplexy, cerebral palsy, etc. Uses for edema, cerebral infarction, and neurological impairment.

Background technique

At present, cardiovascular disease has become one of the main causes of human death worldwide, and the formation of thrombus plays a vital role in the pathogenesis of cardiovascular disease. The coagulation pathway mainly involves the formation of venous and arterial thrombus, which can be considered to include three stages: initiation, expansion, and termination. The coagulation system formed by coagulation factors plays an important role in the process of thrombus formation.

Anticoagulants are a class of drugs that prevent blood clotting and can be used to prevent venous thrombosis, myocardial infarction, and ischemic stroke. Depending on the mechanism of action and source, anticoagulants mainly fall into the following categories:

Coumarins are a class of vitamin K antagonists, and the typical drug is warfarin;

Heparin and its derivatives, including heparin, low molecular weight heparin, fondaparinux sodium, etc.;

Direct factor Xa inhibitors, such as rivaroxaban, apixaban, edoxaban (just launched in 2015);

Direct thrombin inhibitors, such as: dabigatran cool, argatroban, bivalirudin, hirudin, etc.

Traditional anticoagulant drugs such as heparin and vitamin K antagonists (VKAs) have many defects that limit their wide clinical use.

FXa is a trypsin-like serine protease located at the junction of the extrinsic and intrinsic coagulation pathways. After binding to its cofactor Va (FVa) and calcium ions on the surface of phospholipids, FXa converts prothrombin to thrombin, the last enzyme produced in the coagulation cascade. Thrombin further activates platelets and at the same time converts fibrinogen into fibrin, which ultimately leads to thrombus formation. FXa is a key coagulation factor in the coagulation pathway, and inhibiting its activity can reduce the generation of thrombin and thus inhibit the formation of thrombus. Because it does not affect the activity of existing thrombin, the risk of bleeding caused by selective FXa inhibitors is relatively small, and compared with traditional anticoagulant drugs, it has better curative effect and safety, has a wide spectrum of diseases for treatment, has a stable dose-effect relationship, and is convenient to take orally. There will be no drug-drug interactions, and there is no need for clinical monitoring. Therefore, FXa is a very important and clear target of new anticoagulants.

Bayerane-type tetracyclic diterpene compound isosteviol has a wide range of biological activities in anti-tumor, regulating blood fat, lowering blood sugar, anti-inflammation, etc., and its raw material stevioside is relatively cheap and easy to obtain, and has low safety and toxicity. Structural transformation and modification of lead compounds is of great significance for the discovery of active compounds that can be applied to the market.

The present invention mainly modifies the structure of the 19-position carboxyl group and the 16-position carbonyl group of isosteviol through a series of reactions such as esterification, oximation, etherification, and Heck. a more active compound. Structural modification: Ethyl esterification of the 19-position hydroxyl group, oximation of the 16-position carbonyl group, and then structural modification of the oxime hydroxyl group. Different structural fragments are connected through etherification reaction and Heck reaction to obtain compounds with general structure and series of compounds.

Technical solutions

The present invention relates to a tetracyclic diterpene compound, the general structural formula is:

R1 is: -H,

any one or more of

R2 is: -COOEt;

n is: 0-2.

R2 is: -COOEt;

n is: 0-2;

The present invention relates to tetracyclic diterpene compound, the structure is:

The present invention relates to tetracyclic diterpene compounds, numbered as compound cp-2 series, with the following structure:

The present invention relates to tetracyclic diterpene compounds, numbered as compound cp-3 series, with the following structure:

The invention relates to a tetracyclic diterpene compound used for preparing blood coagulation factor inhibitors.

The invention relates to a composition in which tetracyclic diterpene compounds are used to prepare any one or more of anticoagulant and antithrombotic.

The invention relates to a pharmaceutical composition for preparing a tetracyclic diterpene compound for treating any one or more diseases of cerebral ischemia, cerebral apoplexy, cerebral edema, cerebral infarction and nerve function damage.

The invention relates to a pharmaceutical composition for inhibiting cerebral ischemia prepared by a tetracyclic diterpene compound.

The invention relates to a pharmaceutical composition prepared by a tetracyclic diterpene compound for inhibiting any one or more diseases of nerve function damage caused by cerebral ischemia and cerebral infarction.

The invention relates to a pharmaceutical composition prepared by a tetracyclic diterpene compound for reducing brain water content and alleviating any one or more diseases in cerebral edema.

The present invention relates to formulations of tetracyclic diterpenoids containing 0.01-500 mg of active compound per formulation unit.

The present invention relates to preparations of tetracyclic diterpenoids containing 0.1-200 mg of active compound per preparation unit.

The present invention relates to formulations of tetracyclic diterpenoids containing 1-100 mg of active compound per formulation unit.

The invention relates to preparations of tetracyclic diterpene compounds, and the preparation types include tablets, capsules, granules, pellets, sustained-release capsules, freeze-dried powder injections, injections and the like.

The compound of the invention is aimed at anticoagulant and antithrombotic, and has good market application prospect.

The compound of the present invention has good market application prospects for the treatment of cerebral ischemia, cerebral apoplexy, cerebral edema, cerebral infarction and nerve function damage.

Pharmacodynamic test of the present invention

Drug efficacy test 1: In vitro study on the inhibitory effect of compounds on blood coagulation factor FXa

1. Purpose of the test

According to the requirements of the guidelines for preclinical research of new western medicine, the in vitro inhibitory effect and IC ₅₀ of the newly synthesized series of compounds on blood coagulation factor Xa (FXa) were studied.

experiment material

2.1 Test drugs

The newly synthesized series of compounds and positive drugs were provided by Peking University Health Science Center.

2.2 Test reagents

Human FXa, Kordia Company (purchased by Jinan Xinlianxin Biotechnology Co., Ltd.); S-2765 chromogenic substrate, Boatman Biotech Company (purchased by Jinan Xinlianxin Biotechnology Co., Ltd.); bovine serum albumin (BSA), Sinopharm; DMSO, tris hydrochloride (Tris), Sigma.

2.3 Test equipment

FORMA 700 ultra-low temperature refrigerator, Thermo Company; YC-300L drug storage cabinet, Zhongke Meiling Low Temperature Technology Co., Ltd.; Direct-Q with pump ultrapure water instrument, Millopore Company; M200 full-wavelength microplate reader, Tecan Company .

3. Test method

3.1 Compound concentration selection

Positive drug: 0.1nM, 1nM, 10nM, 100nM, 1000nM.

Compound: Set the concentration of the compound to be tested to 1nM, 10nM, 100nM, 1000nM, 10000nM or 10nM, 100nM, 1000nM, 10000nM, 100000nM according to the preliminary screening results.

3.2 Experimental steps

3.2.1 Preparation of BSA Buffer (Bovine Serum Albumin Buffer)

Get 0.005mol (0.605g) Tris, 0.01mol (0.58g) NaCl, 0.10g BSA, add 100ml triple distilled water, shake up, and obtain final concentration and be 0.05M Tris-0.1M NaCl-0.1%BSA buffer solution (pH= 7.4), spare.

3.2.2 Preparation of compound solution

Take 10 μl of the compound, add 100 nl of DMSO, vortex, dissolve and mix to obtain a 10 ⁵ mol/l compound mother solution. Quantitatively dilute to the specified concentration according to the doubling dilution method.

3.2.3 Determination of FXa inhibitory activity

In a 96-well plate, add 10 μl of the above-mentioned prepared different concentrations of the drug solution (add 0.1% DMS010 μl to the blank group), and 25 μl of 0.003 IU/mL human FXa, and 40 μl of BSA buffer (pH 7.4 )well mixed. Incubate at 37°C for 15 min. Then add 40 μl of chromogenic substrate S-2765, shake for 10 seconds, and incubate at room temperature for 1 hour. The absorbance of each well at 405 nm was measured with a microplate reader.

3.3 Statistics

FXa inhibitory activity=1-[(OD/min)sample/(OD/min)control]. The IC ₅₀ value was calculated and analyzed by SPSS19.0 software.

