CN101235031A - Oriented synthesis and crystal structure of 21(S) argatroban, and preparation for monohydrate thereof - Google Patents

Abstract

The invention relates to a 21(S) argatroban rational synthesis method, a corresponding crystal structure and a hydrate preparation method, wherein the rational synthesis uses (3S)-1, 2, 3, 4-tetrahydrochysene-3-methyl-8-quinolinesulfonyl chloride as raw material to prepare single diastereoisomer 21(S) argatroban, and uses single crystal and polycrystalline powder X diffraction method to determine the absolute configuration as 21(S) and the crystal systems I, II, and the 21(S) argatroban is soluble in hot water, via controlling the cooling speed, a 21(S) argatroban hydrate is prepared. Animal tests prove that the anticoagulant function of the 21(S) argatroban hydrate is 2-3 powers of 21(R) argatroban hydrate.

Description

21 (S) argatroban orientation is synthesized the preparation with crystalline structure and monohydrate

Technical field

The invention belongs to medical technical field, particularly relate to the preparation of a kind of 21 (S) argatroban directed synthetic and crystalline structure and monohydrate.

Background technology

Argatroban has carried out reporting [US4101653] in 1978 by S.Okamoto of Japanese Misubishi chemical company etc. first as a kind of thrombin inhibitors.Japan ratified the thrombin inhibitors [Hijikata-Okunomiya, A., et al., Thromb.Hemostasis, 1992,18,135] that this medicine uses as non-enteron aisle in 1992.Argatroban-(2R, 4R)-1-[(2S)-5-[(aminoiminomethyl) amino]-1-oxo-2-[[(1,2,3,4-tetrahydro-3-methyl-8-quinolinyl) sulfonyl] amino] pentyl]-4-methyl-2-piperidinecarboxylic acid---its chemical ingredients is the mixture of 21 (R) and 21 (S) argatroban, and both ratios are 64～65 usually: 36～35[US 6 440 417, Cossy.J., et al, Bioorganic ﹠amp; Medicine Chemistry Letters, 11 (2001), 1989-1992, Journal ofPharmaceutical Sciences, Vol.82, No.6,672 (1993)].

X=CH3, Y=H, 21 (S) argatroban (VIII)

X=H, Y=CH3,21 (R) argatroban

Argatroban is the arginic derivative of L-, and it interacts with the thrombin activity site, is the competitive inhibitor of zymoplasm.This medicine is the activity of deactivation zymoplasm (factor IIa) directly; To the not directly effect of generation of zymoplasm, its effect does not rely on intravital antithrombin; Its not only can deactivation blood in the zymoplasm of free state, but also can deactivation and the zymoplasm that combines of fibrinous thrombus; The positive regeeration of blocking-up blood coagulation waterfall; Can suppress platelet aggregation response during extremely low concentration, indirectly the generation of Trombin inhibiting by thrombin induction.In addition, the molecular weight of argatroban is little, and it can enter into thrombus inside, therefore directly deactivation with fibrinous thrombus bonded zymoplasm, and still can bring into play anti thrombotic action to those outmoded thrombus.In addition, argatroban can also reduce the level of TAT in the blood plasma (thrombin-antithrombin complex) greatly, effectively improves patient's hypercoagulative state, therefore has extraordinary clinical effectiveness in chronic thrombotic disease.

Argatroban has caused that in the widespread use of diseases such as countries in the world treatment thrombus the medical worker is to two diastereomers 21 of argatroban (S) argatroban-(2R, 4R)-1-[(2S)-5-[(aminoiminomethyl) amino]-1-oxo-2-[[[(3S)-1,2,3,4-tetrahydro-3-methyl-8-quinolinyl] sulfonyl] amino] pentyl]-4-methyl-2-piperidinecarboxylic acid and 21 (R) argatroban-(2R, 4R)-1-[(2S)-5-[(aminoiminomethyl) amino]-1-oxo-2-[[[(3R)-1,2,3,4-tetrahydro-3-methyl-8-quinolinyl] sulfonyl] amino] pentyl]-the showing great attention to of 4-methyl-2-piperidinecarboxylic acid, relate generally to 21 (S) and 21 (R) argatroban separate or stereoselectivity is synthetic and the two bioactive difference.In recent years, the countries in the world health organization is more and more stricter to the requirement and the regulation of pharmaceutical cpd, except the high-content of the total content of stipulating various impurity and single impurity, isomer or the optical isomer that allows in the pharmaceutical cpd carried out isolation identification one by one, and strict its content of control.The raceme medicine is the mixture of optical isomer, and from reducing treatment consumption and the minimizing angle to the human body untoward reaction, it is a development trend that high reactivity single component optics live body replaces raceme as medicine.

