CN106214681B - Application and its synthetic method of the compound on the medicine for suppressing kallikrein KLK7 is prepared - Google Patents
Application and its synthetic method of the compound on the medicine for suppressing kallikrein KLK7 is prepared Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4245—Oxadiazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/425—Thiazoles
- A61K31/426—1,3-Thiazoles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
Abstract
The invention belongs to biomedical sector, more particularly, to application of the compound on the medicine for suppressing kallikrein KLK7 is prepared and its synthetic method.Application of the compound on the medicine for suppressing kallikrein KLK7 is prepared, the structural formula of the compound are：The compound provided by the invention for suppressing kallikrein KLK7 can effectively suppress the activity of KLK7, its synthetic method is simple and practicable, and the pharmaceutical preparation for suppressing KLK7 activity can be prepared into by adding appropriate auxiliary material and auxiliary agent.
Description
Technical field
The invention belongs to biomedical sector, and the medicine of suppression kallikrein KLK7 is being prepared more particularly, to compound
On application and its synthetic method.
Background technology
Atopic dermatitis (Atopic Dermatitis, AD) is a kind of chronic inflammatory skin of wide-scale distribution, feature
It is drying, flaky skin, inflammation, percutaneous permeability increase, the skin of patient AD is to common and harmless to normal person in environment
Factor also can be very sensitive so as to being susceptible to surface infection.In the hair of the atopic dermatitis of past 30 Nian Jian industrialized countries
Sick rate increases 2 to 3 times, it is estimated that the atopic dermatitis illness rate of children has reached 15-30%, and the atopic dermatitis of adult is suffered from
Sick rate has reached 2-10%.Due to the complexity of pathogenesis, few, but skin barrier function is solved to the pathogenesis of AD
Disorder has proven to be one of key factor in AD evolutions.The stabilization of skin barrier depends on the cutin shape of stratum granulosum
Into cell differentiation (i.e. furfur) is come off for horn cell and skin surface horn cell between balance, the furfur process bag of skin
Include by the degraded of the KLKs desmosomes participated in, since desmosome is the Fibronectin of horn cell, the degraded of desmosome can then cause cutin
Cell detachment and cause the desquamation of skin.Evidence shows that the imbalance of local or temporary KLKs activity control, is AD patient skin screens
Barrier stablizes one of the main reason for missing.
KLKs (Kallikreins, kallikrein) is with trypsase or the specific silk ammonia of chymotrypsin
Pepsin, its encoding gene are positioned at 19q13.4, encode 15 secreting type serine protease KLK 1-KLK 15.
Proving, KLKs is present in the corneocyte of normal person, has now been found that, at least 8 kinds of KLK (KLK 5, KLK 6, KLK 7,
KLK 8, KLK 10, KLK 11, KLK 13 and KLK 14) it is present in the cuticula and sweat of people, wherein KLK7 accounts for total amount
40%.KLK7 is probably a key enzyme of skin injury, and in patient AD it may to participate in a Systemic inflammation anti-
Should, it is the most prominent in the cuticula KLK7 rises of AD, this prompting KLK7 be probably in the pathogenesis of AD it is vital, because
This, suppresses treatment of the activity of KLK7 for AD and seems mostly important.
LEKTI (Lympho-epithelial Kazal-type-related inhibitor, LEKTI) is by SPINK5
The member of the serpin family of gene code, LEKTI precursors include a signal peptide and be spaced apart 15
A serine protease suppresses domain (D1-D15), several protein fragments is hydrolyzed to by furin rapidly after expression, wherein wrapping
The LEKTI in the domain containing D6-D9 suppression can suppress the activity of the enzymes such as trypsase, chymotrypsin, KLK5 and KLK7.LEKTI's
Endogenous target includes at least KLK5, KLK7 and KLK14, they participate in the furfur process of skin jointly.In patient AD, LEKTI expression
The horizontal serious abnormal rise for reducing, result in KLK7 activity.Although LEKTI is the natural inhibitor of KLK7 in epidermis, make
To treat the externally applied drug of AD, it cannot pass through keratoderma, and the characteristic of its macro-molecular protein also results in absorption and expands
The problem of scattered speed is excessively slow, degradation speed is too fast, thus act on very limited.Up to the present, also without a kind of effective
KLK7 inhibitor is applied to clinic.
The content of the invention
Based on this, the present invention provides application and its synthesis of the compound on the medicine for suppressing kallikrein KLK7 is prepared
Method.