4. Test results

Table 1. In vitro activity of compounds vs Factor Xa

As shown in Table 1, the IC ₅₀ of the positive control compounds Rivaroxaban and Edoxaban were 0.7±0.08nM and 3.4±0.43nM, respectively. Compounds with IC ₅₀ <1μM include cp-2-11(55.4±4.7nM), cp-2-16(29.8±3.2nM), cp-2-20(451.7±52.7nM), cp-3-13(421.6 ±39.7nM)

Drug efficacy test 2. In vitro study on the inhibitory effect of the compound on various blood coagulation factors and the influence of aPTT and PT

1. Purpose of the test

According to the requirements of the guidelines for the preclinical research of new western medicines, study the effect of newly synthesized series of compounds on blood coagulation factor Xla (FXIa), blood coagulation factor VIIa (FVIIa), blood coagulation factor XIIa (FXIIa), blood coagulation factor IXa (FIXa), thrombin (Thrombin ), plasma kallikrein (Plasma Kallikrein, PK), trypsin (Trypsin), chymotrypsin (Chymotrypsin) in vitro inhibition and K _i . The effects of compounds on activated partial thromboplastin time (activated partial thromboplastin time, aPTT) and plasma prothrombin time (prothrombin time, PT) in vitro were also determined.

2. Test material

2.1 Test drugs

Two newly synthesized compounds were provided by Peking University Health Science Center.

2.2 Test reagents

Human thrombin, FXIIa (American Diagnostica Inc., Greenwich, CT, USA); Human FIXa, FXIa, PK (Haematologic Technologies Inc., Essex Junction, VT, USA); Human trypsin, chymotrypsin (Sigma Chemical Co., St Louis, MO, USA); Human FVIIa (Novo Pharmaceuticals, Bagsvaerd, Denmark); Chromogenic Substrate S-2222, Chromogenic Substrate S-2238, Chromogenic Substrate S-230, Chromogenic Substrate S-2366, Chromogenic Substrate Substrate S-2288, chromogenic substrate CS PSA (DiaPharma, West Chester, Ohio, USA); Spectrozyme FIXa, FXIIa specific chromogenic substrate (American Diagnostica Inc., Greenwich, CT, USA); bovine serum albumin (BSA), DMSO, Tris hydrochloride (Tris), PEG8000 (Sigma Chemical Co., St Louis, MO, USA); Dade Actin FS kit, Thromboplastin C-Plus kit (Dade Behring, Marburg, Germany).

2.3 Test equipment

FORMA 700 ultra-low temperature refrigerator, Thermo Company; YC-300L drug storage cabinet, Zhongke Meiling Low Temperature Technology Co., Ltd.; Direct-Q with pump ultrapure water instrument, Millopore Company; M200 full-wavelength microplate reader, Tecan Company ; Sysmex CA-6000 automatic coagulation instrument, Dade-Behring Company.

3. Test method

3.1 Compound concentration selection

Serine Protease Selectivity test: 1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM.

aPTT and PT test: 1nM, 10nM, 100nM, 1000nM, 10000nM, 100000nM, 1000000nM.

3.2 Experimental steps

3.2.1 Preparation of buffer solution

FXIa, FXIIa and chymotrypsin activity assay working solution: 50 mM HEPES, 145 mM NaCl, 5 mM KCl and 0.1% PEG 8000 (pH 7.4).

Thrombin, trypsin and PK activity assay working solution: 100mM Na ₃ PO ₄ , 200mM NaCl and 0.5% PEG 8000 (pH 7.5).

FVIIa activity assay working solution: 50 mM HEPES, 150 mM NaCl, 5 mM CaCl ₂ and 0.1% PEG8000 (pH 7.4).

FIXa activity assay working solution: 50 mM Tris, 100 mM NaCl, 5 mM CaCl ₂ , 0.5% PEG 8000 and 2% DMSO (pH 7.4).

3.2.2 Determination of enzyme activity

Take 1 μmol of the compound, add 100 μl DMSO, shake, dissolve and mix to obtain a 10 mmol/l compound mother solution. Quantitatively dilute to the specified concentration according to the doubling dilution method. In a 96-well plate, add 10 μl of the above-mentioned prepared different concentrations of the drug solution (add 0.1% DMS010 μl to the blank group), and 25 μl of 0.003 IU/mL FVIIa, FIXa, FXIa, FXIIa, chymotrypsin , thrombin, trypsin and PK were mixed evenly with 40 μl of different working buffers. Incubate at 37°C for 15 min. Then add 40 μl of chromogenic substrates S-2222 (Trypsin), S-2238 (Thrombin), S-2302 (PK), S-2366 (FXIa), S-2288 (FVIIa), CS PSA (Chymotrypsin) or Spectrozyme FIXa, FXIIa specific chromogenic substrate, the final concentration is 10mM, after shaking for 10s, incubate at room temperature for 1h. The absorbance of each well at 405 nm was measured with a microplate reader.

3.2.3 Determination of aPTT and PT

1 ml of venous blood was collected from healthy volunteers in vacuum blood collection tubes (vacuum blood collection tubes containing 0.11 mM sodium citrate, Becton Dickinson, Mountainview, CA, USA), centrifuged at 2000 g, 4°C for 10 min to prepare plasma. Mix 50 μl of plasma and compound mother solution in different proportions and incubate at 37°C for 1 min, then add 50 μl of Dade Actin FS reagent, add CaCl ₂ after 2 min, and measure coagulation time with an automatic coagulation analyzer. 50 μl of plasma and compound mother solution were mixed in different proportions and incubated at 37°C for 3 min, then 100 μl of Thromboplastin C-Plus reagent was added, and the coagulation time was measured by an automatic coagulation analyzer. Calculate the ratio of PT or aPTT of different concentration compound groups to the Control group, and make a standard curve. The results are expressed as the compound concentration (EC _2x ) required to prolong the coagulation time to 2 times of the Control group.

3.3 Statistics

Inhibitory activity = 1 - [(OD/min) sample/(OD/min) control]. The IC ₅₀ value is calculated and analyzed by SPSS19.0 software, K _i =IC ₅₀ /(1+[s]/K _m ), where [s] is the substrate concentration, and K _m is the Michaelis constant.

4. Test results

Table 2. Serine Protease Selectivity for compound cp-2-11

As shown in Table 2, the inhibitory effect of cp-2-11 compounds on thrombin (K _i =517±49nM), plasma kallikrein (K _i =277±45nM) and trypsin (K _i =331±52nM) Stronger, but the weakest inhibitory effect on chymotrypsin (K _i =12378±1603nM). Table 3. Serine Protease Selectivity for compound cp-2-16

As shown in Table 3, the inhibitory effects of cp-2-16 compounds on FXIa (K _i =1697±158nM), plasma kallikrein (K _i =2205±285nM) and trypsin (K _i =2130±244nM) were relatively Stronger, but except for FXIa inhibitory effect stronger than cp-2-11 compound, other enzyme inhibitory effects are significantly weaker than cp-2-11 compound.

Table 4. aPTT and PT Data for Compound cp-2-11 and cp-2-16

As shown in Table 4, the concentration of cp-2-11 compound that can prolong aPTT and PT by two times is 2 μM and 47 μM, respectively, and the concentration of cp-2-16 compound that can prolong aPTT and PT by two times is 4 μM and 54 μM, respectively , suggesting that the inhibitory effect of cp-2-11 compound on coagulation system in vivo and in vitro is significantly stronger than that of cp-2-16 compound.

Pharmacological Experiment 3: Experimental Study on the Antithrombotic Effect of cp-2-11 and 16

experiment material

Animals: Wistar rats, weighing 210-230g, male and female, purchased from Changchun Yisi Experimental Animal Technology Co., Ltd., production certificate number: SCXK (Ji)-2011-0003, quality certificate number: 201600014639, experimental animals License number: SYXK (Ji) 2015-0009; Wistar rats, weighing 220-250g, male and female, purchased from Liaoning Changsheng Biotechnology Co., Ltd., production certificate number: SCXK (Liao)-2015-0001, quality Certificate number: 211002300015398, 211002300014521, laboratory animal use license number: SYXK (Ji) 2015-0009. KM mice, weighing 19-21 g, half male and half male, were purchased from Changchun Institute of Biological Products Co., Ltd., animal license number: SCXK (Ji)-2011-0003. Drug: Test product cp-2, colorless oily liquid, batch number: 20160421, positive control drug Edoxaban, white powder, batch number: 20160406, all provided by the State Key Laboratory of Natural Medicine and Biomimetic Medicine, Peking University, positive The control drug, clopidogrel bisulfate tablets, was produced by Shenzhen Xinlitai Pharmaceutical Co., Ltd., batch number: AA20150316.