The influence experiment of the injection liquid of 21 (S) and 21 (R) argatroban to the normal dogs coagulation indexes reported in the bright outstanding Science and Technology Ltd. of China Song Hong seas in 2006 etc., experimental result shows: 21 (S) argatroban of single component is to prolonging normal dogs whole blood clotting time (CT) effect significantly, and its action effect is the twice of 21 (R) argatroban; 21 (S) argatroban is to prolonging normal dogs recalcification time (RT) effect significantly, and its action effect is three times of 21 (R) argatroban; 21 (S) argatroban is to prolonging normal dogs prothrombin time (PT) effect significantly, and its action effect is the twice of 21 (R) argatroban; 21 (S) argatroban is to prolonging normal dogs KPTT (APTT) effect significantly, and its action effect is the twice of 21 (R) argatroban.21 (S) argatroban is to prolonging normal dogs thrombin time (TT) effect significantly, and its action effect is slightly stronger than 21 (R) argatroban; 21 (S) argatroban has the reduction effect to normal dogs platelet adhesion rate and platelet aggregation rate.Obvious 21 (S) will be better than argatroban and 21 (R) argatroban [CN101032485A] greatly as thrombin inhibitors.

The mask work of two diastereomers 21 of argatroban (S) and 21 (R) has attracted numerous investigators.Because the two physico-chemical property is very similar, so separating difficulty is very big.Rawson in 1993, Thomas E.; VanGorp, Kimmie A.; Yang, Janet etc. separate with column chromatography with high pressure lipuid chromatography (HPLC) first and have obtained single 21 (S) and 21 (R) argatroban [Journal of Pharmaceutical Sciences vol.82, No.6,672]; ThibaudeauKaren etc. have reported Protein A chromatographic separation method [US6440417].But, therefore there is not the industrialization practical value because the fractional dose of above-mentioned these methods is little, efficient is low.The method [CN1951936A] that the employing recrystallization method separates 21 (S) and 21 (R) argatroban has been reported in the bright outstanding Science and Technology Ltd. in Chinese Tianjin Song Hong seas in 2006 etc., thereby make batch process 21 (S) argatroban become possibility, but this method yield is low, trivial operations, cost are higher, and produce 21 (R) argatroban byproduct that contains a small amount of 21 (S) in a large number, considering from the suitability for industrialized production angle, still is not a kind of ideal method.But up to the present do not find the directed synthetic report that reaches the monohydrate preparation of 21 (S) argatroban as yet.

Summary of the invention

The object of the present invention is to provide a kind of operating process is simple relatively, cost is low and yield is high 21 (S) the argatroban directional synthesis method and the preparation and the research of relevant crystalline structure of monohydrate.

21 (S) provided by the invention argatroban monohydrate has the chemical structural formula shown in the following formula I---(2R, 4R)-1-[N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid monohydrate:

The directional synthesis method of above-mentioned 21 (S) provided by the invention argatroban comprises the following step (see figure 1) of carrying out in order:

1) with (2R, 4R)-1-[N ^G-nitro-L-arginyl]-4-methyl-Pipecolic Acid carbethoxy hydrochloride is as raw material, and it is dissolved in the methylene dichloride, as acid binding agent, drips (3S)-1,2,3 with triethylamine, 4-tetrahydrochysene-3-methyl-8-quinoline sulfuryl chloride, reaction make (2R, 4R)-1-[N ^G-nitro-N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid ester VI;

