CN103772270A - Metabolin marker of 2-hydroxyl radical tetrahydro-thiophene pyridine derivative with optical activity as well as preparation and application thereof - Google Patents
Metabolin marker of 2-hydroxyl radical tetrahydro-thiophene pyridine derivative with optical activity as well as preparation and application thereof Download PDFInfo
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- JBSAZVIMJUOBNB-WUJWULDRSA-N COC([C@H](c1ccccc1Cl)N(CCC1S2)CC1=CC2=O)=O Chemical compound COC([C@H](c1ccccc1Cl)N(CCC1S2)CC1=CC2=O)=O JBSAZVIMJUOBNB-WUJWULDRSA-N 0.000 description 1
- QQCDLVPXDMRAHK-GDAGCXPBSA-N COC([C@H](c1ccccc1Cl)N(CCN1)C/C1=C/C(O)=O)=O Chemical compound COC([C@H](c1ccccc1Cl)N(CCN1)C/C1=C/C(O)=O)=O QQCDLVPXDMRAHK-GDAGCXPBSA-N 0.000 description 1
- ORAPWJHPBUYZNV-SRFYIBEKSA-N COC([C@H](c1ccccc1Cl)N(CC[C@@H]1SC)C/C1=C/C(O)=O)=O Chemical compound COC([C@H](c1ccccc1Cl)N(CC[C@@H]1SC)C/C1=C/C(O)=O)=O ORAPWJHPBUYZNV-SRFYIBEKSA-N 0.000 description 1
- 0 COC([C@](c1ccccc1Cl)N(CC[C@]1*CS)CC1=CC(O)=O)=O Chemical compound COC([C@](c1ccccc1Cl)N(CC[C@]1*CS)CC1=CC(O)=O)=O 0.000 description 1
- MYWQPJTWYSFPBU-VAKJNQMVSA-N C[C@H](CCN(C1)[C@H](C(OC)=O)c(cccc2)c2Cl)/C1=C\C(O)=O Chemical compound C[C@H](CCN(C1)[C@H](C(OC)=O)c(cccc2)c2Cl)/C1=C\C(O)=O MYWQPJTWYSFPBU-VAKJNQMVSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/72—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, directly attached to ring carbon atoms
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/72—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with hetero atoms or with carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D211/74—Oxygen atoms
- C07D211/76—Oxygen atoms attached in position 2 or 6
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
Abstract
The invention relates to the field of medicines, in particular to a novel metabolin marker and application of the metabolin marker to develop or prepare a medicine containing the 2-hydroxyl radical tetrahydro-thiophene pyridine derivative with the optical activity. The medicines can be used for preventing or treating diseases caused by thrombus. The invention further provides a preparation method of the novel metabolin marker.
Description
Technical field
The present invention relates to field of medicaments, the purposes aspect the medicine that is specifically related to novel metabolite mark and contains optical activity 2-hydroxy tetrahydro thienopyridine derivative in development or preparation, described medicine can be used for prevention or the disease that caused by thrombus for the treatment of.The present invention also provides the preparation method of novel metabolite mark.
In this patent, please require the Chinese patent application right of priority of (application number 201210395415.4, the applying date: on October 17th, 2012, invention and created name: dimension card Gray's metabolite mark, Preparation Method And The Use).
Background technology
Clopidogrel (Clopidogrel) is as irreversible P2Y
12receptor antagonist is current most widely used anti-platelet aggregation medicinal.Be used for the treatment of clinically atheromatosis, acute coronary syndrome and thrombotic complications etc.Clinical study is found, clopidogrel oral administration biaavailability is very low, and onset is slow, hematoblastic inhibition is had to shortcoming (Cardiovascular Drug Reviews, 1993 of delayed action, 11,180), and have " clopidogrel Resistant " phenomenon (Circulation, 2004,109,166).
Prasugrel (Prasugrel) is the oral antiplatelet drug of new listing, is also an irreversible P2Y
12receptor antagonist.Compared with clopidogrel, although prasugrel can be more fast, anticoagulant more effectively, it has larger bleeding risk (N Engl J Med, 2007,357,2001).Other untoward reactions of prasugrel are thrombopenia and neutrophil leucocyte minimizing etc.
ADZ6140 is the oral reversible P2Y of first listing
12receptor antagonist.Compared with clopidogrel, that ADZ6140 has is rapid-action (peak action occur in medication after 2～4h), to the significant advantage of hematoblastic restraining effect, but the irrelevant main hemorrhage rate of ADZ6140 and CABG is higher than clopidogrel group.In addition, other common adverse reactions of ADZ6140 have expiratory dyspnea and bradyarrhythmia etc.
Chinese patent application 201010624329.7 discloses optically active 2-hydroxy tetrahydro thienopyridine derivative, wherein, representative compound dimension card Gray (embodiment 5, compound 1-2) has shown the platelet aggregation inhibitory activity stronger than clopidogrel and higher oral administration biaavailability.But the metabolite that not yet has so far any bibliographical information to generate in vivo above-mentioned optically active 2-hydroxy tetrahydro thienopyridine derivative carries out comprehensive quantitative and qualitative analysis research.Existing document (JOURNAL OF PHARMACEUTICAL SCIENCES2013,102,741; JOURNAL OF MEDICINAL CHEMISTRY 2012,55,3342:CN201010624329.7) just reported relevant thiolactone metabolite and the active metabolite containing free sulfhydryl groups, and for the research of other metabolites without any bibliographical information.
Although optically active 2-hydroxy tetrahydro thienopyridine derivative can overcome clopidogrel Resistant (JOURNAL OF MEDICINAL CHEMISTRY2012,55,3342), but, how, in guaranteeing curative effect of medication, the bleeding risk that farthest reduces such medicine remains a still unsolved crucial difficult problem.In addition, because such medicine and clopidogrel have different pathways metabolisms, therefore, characteristic and/or the exposed amount of the metabolite of optically active 2-hydroxy tetrahydro thienopyridine derivative may be very different compared with clopidogrel, thereby bring very large uncertainty to the efficacy and saferry of medicine.
In a word, in development or prepare optically active 2-hydroxy tetrahydro thienopyridine derivative also have many still unsolved gordian technique difficult problems aspect novel medicament for resisting platelet aggregation.
Summary of the invention
The invention provides the metabolite mark of the active 2-hydroxy tetrahydro of novel optical thienopyridine derivative.Purposes aspect the medicine of the disease that the metabolite mark that the present invention also provides optical activity 2-hydroxy tetrahydro thienopyridine derivative causes at preparation prevention and treatment thrombus.
In the present invention, described optical activity 2-hydroxy tetrahydro thienopyridine derivative is as shown in the formula the compound shown in I or its pharmacy acceptable salt or its solvate:
Wherein, R is R
1cO, PO (OR
2)
2, CH
2oPO (OR
2)
2or COOR
1; R
1be straight or branched alkyl, phenyl, styryl, 4-Vinyl phenol base, 4-hydroxyl-3-methoxyl-styrene or the 3-pyridyl of 1～10 carbon; R
2for hydrogen, sodium or potassium.
