CN106565685B - Antitubulin - Google Patents
Antitubulin Download PDFInfo
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- CN106565685B CN106565685B CN201610889855.3A CN201610889855A CN106565685B CN 106565685 B CN106565685 B CN 106565685B CN 201610889855 A CN201610889855 A CN 201610889855A CN 106565685 B CN106565685 B CN 106565685B
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- propylene subunit
- piperazine
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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
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
- C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
Abstract
Of the invention provides a kind of new Antitubulin and its application, which is a series of compounds based on substituted heterocyclic skeleton, using the colchicin binding site in tubulin as target.It has a structure in whichWherein:
Description
Technical field
The present invention relates to drug field, relate in particular to as Antitubulin new series compound and its
Using.
The present invention also relates to the midbody compounds for preparing the compound.
The invention further relates to the pharmaceutical compositions containing the compound.
Background technique
As the main means of oncotherapy, anti-tumor drug is to extend the life span of patient and improve its life matter
Amount is made that comparable contribution.The drug (microtubule inhibitors) of micro-pipe is wherein acted on, and has in tumour medicine and quite weighs
The status wanted.But current clinical medicine, influenced by following bad problem: poor water solubility, be unfavorable for administration and
Allergic reaction, serious toxic side effect and the acquired resistance easily caused causes curative effect reduction, chemical structure complexity to be difficult to
It synthesizes and causes source rare, limit their further uses.Thus, it is badly in need of finding and designs novel tubulin inhibition
The simple micromolecular inhibitor of agent, especially structure.
Micro-pipe (microtubule) is the important component of eukaryocyte, is also important anti-tumor drug action target.It is micro-
Pipe is the chief component of cytoskeleton, is made of alpha-tubulin and 'beta '-tubulin heterodimer, has hollow tubular
The characteristics of structure.In addition, there are also a kind of γ tubulins, it is not the constituent of micro-pipe, but participates in the assembling of micro-pipe.
Micro-pipe has the kinetic characteristics of polymerization and depolymerization, conveys in cellular morphology, cell division, signal transduction and substance
It plays an important role Deng during.Micro-pipe becomes spindle in prophase of cell division polymerization, and spindle is led in mitosis
Draw chromosome to move into two daughter cells to the two poles of the earth, completes cell Proliferation.Since micro-pipe has extremely in cell division
Important role has become one of the important target spot of anti-tumor drug research.The tubulin for acting on microtubule system inhibits
Agent also has become a kind of effective antitumour drug.
There are two types of classification methods for Antitubulin.One is be divided into two types according to the difference of mechanism of action: 1.
Inhibit the tubulin depolymerizing agent of tubulin polymerization；2. promoting the Tubulin polymerization agent of tubulin polymerization.Another kind point
Class method is to be divided into 3 classes according to the difference of Antitubulin and tubulin action site: 1. acting on colchicin position
The Antitubulin of point；2. acting on the Antitubulin in vincaleukoblastinum site；3. acting on the micro- of taxol site
Tubulin inhibitor.
Existing research shows that in tubulin (tubulin), there are three main drug binding sites: taxol combines
Site (Taxol site), vincaleukoblastinum binding site (Vinblastine sitc) and colchicin binding site
(Colchieinesite).But in these three sites, colchicin site is conducive to due to the small volume of itself binding cavity
The research of small molecule, anti-tumor inhibitor.
Chinese patent application CN 1684955 is related to the compound dehydrogenation phenyl A Xi of a kind for the treatment of cancer and fungal infection
This fourth (plinabulin) (Formula II), is cell cycle inhibitor, as tumor growth inhibitors or fungistat.
Chinese patent application CN1934101A is related to the purposes of above compound, close for reducing blood vessel hyperplasia and blood vessel
Degree acts on tumor vessel, but plinabulin exclusive use is weaker to the inhibiting effect of tumour, dry to the function of immune system
It disturbs larger.
Summary of the invention
It is an object of the present invention to provide a kind of new Antitubulins, are based on substituted heterocycle to be a series of
The compound of skeleton, using the colchicin binding site in tubulin as target.
By the structural property for binding site of analyzing and researching, and the combination of itself and inhibitor is simulated, determines each subprovince domain
Property, key effect residue and potential binding site；Start with from the shared group in inhibitor structure, gradually expands shared
The property of group, to assume the stay in place form of inhibitor.It is ground again from activity conformation and in conjunction with existing structure-activity relationship
Study carefully, propose the pharmacophore model of inhibitor and influences active part-structure factor.On this basis, carry out novel micro-
Design, synthesis, structure of modification and the external activity research of pipe inhibitor.
It is a further object to provide a kind of intermediates for preparing above compound.