2) intermediate VI acidication in basic solution is got intermediate VII:

3) with intermediate VII palladium carbon catalytic hydrogenation denitration base protection in alcohol, get single diastereomer anhydrous 21 (S) argatroban VIII:

4) above-mentioned anhydrous 21 (S) argatroban VIII is mixed with distilled water, under 80～100 ℃ temperature, heated 0.5～1.0 hour, all dissolve to solid, after cooling afterwards again low temperature place a few hours, until separating out crystal, filter at last, washing, vacuum-drying and obtain 21 shown in the following formula I (S) argatroban monohydrate:

Hydrogenation pressure is 1.0～100kg/cm in the described step 3) ², temperature of reaction is 25～100 ℃, alcohol is any in methyl alcohol or the ethanol.

The weight ratio of anhydrous 21 (S) argatrobans and distilled water is 1: 30～150 in the described step 4), and the vacuum-drying temperature is 40～60 ℃.

Speed when cooling in the described step 4) is: 80～100 ℃ → 50 ℃ 2～4 hours;

50 ℃ → 0 ℃ 2～4 hours;

The low temperature laying temperature is 0 ℃, and be 5～10 hours storage period.

The water-content of described 21 (S) argatroban monohydrate is 3.3～3.8%.

VIII gets crystal formation I with the distilled water recrystallization with described anhydrous 21 (S) argatroban.

VIII gets crystal form II with the methanol-water recrystallization with described anhydrous 21 (S) argatroban.

The directional synthesis method of 21 (S) provided by the invention argatroban be with (2R, 4R)-1-[N ^G-nitro-L-arginyl]-4-methyl-Pipecolic Acid carbethoxy hydrochloride with (3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline sulfuryl chloride condensation get (2R, 4R)-1-[N ^G-nitro-N ²-[(3S)-1; 2; 3; 4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-the L-arginyl]-4-methyl-Pipecolic Acid ester; get single diastereomer 21 (S) argatroban through alkaline hydrolysis and catalytic hydrogenation denitration base then; 21 (S) argatroban is dissolved in hot water, and controlled chilling speed makes 21 (S) argatroban monohydrate.Animal experiment proves that the blood coagulation resisting function of 21 (S) argatroban monohydrate is 2～3 times of 21 (R) argatroban monohydrate.21 (S) argatroban is obtained crystal formation I with the distilled water recrystallization, through monocrystalline and polycrystal powder X diffraction approach, confirm that absolute configuration is 21 (S), crystalline structure is a rhombic system, No. 19 spacers.21 (S) argatroban is obtained crystal form II with the methanol-water recrystallization.

Description of drawings

Fig. 1 is 21 (S) argatroban monohydrate synthetic route chart.

Fig. 2 is 21 (S) provided by the invention argatroban crystal formation I monocrystalline X-diffraction space structure figure.

Fig. 3 is 21 (S) provided by the invention argatroban crystal formation I monocrystalline X-diffraction structure cell figure.

Fig. 4 is 21 (S) provided by the invention argatroban crystal formation I polycrystal powder X-diffractogram.

Fig. 5 is 21 (S) provided by the invention argatroban crystal form II polycrystal powder X-diffractogram.

Fig. 6 is 21 (S) provided by the invention argatroban monohydrate differential thermal analysis curve.

Embodiment

Embodiment 1:

1) in 500ml four-hole reaction flask, add 200ml chloroform and 25g (0.061mole) (2R, 4R)-1-[N ^G-nitro-L-arginyl]-4-methyl-Pipecolic Acid carbethoxy hydrochloride; be cooled to 5 ℃; add 18.5g (0.18mole) triethylamine then, and drip 14.7g (0.061mole) (3S)-1,2; 3; 4-tetrahydrochysene-3-methyl-8-quinoline sulfuryl chloride stirred 3 hours under the room temperature, and TLC follows the tracks of reaction; to (2R, 4R)-1-[N ^G-nitro-L-arginyl]-4-methyl-Pipecolic Acid carbethoxy hydrochloride disappears, reaction finishes, with 50ml washing twice, anhydrous magnesium sulfate drying, steaming desolventizes, column chromatography for separation, the yellow solid shape (2R, 4R)-1-[N ^G-nitro-N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid ester VI 32.5g, yield is 92.1%;