Technical solution of the present invention is：
Application of the compound on the medicine for suppressing kallikrein KLK7 is prepared, the structural formula of the compound are：
The method of the synthesis compound, the synthetic route are as follows：
Wherein：
The synthesis of material three：Ethanol, material one, the formalin of mass concentration 37%, hydrogen-oxygen are added into reaction bulb
Change sodium and material two, reaction, reaction finishes, and ethyl acetate extracts three times, and the dosage of each ethyl acetate is foregoing ethanol body
Long-pending 1~5 times, merges organic phase, distills, and cools down, and filters, and washing, ethanol washes, dry material three；
The synthesis of material four：Material three, aceticanhydride are added into reaction bulb, controlled at -12~-10 DEG C, quality is added dropwise
The fuming nitric aicd that concentration is 90% reacts, and reaction finishes, rises to 25 DEG C, pour into frozen water, and the volume of frozen water is reaction solution 5-10
Times, filtering, is first washed, then washed with frozen water with 1~5% sodium bicarbonate solution, dry material four；
The synthesis of material five：Addition ethanol, water, acetic acid, iron powder, ammonium chloride and material four into reaction bulb, back flow reaction,
Ethanol is added, second of quality for adding ethanol is 5~10 times of material four, is filtered while hot, filtrate is cooled to 25 DEG C, pours into
In frozen water, the volume of frozen water is 10~20 times of filtrate, is filtered, washing, dry material five；
The synthesis of material seven：Material five, material six, dimethylformamide, potassium carbonate are added into reaction bulb, reaction is cold
But to 25 DEG C, reaction solution is added in frozen water, the quality of frozen water is 10~20 times of reaction solution, is filtered, washing, dry thing
Matter seven.
The synthesis of material eight：The mixture of material seven and pyridine is added into reaction bulb, then phosphorus oxychloride is added dropwise, heating is anti-
Should, reaction solution is cooled down, adds chloroform, is stirred, cooling, adds cold water, separates organic phase, cold water washes chloroform layer as neutrality, dense
Contracting removes chloroform, dry material eight.
The synthesis of compound I：Dimethylformamide, material eight, potassium carbonate and material nine are sequentially added in reaction bulb, added
Thermal response, cooling, reaction solution is poured into water, and is filtered, filter cake water wash, dry compound I.
Preferably, the matter of the material two, the formalin of mass concentration 37%, material one, sodium hydroxide and ethanol
Amount is than being 1:1~3:1~3:1~2:When reaction 2-6 is small at 5~30,25~60 DEG C.
Preferably, the material three, aceticanhydride, the mass ratio of nitric acid are 1.0:0.5~5.0:0.5~2.0, -5~-10
When reaction 4-8 is small at DEG C.
Preferably, the material four, ethanol, water, acetic acid, iron powder, ammonium chloride mass ratio are 1:2.5~5.0:2.0~
5.0:0.05~0.15:1.0~2.0:0.02~0.1, when back flow reaction 2-6 is small after.
Preferably, the material five, material six, potassium carbonate, the molar ratio of dimethylformamide are 1.0:1.0~2.0:
1.0~1.5:10.0~30.0, be warming up to 80-100 DEG C reaction 4-6 it is small when.
Preferably, the material seven, pyridine, the molar ratio of phosphorus oxychloride are 1.0:1.0~1.5:1.5~2.5, finish,
Be warming up to 100~120 DEG C of reaction 3-6 it is small when, reaction solution is cooled to 50 DEG C, adds chloroform, the volume of chloroform is the 40 of pyridine
~60 times, when stirring 1~2 is small, 0~5 DEG C is cooled to, adds 5~10 DEG C of cold water, the volume of cold water is the 0.6~2.0 of chloroform
Times, separate organic phase, cold water washes chloroform layer as neutrality, and concentration removes chloroform, dry material eight.
Preferably, the material eight, material nine, potassium carbonate, the molar ratio of dimethylformamide are 1.0:2.0~4.0:
2.0~4.0:20.0~50.0,50~80 DEG C are heated to, reacts 1~4h, cooling, reaction solution is poured into water, the volume of water is
10~20 times of reaction solution, filter, filter cake water wash, dry compound I.
The application of the compound or the method on the medicine for suppressing kallikrein KLK7 is prepared, it is described to prepare suppression
The medicine of application on the medicine of kallikrein KLK7 processed includes：
In one or more of mixtures.
Preferably, the medicine for preparing the application on the medicine for suppressing kallikrein KLK7 includes：Compound I, change
Compound II, compound III, the molar ratio of compound IV and compound V are 1:1~4:1~5：1~3：1~2.