Reagent: Chloral hydrate, produced by Sinopharm Chemical Reagent Co., Ltd., batch number: 20150724, preparation method: Weigh 9.0g of chloral hydrate, add 0.9% normal saline to dissolve to 300ml, 10ml/kg for later use. Adenosine diphosphate (ADP, molecular weight is 427), content 95%, produced by Dalian Meilun Biotechnology Co., Ltd., batch number: 20160326, preparation method: accurately weigh ADP11.3mg and add physiological saline to dissolve to 50ml, and its concentration is 0.5 mM; Tween-80, produced by Tianjin Huadong Reagent Factory, batch number: 20150722; sodium chloride injection, provided by Liaoning Minkang Pharmaceutical Co., Ltd., batch number: 20160506. Activated partial thromboplastin time (APTT), lot number: 1121892; prothrombin time (PT), lot number: 105295; thrombin time (TT), lot number: 121165; fibrinogen (FIB), lot number: 132105;

Instruments: YLS-14B Small Animal Thrombosis Apparatus, Jinan Yiyan Technology Development Co., Ltd.; In Vitro Thrombosis Apparatus, LBY-NJ2 Platelet Aggregation Apparatus, Beijing Prison Instruments Co., Ltd.; BT5-3 Low-speed Desktop Centrifuge, Beijing Times Beili Centrifuge Co., Ltd.; XT-2000i automatic five-differentiation blood cell analyzer, Japan Xisenmeikang Medical Electronics Co., Ltd.; FB-40 semi-automatic blood coagulation analyzer, Shanxi Sen Industrial Co., Ltd.

Test Method and Results

Antithrombotic effect

1. Effect on the time of thrombus formation in rats

Take 50 Wistar rats, half male and half male. After adapting to the environment in a clean observation room for 3 days, they are randomly divided into five groups, 10 rats in each group. The first group is the blank control group, and the second group is clopidogrel bisulfate. Tablet 10mg/kg, the third group was edoxaban 10mg/kg, the fourth group was cp-2-11 10mg/kg, and the fifth group was cp-2-16 10mg/kg. The volume was 10ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, they were anesthetized with 3% chloral hydrate 0.3g/kg ip, and the common carotid artery was isolated. The time of thrombus formation in rats was measured with a YLS-14B small animal thrombosis instrument. Experimental conditions: stimulation current 1.2mA, stimulation time 5min, recording 30min. During the measurement, the stimulating and temperature-controlling electrodes are fully in contact with the common carotid artery. After the current is stable, the stimulation is started. The time of 100% thrombus formation within 30 minutes is taken as the thrombus formation time in rats. If the thrombus does not reach 100% formation within 30 minutes, then The formation time was recorded as "30"; at the same time, the maximum percentage of thrombus formation within 30 minutes and the 100% formation rate of thrombus within 30 minutes were recorded. The results are shown in Table 5.

Table 5. Effects on the time of thrombus formation in rats ( n=10)

Note: Compared with the blank control group *P<0.05; **P<0.01; ***P<0.001 (the same as the following table)

From the results in Table 5, it can be seen that after intragastric administration, cp-2-11 10mg/kg, cp-2-16 10mg/kg, clopidogrel bisulfate tablets 10mg/kg and edoxaban 10mg/kg can all significantly Prolong the time of thrombus formation in rats; cp-2-11, cp-2-16 and clopidogrel bisulfate tablets can also significantly reduce the maximum percentage of thrombus formation in rats within 30 minutes and the 100% formation rate of thrombus within 30 minutes, compared with blank control There were significant differences in group comparisons. Moreover, cp-2-11 and cp-2-16 have similar antithrombotic effects, and are stronger than the positive control drugs clopidogrel bisulfate tablets and edoxaban.

2. Effects on thrombosis in vitro in rats

Get 50 Wistar rats, half male and half female, and randomly divide them into five groups, 10 rats in each group. Doxaban 10mg/kg, the fourth group was cp-2-11 10mg/kg, and the fifth group was cp-2-16 10mg/kg. The volume was 10ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, anesthetize with 3% chloral hydrate 0.3g/kg ip, take 3.0ml of blood from the abdominal aorta, inject 1.8ml of it into the siliconized in vitro thrombus rubber tube, and connect the two ends of the tube , form a ring, place it on an in vitro thrombus formation instrument and rotate for 15 minutes, measure the length of the thrombus, and weigh the wet weight and dry weight of the thrombus. The results are shown in Table 6.

Table 6. Effects on thrombus length, wet weight and dry weight in vitro of rats ( n=10)

It can be seen from the results in Table 6 that cp-2-11 10mg/kg can significantly shorten the length of thrombus and reduce the wet weight and dry weight of thrombus after intragastric administration; cp-2-16 10mg/kg can significantly reduce the wet weight of thrombus Thrombus length and dry weight have a certain inhibitory effect; Clopidogrel bisulfate tablet 10mg/kg can significantly shorten the thrombus length, reduce the thrombus dry weight, and have a certain inhibitory effect on the thrombus wet weight. Edoxaban 10mg/kg can significantly shorten the length of thrombus, reduce the wet weight and dry weight of thrombus. The comprehensive effect of cp-2-11 is stronger than that of clopidogrel bisulfate tablets and edoxaban.

2. Effects on platelet aggregation and adhesion rate in normal rats

1. Effects on platelet aggregation in normal rats

Take 50 Wistar rats and randomly divide them into five groups, 10 in each group. The first group is the blank control group, the second group is clopidogrel bisulfate tablets 10mg/kg, and the third group is edoxaban 10mg /kg, the fourth group was cp-2-11 10mg/kg, and the fifth group was cp-2-16 10mg/kg. The volume was 10ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, anesthetize with 3% chloral hydrate 0.3g/kg ip, take 4.5ml of blood from the abdominal aorta, inject it into a test tube containing 0.5ml of sodium citrate, shake it well, and put it in the BT5-3 type Centrifuge in a low-speed desktop centrifuge at 1200 rpm for 10 minutes, slowly absorb 0.6ml of the upper layer of platelet-rich plasma (PRP), centrifuge the remaining plasma again, and centrifuge at 3300 rpm for 10 minutes, and absorb the upper layer of platelet-poor plasma (PPP). Use a micro-sampler to draw 300 μl of PPP and PRP into each cuvette, preheat it in the LBY-NJ2 platelet aggregation instrument for 5 minutes, adjust to zero with PPP, then adjust the PRP to 30±5, and add 0.5mM 6 μl of ADP inducer, record the platelet aggregation rate and the maximum aggregation rate at 1, 3, and 5 minutes. The results are shown in Table 7.

Table 7. Effects on platelet agglutination of normal rats administered in vivo ( n=10)

It can be seen from the results in Table 7 that cp-2-11 10mg/kg can significantly reduce the platelet aggregation rate and maximum aggregation rate for 3min, 5min, compared with the blank control group, and has a significant effect on the aggregation rate of 1min. A certain reduction effect; cp-2-16 10mg/kg has a certain reduction effect on platelet aggregation rate and maximum aggregation rate at 1min, 3min, 5min, but there is no significant difference compared with the blank control group. Positive control drugs clopidogrel bisulfate tablets 10mg/kg and edoxaban 10mg/kg can significantly reduce platelet aggregation rate and maximum aggregation rate in 3min, 5min, compared with the blank control group, there are significant differences, edoxaban 10mg/kg also significantly reduces the 1min aggregation rate of platelets, and clopidogrel bisulfate tablets 10mg/kg has a tendency to reduce the 1min aggregation rate.