2) in 500ml four-hole reaction flask, add 100ml ethanol and 30g (0.052mole) intermediate VI, stir and add 100ml 1N NaOH down, stirred under the room temperature 24 hours, TLC follows the tracks of reaction, disappears to intermediate VI, reaction finishes, transfer pH ≈ 11 with 1N NaOH, with 100ml ethyl acetate and the washing of 100ml chloroform, water 1N hcl acidifying, separate out solid, filter.The 20ml washing, drying gets white solid intermediate VII 27g, and yield is 94.7%;

3) in the 100ml autoclave, add 1g intermediate VII, 0.02g triphenyl phosphorus, 0.2g 5%pd/c, 13ml dehydrated alcohol and 4ml glacial acetic acid, 65.70 ℃ of the outer temperature of control autoclave.Feed hydrogen, the 10atm that keep-ups pressure reacted 24 hours, and stopped reaction removes by filter palladium carbon.Precipitation, with the dissolving of 20ml methylene dichloride, saturated sodium bicarbonate is regulated pH ≈ 7, divides water-yielding stratum, with the washing of 10ml saturated sodium-chloride, leave standstill again, tell organic layer, anhydrous magnesium sulfate drying, filter, remove methylene dichloride, get white solid anhydrous 21 (S) argatroban VIII0.75g, yield is 82%;

4) above-mentioned 1g anhydrous 21 (S) argatroban VIII is joined in the 100ml distilled water, stir, in 0.5～1.0 hour time, be warming up to 100 ℃, this moment, solid all dissolved, cool then, speed of cooling is to reduce to 50 ℃ in about 2 hours, reduces to 0 ℃ in 2 hours again.Placed 10 hours down at 0 ℃, until separating out crystallization, filter then, washing, and vacuum-drying under 40～50 ℃ temperature get 21 (S) argatroban monohydrate 0.73g, and mp:227-229 ℃, water content is 3.8% (karl Fischer method).Differential thermal analysis shows that 21 (S) argatroban monohydrate loses crystal water in the time of 82 ℃, the crystal water of 21 (S) argatroban monohydrate volatilization (see figure 6) in the time of 100 ℃.

Embodiment 2:

1g21 (S) argatroban is joined in the 150ml distilled water, stir, in 0.5～1.0 hour time, be warming up to 95 ℃, this moment, solid all dissolved, and cooled then, and speed of cooling is at the uniform velocity to reduce to 80 ℃ earlier in 2 hours, reduce to 50 ℃ in 1 hour then, reduce to 0 ℃ in 2 hours again.Placed 8 hours down at 0 ℃, until separating out crystallization, filter then, washing, and vacuum-drying under 50～60 ℃ temperature get 21 (S) argatroban monohydrate 0.65g, and water content is 3.4% (karl Fischer method).

Embodiment 3:

VIII gets crystal formation I with the distilled water recrystallization with 1g anhydrous 21 (S) argatroban, through monocrystalline and polycrystal powder X diffraction, confirms that 21 of its absolute configurations are S body (see figure 2), and crystal formation I is a rhombic system, P2 (1) 2 (1) 2 (1) spacer (No. 19 spacers).Unit cell parameters: a=9.372 , b=15.503 , c=21.735 , α=β=γ=90 ℃ (see figure 3).Crystal formation I polycrystal powder X diffraction data and single crystal X diffraction simulated data match (seeing Fig. 4, table 1).