Advantage of the present invention：
1st, the compound provided by the invention for suppressing kallikrein KLK7 can effectively suppress the activity of KLK7, it is closed
Simple and practicable into method, the pharmaceutical preparation for suppressing KLK7 activity can be prepared into by adding appropriate auxiliary material and auxiliary agent.
2nd, lacked using what synthetic method accessory substance of the present invention produced, impurity content as low as 0.05%~0.08%.
3rd, the compounds of this invention and the combination of other compounds, it is more efficient for the activity suppression of kallikrein KLK7.
4th, the serine protease mainly expressed in skin further includes KLK5, KLK14, Matriptase with
Elastase2.The compounds of this invention compound is constant under 100 μM of concentration to above-mentioned serine protease to reveal suppression work
Property, show that its KLK7 of formula has good specificity.
5th, the compounds of this invention is in pH8,150mM NaCl, and 72h is incubated in 37 DEG C of buffer solution, its suppression to KLK7 is lived
Property remain unchanged, show that the suppression of the compounds of this invention is efficient, activity is strong, and its inhibition is stablized.
Brief description of the drawings
Fig. 1：The concentration of the compounds of this invention I and the relation curve of inhibiting rate.
Fig. 2：For the H of the compounds of this invention I1Nuclear-magnetism figure.
Fig. 3：For the C of the compounds of this invention I13Nuclear-magnetism figure.
Fig. 4：7 compound concentration of embodiment and inhibiting rate relation curve.
Embodiment
The present invention is further illustrated with reference to embodiment, but the scope of protection of present invention is not limited to implement
The scope of example statement.
Embodiment 1
Application of the compound on the medicine for suppressing kallikrein KLK7 is prepared, the structural formula of the compound are：
Embodiment 2
The method of compound described in synthetic example 1, the synthetic route are as follows：
Wherein：
The synthesis of material three：Ethanol, material one, the formalin of mass concentration 37%, hydrogen-oxygen are added into reaction bulb
Change sodium and material two, reaction, reaction finishes, and ethyl acetate extracts three times, and the dosage of each ethyl acetate is foregoing ethanol body
Long-pending 1~5 times, merges organic phase, distills, and cools down, and filters, and washing, ethanol washes, dry material three；
The synthesis of material four：Material three, aceticanhydride are added into reaction bulb, controlled at -12 DEG C, mass concentration, which is added dropwise, is
90% fuming nitric aicd reaction, reaction finishes, rises to 25 DEG C, pour into frozen water, and the volume of frozen water is 5-10 times of reaction solution, mistake
Filter, is first washed, then washed with frozen water with 1~5% sodium bicarbonate solution, dry material four；
The synthesis of material five：Addition ethanol, water, acetic acid, iron powder, ammonium chloride and material four into reaction bulb, back flow reaction,
Ethanol is added, second of quality for adding ethanol is 5 times of material four, is filtered while hot, filtrate is cooled to 25 DEG C, pours into frozen water
In, the volume of frozen water is 10 times of filtrate, is filtered, washing, dry material five；
The synthesis of material seven：Material five, material six, dimethylformamide, potassium carbonate are added into reaction bulb, reaction is cold
But to 25 DEG C, reaction solution is added in frozen water, the quality of frozen water is 10 times of reaction solution, is filtered, washing, dry material
Seven.
The synthesis of material eight：The mixture of material seven and pyridine is added into reaction bulb, then phosphorus oxychloride is added dropwise, heating is anti-
Should, reaction solution is cooled down, adds chloroform, is stirred, cooling, adds cold water, separates organic phase, cold water washes chloroform layer as neutrality, dense
Contracting removes chloroform, dry material eight.
The synthesis of compound I：Dimethylformamide, material eight, potassium carbonate and material nine are sequentially added in reaction bulb, added
Thermal response, cooling, reaction solution is poured into water, and is filtered, filter cake water wash, dry compound I.
The material two, the formalin of mass concentration 37%, material one, the mass ratio of sodium hydroxide and ethanol are 1:
1:1:1:When reacting 2 at 5,25 DEG C.
The material three, aceticanhydride, the mass ratio of nitric acid are 1:0.5:0.5, when reaction 4 is small at -5 DEG C.
The material four, ethanol, water, acetic acid, iron powder, ammonium chloride mass ratio are 1:2.5:2:0.05:1:0.02, reflux is anti-
Answer 2 it is small when.
The material five, material six, potassium carbonate, the molar ratio of dimethylformamide are 1:1:1:10, it is warming up to 80 DEG C instead
Answer 4 it is small when.