2. Effects on platelet aggregation of normal rats administered in vitro

Take 20 Wistar rats, half male and half female, weighing 220-250g. After adapting to the environment in a clean observation room for 3 days, the rats were anesthetized with 3% chloral hydrate, 10ml/kg, and 6ml of abdominal aorta was put into 3.8% In a test tube anticoagulated with sodium citrate, shake it steadily for 10 times, centrifuge in a BT5-3 low-speed desktop centrifuge at 1200 rpm for 10 minutes, slowly absorb 1.5ml of platelet-rich plasma (PRP) in the upper layer, and dissolve the remaining The plasma was centrifuged again at 3300 rpm for 10 minutes, and the upper platelet-poor plasma (PPP) was absorbed. Use a micro-sampler to draw 300 μl of PPP and PRP into each cuvette, adjust to zero with PPP, adjust PRP to 30±5, add 10 μl of liquid medicine to PRP at the same time, preheat in LBY-NJ2 platelet aggregation instrument for 5 minutes, Add 6μl of 0.5mM ADP inducer with a micro-sampler, add 6μl of normal saline to the blank control group, and record the platelet aggregation rate and maximum aggregation rate at 1, 3, and 5 minutes. The results are shown in Table 8.

Table 8. Effects on rat platelet aggregation in vitro ( n=10)

It can be seen from the results in Table 4 that cp-2-11 and cp-2-16 10 μg/ml in vitro can reduce the platelet aggregation rate and maximum aggregation rate at 1 min, 3 min and 5 min; but there is no significant difference compared with the blank control group ; Positive control drugs clopidogrel bisulfate tablets μg/ml and edoxaban μg/ml also had a certain reduction effect on platelet aggregation rate and maximum aggregation rate at 1min, 3min and 5min, but the effect of cp-2-11 Slightly stronger.

3. Effects on platelet adhesion rate in normal rats

Take 50 Wistar rats and randomly divide them into five groups, 10 in each group. The first group is the blank control group, the second group is clopidogrel bisulfate tablets 10mg/kg, and the third group is edoxaban 10mg /kg, the fourth group was cp-2-11 10mg/kg, and the fifth group was cp-2-16 10mg/kg. The volume was 10ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, anesthetize with 3% chloral hydrate 0.3g/kg ip, take 4.5ml of blood from the abdominal aorta, inject it into a test tube containing 0.5ml of sodium citrate, shake well, take 1ml into a circle In the adherent sphere, the in vitro thrombosis and platelet adhesion dual-purpose instrument was used to rotate for 15 minutes, and the automatic five-differential hematology analyzer was used to measure the number of platelets before and after adhesion, and the adhesion rate was calculated. The results are shown in Table 5.

Table 9. Effect on normal rat platelet adhesion rate ( n=10)

It can be seen from the results in Table 9 that cp-2-11 10mg/kg had no effect on the platelet adhesion rate after intragastric administration, but cp-2-16 10mg/kg significantly increased the platelet adhesion rate; Gray tablet 10mg/kg can reduce the platelet adhesion rate to a certain extent, while edoxaban can increase it to a certain extent.

3. Anticoagulant effect

1. Effect on bleeding time of mice

Take 50 KM mice and randomly divide them into four groups, 10 in each group. The first group is the blank control group, the second group is clopidogrel bisulfate tablets 15mg/kg, and the third group is edoxaban 15mg /kg, the fourth group was cp-2-11 15mg/kg and the fifth group was cp-2-16 15mg/kg. The volume was 20ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, the tip of the tail of the mouse was cut off by 3mm, and then the tail was put into 37°C physiological saline, and the bleeding time was recorded. The results are shown in Table 10.

Table 10. Effects on bleeding time in mice

It can be seen from the results in Table 10 that, once intragastric administration, cp-2-11 10mg/kg, cp-2-16 10mg/kg and positive control drugs clopidogrel bisulfate tablet 10mg/kg, edoxaban 10mg/kg Both can significantly prolong the bleeding time of mice, and there are significant differences compared with the blank control group. The effect of cp-2-11 is stronger than that of cp-2-16, and the effects of cp-2-16, clopidogrel bisulfate tablets and edoxaban are similar.

2. Effect on coagulation time of mice

Take 50 KM mice and randomly divide them into five groups, 10 in each group. The first group is the blank control group, the second group is clopidogrel bisulfate tablets 15mg/kg, and the third group is edoxaban 15mg /kg, the fourth group was cp-2-11 15mg/kg, and the fifth group was cp-2-16 15mg/kg. The volume was 20ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after the administration, the right eyeball of the mouse was removed, the first drop of blood was discarded, and two drops of blood were taken on a glass slide. One drop of blood was picked with a needle every 30 seconds until blood streaks appeared, and the other drop of blood was used for the last drop. Repeat the test and record the coagulation time. The results are shown in Table 11.

The impact of table 11 on mouse coagulation time

It can be seen from the results in Table 7 that, after a single intragastric administration, both cp-2-11 15mg/kg and edoxaban 15mg/kg can significantly prolong the coagulation time of mice, and there is a significant difference compared with the blank control group; cp-2-11 2-16 15mg/kg and Clopidogrel Bisulfate Tablets 15mg/kg have a certain effect on prolonging the coagulation time of mice, but there is no significant difference compared with the blank control group.

3. Effects on coagulation factors in normal rats

Take 50 Wistar rats and randomly divide them into five groups, 10 in each group. The first group is the vehicle control group, the second group is clopidogrel bisulfate tablets 10mg/kg, and the third group is edoxaban 10mg /kg, the fourth group was cp-2-11 10mg/kg, and the fifth group was cp-2-16 10mg/kg. The volume was 10ml/kg for intragastric administration once, and the blank control group was given the same volume of vehicle. One hour after administration, anesthetize with 3% chloral hydrate 0.3g/kg ip, take 4.5ml blood from the abdominal aorta, inject it into a test tube containing 0.5ml sodium citrate, shake well, and adopt FB-40 type Semi-automatic blood coagulation analyzer, according to kit method determination (prothrombin time) PT, activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB) content. The results are shown in Table 12.

Table 12. Effects on normal rat coagulation factors ( n=10)

It can be seen from the results in Table 12 that cp-2-11 10mg/kg can significantly prolong APTT and TT, and has a certain prolonging effect on PT, but has a slight downward trend on Fib; cp-2-16 10mg/kg can significantly prolong PT, APTT and TT have a certain prolonging effect, and have a slight downward trend to Fib. Positive control drugs clopidogrel bisulfate tablet 10mg/kg, edoxaban 10mg/kg can significantly prolong PT, APTT, TT, and can significantly reduce the Fib content.

Study on dosage forms of cp-2-11

Tablet preparation

cp-2-11 1-200g; microcrystalline cellulose 1-200g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; Magnesium acid 1-20g, granulated, dried at 60°C, granulated with a 12-mesh sieve, pressed into tablets; 1000 pieces; each piece contains 1-200mg cp-2-11

Preparation of capsules

cp-2-11 1-200g; microcrystalline cellulose 1-200g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; 12 Granulate with a mesh sieve, dry at 60°C, granulate with a 14-mesh sieve, and fill into empty capsules; 1000 capsules are obtained; each capsule contains 1-200mg cp-2-11.

Preparation of granules

cp-2-11 1-200g; starch 1-200g; poloxamer 1-50g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-50g; 25% starch slurry 10-2000ml; powdered sugar 10- 50g; Magnesium stearate 1-20g, granulated with 14 mesh sieve, dried at 60°C, granulated with 14 mesh sieve, packed into bags; 1000 bags; each bag contains 1-200mg of compound cp-2-11.

Preparation of extended-release tablets

cp-2-11 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; Magnesium stearate 1-20g, Talc powder 1-200g; Granulate with 14 mesh, dry at 60℃, granulate with 12 mesh sieve, press into tablets; 1000 pieces; each tablet contains 1-200mg compound cp -2-11.

Preparation of pellets

cp-2-11 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; roll pellets, dry at 60°C to make pellets, fill in empty capsules; 1000 capsules; each capsule contains 1-200mg of compound cp-2-11.

Preparation of sustained-release capsules

cp-2-11 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; magnesium stearate 1-20g, granulated with 14 mesh, dried at 60°C, granulated with 12 mesh sieve, pressed into tablets; 1000 pieces; each tablet contains 1-200mg of compound cp-2-11.

Preparation of freeze-dried powder injection

cp-2-11 1-200g; poloxamer 0.5-10g; add 1.0-10.0g glutamic acid, add 1-30.0g mannitol, add water for injection, heat to dissolve, dilute to 50-5000ml, filter and filtrate Ultrafiltration, sub-package, freeze-drying, and capping after completion. Made 1000 pieces, each containing 1-200mg of compound FHZ-1. The above-mentioned freeze-drying is divided into four stages: (1) pre-freezing for 3.6 hours at a temperature of -33°C; (2) drying under reduced pressure for 14 hours at a temperature of -36°C; (3) heating and drying for 4 hours at a temperature of -13°C ℃; (4) Secondary heating and drying for 4 hours, the temperature is 30 ℃.