Table 1 21 (S) argatroban crystal formation I monocrystalline X-diffraction simulated data and the contrast of polycrystal powder X-diffraction data

The single crystal diffraction simulated data		Powder diffraction data
				2-theta	d-spacing	2-theta	d-spacing
7.00	12.6215	6.98	12.6536
				8.14	10.8673	8.12	10.8795

9.94	8.8988	9.94	8.8912
				10.28	8.6057	10.26	8.6146
11.03	8.0202	11.08	7.9788
				11.76	7.5242	11.74	7.5317
12.47	7.0971	12.46	7.0981
				13.72	6.4531	13.70	6.4583
14.03	6.3108	14.04	6.3026
				14.83	5.9732	14.80	5.9806
15.38	5.7596	15.42	5.7415
				16.49	5.3761	16.48	5.3746
16.94	5.2346	16.90	5.2419
				19.62	4.5254	19.62	4.5209
20.21	4.3928	20.22	4.3881
				20.64	4.3028	20.68	4.2915
21.23	4.1855	21.22	4.1835
				22.17	4.0194	22.14	4.0117
22.55	3.9435	22.52	3.9449
				23.46	3.7915	23.40	3.7985
24.86	3.5816	24.86	3.5786
				25.25	3.5274	25.24	3.5256
26.07	3.4175	26.04	3.4190
				26.96	3.3067	26.98	3.3020
27.78	3.2107	27.78	3.2087
				28.87	3.0921	28.88	3.0890
29.34	3.0445	29.34	3.0416
				30.88	2.8955	30.88	2.8933
32.42	2.7612	32.42	2.7593
				32.97	2.7168	32.98	2.7137
33.67	2.6615	33.66	2.6604

Embodiment 4:

VIII is dissolved in the 95% methanol-water solution with 1g anhydrous 21 (S) argatroban, and cooling is placed, until separating out crystal, filter then, washing, and under 60-70 ℃, carry out drying, get the 0.7g white solid, crystal formation is a crystal form II, and its polycrystal powder X diffractogram is seen Fig. 5, and diffraction data sees Table 2.

Table 2 21 (S) argatroban crystal form II polycrystal powder X-diffraction data

WJ07-S-070401		WJ07-S-070501
				2-theta	d-spacing	2-theta	d-spacing
7.56	11.6841	7.58	11.6533
				9.28	9.5220	9.28	9.5220
10.32	8.5646	10.34	8.5481

11.04	8.0076	11.06	7.9932
				11.96	7.3937	11.98	7.3814
14.42	6.1374	14.46	6.1205
				15.18	5.8318	15.22	5.8165
15.88	5.5763	15.92	5.5623
				16.76	5.2854	16.78	5.2791
17.82	4.9733	17.86	4.9623
				18.66	4.7513	18.68	4.7462
19.12	4.6380	19.18	4.6236
				19.52	4.5439	19.56	4.5347
20.08	4.4184	20.12	4.4097
				20.38	4.3540	20.42	4.3456
20.88	4.2509	20.90	4.2468
				21.68	4.0958	21.74	4.0846
22.28	3.9868	22.30	3.9833
				22.68	3.9174	22.72	3.9106
23.34	3.8081	23.36	3.8094
				24.18	3.6777	24.20	3.6747
24.78	3.5900	24.82	3.5843
				25.98	3.4268	26.02	3.4216
26.42	3.3707	26.44	3.3682
				27.22	3.2734	27.22	3.2734
27.70	3.2178	27.72	3.2155
				28.06	3.1773	28.10	3.1729
28.84	3.0932	28.84	3.0932
				29.66	3.0095	29.72	3.0035
30.78	2.9025	30.82	2.8988
				33.62	2.6635	32.62	2.6635
36.54	2.4571	36.58	2.4545

Embodiment 5:

21 (S) and 21 (R) argatroban are tested the normal dogs Blood clotting

Test materials

1, trial-product:

21 (S) argatroban monohydrate of preparing among the embodiment 1～2;

21 (R) argatroban monohydrate;

Colourless clear liquid, specification: 20ml:10mg stores: shading, the airtight preservation of room temperature.