The present invention also provides the pharmaceutical compositions comprising above-mentioned Antitubulin.
According to an aspect of the present invention, a kind of Antitubulin has formula (I) structure:
Wherein:
N independently indicates 0~5 integer, and condition is n≤5, and A indicates that monosubstituted or polysubstituted group, the group are selected from
H, C1-C20 alkyl, C1-C20 alkyl, C1-C20 acylamino-, C1-C20 acyloxy, C1-C20 alkanoyl, C1-C20 alcoxyl carbonyl
Base, C1-C20 alkoxy, C1-C20 alkylamino, C1-C20 alkane carboxylic amino, aroyl, aralkanoyl, carboxyl, cyano, halogen, hydroxyl
Base, nitro and methylthiophene base.
It is further preferred that n independently indicates 0~2 integer in above-mentioned formula (I), condition is n≤2, and A indicates monosubstituted or more
Substituted group, the group are selected from H, vinyl, methyl, trifluoromethoxy, methoxyl group, cyano, halogen and benzoyl.
Representative compound of the invention includes:
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- ethylene
Base phenyl) -2- propylene subunit) piperazine-2,5-dione (9)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- fluorobenzene
Base) -2- propylene subunit) piperazine-2,5-dione (10)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- phenyl third
Alkene subunit) piperazine-2,5-dione (11)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (2,3- bis-
Aminomethyl phenyl) -2- propylene subunit) piperazine-2,5-dione (12)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- trifluoro
Methoxyphenyl) -2- propylene subunit) piperazine-2,5-dione (13)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (2,5- bis-
Fluorophenyl) -2- propylene subunit) piperazine-2,5-dione (14)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- methoxy
Base phenyl) -2- propylene subunit) piperazine-2,5-dione (15)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3,5- bis-
Methoxyphenyl) -2- propylene subunit) piperazine-2,5-dione (16)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- chlorobenzene
Base) -2- propylene subunit) piperazine-2,5-dione (17)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- itrile group
Phenyl) -2- propylene subunit) piperazine-2,5-dione (18)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- benzene first
Aminosulfonylphenyl) -2- propylene subunit) piperazine-2,5-dione (19)；
According to another aspect of the present invention, the intermediate for preparing the compounds of this invention is provided, there is formula (B) structure:
Hereafter illustrate the preparation method of the compounds of this invention by taking representative compound 9 of the invention as an example.
Synthetic route 1 of the present invention is as follows:
According to the method for route 1, the present invention has synthesized following some representative compounds:
Table one: structural formula of compound
According to another aspect of the invention, a kind of pharmaceutical composition is provided, formula (I) compound containing therapeutically effective amount with
And pharmaceutically acceptable carrier and/or auxiliary material, for treating various cancers, infection, inflammation and routine propagation disease, or control
Treat the Other diseases such as psoriasis and other skin diseases characterized by there is rapid proliferative cell.The therapeutically effective amount refers to
The amount for leading to formula (I) compound contained by Pharmaceutical composition is enough to generate clinically desirable response to treatment, such as makes the tumour of drug user
Size is reduced in clinically-acceptable range.
In a specific embodiment, the compound of the present invention or pharmaceutical composition are for treating sarcoma, cancer and/or white
Blood disease.It can include fiber individually or as the Exemplary conditions of a part application of therapeutic scheme by the compound or composition
Sarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphatic vessel meat
Tumor, lymphangioendothelial sarcoma, synovialoma, celiothelioma, Ewing' s tumor, liomyoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, cream
Gland cancer, oophoroma, prostate cancer, squamous cell carcinoma, basal-cell carcinoma, syringocarcinoma, carcinoma of sebaceous glands, mastoid process cancer, papillary adenocarcinoma,
Cystadenocarcinoma, cephaloma, bronchiolar carcinoma, clear-cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma,
Wilm'stumor, cervix cancer, orchioncus, lung cancer, Small Cell Lung Cancer, bladder cancer, epithelioma, glioma, star are thin
Born of the same parents' tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, auditory nerve tumor, few prominent mind
Through glioma, meningioma, melanoma, neuroblastoma and retinoblastoma cell cancer.
In certain specific embodiments, the compound of the present invention or composition for treat such as breast, prostate,
The illness for the cancer that kidney, bladder or colonic tissue are formed.
In other specific embodiments, the compound of the present invention or pharmaceutical composition occur for treating in adipose tissue
Proliferative or tumor disease, such as adipose cell tumors, that is, lipoma, inosteatoma, at lipoblastoma, liposis, palm fibre
Color lipoma (hibemomas), hemangioma and/or embryonal-cell lipoma.
In other specific embodiments, infective agent and parasitic animal and plant can also be controlled (such as with the composition and compound
Bacterium, trypanosoma, fungi etc.).