The material seven, pyridine, the molar ratio of phosphorus oxychloride are 1:1:1.5, finish, be warming up to 100 DEG C reaction 3 it is small when,
Reaction solution is cooled to 50 DEG C, adds chloroform, the volume of chloroform is 40 times of pyridine, when stirring 1 is small, is cooled to 0 DEG C, adds 5
DEG C cold water, the volume of cold water is 0.6 times of chloroform, separates organic phase, and cold water washes chloroform layer as neutrality, and concentration removes chloroform,
Dry material eight.
The material eight, material nine, potassium carbonate, the molar ratio of dimethylformamide are 1:2:2:20,50 DEG C are heated to, instead
1h is answered, cools down, reaction solution is poured into water, the volume of water is 10 times of reaction solution, is filtered, filter cake water wash is dry to change
Compound I.
Embodiment 3
The method of compound described in synthetic example 1, the synthetic route is described in embodiment 2：
The synthesis of material three：Ethanol, material one, the formalin of mass concentration 37%, hydrogen-oxygen are added into reaction bulb
Change sodium and material two, reaction, reaction finishes, and ethyl acetate extracts three times, and the dosage of each ethyl acetate is foregoing ethanol body
Long-pending 5 times, merge organic phase, distill, and cool down, and filter, and washing, ethanol washes, dry material three；
The synthesis of material four：Material three, aceticanhydride are added into reaction bulb, controlled at -10 DEG C, mass concentration, which is added dropwise, is
90% fuming nitric aicd reaction, reaction finish, rise to 25 DEG C, pour into frozen water, and the volume of frozen water is 10 times of reaction solution, filtering,
First washed with 1~5% sodium bicarbonate solution, then washed with frozen water, dry material four；
The synthesis of material five：Addition ethanol, water, acetic acid, iron powder, ammonium chloride and material four into reaction bulb, back flow reaction,
Ethanol is added, second of quality for adding ethanol is 10 times of material four, is filtered while hot, filtrate is cooled to 25 DEG C, pours into ice
In water, the volume of frozen water is 20 times of filtrate, is filtered, washing, dry material five；
The synthesis of material seven：Material five, material six, dimethylformamide, potassium carbonate are added into reaction bulb, is cooled to 25
DEG C, reaction solution is added in frozen water, the quality of frozen water is 20 times of reaction solution, is filtered, washing, dry material seven.
The synthesis of material eight：The mixture of material seven and pyridine is added into reaction bulb, then phosphorus oxychloride is added dropwise, heating is anti-
Should, reaction solution is cooled down, adds chloroform, is stirred, cooling, adds cold water, separates organic phase, cold water washes chloroform layer as neutrality, dense
Contracting removes chloroform, dry material eight.
The synthesis of compound I：Dimethylformamide, material eight, potassium carbonate and material nine are sequentially added in reaction bulb, added
Thermal response, cooling, reaction solution is poured into water, and is filtered, filter cake water wash, dry compound I.
The material two, the formalin of mass concentration 37%, material one, the mass ratio of sodium hydroxide and ethanol are 1:
3:3:2:When reaction 6 is small at 30,60 DEG C.
The material three, aceticanhydride, the mass ratio of nitric acid are 1:5:2, when reaction 8 is small at -10 DEG C.
The material four, ethanol, water, acetic acid, iron powder, ammonium chloride mass ratio are 1:5:5:0.15:2:0.1, back flow reaction 6
After hour.
The material five, material six, potassium carbonate, the molar ratio of dimethylformamide are 1:2:1.5:30, it is warming up to 100 DEG C
React 6 it is small when.
The material seven, pyridine, the molar ratio of phosphorus oxychloride are 1:1.5:2.5, finish, it is small to be warming up to 120 DEG C of reactions 6
When, reaction solution is cooled to 50 DEG C, adds chloroform, the volume of chloroform is 60 times of pyridine, when stirring 2 is small, is cooled to 5 DEG C, adds
Enter 10 DEG C of cold water, the volume of cold water is 2 times of chloroform, separates organic phase, and cold water washes chloroform layer as neutrality, and concentration removes dechlorination
It is imitative, dry material eight.
The material eight, material nine, potassium carbonate, the molar ratio of dimethylformamide are 1:4:4:50,80 DEG C are heated to, instead
4h is answered, cools down, reaction solution is poured into water, the volume of water is 20 times of reaction solution, is filtered, filter cake water wash is dry to change
Compound I.