2, reagent:

0.9% sodium chloride injection, specification: 500ml, lot number: D510150401 is produced by Jinan three nine-day periods after the winter solstice benefit people pharmaceutical Co. Ltd; Sodium Citrate, analytical pure, specification: 500g, lot number: 20021105, produce by Shandong Boshan chemical reagent factory, be made into 3.2% solution during use with physiological saline;

Prothrombin time, thrombin time, activated partial thromboplastin time test kit provide by Shanghai Sun Bio-Tech Co., Ltd.;

ADP: giving birth to scientific ﹠ trading Co., Ltd. by Beijing Orient Puli provides.

3, animal:

Common domesticated dog (Canis familiaris Linne), 18, male and female half and half, 10-15kg is bought by market.

4, key instrument equipment: the full-automatic blood counting instrument of HEMAVET-950 type, produce by Drew Scientific company;

C2000 type coagulo meter is given birth to scientific ﹠ trading Co., Ltd. by Beijing Orient Puli and is produced;

LBY-NJ2 type platelet aggregation instrument is given birth to scientific ﹠ trading Co., Ltd. by Beijing Orient Puli and is produced;

The platelet adhesion reaction instrument is given birth to scientific ﹠ trading Co., Ltd. by Beijing Orient Puli and is produced;

Test method:

1, animal grouping:

Get 18 of healthy common domesticated dogs (Canis familiaris Linne), be divided into three groups at random, be respectively normal control group, argatroban injection liquid S body, three test group of R body, every group of 6 animals.

2, dosage is selected and foundation:

The clinical usage of trial-product: usually adult in 2 days of beginning 6 (argatroban 60mg) on the 1st is diluted with the transfusion of appropriate amount, through lasting intravenous drip in 24 hours.Thereafter in 5 days 2 (argatroban 20mg) on the 1st, with the transfusion dilution of appropriate amount, every day each 1 time sooner or later, each 1 (argatroban 10mg), 1 intravenous drip in 3 hours.

With reference to the clinical consumption of argatroban injection liquid, in per day for adults 15mg, can get the dog dosage by adult 70kg and 12kg dog dose,equivalent Reduced Coefficient Method and be: people's consumption * 0.32/12 tentatively is decided to be 0.4mg/kg.

3, animal soup preparation:

0.4mg/kg dosage solution: get corresponding need testing solution 0.8ml (0.5mg/ml testing drug)+0.9% sodium chloride injection 9.2ml;

4, process of the test:

Getting indexs such as hematometry whole blood clotting time (CT), recalcification time (RT), prothrombin time (PT), thrombin time (TT), KPTT (APTT), platelet adhesion rate, platelet aggregation rate before every animals administer is worth before as medicine, mensuration finishes, every treated animal gives corresponding animal soup by the 10ml/kg volume, once a day, for three days on end, got blood in 15 minutes after the administration in the 3rd day and survey above blood coagulation index of correlation, test result sees Table 3～9.

Wherein, the measuring method of whole blood clotting time: get 3 in test tube, label is 1,2, No. 3.Successfully extract dog venous blood 3ml with disposable sterilized injector, flow into syringe needle from blood and pick up counting, go slowly to inject blood 1ml along the every pipe of tube wall behind the syringe needle, put in 37 ℃ of water-baths.At regular intervals first pipe is tilted 1 time, till test tube being inverted blood and not being flowed; Observe second pipe more successively, after second pipe solidifies, observe the 3rd pipe again.With the 3rd pipe setting time is the clotting time.

The recalcification time method: get Sodium Citrate anticoagulation 1ml, centrifuging and taking blood plasma 0.2ml adds the calcium chloride solution 0.2ml of 0.025mol/L, and mixing is placed on 37 ℃ of water-baths, from adding the calcium timing, is recalcification time until the time of solidifying.

Instrument measuring is adopted in other test.

5, data statistics and result judge:

Each index is with mean ± standard deviation before every treated animal medicine and behind the medicine

Expression.Changing value behind changing value and the blank group medicine behind each medicine group medicine is carried out the t check, to judge the effect of medicine.