Preparation of Injections

cp-2-11 1-200g; poloxamer 1-20g; add 1-20g glutamic acid, add water for injection, heat to dissolve, dilute to 100-5000ml, filter, filtrate ultrafiltration, filling and sterilization. 1000 pieces were prepared, each containing 1-200 mg of compound cp-2-11. Study on dosage forms of cp-2-16

Tablet preparation

cp-2-16 1-200g; microcrystalline cellulose 1-200g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; Magnesium acid 1-20g, granulated, dried at 60°C, granulated with a 12-mesh sieve, pressed into tablets; 1000 pieces; each tablet contains 1-200mg cp-2-16

Preparation of capsules

cp-2-16 1-200g; microcrystalline cellulose 1-200g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; 12 Granulate with a mesh sieve, dry at 60°C, granulate with a 14-mesh sieve, and fill into empty capsules; 1000 capsules are obtained; each capsule contains 1-200mg cp-2-16.

Preparation of granules

cp-2-16 1-200g; starch 1-200g; poloxamer 1-50g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-50g; 25% starch slurry 10-2000ml; powdered sugar 10- 50g; Magnesium stearate 1-20g, granulated with 14 mesh sieve, dried at 60°C, granulated with 14 mesh sieve, packed into bags; 1000 bags; each bag contains 1-200mg of compound cp-2-16.

Preparation of extended-release tablets

cp-2-16 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; Magnesium stearate 1-20g, Talc powder 1-200g; Granulate with 14 mesh, dry at 60°C, granulate with 12 mesh sieve, press into tablets; 1000 pieces; each tablet contains 1-200mg compound cp -2-16.

Preparation of pellets

cp-2-16 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; roll pellets, dry at 60°C to make pellets, fill in empty capsules; 1000 capsules; each capsule contains 1-200mg of compound cp-2-16.

Preparation of sustained-release capsules

cp-2-16 1-200g; microcrystalline cellulose 1-500g; poloxamer 1-200g; 95% ethanol 1-500ml; hydroxypropyl cellulose 1-100g; 25% starch slurry 10-2000ml; micropowder Silica gel 1-100g; magnesium stearate 1-20g, granulated with 14 mesh, dried at 60°C, granulated with 12 mesh sieve, pressed into tablets; 1000 pieces; each tablet contains 1-200mg of compound cp-2-16.

Preparation of freeze-dried powder injection

cp-2-16 1-200g; poloxamer 0.5-10g; add 1.0-10.0g glutamic acid, add 1-30.0g mannitol, add water for injection, heat to dissolve, dilute to 50-5000ml, filter and filtrate Ultrafiltration, sub-package, freeze-drying, and capping after completion. Made 1000 pieces, each containing 1-200mg of compound FHZ-1. The above-mentioned freeze-drying is divided into four stages: (1) pre-freezing for 3.6 hours at a temperature of -33°C; (2) drying under reduced pressure for 14 hours at a temperature of -36°C; (3) heating and drying for 4 hours at a temperature of -13°C ℃; (4) Secondary heating and drying for 4 hours, the temperature is 30 ℃.

Preparation of Injections

cp-2-16 1-200g; poloxamer 1-20g; add 1-20g glutamic acid, add water for injection, heat to dissolve, dilute to 100-5000ml, filter, filtrate ultrafiltration, filling and sterilization. Made 1000 pieces, each containing 1-200mg of compound cp-2-16.

specific embodiment

Embodiment 1: numbered as cp-1-3,

Molecular formula: C ₂₂ H ₃₄ O ₃ , molecular weight: 346.25, structural formula as follows:

H Spectrum ¹ H NMR (400MHz, CDCl3): 4.11 (q, J=7.0Hz, 2H), 2.65 (dd, J=18.5, 3.2Hz, 1H), 2.19 (d, J=13.3Hz, 1H), 2.04 -1.33(m, 14H), 1.27(t, J=7.1Hz, 3H), 1.23-1.21(m, 1H), 1.2(s, 3H), 1.16-1.0(m, 2H), 0.99(s, 3H ), 0.97-0.86 (m, 1H), 0.73 (s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ222.5, 177.3, 60.0, 57.0, 54.7, 54.3, 48.7, 48.5, 43.7, 41.6, 39.9, 39.5, 38.0, 37.9, 37.3, 28.9, 21.7, 20.3, 19.9 , 19.0, 14.1, 13.4

IR IR (KBr): 2956, 2928, 2845, 1728, 1465, 1380, 1227, 1151, 1095, ^1026cm-1

Weigh 18g of isosteviol and 15g of KOH, add to 100ml of DMSO, stir for 10min, add 6ml of C ₂ H ₅ Br and a small amount of crown ether, react at 40°C for 4h, cool to room temperature after the reaction, pour the reaction solution into 1000ml of cold water, and let stand for 30min , suction filtered, and the precipitate was washed 3 times with water.

Embodiment 2: the number is cp-1-4,

Molecular formula: C ₂₂ H ₃₆ O ₃ , molecular weight: 348.27, structural formula as follows:

H spectrum data: ¹ H NMR (400MHz, CDCl ₃ ): 4.1(q, J=7.1Hz, 2H), 3.87(dd, J=10.6, 4.8Hz, 1H), 2.18(d, J=13.6Hz, 1H ), 1.89-1.29(m, 14H), 1.27(t, J=7.1, Hz, 3H), 1.18(s, 3H), 1.08-0.95(m, 4H), 0.93(s, 3H), 0.9-0.83 (m, 1H), 0.77 (s, 3H)

C spectrum data: ¹³ C NMR (100MHz, CDCl ₃ ): δ177.6, 80.6, 59.9, 57.2, 55.8, 55.2, 43.7, 42.8, 42.0, 41.8, 40.0, 38.1, 38.0, 33.7, 29.0, 24.9, 21.8, 20.5, 19.0, 14.1, 13.4

Infrared data: IR(KBr): 3533.1, 2981.2, 2940.2, 2837.6, 1700.6, 1466.0, 1320.1, 1230.3, 1178.9, ^{1047.9cm -1}

Weigh 1 mmol of compound cp-1-3, add it into 10 ml of absolute ethanol solution under ice bath, slowly add 1.5 mmol of NaBH ₄ , and react under ice bath for 1 h. After the reaction, a small amount of water was added to continue the reaction for 10 min, filtered, evaporated to dryness, and recrystallized from chloroform-methanol to obtain colorless crystals.

Embodiment 3: the number is cp-1-5

Molecular formula: C ₂₂ H ₃₅ NO ₃ , molecular weight: 361.26, structural formula as follows:

H spectrum data: ¹ H NMR (400MHz, CDCl ₃ ): 4.11(m, 2H), 2.99(dd, J=18.6, 3.0Hz, 1H), 2.19(d, J=13.4Hz, 1H), 2.01(d , J=18.6Hz, 1H), 1.93-1.58(m, 7H), 1.52-1.39(m, 4H), 1.29(t, J=7.1Hz, 3H), 1.26-1.21(m, 2H), 1.20( s, 3H), 1.13(s, 3H), 1.12-0.85(m, 4H), 0.79(s, 3H)

C spectrum data: ¹³ C NMR (100MHz, CDCl ₃ ): δ177.4, 171.0, 60.0, 57.1, 56.3, 54.9, 43.8, 43.7, 40.9, 40.6, 40.0, 39.5, 38.1, 38.0, 36.8, 28.9, 22.1, 21.7, 20.4, 19.0, 14.1, 13.4

Infrared data: IR(KBr): 3305.8, 2943.2, 2847.1, 1723.0, 1450.0, 1376.5, 1233.5, 1179.2, ^{1028.2cm -1}

Synthesis:

Weigh 1 mmol of compound cp-1-3, dissolve it in 5 ml of absolute ethanol, add 1.5 mmol of hydroxylamine hydrochloride and 1.5 mmol of NaHCO ₃ , react at 60°C for 2 hours, evaporate the solvent to dryness after the reaction, dissolve with CH ₂ Cl ₂ , wash with water, and saturate Washed with NaCl, treated with anhydrous MgSO4, and evaporated to dryness to obtain a white powder