In the present invention, preferred formula I compound has following structure:
The inventor is surprised to find, and the compound shown in the formula I in vivo metabolite of exposed amount maximum is different from existing document (JOURNAL OF PHARMACEUTICAL SCIENCES2013,102,741; JOURNAL OF MEDICINAL CHEMISTRY2012,55,3342; CN201010624329.7) any metabolite of reporting, it has as shown in the formula the novel texture shown in II:
Above-mentioned formula II compound can be respectively two diastereomers, can be also the mixture of two diastereomers.Specifically, two diastereomers of formula II compound representative are respectively as shown in the formula the compound shown in II-1 and formula II-2:
As everyone knows, efficacy and saferry to medicine is produced tremendous influence by the medicine in vivo metabolite of exposed amount maximum, and therefore, the unexpected discovery of the present invention is significant to the medicament for resisting platelet aggregation shown in development or preparation formula I.
The inventor shows the dynamic metabolism research of compound shown in formula I, first compound shown in formula I generates the thiolactone shown in formula III through enteron aisle esterase hydrolyzed in vivo, through the active metabolite shown in peroxidation open loop production IV, formula IV compound is again through the sulphur methyl ether metabolite shown in Hypermethylation production II again.
Result of study of the present invention shows first, aspect the medicament for resisting platelet aggregation shown in development or preparation formula I, metabolite shown in formula II is the drug metabolite mark that must detect, monitor and regulate and control quantitative and qualitative analysis, therefore, compound shown in formula II has important use aspect the medicament for resisting platelet aggregation shown in development or preparation formula I.
The pharmacy acceptable salt of formula I compound of the present invention can be its hydrochloride, hydrobromate, hydriodate, nitrate, perchlorate, vitriol, phosphoric acid salt, mesylate, three fluoro mesylates, esilate, benzene sulfonate, tosilate, acetate, malate, fumarate, succinate, Citrate trianion, tartrate, oxalate, maleate, lactic acid salt, mandelate, pamoic acid (palmoxiric acid) salt, glycinate, lysine salt, arginic acid salt, ornithine salt, glutaminate or aspartate.
Described formula I compound or its pharmacy acceptable salt have good platelet aggregation restraining effect and thrombosis restraining effect as the medicine of effective constituent, therefore can be used as preventive or the therapeutical agent of the disease being caused by thrombus.The thrombotic disease that thrombus after the atherosclerosis that the disease that described thrombus causes the is stable or postoperative restenosis of unstable angina pectoris, cerebral ischemia attack, interventional cardiac procedures etc. causes, thrombotic disease, the thrombolysis that diabetes occur together forms that dementia, tip artery disease, hemodialysis or atrial fibrillation that disease, infarct, ischemic cause occur together or blood vessel prosthesis again or uses Aorta-coronary artery bypass to produce.
The invention provides formula II the compound purposes aspect the medicament for resisting platelet aggregation shown in development or preparation formula I, the especially curative effect in the lysis that the medicament for resisting platelet aggregation shown in prediction and controlling type I is preventing and treatment is caused by thrombus and the purposes aspect security.Especially, described purposes is to predict and curative effect and the bleeding risk of the medicament for resisting platelet aggregation shown in controlling type I by detecting formula II compound exposed amount in vivo.
The present invention also provides metabolite mark and then the curative effect of prediction and regulating medicine and the method for bleeding risk of formula II compound as the medicament for resisting platelet aggregation shown in formula I, the method is to pass judgment on the pharmacokinetics behavior of medicine by detecting formula II compound exposed amount in vivo, and predict the exposed amount of active metabolite, and then the curative effect of prediction and regulating medicine and bleeding risk, this is because the curative effect of antiplatelet drug and bleeding risk are closely-related with the exposed amount of its active metabolite.
By deep drug disposition dynamic metabolism research, the inventor is surprised to find, in rat and monkey body, the major metabolite of the medicament for resisting platelet aggregation shown in formula I (dimension card Gray: R=MeCO) in administration after 4 hours is the sulphur methyl ether shown in formula II.In human body, the major metabolite of medicament for resisting platelet aggregation shown in formula I (dimension card Gray: R=MeCO) in administration after 1 hour is also the sulphur methyl ether shown in formula II, especially administration after 6 hours the sulphur methyl ether shown in formula II be almost unique major metabolite that can detect.And on the other hand, in dog body, sulphur methyl ether shown in formula II is also one of major metabolite of medicine, but topmost metabolite is that (structure is through mass spectrum and nuclear magnetic data confirmation as shown in the formula V compound, and with synthetic standard substance contrast confirmation), therefore, formula V compound be in the time carrying out medicine generation in dog body and poison for dynamics research the drug metabolite mark that must detect quantitative and qualitative analysis.
Result of study of the present invention shows, if coming curative effect and the bleeding risk of the medicament for resisting platelet aggregation shown in prediction type I, the exposed amount of the active metabolite shown in the formula of employing IV there is very large uncertainty, because the exposed amount of this active metabolite is minimum, and its metabolic stability and chemical stability be non-constant all, in the blood plasma of getting after blood, can decompose rapidly (inventor's discovery, even if the blood plasma taking out is cooling with ice bath, in 10 minutes, still there is the active metabolite degraded that exceedes 25%), detect again after quick derivatization even if carry out, its circulation ratio is also very poor, can bring very large error.By further research, be surprisingly found out that, when adopting the sulphur methyl ether metabolite shown in formula II when detecting index, to there is good accuracy and circulation ratio.Its reason is: the medicament for resisting platelet aggregation shown in (1) formula I can generate the formula II metabolite of significant quantity in all kind bodies, is beneficial to detection by quantitative; (2) metabolic stability of formula II metabolite and chemical stability are all very good, without detecting after derivatize, easy to detect again, and favorable reproducibility; (3) inventor has proved that the concentration of active metabolite in laboratory animal blood plasma (compound shown in formula IV) and the concentration of sulphur methyl ether metabolite (compound shown in formula II) have extraordinary dependency by experiment.Therefore, can measure easily and accurately the exposed amount of active metabolite by the exposed amount that detects metabolite shown in formula II, and then can predict and curative effect and the security of regulating medicine, especially can predict the bleeding risk with regulating medicine.
The present invention also provides the preparation method of formula II compound (dimethyl sulfide metabolite), as shown in following reaction formula.
Specifically comprise the following steps:
(1) formula VI compound reacts production VII compound with Methyl disulfide, and wherein, P is Boc or trityl-protecting group;
(2) formula VII compound production VIII compound under the effect of deprotection agent, the deprotection agent adopting is hydrochloric acid, acetic acid or trifluoracetic acid;
(3) formula VIII compound or its salt reacts under the effect of alkali with formula IX compound, production X compound, wherein, R
3be the phenyl of alkyl, trifluoromethyl, pentafluoroethyl group, seven fluoropropyls, phenyl or the Z replacement of 1～6 carbon, wherein Z is alkyl, halogen, itrile group, nitro or the trifluoromethyl of 1～3 carbon, and Z group is positioned at 2,3 or 4 of phenyl ring; The alkali adopting is triethylamine, 1,8-diazacyclo [5,4,0] 11 carbon-7-alkene (DBU), pyridine, 4-N, N-lutidine (DMAP), diisopropylethylamine, diisopropylamine lithium, salt of wormwood, sodium carbonate, saleratus, sodium bicarbonate, potassium tert.-butoxide or sodium tert-butoxide;
(4) formula X compound reacts under the effect of alkali with formula XI compound, production XII compound, wherein, R
4and R
5it is respectively the straight or branched alkyl of 1～6 carbon;
(5) formula XII compound is through alkalescence or acid selective hydrolysis production II compound.