Table 3: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs whole blood clotting time (CT)

Group	Dosage (mg/kg)	Setting time (S) before the medicine	The medicine after coagulation time (S)	Setting time changing value (S)
					The normal control group	-	390.0±70.1	375.2±84.1	-14.8±29.5
The S body	0.4	390.8±69.3	551.8±76.1	161.0±102.0**
					The R body	0.4	398.7±95.6	495.8±91.4	97.2±47.9**

* compare with the normal control group P＜0.05 * * P＜0.01.

The result shows, compares with the normal control group, and argatroban injection liquid S body and R body all can obviously prolong whole blood clotting time (CT), and wherein the action effect of S body is about the twice of R body.

Table 4: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs recalcification time (RT)

Group	Dosage (mg/kg)	Recalcification time (S) before the medicine	Recalcification time behind the medicine (S)	Recalcification time changing value (S)
					The normal control group	-	126.3±39.4	138.5±30.9	12.2±18.1
The S body	0.4	143.2±33.4	196.5±31.4	53.3±30.5*
					The R body	0.4	135.3±33.8	154.3±37.1	19.0±11.6

* compare with the normal control group P＜0.05 * * P＜0.01.

The result shows, compares with the normal control group, and argatroban injection liquid S body and R body all can obviously prolong recalcification time (RT), and wherein the action effect of S body is about three times of R body.

Table 5: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs prothrombin time (PT)

Group	Dosage (mg/kg)	PT (S) before the medicine	PT behind the medicine (S)	PT changing value (S)
					The normal control group	-	6.2±0.3	8.2±1.2	2.0±1.0
The S body	0.4	5.6±0.3	9.6±0.7	4.0±0.7**
					The R body	0.4	6.6±0.3	9.0±0.6	2.4±0.7

* compare with the normal control group P＜0.05 * * P＜0.01.

The result shows that argatroban injection liquid S physical efficiency obviously prolongs dog prothrombin time (PT), compares with the normal control group, and significant difference (P＜0.05 or P＜0.01) is arranged; The R body also has certain effect, but compares there was no significant difference (P＞0.05) with the normal control group.The action effect of S body is about the twice of R body.

Table 6: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs thrombin time (TT)

Group	Dosage (mg/kg)	TT (S) before the medicine	TT behind the medicine (S)	TT changing value (S)
					The normal control group	-	10.4±0.6	12.8±1.8	2.4±2.0
The S body	0.4	11.1±0.5	53.3±19.2	42.2±19.2**
					The R body	0.4	10.1±0.7	43.1±3.3	33.0±3.7**

* compare with the normal control group P＜0.05 * * P＜0.01.

The result shows that argatroban injection liquid R body and S body all can both obviously prolong dog thrombin time (TT), compares with the normal control group, and significant difference (P＜0.01) is arranged, and wherein the action effect of S body slightly is better than the R body.

Table 7: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs KPTT (APTT)

Group	Dosage (mg/kg)	APTT (S) before the medicine	APTT behind the medicine (S)	APTT changing value (S)
					The normal control group	-	20.1±1.4	21.0±1.9	1.0±3.0
The S body	0.4	20.0±1.0	24.5±0.8	4.6±1.4*
					The R body	0.4	19.8±0.4	22.3±2.2	2.6±1.8

* compare with the normal control group P＜0.05 * * P＜0.01.

The result shows that argatroban injection liquid S physical efficiency obviously prolongs dog KPTT (APTT), compares with the normal control group, and significant difference (P＜0.05) is arranged; The R body also has certain effect, but compares there was no significant difference (P＞0.05) with the normal control group.The action effect of S body is about the twice of R body.

Table 8: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs platelet adhesion rate

Group	Dosage (mg/kg)	Adhesion rate (%) before the medicine	Adhesion rate behind the medicine (%)	Adhesion rate changing value (%)
					The normal control group	-	30.1±5.3	29.5±5.1	-0.6±3.4
The S body	0.4	29.6±4.1	24.2±3.8	-5.4±4.4
					The R body	0.4	29.0±4.9	24.7±4.6	-4.3±6.6

Compare with the normal control group P＞0.05.

The result shows that argatroban injection liquid R body and S body all have certain reduction effect to platelet adhesion rate, but compares with the normal control group, there was no significant difference (P＞0.05).