Embodiment 4
The method of compound described in synthetic example 1, the synthetic route is described in embodiment 2：
The synthesis of material three：Ethanol, material one, the formalin of mass concentration 37%, hydrogen-oxygen are added into reaction bulb
Change sodium and material two, reaction, reaction finishes, and ethyl acetate extracts three times, and the dosage of each ethyl acetate is foregoing ethanol body
Long-pending 2.5 times, merge organic phase, distill, and cool down, and filter, and washing, ethanol washes, dry material three；
The synthesis of material four：Material three, aceticanhydride are added into reaction bulb, controlled at -11 DEG C, mass concentration, which is added dropwise, is
90% fuming nitric aicd reaction, reaction finish, rise to 25 DEG C, pour into frozen water, and the volume of frozen water is 8 times of reaction solution, filtering, first
Washed with 4% sodium bicarbonate solution, then washed with frozen water, dry material four；
The synthesis of material five：Addition ethanol, water, acetic acid, iron powder, ammonium chloride and material four into reaction bulb, back flow reaction,
Ethanol is added, second of quality for adding ethanol is 8 times of material four, is filtered while hot, filtrate is cooled to 25 DEG C, pours into frozen water
In, the volume of frozen water is 15 times of filtrate, is filtered, washing, dry material five；
The synthesis of material seven：Material five, material six, dimethylformamide, potassium carbonate are added into reaction bulb, reaction is cold
But to 25 DEG C, reaction solution is added in frozen water, the quality of frozen water is 15 times of reaction solution, is filtered, washing, dry material
Seven.
The synthesis of material eight：The mixture of material seven and pyridine is added into reaction bulb, then phosphorus oxychloride is added dropwise, heating is anti-
Should, reaction solution is cooled down, adds chloroform, is stirred, cooling, adds cold water, separates organic phase, cold water washes chloroform layer as neutrality, dense
Contracting removes chloroform, dry material eight.
The synthesis of compound I：Dimethylformamide, material eight, potassium carbonate and material nine are sequentially added in reaction bulb, added
Thermal response, cooling, reaction solution is poured into water, and is filtered, filter cake water wash, dry compound I.
The material two, the formalin of mass concentration 37%, material one, the mass ratio of sodium hydroxide and ethanol are 1:
2:2:1.5:When reaction 5 is small at 25,25~60 DEG C.
The material three, aceticanhydride, the mass ratio of nitric acid are 1:3:1.5, when reaction 6 is small at -8 DEG C.
The material four, ethanol, water, acetic acid, iron powder, ammonium chloride mass ratio are 1:3:3:0.1:1.5:0.08, reflux is anti-
Answer 5 it is small when after.
The material five, material six, potassium carbonate, the molar ratio of dimethylformamide are 1:1.8:1.3:20, it is warming up to 95
DEG C reaction 5 it is small when.
The material seven, pyridine, the molar ratio of phosphorus oxychloride are 1:1.2:1.8 finishing, it is small to be warming up to 110 DEG C of reactions 5
When, reaction solution is cooled to 50 DEG C, adds chloroform, the volume of chloroform is 50 times of pyridine, when stirring 1.5 is small, is cooled to 4 DEG C,
8 DEG C of cold water is added, the volume of cold water is 0.8 times of chloroform, separates organic phase, and cold water washes chloroform layer as neutrality, and concentration removes
Chloroform, dry material eight.
The material eight, material nine, potassium carbonate, the molar ratio of dimethylformamide are 1:3:3:30,60 DEG C are heated to, instead
3h is answered, cools down, reaction solution is poured into water, the volume of water is 15 times of reaction solution, is filtered, filter cake water wash is dry to change
Compound I.
Embodiment 5
1) compound I is dissolved in DMSO, is configured to the mother liquor of concentration 10mM；
2) compound shown in Formulas I is added in KLK7 activity buffer liquids, is configured to 6.25 μM, 12.5 μM, 25 μM, 50 μM,
100 μM, 200 μM of solution, and acted on 15 minutes at 37 DEG C；
3) fluorogenic substrate MCa-R-P-K-P-V-E-Nval-W-R-K (Dnp)-NH is added2, the work of survey KLK7 at 37 DEG C
Property 15 minutes, using fluorescence microplate reader detect fluorescent value, according to the size of fluorescent value calculate KLK7 activity.
Negative control group：DMSO is added in the activity buffer liquid of KLK7, other operating conditions are identical with experimental group.
The activity of the KLK7 obtained according to above-mentioned experimental group and negative control group calculates inhibiting rates of the compound I to KLK7,
The formula for calculating inhibiting rate is %inhibition=(1-a/b) × 100%, and result of calculation is as shown in table 1；Wherein, a is to be measured
The enzyme activity of sample, b are the enzyme activity of negative control.