Accompanying drawing explanation
Fig. 1 be the metabolite profile of dimension card Gray in monkey blood plasma (from top to bottom respectively representative take medicine after 0.5,1,4 and the blood sample of 8h);
Fig. 2 be the metabolite profile of dimension card Gray in dog plasma (from top to bottom respectively representative take medicine after 0.5,1,4 and the blood sample of 8h);
Fig. 3 be the metabolite of dimension card Gray in rat plasma general (from top to bottom respectively representative take medicine after 0.5,1,4 and the blood sample of 8h);
Fig. 4 be the Q-TOF mass spectrometric detection metabolite profile in blood plasma after the oral dimension card of volunteer Gray (from top to bottom respectively representative take medicine after 1,2 and the blood sample of 6h);
Fig. 5 is the hydrogen nuclear magnetic resonance spectrogram of formula II compound;
Fig. 6 is the carbon-13 nmr spectra figure of formula II compound;
Fig. 7 is the hydrogen nuclear magnetic resonance spectrogram of formula V compound;
Fig. 8 is the carbon-13 nmr spectra figure of formula V compound.
Embodiment
Below by specific embodiment, content of the present invention is described.In the present invention, the embodiment of the following stated is in order better to set forth the present invention, is not for limiting the scope of the invention.
The pharmacokinetic of dimension card Gray in cynomolgus monkey, beasle dog and SD rat body
1. test objective: identify that cynomolgus monkey, beasle dog and SD rat oral gavage tie up the meta-bolites in blood plasma after card Gray, comparison drug disposition metabolism behavior difference.
2. sample collecting: 2 of healthy cynomolgus monkeys, each 1 of male and female, body weight 3～4kg, ties up card Gray by 5mg/kg gavage.2 of healthy beasle dogs, male, body weight 8～10kg, ties up card Gray by 5mg/kg gavage.2 of healthy SD rats, male, body weight 180～220g, ties up card Gray by 10mg/kg gavage.Fasting 12h before test, freely drinks water, administration on an empty stomach in morning, the unified feed of 2h after administration.Before administration and after administration 0.5,1,4 and 8h extracting vein blood 0.5ml, after whole blood collection, add immediately in the EDTA anticoagulant tube that is placed with 25 μ l derivatization reagents (3 '-methoxyl group bromoacetophenone (BMP) acetonitrile solution), as early as possible test tube is put upside down gently and mixed 5～6 times, room temperature is placed 10min, the centrifugal 10min of 3500rpm, separated plasma is put in the anti-freezing plastic test tube that posts label, puts into-80 ℃ of refrigerator and cooled and freezes preservation.
3. instrument and condition
Instrument: the U.S. Synapt of Waters company type quadrupole-flight time tandom mass spectrometer (Q-TOF MS), be furnished with electron spray ionisation source (ESI source) and Acquity UPLC liquid chromatographic system.
UPLC condition: chromatographic column is ACQUITY
tMhSS T3C
18post (2.1 × 100mm I.D., 1.8 μ m particle diameters), Waters company of the U.S.; Column temperature is 45 ℃; Flow velocity is 0.45mL/min; Moving phase is that acetonitrile/5mM ammonium acetate is containing 0.05% formic acid gradient elution.
Mass spectrum condition: ion source is electron spray ionisation source (ESI), adopt positive scan mode to detect, desolventizing gas (nitrogen) flow velocity is 700L/h, desolventizing temperature degree is 350 ℃, source temperature is 100 ℃, capillary voltage is 2700V, and when low-yield scanning, transmitting collision energy is 4eV, and trap collision energy is 6eV; When high-energy scanning, transmitting collision energy is 12eV, and trap collision energy is 10-20eV.Choose the leucine enkephalin (m/z556.2771) of 200ng/mL and proofread and correct external standard as mass-to-charge ratio, flow velocity is 10 μ L/min.
Data processing: data gathering adopts the Masslynx V4.1 software of Waters company, data analysis adopts MetaboLynx software and MassFragment
tMsoftware.
The processing of biological sample: the plasma sample gathering before drawing medicine and after administration, merges according to kind and sampling time point.Get and merge plasma sample 400 μ L, add 800 μ L acetonitriles, eddy current mixing 1min, centrifugal 5min (14000rpm), take out whole supernatant liquors and be placed in 10mL test tube, under 40 ℃ of airflows, dry up, with 80 μ L acetonitriles: water (1:9, v/v) dissolve, get 10 μ L and carry out UPLC/Q-TOF MS analysis.
4. result: Fig. 1～3 have provided the metabolite profile of dimension card Gray in monkey, dog and rat plasma, shown in the structure of metabolite carried out structural identification through mass spectrum, nuclear-magnetism and synthetic standard reference material.Result shows:
(1) after administration, the main metabolites in the monkey blood plasma of starting stage (0.5h), for the active metabolite shown in formula IV (what detect with UPLC/Q-TOF MS is that free sulfhydryl groups and 3 '-methoxyl group bromoacetophenone (BMP) of the active metabolite shown in formula IV reacts obtained derivatize product M15-4), is secondly the sulphur methyl ether M9-2 shown in formula II; And main metabolites in monkey blood plasma after administration 4h is M9-2 (being the sulphur methyl ether shown in formula II).
(2) major metabolite in the dog plasma of initial after administration and late stage is the meta-bolites M4 shown in formula V (structure is through mass spectrum and nuclear magnetic data confirmation, and the synthetic standards product contrast confirmation with embodiment 7), be secondly the sulphur methyl ether M9-2 shown in formula II.
(3) in the rat plasma after administration, dimension card Gray's metabolism behavior is similar to the metabolism behavior in monkey blood plasma.
In sum, result of study of the present invention shows, ties up the metabolism behavior of card Gray in monkey and rat body comparatively approaching; The exposed amount of active metabolite (in M15-4) in dog body is far below other two kinds; In all test kinds, late stage after administration (such as in 8h left and right), the exposed amount of active metabolite (in M15-4) is all very low; In monkey and rat body, the sulphur methyl ether metabolite (M9-2, M9-4) shown in formula II is topmost metabolite; In dog body, the sulphur methyl ether M9-2 shown in the meta-bolites M4 shown in formula V and formula II is two topmost metabolites.
Embodiment 2
The metabolism behavioral study of dimension card Gray in human body
1. test objective: adopt UPLC/Q-TOF MS method qualitative and identify quantitatively the meta-bolites in blood plasma after the oral dimension card of volunteer Gray sheet.
2. materials and methods
Reagent:
| 3 '-methoxyl group bromoacetophenone | Sigma company of the U.S. |
| Acetonitrile (chromatographically pure) | Sigma company of the U.S. |
| Ammonium acetate (chromatographically pure) | Tedia company of the U.S. |
| Formic acid | Fluka company of the U.S. |
Sample collecting: 3 of healthy male volunteers, fasting 12h before test, test took 25mg dimension card Gray with 200ml warm water respectively the same day, after taking medicine, after 2h, can drink water, and after 4h, had meal.Before taking medicine (0h) and take medicine after 1,2 and 6h through getting blood 2mL, after whole blood collection, add immediately in the EDTA anticoagulant tube that is placed with 100 μ L derivatization reagents (3 '-methoxyl group bromoacetophenone (BMP) acetonitrile solution), as early as possible test tube is put upside down gently and mixed 5～6 times, room temperature is placed 10min, the centrifugal 10min of 3500rpm, separated plasma is put in the anti-freezing plastic test tube that posts label, puts into-80 ℃ of refrigerator and cooled and freezes preservation.