Table 9: argatroban injection liquid S body, R body and normal control group soup are to the influence of normal dogs platelet aggregation rate

Group	Dosage (mg/kg)	Aggregation rate (%) before the medicine	Aggregation rate behind the medicine (%)	Aggregation rate changing value (%)
					The normal control group	-	51.2±8.8	45.2±6.2	-6.0±9.0
The S body	0.4	47.1±4.4	42.2±8.6	-4.9±9.9
					The R body	0.4	51.5±9.9	43.5±9.2	-8.0±13.6

Compare with the normal control group P＞0.05.

The result shows that argatroban injection liquid R body and S body all have certain reduction effect to platelet aggregation rate, but compares with the normal control group, there was no significant difference (P＞0.05).

Test-results shows, 21 (S) argatroban monohydrate is than 21 (R) argatroban monohydrate significant prolongation whole blood clotting time (CT) more; Significant prolongation recalcification time (RT) more; Can more obvious prolongation dog thrombin time, can more obvious prolongation dog KPTT (APTT), prolong dog thrombin time (TT) more by force, above-mentioned test 21 (S) argatroban is compared with the normal control group all significant difference.The two all has the reduction effect to platelet adhesion rate and aggregation rate, but there was no significant difference.Test-results shows that 21 (S) argatroban monohydrate and 21 (R) argatroban monohydrate relatively have more significant blood coagulation resisting function, and single component 21 (S) argatroban will improve effect or reduce consumption as antithrombotic drug.

Claims (5)

1. 21 (S) argatroban monohydrate with chemical structural formula I---(2R, 4R)-1-[N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid monohydrate, it is characterized in that this compound contains a crystal water.

2. the preparation method of 21 (S) as claimed in claim 1 argatroban monohydrate is characterized by:

(1) 21 (S) argatroban mixes with water, heated 0.5-1.0 hour down at 80-100 ℃, and the solid dissolving, controlled chilling speed is separated out crystallization, gets 21 (S) argatroban monohydrate;

When (2) preparing 21 (S) argatroban monohydrate, 21 (S) argatroban: water=1: 30～150 (w/w)

When (3) preparing 21 (S) argatroban monohydrate,

Speed of cooling: 80～100 ℃ → 40-50 ℃ 2～4 hours;

40-50 ℃ → 0 ℃ 2 hours;

Placed 5～10 hours for 0 ℃;

When (4) preparing 21 (S) argatroban monohydrate, 40-50 ℃ of crystal drying temperature;

(5) 21 (S) argatroban monohydrate that makes, its water content is 3.3-3.8%.

3. according to claim 2, the directional synthesis method of 21 (S) argatroban is characterized by:

(1) with (2R, 4R)-1-[N ^G-nitro-L-arginyl]-4-methyl-Pipecolic Acid carbethoxy hydrochloride is as raw material, with (3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline sulfuryl chloride react intermediate VI---(2R, 4R)-1-[N ^G-nitro-N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid ester:

R＝CH ₃，C ₂H ₅

(2) with intermediate VI hydrolysis in aqueous sodium hydroxide solution, acidifying gets intermediate VII---(2R, 4R)-1-[N ^G-nitro-N ²-[(3S)-1,2,3,4-tetrahydrochysene-3-methyl-8-quinoline alkylsulfonyl]-L-arginyl]-4-methyl-Pipecolic Acid:

(3) intermediate VII gets argatroban VIII through palladium carbon catalytic hydrogenation:

4. according to claim 3, anhydrous 21 (S) argatroban VIII, confirm through monocrystalline and polycrystal powder diffraction: 21 absolute configurations are the S type, and crystalline structure is crystal formation I: rhombic system, No. 19 spacers---P2 (1) 2 (1) 2 (1); Anhydrous 21 (S) argatroban methanol-water recrystallization, crystal is a crystal form II through the crystal formation of polycrystal powder X diffraction proof.

5. according to claim 1, the blood coagulation resisting function of 21 (S) argatroban monohydrate is 2～3 times of 21 (R) argatroban monohydrate.

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