1 compound I of table to the inhibiting rate of KLK7 activity under various concentrations, draw shown in Formulas I by the result according to table 1
The concentration of compound and the graph of a relation of inhibiting rate, carry out curve fitting to obtain chemical combination shown in Formulas I using KaleidaGraph softwares
The concentration of thing and the relation curve of inhibiting rate, as shown in Figure 1.
With the increase of compound I thing concentration it can be seen from the result of table 1 and Fig. 1, to the inhibiting rate of KLK7 activity by
Cumulative height, when the concentration of compound I is 200 μM, reaches 100% to the inhibiting rate of KLK7 activity.
Therefore, compound I has KLK7 excellent inhibition, which adds suitably auxiliary as active component
Material and auxiliary agent can be prepared into the pharmaceutical preparation for suppressing KLK7 activity.
Compound I is under various concentrations to the inhibiting rate of KLK7 activity
The preparation method letter of the compound provided by the invention for suppressing kallikrein KLK7 it can be seen from foregoing description
It is single easy, it can effectively suppress the activity of KLK7, the medicine for suppressing KLK7 activity can be prepared into by adding appropriate auxiliary material and auxiliary agent
Thing preparation.
Embodiment 6
The medicine for preparing the application on the medicine for suppressing kallikrein KLK7 includes：
The compound I, compound II, compound III, the molar ratio of compound IV and compound V are 1:1~4:1~
5：1~3：1~2.
Embodiment 7
It is 1 in molar ratio in 6 composition of embodiment:1:1：1：After 1 mixing, tested according to the method for embodiment 5 different
To the inhibiting rate of KLK7 activity under concentration, 2 and Fig. 4 are the results are shown in Table.
2 mixture of table is under various concentrations to the inhibiting rate of KLK7 activity
By table 2 the results show that formula compound I, compound II, compound III, compound IV and compound V combination effects
It is more notable than effect is used alone, in the case of same additive amount, to KLK7 when the drug effect of mixture is than being used alone
Maximum inhibition higher, it may be possible to which several molecules in inhibiting mechanism are different, and collaboration use dramatically increases drug effect.
Embodiment 8
The method for synthesizing compound II, the synthetic route are as follows：
Wherein：
The synthesis of material 12：By material ten, material 11, POCl3Add in the round-bottomed flask equipped with reflux condensing tube,
Round-bottomed flask is positioned over to be cleaned by ultrasonic in instrument and is reacted, decompression steams remaining POCl3, residue is poured into trash ice, trash ice
Quality is 20 times of residue, separates out precipitation, is filtered, and solid is washed with 1~5% NaOH solution, is washed to neutrality, obtains material
12；
The synthesis of material 13：Adding dichloromethane and material 12 in reaction bulb, salt ice bath is cooled to -15 DEG C, lucifuge,
Under nitrogen protection, the anhydrous methylene chloride solution of 20wt% Boron tribromides is added dropwise, after dripping, temperature reaction, reaction knot
Reaction solution is poured into frozen water after beam, the quality of frozen water is 5 times of reaction solution, separates organic phase, water is mutually extracted with ethyl acetate
Three times, the volume of each ethyl acetate is 0.5 times of water phase, merges organic phase, and organic phase is dried with sodium sulphate, steams solvent,
Obtain material 13；
The synthesis of material 14：Monoxone is mixed into obtain solution A with 5wt% sodium hydrate aqueous solutions, by material 13 with
5wt% sodium hydrate aqueous solutions mix to obtain solution B, and solution A is added in reaction bulb, then solution B is added dropwise, and are added dropwise, and rise
Temperature reaction, cooling, adds 5wt% hydrochloric acid to be acidified to pH=1, filtering, washing, dry material 14；
The synthesis of material 15：Connect in reaction bulb equipped with stirring rod, thermometer, reflux condensing tube, its reflux condensing tube
Hydrogen chloride absorption device is connect, sequentially adds thionyl chloride and material 14, stirs temperature reaction, depressurizes and steams low-boiling-point substance, in bottle
Residue is material 15, is directly used in the next step without isolation；
The synthesis of compound II：Material 15 made from step, adds triethylamine, dichloromethane on being added in reaction bulb
With material 16, stirring reaction, decompression steams solvent, cools down, and filters, and washing, acetone washes to obtain compound II.
The material ten, material 11, POCl3Mass ratio be 1:1:3, it is 50 DEG C to be cleaned by ultrasonic reaction temperature in instrument,
Power is 350W, ultrasonication 60 minutes.