Embodiment 4: numbered as cp-2-1

Molecular formula C ₂₉ H ₄₀ ClNO ₃ : Molecular weight: 485.27, the structure is as follows:

H Spectrum ¹ H NMR (400MHz, CDCl ₃ ): 7.36 (br s, 1H), 7.32-7.18 (m, 3H), 5.06 (s, 2H), 4.22-4.0 (m, 2H), 2.95 (dd, J =18.6, 2.9Hz, 1H), 2.19(d, J=13.2Hz, 1H), 1.95(d, J=18.6Hz, 1H), 1.91-1.55(m, 7H), 1.51-1.33(m, 4H) , 1.28(t, J=7.1, 3H), 1.26-1.20(m, 1H), 1.19(s, 3H), 1.18-1.12(m, 1H), 1.10(s, 3H), 1.09-0.83(m, 4H), 0.76(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl ₃ ): δ177.4, 170.6, 140.9, 134.1, 129.5, 127.9, 127.5, 125.8, 74.5, 60.0, 57.2, 56.4, 54.9, 43.8, 43.7, 40.9, 40.7, 40.0, 39.6, 38.1, 38.0, 37.8, 28.9, 22.2, 21.7, 20.4, 19.0, 14.2, 13.4

IR IR (KBr): 3041.6, 2931.32, 2847.0, 1711.4, 1650.1, 1600.8, 1467.6, 1377.3, 1237.2, 1185.2, 1051.9, 881.7, 777.1 ^{, 682.0cm-1}

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol 3-chlorobenzyl bromide, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane dissolves , washed with water, washed with saturated NaCl, treated with anhydrous MgSO ₄ , and silica gel column (PE: EA 50: 1) to obtain the compound.

Embodiment 5: the number is cp-2-2

Molecular formula C ₂₉ H ₄₀ FNO ₃ : Molecular weight 469.30: The structure is as follows:

H Spectrum ¹ H NMR (400MHz, CDCl3): 7.36-7.29 (m, 2H), 7.05-6.97 (m, 2H), 4.19-4.0 (m, 2H), 2.91 (dd, J=18.6, 2.9Hz 1H) , 2.18(d, J=13.2Hz 1H), 1.92(d, J=18.6Hz 1H), 1.88-1.51(m, 7H), 1.26(t, J=7.1Hz 3H), 1.23-1.18(m, 1H ), 1.18(s, 3H), 1.16-1.10(m, 1H), 1.09(s, 3H), 1.08-0.82(m, 4H), 0.74(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.3, 170.1, 162.3, 134.4, 129.7, 115.0, 74.6, 59.9, 57.2, 56.4, 54.9, 43.8, 43.7, 40.9, 40.7, 40.0, 39.6, 38.0, 37.9 , 37.7, 28.9, 22.3, 21.7, 20.4, 19.0, 14.1, 13.4

F Spectrum ¹⁹ F NMR (376.3MHz, CDCl3) δ-114.9

IR IR (KBr): 3041.6, 2921.1, 2870.8, 2847.9, 1719.9, 1652.7, 1602.6, 1510.4, 1466.6, 1376.3, 1222.9, 1185.8,

IR IR (KBr): 3041.6, 2921.1, 2870.8, 2847.9, 1719.9, 1652.7, 1602.6, 1510.4, 1466.6, 1376.3, 1222.9, 1185.8, 1041.4, 833.9cm ^-1

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol 4-fluorobenzyl bromide, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane dissolves , washed with water, washed with saturated NaCl, treated with anhydrous MgSO ₄ , and silica gel column (PE: EA 50: 1) to obtain the compound.

Embodiment 6, the number is cp-2-11

Molecular formula: C ₂₇ H ₃₉ NO ₃ S, molecular weight: 457.27, structure as follows:

H spectrum ¹ H NMR (400MHz, CDCl3): 7.28-7.25 (m, 1H), 7.24-7.21 (m, 1H), 7.13-7.09 (m, 1H), 5.08 (s, 2H), 4.19-4.0 (m , 2H), 2.9(dd, J=18.6, 3.1Hz, 1H), 2.18(d, J=13.3Hz, 1H), 1.92(d, 18.6Hz, 1H), 1.88-1.54(m, 7H), 1.47 -1.36(m, 4H), 1.27(t, J=7.1Hz, 3H), 1.24-1.19(m, 1H), 1.18(s, 3H), 1.17-1.13(m, 1H), 1.12(s, 3H ), 1.08-0.83(m, 4H), 0.74(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.3, 169.9, 139.6, 127.7, 125.5, 123.0, 70.6, 59.9, 57.2, 56.4, 55.0, 43.8, 43.7, 40.9, 40.7, 40.0, 39.6, 38.0, 37.7 , 28.9, 22.3, 21.7, 21.7, 20.4, 19.0, 14.2, 13.4

IR IR (KBr): 3103.3, 2936.3, 2846.7, 1719.9, 1655.6, 1544.8, 1451.9, 1376.4, 1231.9, 1179.0, 1028.4, ^637.2cm-1

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol3-bromomethylthiophene, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane Dissolve, wash with water, wash with saturated NaCl, treat with anhydrous MgSO ₄ , and use a silica gel column (PE:EA 50:1) to obtain the compound.

Embodiment 7, the number is cp-2-13

Molecular formula: C ₂₈ H ₄₆ N ₂ O ₄ , molecular weight: 474.35, structure as follows:

H Spectrum ¹ H NMR (400MHz, CDCl3): 4.12(t, J=5.7Hz, 2H), 4.10-3.96(m, 2H), 3.64(t, J=4.6Hz, 4H), 2.82(dd, 18.5, 2.9Hz, 1H), 2.65-2.53(m, 2H), 2.53-2.41(m, 4H), 2.11(d, J=13.2Hz, 1H), 1.83(d, J=18.5Hz, 1H), 1.80- 1.47(m, 7H), 1.40-1.30(m, 4H), 1.21(t, J=7.1Hz, 3H), 1.18-1.13(m, 2H), 1.12(s, 3H), 1.03(s, 3H) , 1.01-0.77(m, 4H), 0.68(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.2, 169.3, 71.3, 66.9 (2C), 59.9, 57.6, 57.1, 56.4, 54.9, 54.1 (2C), 43.6, 43.5, 40.9, 40.6, 39.9, 39.6 , 38.0, 37.9, 37.6, 28.9, 22.3, 21.6, 20.4, 18.9, 14.1, 13.3

IR IR (KBr): 2953.9, 2847.5, 1719.8, 1655.4, 1450.2, 1363.2, 1230.7, 1179.0, 1149.6, ^1033.7cm-1

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol 4-(2-chloroethyl)morpholine, react for 10h, add 1ml ice water and continue stirring for 10min, evaporate to dryness after the reaction is completed Solvent, dichloromethane dissolved, washed with water, washed with saturated NaCl, anhydrous MgSO ₄ treatment, silica gel column (PE: EA 20: 1) to obtain the compound

Embodiment 8, the number is cp-2-16

Molecular formula: C ₂₇ H ₄₃ NO ₄ , molecular weight: 445.32, structure as follows:

H spectrum 1H NMR (400MHz, CDCl3): 4.65-4.61 (m, 2H), 4.33-4.30 (m, 2H), 4.18-4.01 (m, 4H), 2.9 (dd, J=18.6, 3.1Hz, 1H) , 2.17(d, J=13.2Hz, 1H), 1.89(d, J=18.5Hz, 1H), 1.86-1.53(m, 7H), 1.47-1.35(m, 4H), 1.33(s, 3H), 1.26(t, J=7.1, 3H), 1.24-1.18(m, 2H), 1.17(s, 3H), 1.07(s, 3H), 1.06-0.81(m, 4H), 0.75(s, 3H)

C spectrum 13C NMR (100MHz, CDCl3): δ177.3, 169.6, 80.3, 80.2, 77.7, 59.9, 57.2, 56.3, 54.9, 43.7, 43.6, 40.9, 40.7, 40.3, 39.9, 39.6, 38.1, 38.0, 37.3, 29.0, 22.2, 21.6, 21.5, 20.4, 19.0, 14.1, 13.3

IR IR (KBr): 2930.9, 2849.1, 1721.9, 1452.7, 1379.1, 1231.7, 1152.3 ^{, 1042.7cm-1}

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol 3-chloromethyl-3-methyloxetane, react for 10h, add 1ml ice water and continue stirring for 10min, react After completion, evaporate the solvent to dryness, dissolve in dichloromethane, wash with water, wash with saturated NaCl, treat with anhydrous MgSO ₄ , and use a silica gel column (PE:EA40:1) to obtain the compound.