Instrument: the U.S. Synapt of Waters company type quadrupole-flight time tandom mass spectrometer (Q-TOF MS), be furnished with electron spray ionisation source (ESI source) and Acquity UPLC liquid chromatographic system.
UPLC condition: chromatographic column is ACQUITY
tMhSS T3C
18post (2.1 × 100mm I.D., 1.8 μ m particle diameters), Waters company of the U.S.; Column temperature is 45 ℃; Flow velocity is 0.45mL/min; Moving phase is that acetonitrile/5mM ammonium acetate is containing 0.05% formic acid gradient elution.
Mass spectrum condition: ion source is electron spray ionisation source (ESI), adopt positive scan mode to detect, desolventizing gas (nitrogen) flow velocity is 700L/h, desolventizing temperature degree is 350 ℃, source temperature is 100 ℃, capillary voltage is 2700V, and when low-yield scanning, transmitting collision energy is 4eV, and trap collision energy is 6eV; When high-energy scanning, transmitting collision energy is 12eV, and trap collision energy is 10-20eV.Choose the leucine enkephalin (m/z556.2771) of 200ng/mL and proofread and correct external standard as mass-to-charge ratio, flow velocity is 10 μ L/min.
Data processing: data gathering adopts the Masslynx V4.1 software of Waters company, data analysis adopts MetaboLynx software and MassFragment
tMsoftware.
The processing of biological sample: the plasma sample gathering before drawing medicine and after administration, merges according to group and sampling time point.Get and merge plasma sample 400 μ L, add 800 μ L acetonitriles, eddy current mixing 1min, centrifugal 5min (14000rpm), take out whole supernatant liquors and be placed in 10mL test tube, under 40 ℃ of airflows, dry up, with 80 μ L acetonitriles: water (1:9, v/v) dissolve, get 10 μ L and carry out UPLC/Q-TOF MS analysis.
3. result: utilize Metabolynx software to carry out MDF processing to the plasma sample data before and after volunteer's administration, the oral dimension of volunteer is blocked the Q-TOF mass spectrometric detection metabolite profile in blood plasma after each thunder and seen Fig. 4.
Metabolite identification in dimension card Gray blood plasma: the thiolactone shown in original shape medicine and formula III do not detected in the experimenter's blood plasma after oral dimension card Gray sheet, detect altogether comprise the active metabolite shown in formula IV and corresponding cyclic olefinic bond metabolite (detect with UPLC/Q-TOF MS be the metabolite shown in formula IV and accordingly free sulfhydryl groups and 3 '-methoxyl group bromoacetophenone (BMP) of cyclic olefinic bond metabolite react obtained derivatize product, with M15-1, M15-3 and M15-4 meter) at interior 7 kinds of meta-bolitess (seeing Fig. 4), wherein, the meta-bolites of exposed amount maximum is the sulphur methyl ether M9-2 shown in formula II.Relevant meta-bolites information is in table 1.
Meta-bolites relevant information in blood plasma after the oral 25mg dimension of table 1. experimenter card Gray
4. conclusion
Result of study of the present invention shows: in subject, in administration, the major metabolite after 1 hour is the sulphur methyl ether shown in formula II to dimension card Gray, in administration, after 6 hours, the sulphur methyl ether shown in formula II is almost unique major metabolite (seeing Fig. 4) that can detect.Therefore, the inventor is surprised to find, medicine dynamic metabolism to this type of medicine research and the efficacy and saferry evaluation of existing bibliographical information have very large careless omission, i.e. the metabolite of exposed amount maximum in vivo of the medicament for resisting platelet aggregation shown in formula I--the sulphur methyl ether shown in formula II has been ignored in correlative study in the past completely! Result of study of the present invention shows, aspect the medicament for resisting platelet aggregation shown in development formula I, sulphur methyl ether shown in formula II is the index compound that must further investigate (comprising safety evaluation), especially pharmacokinetic aspect in vivo, the sulphur methyl ether shown in formula II is the significant metabolite that must carry out quantitative and qualitative analysis research.
Embodiment 3
The pharmacokinetic of formula I-3 compound in SD rat body
1. test objective: the meta-bolites after evaluation SD rat oral gavage giving construction I-3 compound in blood plasma, study its drug disposition metabolism behavior.
2. sample collecting: 2 of healthy SD rats, male, body weight 180～220g, by 10mg/kg gavage giving construction I-3 compound.Fasting 12h before test, freely drinks water, administration on an empty stomach in morning, the unified feed of 2h after administration.Before administration and after administration 1,4 and 8h extracting vein blood 0.5ml, after whole blood collection, add immediately in the EDTA anticoagulant tube that is placed with 25 μ l derivatization reagents (3 '-methoxyl group bromoacetophenone (BMP) acetonitrile solution), as early as possible test tube is put upside down gently and mixed 5～6 times, room temperature is placed 10min, the centrifugal 10min of 3500rpm, separated plasma is put in the anti-freezing plastic test tube that posts label, puts into-80 ℃ of refrigerator and cooled and freezes preservation.
3. instrument and condition
Instrument: the U.S. Synapt of Waters company type quadrupole-flight time tandom mass spectrometer (Q-TOF MS), be furnished with electron spray ionisation source (ESI source) and Acquity UPLC liquid chromatographic system.
UPLC condition: chromatographic column is ACQUITY
tMhSS T3C
18post (2.1 × 100mm I.D., 1.8 μ m particle diameters), Waters company of the U.S.; Column temperature is 45 ℃; Flow velocity is 0.45mL/min; Moving phase is that acetonitrile/5mM ammonium acetate is containing 0.05% formic acid gradient elution.
Mass spectrum condition: ion source is electron spray ionisation source (ESI), adopt positive scan mode to detect, desolventizing gas (nitrogen) flow velocity is 700L/h, desolventizing temperature degree is 350 ℃, source temperature is 100 ℃, capillary voltage is 2700V, and when low-yield scanning, transmitting collision energy is 4eV, and trap collision energy is 6eV; When high-energy scanning, transmitting collision energy is 12eV, and trap collision energy is 10-20eV.Choose the leucine enkephalin (m/z556.2771) of 200ng/mL and proofread and correct external standard as mass-to-charge ratio, flow velocity is 10 μ L/min.
Data processing: data gathering adopts the Masslynx V4.1 software of Waters company, data analysis adopts MetaboLynx software and MassFragment
tMsoftware.
The processing of biological sample: the plasma sample gathering before taking respectively administration and after administration.Get plasma sample 400 μ L, add 800 μ L acetonitriles, eddy current mixing 1min, centrifugal 5min (14000rpm), take out whole supernatant liquors and be placed in 10mL test tube, under 40 ℃ of airflows, dry up, with 80 μ L acetonitriles: water (1:9, v/v) dissolve, get 10 μ L and carry out UPLC/Q-TOF MS analysis.
4. result: after SD Oral Administration in Rats 10mg formula I-3 compound, in blood plasma, main metabolites relevant information is as shown in table 2.