The dichloromethane, material 12,20wt% Boron tribromides dichloromethane solution mass ratio be 20:1:6, rise
When reaction 2 is small at warm to 25 DEG C, 25 DEG C.
The molar ratio of monoxone and sodium hydroxide is 1 in the solution A:1, material 13 and hydroxide in the solution B
The molar ratio of sodium is 1:1, solution A is added in reaction bulb, then solution B is added dropwise, be added to material 13 in reaction bulb with
Chloroacetic molar ratio is 1:1, be warming up to 60 DEG C reaction 1 it is small when, be cooled to 25 DEG C.
The molar ratio of the material 14 and thionyl chloride is 1:1, stirring is warming up to 60 DEG C, when reaction 5 is small at 60 DEG C.
The material 15, material 16, the molar ratio 1 of triethylamine and dichloromethane:1:1:5, stir 2 at 0~25 DEG C
Hour.
Embodiment 9
The method for synthesizing compound III, the synthetic route are as follows：
Wherein：
The synthesis of material 18：Material 17, KOH, water, carbon disulfide are added to reaction bulb, it is heated to flowing back, instead
Answer 2 it is small when, be cooled to 25 DEG C, filtering, filtrate separates out material 18 with 1wt% salt acid for adjusting pH value, filtering, washing, dry thing
Matter 18；
The synthesis of material 20：Addition material 18, material 19, toluene and potassium carbonate into reaction bulb, back flow reaction,
Cold filtration, washes solid, filtrate decompression concentration, residue silica gel column chromatography, hexane-ethylacetate elution, obtains with dichloromethane
Material 20.
The synthesis of compound III：Material 20,4- fluorine benzyl chloride, NaOH, acetonitrile are added into reaction bulb, back flow reaction is cold
But, decompression steams solvent and obtains residue, and residue with Ethyl acetate extracts three times, and the volume of each ethyl acetate is residue
2 times, organic phase washing, then dried with anhydrous sodium sulfate, filter, filtrate decompression steams solvent afforded crude material, and crude product is tied again with acetonitrile
It is brilliant to obtain compound III.
The material 17, carbon disulfide, KOH, the molar ratio of water are 1:1:1:5.
The material 18, material 19, potassium carbonate, the molar ratio of toluene are 1:1:1:5, back flow reaction, cools down, described
Hexane-ethylacetate volume ratio is 2:1.
When reflux time is 4 small during the material 20 synthesizes, 25 DEG C are cooled to.
The material 20,4- fluorine benzyl chloride, NaOH, the molar ratio of acetonitrile are 1:1:1:10, when back flow reaction 4 is small.
Embodiment 10
The method for synthesizing compounds Ⅳ, the synthetic route are as follows：
Wherein：
The synthesis of material 23：Material 21, material 22, glacial acetic acid are added to reaction bulb, are heated to back
When stream reaction 4-~8 are small, cool down, filter, washing, dichloromethane is washed, and obtains material 23；
The synthesis of material 24：The mixture of material 23 and pyridine is added into reaction bulb, then trichlorine oxygen is added dropwise
Phosphorus, finishes, temperature reaction, and reaction solution is cooled to 50 DEG C after reaction, adds chloroform, stirring reaction, is cooled to 5 DEG C, adds
Enter cold water, separate organic phase, cold water washes chloroform layer as neutrality, and concentration removes chloroform, dry material 24；
The synthesis of compounds Ⅳ：Material 24, material 25, toluene, potassium carbonate are added into reaction bulb, reflux is anti-
Answer 4 it is small when, be cooled to 25 DEG C, filtering, washes solid, filtrate decompression is concentrated to give crude product, and crude product is obtained with Gossypol recrystallized from chloroform with dichloromethane
Compounds Ⅳ.
The material 21, material 22, the molar ratio of glacial acetic acid are 1:1:1.5, when back flow reaction 4 is small, cooling
To 25 DEG C.