Pharmaceutical composition of the invention can be administered by approach such as intravenous, intradermal, intramuscular, subcutaneous, oral or suckings,
The dosage form of its Pharmaceutical composition can be gastrointestinal agents preparation such as tablet, capsule, pill etc., be also possible to parenteral administration
Preparation such as injection, external preparation etc..
In addition, this method includes to cancer or phase the present invention also provides the method for treating antitumor and related disease
The patient for closing the patient's condition uses the compound of the logical formula (I) of therapeutically effective amount.
The present invention also provide in patient adjust microtubule polymerization method, this method include dosage treatment effective amount at least
A kind of the compounds of this invention or its pharmaceutically acceptable prodrug, salt, hydrate, solvate, crystal form or diastereomeric different
Structure body.
The compound of the present invention can test with chemotherapeutic agent application for the treatment of tumour and show the compounds of this invention and chemotherapy
Agent can increase tumor inhibitory effect when being used in combination, while reduce the toxicity of chemotherapeutics.Most preferred compound is compound
(9), chemotherapeutics can be conventional chemotherapy agent used in general therapeutic tumour, in a preferred embodiment, by compound (9)
It is used in combination with docetaxel and achieves extraordinary therapeutic effect.
The compound of the present invention can inhibit the growth and proliferation of cancer cell in combination with tubulin, to corresponding white thin
Born of the same parents and granulocyte are almost without influence, smaller to function of immune system interference when fighting tumor proliferation, considerably improve antitumor
The drawbacks of clinical drug uses.This compound can also be used to treat other illnesss relevant to hyper-proliferative.
Detailed description of the invention
Fig. 1 is the tumor proliferation inhibiting effect of the compound of the present invention；
The antitumor action and safety experiment that Fig. 2 is compound (9) are as a result, in figure:
A: compound inhibits nude mouse the inhibiting effect of tumor proliferation；B: when compound inhibits nude mouse tumor proliferation
Influence to the weight of animals；C: compound (9) and with docetaxel combined continuous administration after for SD Polymorphonuclear Leukocyte
The influence of counting；
Fig. 3 is the influence that the compound of the present invention counts Polymorphonuclear Leukocyte.
Specific embodiment
The compound of the present invention structure of modification be on the basis of using natural products Phenylahistin as lead compound,
And the structure-activity relationship reported is combined to carry out corresponding structure of modification.According to vinylogy principle, it is inserted between piperazine and phenyl ring
Different substituent groups like piperazine diene and phenyl ring are connected directly, then is introduced into phenyl ring on Electrical distribution by one double bond
On, active similar or active stronger compound may be obtained；And a diene key is inserted between piperazine ring and pyrazoles
Keep compound more stable, it is not easy to go bad at normal temperature, therefore the stability of the compounds of this invention is more preferable, safety is higher.
The present invention is hereafter further illustrated with specific embodiment, but any restrictions are not formed to the scope of the present invention.
Universal method: fusing point is measured in RT-1 melting point apparatus (Tianjin analysis instrument factory) equipment, temperature is calibrated.Bruker
1HNMR spectrum is recorded in AV400 (400MHz) spectrometer and carries out it with parts per million (ppm) downfield of TMS
Explanation.Infrared spectroscopy is recorded on Nicolet Magna 550FT-IR Fu Liye spectrophotometer.With
HP1100Esquire2000 liquid chromatography/mass spectrometry combined instrument records mass spectrum.UV is recorded in Shimadzu UV2410 ultraviolet specrophotometer
Spectrum.TLC is carried out in the efficient plate of silica GF254 (Zhifu silica gel development experiments factory, Yantai City).WZZ-1S polariscopy instrument (on
Extra large precision scientific instrument Co., Ltd) on carry out polariscopy.