Main metabolites relevant information in blood plasma after table 2.SD Oral Administration in Rats 10mg formula I-3 compound
4. conclusion: pharmacokinetic result shows, starting stage after oral administration formula I-3 compound, main metabolites in rat plasma, for the active metabolite shown in formula IV (what detect with UPLC/Q-TOF MS is that free sulfhydryl groups and 3 '-methoxyl group bromoacetophenone of the active metabolite shown in formula IV reacts obtained derivatize product M15-4), is secondly the sulphur methyl ether M9-2 shown in formula II; And main metabolites in rat plasma after administration 4h is M9-2 (being the sulphur methyl ether shown in formula II).This result of study has further proved that formula I compound major metabolite is in vivo the sulphur methyl ether shown in formula II.
Embodiment 4
Dimension card Gray's active metabolite and the plasma concentration correlation research of sulphur methyl ether metabolite
1. test objective: prove after the oral dimension card of beasle dog Gray, the concentration of the concentration of active metabolite in its blood plasma (compound shown in formula IV) and sulphur methyl ether metabolite (compound shown in formula II) has dependency.
2. administration, sampling and sample treatment
Beasle dog, 6, purchased from Xin Gang laboratory animal field, Shanghai, under test conditions, adapt to use after one week, fasting in first 12 hours of administration and administration 12 hours, duration of test is freely drunk water.6 beasle dogs are divided into 3 groups, and the dosage of each group is followed successively by: 25mg, 50mg and 75mg.For three dosage groups oral capsule of tieing up card Gray respectively, before administration, after 30min and administration 0.25,0.5,1,2,4,8,12,24,30,36h, at side forelimb venous blood sampling 2.5mL, immediately in the centrifugal 1min of 4000pm, isolates blood plasma 400 μ L.The blood plasma that centrifuging and taking is obtained is divided into two equal portions, add respectively the acetic acid of 600 μ L acetonitriles and 20 μ L50%, vortex mixed is after 30 seconds, and the centrifugal 5min of 12000r/min takes out supernatant liquor, portion is placed in-70 ℃ of Refrigerator stores, for the mensuration of sulphur methyl ether metabolite (compound shown in formula II); From another part, take out the supernatant liquor of 200 μ L, add the 20 μ L derivatization reagent bromo-3 '-methoxyacetophenones of 2-(BMP), vortex concussion 3min is placed on-70 ℃ of Refrigerator stores, for the mensuration of the BMP derivative (AM-BMP) of active metabolite (compound shown in formula IV).
3, the LC-MS/MS detection method of metabolite
The method for measurement of concentration of metabolite adopts LC-MS/MS method.Adopt compound R-95913 as interior mark.Because active metabolite (compound shown in formula IV) is very unstable, its pharmacokinetic parameter carrys out indirect measurement by detecting its BMP derivative (AM-BMP).Sulphur methyl ether metabolite (compound shown in formula II) character is highly stable, can directly detect.Instrument system comprises LC-10ADvp (Shimadzu company) liquid chromatographic system, PerkinElmer series200 automatic sample handling system (perkin elmer Instrument Ltd. of the U.S.) and the triple level Four bar of API4000 detector (u.s.a. applied biosystem company limited), function software is Analyst1.5.1.Be below the concrete detection method of metabolite:
Chromatographic column: Luna Phenyl-Hexyl, 5 μ m, (50mm × 2.0mm)
Moving phase: adopt isocratic elution, condition sees the following form:
| Time (min) | A | B |
| 4 | 20 | 80 |
The aqueous solution of A:5mM ammonium acetate, B: methanol solution
Speed: 400 μ L/min
Column temperature: 20 ℃
Sample size: 5 μ L
Mass spectrum condition:
Scan type: the multistage detection of positive ion
Ion source: Turbo spray ionization mode: ESI
Atomization gas: 20 curtain gas: 10 collision gas: 4
Heating assisted gas: 20 ion voltages: 5500v temperature: 200 ℃
4. method of calculation: utilize DAS2.0 pharmacokinetics professional software to carry out computing with statistical moment method, try to achieve corresponding pharmacokinetic parameters.
5. result: the pharmacokinetic parameter (getting the mean value of each group) of dimension card Gray's active metabolite (compound shown in formula IV) and sulphur methyl ether metabolite (compound shown in formula II) various dose group morning is in Table 3-8.
The pharmacokinetic parameter (dimension card Gray dosage: 75mg) of table 3. active metabolite (formula IV compound) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-t | μg/L*h | 729.78 |
| AUC 0∞ | μg/L*h | 744.75 |
| t 1/2 | h | 7.51 |
| C max | μg/L | 446.83 |
The pharmacokinetic parameter (dimension card Gray dosage: 50mg) of table 4. active metabolite (formula IV compound) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-1 | μg/L*h | 483.12 |
| AUC 0∞ | μg／L*h | 492.41 |
| t 1/2 | h | 7.44 |
| C max | μg/L | 293.32 |
The pharmacokinetic parameter (dimension card Gray dosage: 25mg) of table 5. active metabolite (formula IV compound) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-1 | μg／L*h | 238.24 |
| AUC 0∞ | μg／L*h | 241.18 |
| t 1/2 | h | 7.48 |
| C max | μg／L | 144..57 |
The pharmacokinetic parameter (dimension card Gray dosage: 75mg) of table 6. sulphur methyl ether metabolite (compound shown in formula II) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-t | μg/L*h | 1968.01 |
| AUC 0∞ | μg／L*h | 2141.57 |
| t 1/2 | h | 10.04 |
| C max | μg／L | 482.5 |
The pharmacokinetic parameter (dimension card Gray dosage: 50mg) of table 7. sulphur methyl ether metabolite (compound shown in formula II) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-t | μg／L*h | 1290.14 |
| AUC 0∞ | μg／L*h | 1411.34 |
| t t/2 | h | 10.15 |
| C max | μg/L | 315.23 |
The pharmacokinetic parameter (dimension card Gray dosage: 25mg) of table 8. sulphur methyl ether metabolite (compound shown in formula II) in beasle dog body
| Parameter | Unit | Mean value |
| AUC 0-t | μg／L*h | 635.47 |
| AUC 0∞ | μg／L*h | 690.4 |
| t 1／2 | h | 9.97 |
| C max | μg／L | 149.33 |
6. conclusion: result of study shows, after the oral dimension card of beasle dog Gray, the concentration of the concentration of active metabolite in its blood plasma (compound shown in formula IV) and sulphur methyl ether metabolite (compound shown in formula II) has extraordinary dependency, that is to say, exposed amount in vivo of sulphur methyl ether metabolite (compound shown in formula II) and the exposed amount of active metabolite (compound shown in formula IV) have good dependency.Because the chemical stability of sulphur methyl ether metabolite and metabolic stability are far away higher than the stability of active metabolite, therefore, can determine by detecting the exposed amount of sulphur methyl ether metabolite the exposed amount of active metabolite, so more accurate and effective and evaluate expediently the efficacy and saferry of medicine.In addition, this result of study has further proved that sulphur methyl ether metabolite exposed amount is in vivo far longer than the exposed amount of active metabolite, thereby in the time of the medicament for resisting platelet aggregation shown in development or preparation formula I, compound shown in formula II disclosed in this invention (sulphur methyl ether metabolite) is the index compound must quantitative and qualitative analysis detecting, to guarantee the efficacy and saferry of medicine, especially aspect the bleeding risk of regulating medicine, there is important use.
Embodiment 5
Antiplatelet aggregative activity in the body of sulphur methyl ether metabolite
1. test objective
BORN turbidimetry (Nature, 1962,194 (4832): the antiplatelet aggregative activity of 927) measuring the sulphur methyl ether shown in formula II.