The material 23, pyridine, the molar ratio of phosphorus oxychloride are 1:1:1.5, be warming up to 110 DEG C reaction 2 it is small when, will
Reaction solution is cooled to 50 DEG C, adds chloroform, and the volume of the chloroform is 30 times of pyridine, when stirring reaction 1 is small, is cooled to 0 DEG C,
5 DEG C of cold water is added, the volume of cold water is 0.5 times of chloroform.
Embodiment 11
The technique for synthesizing compound V, the synthetic route are as follows：
Wherein：
The synthesis of material 28：Material 26 and material 27 are added in dry round-bottomed flask, heating carries out
Stirring reaction, thin-layer chromatography tracking reaction to raw material disappear, mixture are cooled to 25 DEG C, is poured into mixture of ice and water, filter,
Washing, chloroform are washed, and obtain material 28；
The synthesis of material 29:Ether, lithium aluminium hydride, stirring and dissolving, ice-water bath successively are added in reaction bulb and is cooled to 0
DEG C, material 28 is slow added into, charging finishes, and is reacted under ice-water bath, reaction solution is poured into frozen water, and standing allows sediment
Bottom is parked in, is filtered, chloroform filter wash slag, filtrate separates organic phase, and water is mutually extracted three times with chloroform, and organic phase merges, and decompression is dense
Contract to obtain material 29.
The synthesis of compound V：Material 29, triethylamine, dichloromethane and material 30 are added in the reaction, and stirring is anti-
Should, concentration removes dichloromethane, then adds water, adds water quality for 10 times of concentrate, with chloroform extraction three times, extractant chloroform
Each dosage is 1 times of the volume of foregoing added water, and organic phase merges, and be concentrated under reduced pressure to obtain crude product, and crude product is obtained with Gossypol recrystallized from chloroform
Compound V.
The material 26, the molar ratio of material 27 are 1:1. rising to 80 DEG C is stirred reaction, the frozen water
The quality of mixture is 5 times of material 26.
The material 28, lithium aluminium hydride, the mass ratio of ether are 1:0.5:15, charging rate is advisable with micro- reflux,
When reaction 1.5 is small under the ice-water bath, reaction solution is poured into frozen water, the volume of frozen water is 1 times of reaction solution, extractant chloroform
Each dosage is 1 times of water phase volume.
The material 29, material 30, the molar ratio of triethylamine and dichloromethane are 1:1:1:10,25 DEG C of stirrings
1.5 it is small when.
The above embodiments are only the preferred technical solution of the present invention, and are not construed as the limitation for the present invention, this Shen
Please in embodiment and embodiment in feature in the case where there is no conflict, can mutually be combined.The protection model of the present invention
Enclose the equivalent substitution side of technical characteristic in the technical solution that should be recorded with claim, including the technical solution of claim record
Case is protection domain.Equivalent substitution i.e. within this range is improved, also within protection scope of the present invention.
Claims (2)
1. application of the compound on the medicine for suppressing kallikrein KLK7 is prepared, it is characterised in that the structure of the compound
Formula is：
2. application according to claim 1, it is characterised in that described to prepare on the medicine for suppressing kallikrein KLK7
The medicine of application includes：Compound I and one or more of mixtures in compound II, compound III and compound V；Institute
It is as follows to state compound II, compound III, compound V structure formula：
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| US20030078255A1 (en) * | 2001-03-23 | 2003-04-24 | Pinto Donald Joseph Phillip | 6-5, 6-6, or 6-7 Heterobicycles as factor xa inhibitors |
| WO2003047520A2 (en) * | 2001-12-04 | 2003-06-12 | Bristol-Myers Squibb Company | SUBSTITUTED AMINO METHYL FACTOR Xa INHIBITORS |
| CN103080104A (en) * | 2010-08-04 | 2013-05-01 | 诺瓦提斯公司 | N-((6-amino-pyridin-3-yl)methyl)-heteroaryl-carboxamides as inhibitors of plasma kallikrein |
| WO2015171527A1 (en) * | 2014-05-05 | 2015-11-12 | Global Blood Therapeutics, Inc. | Pyrazolopyridine pyrazolopyrimidine and related compounds |
| CN105330665A (en) * | 2015-11-04 | 2016-02-17 | 衡阳师范学院 | Kallikrein KLK7 inhibiting compound and preparation method and application thereof |
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| US20030078255A1 (en) * | 2001-03-23 | 2003-04-24 | Pinto Donald Joseph Phillip | 6-5, 6-6, or 6-7 Heterobicycles as factor xa inhibitors |
| WO2003047520A2 (en) * | 2001-12-04 | 2003-06-12 | Bristol-Myers Squibb Company | SUBSTITUTED AMINO METHYL FACTOR Xa INHIBITORS |
| CN103080104A (en) * | 2010-08-04 | 2013-05-01 | 诺瓦提斯公司 | N-((6-amino-pyridin-3-yl)methyl)-heteroaryl-carboxamides as inhibitors of plasma kallikrein |
| WO2015171527A1 (en) * | 2014-05-05 | 2015-11-12 | Global Blood Therapeutics, Inc. | Pyrazolopyridine pyrazolopyrimidine and related compounds |
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