Embodiment 9, the number is cp-2-19

Molecular formula: C ₂₅ H ₃₉ NO ₃ , molecular weight: 401.29, structure as follows:

H spectrum ¹ H NMR (400MHz, CDCl3): 6.07 (m, 1H), 5.32-5.14 (m, 2H), 4.54 (d, J=5.1Hz, 2H), 4.2-4.0 (m, 2H), 2.97- 2.88(m, 1H), 2.18(d, J=13.2, 1H), 1.94(d, J=18.6, 1H), 1.90-1.53(m, 8H), 1.48-1.36(m, 4H), 1.27(t , J=7.1Hz, 3H), 1.24-1.19(m, 1H), 1.18, (s, 3H), 1.10(s, 3H), 1.09-0.83(m, 4H), 0.77(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.4, 169.6, 134.8, 116.7, 74.3, 59.9, 57.2, 56.5, 55.0, 43.7, 43.7, 40.9, 40.6, 40.0, 39.6, 38.1, 38.0, 37.5, 29.0 , 22.2, 21.7, 20.5, 19.0, 14.1, 13.4

IR IR (KBr): 3078.9, 2937.4, 2847.7, 1720.7, 1655.4, 1453.5, 1418.6, 1376.5, 1231.9, 1178.7, 1030.7, ^921.7cm-1

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol allyl bromide, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane dissolves , washed with water, washed with saturated NaCl, treated with anhydrous MgSO ₄ , and silica gel column (PE: EA 50: 1) to obtain the compound.

Embodiment 10, the number is cp-2-20,

Molecular formula: C ₂₅ H ₃₉ NO ₄ , molecular weight: 417.29, structure as follows:

H spectrum ^-1 H NMR (400MHz, CDCl3): 4.28-4.20 (m, 1H), 4.20-4.04 (m, 2H), 4.04-3.95 (m, 1H), 3.28-3.21 (m, 1H), 2.93 ( dd, J=18.6, 2.8Hz, 1H), 2.86-2.80(m, 1H), 2.66-2.60(m, 1H), 2.18(d, J=13.3Hz, 1H), 1.95(d, J=18.6Hz , 1H), 1.91-1.55(m, 7H), 1.48-1.37(m, 4H), 1.28(t, J=7.1Hz, 3H), 1.26-1.20(m, 2H), 1.19(s, 3H), 1.10(s, 3H), 1.09-0.83(m, 4H), 0.78(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.4, 170.2, 74.1, 60.0, 57.2, 56.4, 55.0, 50.4, 44.9, 43.8, 43.7, 40.9, 40.7, 40.0, 39.6, 38.1, 38.0, 37.5, 28.9 , 22.2, 21.7, 20.4, 19.0, 14.2, 13.4

IR IR (KBr): 2933.3 2847.1, 1720.2, 1417.2, 1376.5, 1232.2, 1151.4, 1040.5, 869.9, ^{843.1cm -1}

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol R-epichlorohydrin, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane Dissolve, wash with water, wash with saturated NaCl, treat with anhydrous MgSO ₄ , and use a silica gel column (PE:EA 20:1) to obtain the compound.

Embodiment 11, the number is cp-2-21

Molecular formula: C ₂₅ H ₃₉ NO ₄ , molecular weight: 417.29, structure as follows:

H spectrum ¹ H NMR (400MHz, CDCl3): 4.28-4.20 (m, 1H), 4.19-4.01 (m, 2H), 4.01-3.93 (m, 1H), 3.27-3.20 (m, 1H), 2.97-2.87 (m, 1H), 2.85-2.79(m, 1H), 2.65-2.59(m, 1H), 2.17(d, J=13.2Hz, 1H), 1.95(d, J=18.6Hz, 1H), 1.90- 1.55(m, 7H), 1.47-1.36(m, 4H), 1.27(t, J=7.0Hz, 3H), 1.25-1.19(m, 2H), 1.18(s, 3H), 1.09(s, 3H) , 1.07-0.82(m, 4H), 0.76(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.4, 170.2, 74.2, 60.0, 57.2, 56.4, 55.0, 50.4, 44.9, 43.8, 43.7, 40.9, 40.7, 40.0, 39.6, 38.1, 38.0, 37.5, 28.9 , 22.2, 21.7, 20.4, 18.9, 14.1, 13.4

IR IR (KBr): 2930.2, 2847.1, 1720.3, 1453.2, 1376.6, 1232.8, 1179.5, 1040.9, 869.9, ^843.1cm-1

Synthesis:

Take 1mmol cp-1-5, dissolve in 10ml DMF, slowly add 1.5mmol NaH, react for 30min, add 1.5mmol S-epichlorohydrin, react for 10h, add 1ml ice water and continue stirring for 10min, after the reaction is completed, evaporate the solvent to dryness, dichloromethane Dissolved, washed with water, washed with saturated NaCl, anhydrous MgSO4 treatment, silica gel column (PE: EA 20: 1) to obtain the compound

Embodiment 12, the number is cp-3-9

Molecular formula: C ₃₃ H ₄₃ N ₃ O ₃ , molecular weight: 529.33, structure as follows:

H Spectrum ¹ H NMR (400MHz, CDCl3): 8.75-8.65(m, 2H), 7.98-7.77(m, 3H), 6.73(d, J=16.0Hz 1H), 6.54(dt, J=16.0, 5.7Hz , 1H), 4.72(d, J=5.7Hz, 2H), 4.01(m, 2H), 2.90(dd, J=18.5, 2.9Hz, 1H), 2.10(d, J=13.2Hz, 1H), 1.92 (d, J=18.6Hz, 1H), 1.83-1.48(m, 7H), 1.41-1.28(m, 4H), 1.27-1.20(m, 1H), 1.17(t, J=7.1Hz, 3H), 1.15-1.12(m, 1H), 1.09(s, 3H), 1.05(s, 3H), 1.02-0.73(m, 4H), 0.71(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.2, 169.7, 145.2, 144.4, 143.3, 142.7, 138.7, 130.7, 129.7, 129.4, 128.1, 126.9, 73.7, 59.9, 57.1, 56.4, 54.9, 43.8, 43.6 , 40.9, 40.6, 39.9, 39.5, 38.0, 37.9, 37.5, 28.9, 22.3, 21.6, 20.4, 18.9, 14.1, 13.4

IR IR (KBr): 2953.8, 2925.1, 2849.6, 1768.0, 1718.5, 1615.9, 1454.7, 1364.4, 1230.9, 1151.6, ^{1040.5, 892.1cm -1}

Synthesis:

Take 1mmol cp-2-19, dissolve it in 10ml anhydrous acetonitrile, add 0.05mmol Pd(OAc)2, 0.1mmol P(O-Tolyl)3 and 3mmol Et3N, then add 1.5mmol 6-bromoquinoxaline, Ar Gas protection reaction for 12h, filtered after the reaction, and the filtrate was evaporated to dryness, dissolved in dichloromethane, washed with water, washed with saturated NaCl, anhydrous _MgSO4 treatment, silica gel column (PE: EA 50: 1) to obtain the compound

Embodiment 13, the number is cp-3-13

Molecular formula: C ₃₃ H ₄₃ NO ₃ S, molecular weight: 533.30, structure as follows:

H spectrum ¹ H NMR (400MHz, CDCl3): 7.63-7.60 (m, 2H), 7.48-7.37 (m, 2H), 6.77-6.75 (m, 1H), 7.3 (d, J=16.0Hz, 1H), 6.38(dt, J=16.0, 6.2Hz, 1H), 4.74(d, J=6.2Hz, 2H), 4.10(m, 2H), 2.96(dd, J=18.6, 3.0Hz, 1H), 2.19(d , J=13.2Hz, 1H), 1.99(d, J=18.6Hz, 1H), 1.90-1.58(m, 7H), 1.49-1.39(m, 4H), 1.28(t, J=7.1Hz, 3H) , 1.25-1.21(m, 1H), 1.19(s, 3H), 1.14(s, 3H), 1.12-0.84(m, 5H), 0.79(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.4, 169.7, 154.7, 145.4, 132.8, 132.0, 127.7, 124.9, 123.0, 119.3, 111.4, 106.7, 74.3, 60.0, 57.2, 56.5, 55.0, 43.8, 43.7 , 40.9, 40.7, 40.0, 39.6, 38.1, 38.0, 37.6, 28.9, 22.3, 21.7, 20.5, 19.0, 14.2, 13.4

IR IR (KBr): 2926.5, 2847.7, 1718.9, 1655.4, 1452.3, 1362.5, 1230.0, 1152.1, 1028.6 ^{, 899.5cm-1}

Synthesis:

Take 1mmol of cp-2-19, dissolve it in 10ml of anhydrous acetonitrile, add 0.05mmol of Pd(OAc)2, 0.1mmol of P(O-Tolyl)3 and 3mmol of Et3N, then add 1.5mmol of 5-bromobenzo[B] Thiophene was reacted under Ar gas protection for 12 hours, filtered after the reaction, and the filtrate was evaporated to dryness, dissolved in dichloromethane, washed with water, washed with saturated NaCl, treated with anhydrous MgSO ₄ , and silica gel column (PE:EA 40:1) to obtain the compound.

Embodiment 14, the number is cp-3-15

Molecular formula: C ₃₇ H ₄₆ N ₂ O ₃ , molecular weight: 566.35, structure as follows:

H spectrum ¹ H NMR (400MHz, CDCl3): 8.4 (s, 1H), 8.1-8.06 (m, 2H), 7.53-7.23 (m, 5H), 6.82 (d, J=15.8Hz, 1H), 6.44 ( dt, J=15.8, 6.3Hz), 4.82(d, J=6.3Hz), 4.12(m, 2H), 3.03(dd, J=18.6, 2.6Hz, 1H), 2.21(d, J=13.2Hz, 1H), 2.06(d, J=18.6, 1H), 1.94-1.60(m, 7H), 1.54-1.40(m, 4H), 1.28(t, J=7.1Hz, 3H), 1.26-1.23(m, 1H), 1.22(s, 3H), 1.19(s, 3H), 1.15-0.84(m, 5H), 0.82(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.6, 170.0, 140.0, 139.3, 133.9, 128.5, 125.9, 124.6, 123.5, 123.3, 123.1, 120.4, 119.5, 118.7, 110.8, 110.7, 74.7, 60.1, 57 , 56.5, 55.0, 43.9, 43.8, 41.0, 40.7, 40.0, 39.6, 38.1, 38.0, 37.8, 29.0, 22.4, 21.8, 20.5, 19.0, 14.2, 13.5

IR IR (KBr): 3393.8, 2926.6, 2847.4, 1715.7, 1601.6, 1465.2, 1326.2, 1233.0, 1147.1, 1027.3 ^{, 752.9cm-1}

Synthesis:

Take 1mmol cp-2-19, dissolve it in 10ml anhydrous acetonitrile, add 0.05mmol Pd(OAc)2, 0.1mmol P(O-Tolyl)3 and 3mmol Et3N, then add 1.5mmol 3-iodocarbazole, Ar gas Protect the reaction for 12 hours, filter after the reaction, evaporate the filtrate to dryness, dissolve in dichloromethane, wash with water, wash with saturated NaCl, treat with anhydrous MgSO4, and use a silica gel column (PE:EA20:1) to obtain the compound.

Embodiment 15, numbered as cp-3-23, has the following structure:

Molecular formula: C ₃₁ H ₄₀ F ₃ NO ₃ , molecular weight: 531.30, the structure is as follows

H spectrum ¹ H NMR (400MHz, CDCl3): 6.98 (m, 2H), 6.45 (d, J=16.0Hz, 1H), 6.30 (dt, J=16.0, 5.6Hz, 1H), 4.68 (d, J= 5.6Hz, 2H), 4.09(m, 2H), 2.93(dd, J=18.6, 3.1Hz, 1H), 2.17(d, J=13.3Hz, 1H), 1.95(d, J=18.6Hz, 1H) , 1.89-1.56(m, 7H), 1.48-1.37(m, 4H), 1.27(t, J=7.1Hz, 3H), 1.24-1.19(m, 2H), 1.18(s, 3H), 1.10(s , 3H), 1.07-0.83(m, 4H), 0.77(s, 3H)

C spectrum ¹³ C NMR (100MHz, CDCl3): δ177.3, 170.0, 152.5, 150.0, 140.27, 137.7, 133.3, 129.1, 129.0, 110.2, 73.3, 59.9, 57.2, 56.4, 54.9, 43.8, 43.7, 40.9, 40.7 , 40.0, 39.6, 38.1, 38.0, 37.6, 28.9, 22.2, 21.7, 20.4, 19.0, 14.1, 13.4

IR IR (KBr): 2929.1, 2848.4, 2679.8, 1720.7, 1614.2, 1441.4, 1345.9, 1233.7, 1152.0, 1041.6, 869.1, 845.7 ^{, 789.2cm-1}

Synthesis:

Take 1mmol of cp-2-19, dissolve it in 10ml of anhydrous acetonitrile, add 0.05mmol of Pd(OAc)2, 0.1mmol of P(O-Tolyl)3 and 3mmol of Et3N, then add 1.5mmol of 3,4,5-trifluoro Iodobenzene was reacted under Ar gas protection for 12 hours, filtered after the reaction, and the filtrate was evaporated to dryness, dissolved in dichloromethane, washed with water, washed with saturated NaCl, treated with anhydrous MgSO ₄ , and silica gel column (PE: EA 50: 1) to obtain the compound.

Claims (21)

1. A tetracyclic diterpene compound, the general structural formula is: any one or more of R2 is: -COOEt. n=1-2. 2. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 3. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 4. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 5. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 6. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 7. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 8. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 9. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 10. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 11. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 12. according to a kind of tetracyclic diterpene compound of claim 1, structural formula is: 13. a kind of tetracyclic diterpene compound according to claim 1, structural formula is: 14. The pharmaceutical use of a tetracyclic diterpene compound according to any one of claims 1-13, characterized in that: the tetracyclic diterpene compound is used for the preparation of coagulation factor inhibitors. 15. The pharmaceutical use of a tetracyclic diterpene compound according to any one of claims 1-13, characterized in that it is used to prepare any one or more compositions of anticoagulant and antithrombotic. 16. According to the pharmaceutical use of a tetracyclic diterpene compound according to any one of claims 1-13, it is characterized in that: it is used to prepare and treat cerebral ischemia, treat cerebral apoplexy, treat cerebral edema, treat cerebral infarction, and treat neuropathy. Impairment of any one or more combinations of functions. 17. The pharmaceutical use of a tetracyclic diterpene compound according to any one of claims 1-13, characterized in that it is used to prepare any one or more compositions for reducing brain water content and alleviating brain edema. 18. The pharmaceutical use of a tetracyclic diterpene compound according to any one of claims 1-13, characterized in that: for the preparation of the preparation of the tetracyclic diterpene compound, each preparation unit contains 0.01-500 mg of the active compound. 19. According to the pharmaceutical use of a kind of tetracyclic diterpene compound in any one of claims 1-13, it is characterized in that: it is used to prepare the preparation of tetracyclic diterpene compound, and the preparation type is tablet, capsule, granule , pellets, sustained-release capsules, freeze-dried powder injections, injections. 20. A pharmaceutical application of a tetracyclic diterpene compound, characterized in that: Tetracyclic diterpene compounds are used to prepare any one or more compositions of coagulation factor inhibitors, anticoagulant, antithrombotic, treatment of cerebral ischemia, treatment of cerebral apoplexy, treatment of cerebral edema, treatment of cerebral infarction, and treatment of neurological impairment , The structure of the compound of tetracyclic diterpenoid is 21. according to the pharmaceutical use of a kind of tetracyclic diterpene compound of claim 20, it is characterized in that: It is used to prepare preparations of tetracyclic diterpene compounds, and the preparation types are tablets, capsules, granules, pellets, sustained-release capsules, freeze-dried powder injections, and injections.