2. material
Sulphur methyl ether shown in formula II: synthetic sample Compound I I-1/II-2 (embodiment 6), through consistent according to certification structure mutually with dimension card Gray major metabolite M9-2/M9-4 in vivo.
Positive control: dimension card Gray, Wei Kaier Pharmaceutical Technology Co., Ltd provides by Jiangsu.
ADP:Sigma company product, the U.S..
3. method and result
Positive drug and test-compound are made into suspension for animal oral administration with 0.5%CMC-Na (Xylo-Mucine).
Animal: male SD rat, body weight 250g left and right, by Shanghai, western pul-Bi Kai laboratory animal company limited provides.Animal conformity certification number: 2008001605451 ticket number SCXK (Shanghai): SCXK (Shanghai) 2008-0016.
Instrument: whizzer (80-2 table-type low-speed whizzer) and full-automatic platelet aggregation determinator (STELLEX LG-PAPER-1 thrombocyte blood coagulation analysis of agglomeration instrument) etc.
Method: with reference to BORN turbidimetry (Nature, 1962,194 (4832): 927), test-compound is carried out to the pharmacological activity test of platelet aggregation-against.Add short condensation product adenosine diphosphate (ADP) (ADP) to stir to being rich in hematoblastic blood plasma (PRP), make platelet aggregation.Hematoblastic gathering causes the variation of optical density(OD), can detect by spectrophotometer.The platelet aggregation that this experiment can be evaluated test-compound in vivo or treated in vitro causes.
Platelet aggregation inhibitory activity test: male SD rat, body weight 250g left and right, per os gavage is tieed up card Gray and the test-compound (unit for uniform suspension of 0.5%CMC-Na, drug level is 2mg/ml), dosage is 10mg/kg or 3mg/kg, and blank group per os gavage gives the 0.5%CMC-Na of same volume.The impact of hematometry medicine on ADP induced platelet aggregation rate got in administration after 2 hours.Get blood through eye socket, 3.8% Sodium Citrate anti-freezing, whole blood is 9:1 with the ratio of antithrombotics, the centrifugal 7min of 1000rpm prepares platelet rich plasma (PRP).Adjust PRP with platelet poor plasma (PPP), make platelet count remain on 2x106/ml.Get PRP and add in test cup, hatch 10min for 37 ℃, with PRP zeroing, PPP adjusts 100%, take ADP (final concentration is as 5 μ M) as inductor, measures platelet aggregation percentage ratio by turbidimetry with platelet aggregation instrument, carries out statistics comparison with t-check.L-Arginine is calculated as follows: L-Arginine (%)=[1-(delivery tube is assembled percentage/control tube and assembled percentage)] x100%.
Result: record the L-Arginine after Oral Administration in Rats test-compound by turbidimetry, experimental result is as shown in table 9.
Anticoagulant effect after table 9. Oral Administration in Rats test-compound
4. conclusion: result of study shows, under 10mg/kg and 3mg/kg dosage, ties up card Gray and has all shown significant platelet aggregation inhibitory activity; And the sulphur methyl ether shown in formula II has shown weak platelet aggregation inhibitory activity under 10mg/kg and 3mg/kg dosage.
Embodiment 6
(Z) preparation of-2-((S)-1-((S)-1-(2-chloro-phenyl-)-2-methoxyl group-2-carbonyl ethyl)-4-(first sulfydryl) piperidines-3-subunit) acetic acid (II-1) and (Z)-2-((R)-1-((S)-1-(2-chloro-phenyl-)-2-methoxyl group-2-carbonyl ethyl)-4-(first sulfydryl) piperidines-3-subunit) acetic acid (II-2)
In 250mL three-necked bottle, add compound VI-1 (3.98g; 0.020mol); after nitrogen protection; add the anhydrous THF of 25mL and DMPU (25mL); after fully dissolving, in bottle, temperature is down to-78 ℃, slowly drips LDA (20mL; 0.040mol), make bottle interior temperature lower than-70 ℃.LDA finishes, and stirs 1h in-78 ℃ to-50 ℃.Naturally intensification is cooled to 0 ℃ after stirring 2h, adds Methyl disulfide (5.3mL, 0.06mol), in 0 ℃ of stirring 10min, after stirring at room temperature 1h, reacts completely.In reaction solution, add water 30mL, extract by ethyl acetate (30mL × 3), merge organic layer, with 2M hydrochloric acid (30mL) and water (30mL × 2) washing, organic layer anhydrous sodium sulfate drying, filter, vacuum is spin-dried for, and column chromatography purification (sherwood oil: ethyl acetate=40:1), obtains compound VI I-1 (yellow oil, 2.21g), yield is 45.1%.ESI-MSm／z244.3[M-H]
-.
1H?NMR(300MHz，CDCl
3)：δ4.17(m，2H)，3.74(s，1H)，3.46-3.32(m，1H)，3.26-3.17(m，1H)，2.38-2.23(m，1H)，2.11-1.97(m，4H)，1.43(s，9H).
13C?NMR(75MHz，CDCl
3)：δ199.77，154.08，80.44，50.92，49.68，38.71，29.41，28.20，14.97.
Step 2.
In 250mL eggplant-shape bottle, add compound VI I-1 (6.20g, 25.3mmol), dissolve with 60mL THF, add 5mL concentrated hydrochloric acid, 50 ℃ of reaction 1h, vacuum is spin-dried for, and obtains garnet oily matter.Under ice-cold, in this oily resistates, add 20mL toluene, agitator treating, static after, pour out supernatant liquid, the heavy oily matter of lower floor (compound VI II) is directly used in next step reaction.
Step 3.
In 500mL eggplant-shape bottle, add step reaction products therefrom (about 25.3mmol); after 180mL acetonitrile and 20mL dissolve with methanol; add (R)-2-(2-chloro-phenyl-)-2-(4-oil of mirbane sulfonyloxy)-methyl acetate (Compound I X; 8.79g; 22.8mmol) and saleratus (12.7g; 127mmol); stirred overnight at room temperature under nitrogen protection; suction filtration; filtrate vacuum is spin-dried for, and resistates, through column chromatography purification (sherwood oil: ethyl acetate=20:1), obtains compounds X (yellow oil; 3.85g, yield 46.4%).ESI-MS?m／z328.1[M+H]
+.
1H?NMR(300MHz，CDCl
3)：δ7.53-7.44(m，1H)，7.44-7.37(m，1?H)，7.33-7.21(m，2H)，4.85-4.81(m，1H)，3.78-3.76(m，0.5H)，3.70(s，3H)，3.65-3.60(m.0.5H)，3.25-3.05(m，2H)，2.90-2.71(m，2H)，2.47-2.33(m，1H)，2.05-2.03(m，3H)，2.01-1.93(m，1H).
13C?NMR(75MHz，CDCl
3)：δ201.85，171.97，171.85，136.02，135.93，133.95，133.89，131.14，131.12，130.93，130.89，130.79，128.27，128.19，68.73，68.68，58.72，58.40，53.24，51.39，51.35，47.27，47.11，31.69，31.65，16.05.
Step 4.
In 25mL bis-neck bottles, add NaH (270mg, 6.75mmol) and dry toluene (10mL), under ice bath, drip diethyl phosphonyl tert.-butyl acetate (1.6mL, 6.75mmol), under room temperature, stir 20min.Under ice bath, drip the anhydrous toluene solution (5mL) of compounds X (897mg, 2.70mmol), after stirring 5min, react completely, add water (10mL), with methylene dichloride (20mL × 3) extraction, merge organic layer, anhydrous sodium sulfate drying, vacuum is spin-dried for, resistates, through column chromatography purification (sherwood oil: ethyl acetate=20:1), obtains the faint yellow oily matter that polarity is less (109mg, yield 7.9%):
1h NMR (300MHz, CDCl
3): δ 7.64-7.62 (m, 1H), 7.4l-7.38 (m, 1H), 7.30-7.24 (m, 2H), 5.70 (s, 1H), 5.16-5.15 (m, 1H), 4.78 (s, 1H), 3.71 (s, 3H), 3.70-3.57 (m, 1H), 3.07-3.04 (m, 1H), 2.67-2.60 (m, 2H), 2.24-2.15 (m, 1H), 2.02 (s, 3H), 1.81-1.85 (m, 1H), 1.47 (s, 9H). in addition, also separate and obtain the faint yellow oily matter (192mg that polarity is larger, yield 13.9%):
1h NMR (300MHz, CDCl
3): δ 7.62-7.60 (m,
1h), 7.59-7.39 (m, 1H), 7.38-7.36 (m, 2H), 5.53 (s, 1H), 5.14-5.13 (m, 1H), 4.76 (s, 1H), 3.68 (s, 3H), 3.50-3.46 (m, 1H), 2.83-2.70 (m, 3H), 2.28-2.19 (m, 1H), 2.00 (s, 3H), 1.99-1.90 (m, 1H), 1.46-1.43 (m, 9H). two products that above-mentioned separation obtains are compounds X II-1A and XII-1B, and concrete configuration needs further to be confirmed.In addition, be also separated to compounds X III-1 (faint yellow oily matter, 552mg, yield 40.1%):
1h NMR (300MHz, CDCl
3): δ 7.64-7.60 (m, 1H), 7.37-7.35 (m, 1H), 7.27-7.24 (m, 2H), 5.59-5.58 (m, 1H), 4.85-4.83 (m, 1H), 4.47-4.31 (m, 1H), 3.72-3.70 (m, 3H), 3.55-3.45 (m, 1H), 3.28 (s, 1H), 2.86-2.67 (m, 2H), 2.28-2.26 (m, 1H) 2.04 (s, 3H), 1.97-1.84 (m, 1H), (1.44-1.24 m, 9H).
Step 5.
In two independently parallel laboratory test operates, in eggplant-shape bottle, adding respectively the compounds X II-1A and the XII-1B that in step 4, obtain (is respectively 129mg, 0.304mmol), dissolve with THF (4mL) respectively, add respectively concentrated hydrochloric acid (1mL), 50 ℃ are stirred 50min.Vacuum is spin-dried for, with saturated sodium bicarbonate solution tune pH to 7, methylene dichloride (20mL × 3) extraction, merge organic layer, anhydrous sodium sulfate drying, filter, vacuum is spin-dried for, resistates is through column chromatography purification (sherwood oil: ethyl acetate: Glacial acetic acid=40:20:1), obtain respectively following two target compound II-1 and II-2 (because the configuration of compound is not determined, therefore sort uncertain): one of them compound is faint yellow oily matter, 60mg, yield 53.5%:
1h NMR (300MHz, CDCl
3): δ 7.61-7.60 (m, 1H), 7.39 (s, 1H), 7.27 (s, 2H), 5.78 (s, 1H), 5.12 (s, 1H), 4.82 (s, 1H), 3.72 (s, 3H), 3.68-3.56 (m, 1H), 3.10-3.12 (m, 1H), 2.85-2.59 (m, 2H), 2.24-2.15 (m, 1H), 2.02 (s, 3H), 1.88-1.84 (m, 1H).
13c NMR (75MHz, CDCl
3): δ 172.10,171.96,157.93,135.89,134.33,131.00,130.98,130.64,128.29,117.40,68.95,55.44,53.35,47.18,41.07,31.78,15.36; Another compound is white solid, 68mg, and yield 61%:
1h NMR (300MHz, CDCl
3): δ 7.63-7.59 (m, 1H), 7.43-7.29 (m, 1H), 7.33-7.24 (m, 2H), 5.66 (s, 1H), 5.11-5.12 (m, 1H), 4.81 (s, 1H), 3.71 (s, 3H), 3.63-3.54 (m, 1H), 2.96-2.73 (m, 3H), 2.34-2.18 (m, 1H), 2.01 (s, 3H), 1.89-1.94 (m, 1H).
13c NMR (75MHz, CDCl
3): δ 170.95,170.53,157.01,134.62,133.02,129.80,129.49,127.16,115.86,67.78,53.93,52.13,46.41,39.91,30.69,14.16.
(S) preparation of-2-(1-(1-(2-chloro-phenyl-)-2-methoxyl group-2-carbonyl ethyl)-1,2,5,6-tetrahydropyridine-3-yl) acetic acid (V)
In 10mL eggplant-shape bottle, add compounds X IV (215.0mg, 0.50mmol), dissolve with Glacial acetic acid (8.9mL), add zinc powder (487.5mg, 7.5mmol), room temperature reaction 5h, filter, filtrate vacuum is spin-dried for, in resistates, add water (10mL), extract with methylene dichloride (20mL × 3), merge organic layer, extremely neutral with saturated sodium bicarbonate washing, anhydrous sodium sulfate drying, filter, vacuum is spin-dried for, resistates is through column chromatography purification (sherwood oil: ethyl acetate=8:1), obtain compounds X V (yellow oil, 125.7mg, yield 71.6%):
1h NMR (300MHz, CDCl
3): δ 7.68-7.67 (m, 1H), 7.41-7.35 (m, 1H), 7.31-7.19 (m, 2H), 5.65 (s, 1H), 4.79 (s, 1H), 4.10 (q, J=7.1Hz, 2H), 3.70 (s, 3H), 3.09 (dd, J=40.2,15.6Hz, 2H), 2.92 (s, 2H), 2.61 (t, J=5.7Hz, 2H), 2.17 (s, 2H), 1.23 (t, J=7.1Hz, 3H) .ESI-MSm/z352.1[M+H]
+.
Step 2.
In 100mL eggplant-shape bottle, add compounds X V (830mg, 2.36mmo1), dissolve with methyl alcohol (30mL), splash into 30% sodium hydroxide solution (2.5mL), 50 ℃ of heating 40min, TLC demonstration reacts completely.To neutrality, with methylene dichloride (30mL × 3) extraction, merge organic layer with 3M HCl tune pH, anhydrous sodium sulfate drying, filters, and vacuum is spin-dried for.Resistates, through column chromatography purification (sherwood oil: ethyl acetate: Glacial acetic acid=40:40:1), obtains compound V (yellow oil, 254.0mg, yield 33.3%):
1h NMR (500MHz, CDCl
3): δ 7.69-7.63 (m, 1H), 7.41-7.36 (m, 1H), 7.30-7.21 (m, 2H), 5.68 (s, 1H), 4.85 (s, 1H), 3.69 (s, 3H), 3.22 (d, J=15.6Hz, 1H), 3.06 (d, J=15.6Hz, 1H), 2.95 (s, 2H), 2.68 (dt, J=11.0,5.4Hz, 1H), 2.59 (dt, J=11.4,5.7Hz, 1H), 2.18 (s, 2H).
13c NMR (75MHz, CDCl
3): δ 176.11,171.16,134.75,133.33,130.05,129.75,129.41,128.54,127.11,124.40,67.68,52.75,52.15,46.53,40.79,25.46.ESI-MS m/z324.1[M+H]
+.HRMS calcdfor C
16h
18nO
4cl[M-H]
-m/z322.0846, found322.0848.
Claims (10)
1. as shown in the formula the mixture of the compound shown in II, its diastereomer or diastereomer:
2. compound as claimed in claim 1, is characterized in that, described compound is the compound shown in formula II-1 or formula II-2, or the mixture of compound shown in formula II-1 and formula II-2:
3. the compound described in claim 1 or 2 contains as shown in the formula the purposes aspect the medicine of the compound shown in I or its pharmacy acceptable salt or its solvate in development or preparation:
Wherein, R is R
1cO, PO (OR
2)
2, CH
2oPO (OR
2)
2or COOR
1; R
1be straight or branched alkyl, phenyl, styryl, 4-Vinyl phenol base, 4-hydroxyl-3-methoxyl-styrene or the 3-pyridyl of 1～10 carbon; R
2for hydrogen, sodium or potassium.
4. purposes as claimed in claim 3, it is characterized in that, the compound described in claim 1 or 2 be in the time of the medicine that development or preparation contain formula 1 compound or its pharmacy acceptable salt or its solvate the drug metabolite mark that must detect quantitative and qualitative analysis.
5. the purposes as described in claim 3 or 4, it is characterized in that, the described medicine that contains formula I compound or its pharmacy acceptable salt or its solvate is for preventing and treating the disease being caused by thrombus, comprises the thrombosis after atheromatosis, myocardial infarction, apoplexy, ischemia cerebral thrombosis, peripheral arterial disease, acute coronary syndrome or coronary intervention.
6. the purposes as described in claim 3～5 any one, it is characterized in that, require compound described in 1 or 2 exposed amount in vivo can predict easily and accurately and regulate and control to contain curative effect and the bleeding risk of the medicine of formula I compound or its pharmacy acceptable salt or its solvate by test right.
7. the curative effect of medicine and the method for bleeding risk of predicting and regulating and controlling to contain formula 1 compound or its pharmacy acceptable salt or its solvate, the method is to require compound described in 1 or 2 exposed amount in vivo to measure the exposed amount of described active metabolite of drug by test right, and then prediction and regulate and control curative effect and the bleeding risk of described medicine.
8. the purposes as described in claim 3～6 any one or method claimed in claim 7, is characterized in that, described formula I compound is following compound:
9. prepare a method for the compound described in claim 1 or 2, shown in following reaction formula:
Specifically comprise the following steps:
(1) formula X compound reacts production XII compound with formula XI compound, wherein, and R
4and R
5it is respectively the straight or branched alkyl of 1～6 carbon;
(2) formula XII compound is through alkalescence or acid selective hydrolysis production II compound.
As shown in the formula the compound shown in V development or preparation contain described formula I compound or its pharmacy acceptable salt or its solvate medicine aspect purposes, it is characterized in that, compound shown in formula V is in the time of development or the described medicine of preparation, in the time carrying out in dog body that medicine generation and poison are for dynamics research the drug metabolite mark that must detect quantitative and qualitative analysis:
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310435357.8A CN103772270B (en) | 2012-10-17 | 2013-09-24 | Metabolite markers, the preparation method and the usage of the hydroxy tetrahydro thienopyridine derivative of optical activity 2 |
| PCT/CN2013/084814 WO2014059884A1 (en) | 2012-10-17 | 2013-10-07 | Marker of metabolite of optically-active 2-hydroxyltetrathienopyridine derivative, and preparation method and use thereof |
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| CN201210395415 | 2012-10-17 | ||
| CN2012103954154 | 2012-10-17 | ||
| CN201210395415.4 | 2012-10-17 | ||
| CN201310435357.8A CN103772270B (en) | 2012-10-17 | 2013-09-24 | Metabolite markers, the preparation method and the usage of the hydroxy tetrahydro thienopyridine derivative of optical activity 2 |
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| Publication Number | Publication Date |
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| CN103772270A true CN103772270A (en) | 2014-05-07 |
| CN103772270B CN103772270B (en) | 2017-12-26 |
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| WO (1) | WO2014059884A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019200512A1 (en) * | 2018-04-16 | 2019-10-24 | 江苏威凯尔医药科技有限公司 | Instant release pharmaceutical preparation of anticoagulant and preparation method therefor |
| CN111848497A (en) * | 2020-07-28 | 2020-10-30 | 内蒙古医科大学 | Clopidogrel active metabolite derivative, prodrug thereof, preparation method and application thereof |
| JP2020533401A (en) * | 2017-08-24 | 2020-11-19 | ティエンジン インスティテュート オブ ファーマシューティカル リサーチ カンパニー リミテッドＴｉａｎｊｉｎ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｒｅｓｅａｒｃｈ Ｃｏ．， Ｌｔｄ． | Thienopyridine derivative containing unsaturated aliphatic olefinic bond, its preparation method and use |
Citations (1)
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| CN102120744A (en) * | 2010-02-02 | 2011-07-13 | 江苏威凯尔医药科技有限公司 | Optical-activity 2-hydroxytetrahydrothienopyridine derivative, preparation method and application thereof in pharmacy |
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2013
- 2013-09-24 CN CN201310435357.8A patent/CN103772270B/en active Active
- 2013-10-07 WO PCT/CN2013/084814 patent/WO2014059884A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102120744A (en) * | 2010-02-02 | 2011-07-13 | 江苏威凯尔医药科技有限公司 | Optical-activity 2-hydroxytetrahydrothienopyridine derivative, preparation method and application thereof in pharmacy |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020533401A (en) * | 2017-08-24 | 2020-11-19 | ティエンジン インスティテュート オブ ファーマシューティカル リサーチ カンパニー リミテッドＴｉａｎｊｉｎ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｐｈａｒｍａｃｅｕｔｉｃａｌ Ｒｅｓｅａｒｃｈ Ｃｏ．， Ｌｔｄ． | Thienopyridine derivative containing unsaturated aliphatic olefinic bond, its preparation method and use |
| JP7052043B2 (en) | 2017-08-24 | 2022-04-11 | ティエンジン インスティテュート オブ ファーマシューティカル リサーチ カンパニー リミテッド | Thienopyridine derivative containing unsaturated aliphatic olefinic bond, its preparation method and use |
| WO2019200512A1 (en) * | 2018-04-16 | 2019-10-24 | 江苏威凯尔医药科技有限公司 | Instant release pharmaceutical preparation of anticoagulant and preparation method therefor |
| US11478432B2 (en) | 2018-04-16 | 2022-10-25 | Jiangsu Vcare Pharma Tech Co., Ltd. | Instant release pharmaceutical preparation of anticoagulant and preparation method therefor |
| US11878078B2 (en) | 2018-04-16 | 2024-01-23 | Jiangsu Vcare Pharmatech Co., Ltd. | Instant release pharmaceutical preparation of anticoagulant and preparation method therefor |
| CN111848497A (en) * | 2020-07-28 | 2020-10-30 | 内蒙古医科大学 | Clopidogrel active metabolite derivative, prodrug thereof, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2014059884A1 (en) | 2014-04-24 |
| CN103772270B (en) | 2017-12-26 |
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