Embodiment synthesizes formula (I) compound according to route 1
Embodiment 1
Step 1:
Magnesium chips (14.4g, 0.60mol) and dry tetrahydrofuran (1L) are added in 5000ml dry three-necked flask,
1ml1,2- Bromofume initiation reaction is added dropwise.Then the tetrahydro furan of bromoacetaldehyde condensed ethandiol (100g, 0.60mol) is slowly added dropwise
Mutter solution (500ml).After being added dropwise, two hours are stirred to magnesium chips disappearance.Then by 5- tert-butyl -1H- imidazoles -4- formaldehyde
The tetrahydrofuran solution (500ml) of (30g, 0.20mol) is slowly dropped in above-mentioned solution, is stirred overnight.Then concentrated hydrochloric acid is used
It is adjusted to acid (pH=1), and is heated to 60 DEG C and stirs 30 minutes.Revolving removes tetrahydrofuran, and ethyl acetate extraction is added.Have
Machine, which mutually merges, to be washed with water twice, and saturated common salt washing is primary, finally dry with anhydrous sodium sulfate, concentration.Crude by column chromatography
Purifying (volume ratio of petrol ether/ethyl acetate: 2/1) obtains yellow solid (17.8g, yield:50%).ESI-MS:m/z=
179.2(M+H)；1H-NMR:(500MHz,CDCl3)δ9.62(d,1H),7.68(d,1H),7.59(s,1H),6.75(dd,1H),
1.48(s,9H)。
Step 2:
DMF (100ml) is added in 250ml dry three-necked flask, Isosorbide-5-Nitrae-diacetyl-piperazine -2,5- diketone (2.5g,
17.6mmol), 3- (5- tert-butyl -1H- imidazoles -4-) methacrylaldehyde (2.09g, 11.7mmol) and cesium carbonate (5.74g,
17.6mmol).Mixture under nitrogen protection, is stirred at room temperature 16 hours.Reaction solution is diluted to 1.5L with ethyl acetate, it is organic
It is mutually washed 3 times with salt, anhydrous sodium sulfate dries, filters, concentration.Crude by column chromatography purifying be (eluant, eluent DCM/MeOH's
Volume ratio: 200/1 to 30/1) obtain bright yellow solid (1.89g, yield:43%) .ESI-MS:m/z=317.1 (M+H)；1H-NMR:(500MHz,DMSO-d6)δ11.83(s,1H),10.66(s,1H),8.46(s,1H),7.56(d,1H),7.32(t,
1H),7.18(d,1H),6.84(d,1H),6.77(s,1H),4.28(s,2H),1.32(s,9H)。
Step 3:
3- vinylbenzaldehyde (13.2g, 0.10mol) is added in 1L dry single-necked flask, formoxyl methylene triphen
Base phosphine (33.5g, 0.11mmol) and toluene (200ml).Reaction system flows back 17 hours and is then concentrated.Crude by column chromatography
Purifying (eluant, eluent: the volume ratio of petrol ether/ethyl acetate: 100/1 to 80/20) obtain yellow solid (9.6g, yield:
60%) .ESI-MS:m/z=159.2 (M+H).
Step 4:
DMF (100ml) is added in 250ml dry single-necked flask, intermediate B (1.58g, 5mmol), 3- (3- vinyl
Phenyl) methacrylaldehyde (1.59g, 10mmol) and cesium carbonate (3.26g, 10mmol).Reaction system stirs 2 hours at 80 DEG C, cooling
To room temperature, then to entering in ice water.Ethyl acetate extraction, organic phase are washed three times with salt, and anhydrous sodium sulfate dries, filters,
Concentration.Crude product is washed with petrol ether/ethyl acetate (5/1), obtains bright yellow solid (900mg, yield:43%) .ESI-
MS:m/z=415.1 (M+H)；1H-NMR:(500MHz,DMSO-d6)δ11.82(s,1H),10.81(bs,2H),7.78-7.70
(m,3H),7.54-7.49(m,3H),7.40-7.36(m,2H),7.11-7.08(m,1H),6.89-6.86(m,1H),6.78-
6.72 (m, 1H), 6.66-6.64 (m, 1H), 6.52-6.50 (m, 1H), 6.90 (d, J=17Hz, 1H), 5.31 (d, J=11Hz,
1H),1.34(s,9H).
Embodiment 2~11
Step 1~2 preparation intermediate Bs, with step 1~2 in embodiment 1.Other intermediates and finished product preparation such as following table
It is shown:
Table two: other compounds process for production thereof
Embodiment 12:
Experimental method: using HTRF kinEASE TK kit detect 11 compounds to Lyn α, AKT2, KDR, IKK- β,
The inhibitory activity of c-RAF enzyme, by untested compound, μM equal 10 concentration dilutions, control DMSO concentration are 1% from 100 μM to 0.1,
Each concentration is multiple holes.
In Lyn α, AKT2, KDR, IKK- β, c-RAF enzyme carry out inhibitory activity sieve to 11 compounds with 10 μM of concentration
It looks into.As a result as shown in the table:
IC50 value of three: 11 compounds of table to 5 kinds of kinases
The result shows that these compounds there is different degrees of inhibition to make Lyn α, AKT2, KDR, IKK- β, c-RAF
With.
Embodiment 13: cellular level Inhibition test
Cell strain source:
HT-1080 CAKi-1 human fibrosarcoma cell, AGS human gastric adenocarcinoma, A375 Human skin melanoma cell,
Fadu people swallows squamous cell carcinoma, NCI-H1975 human lung adenocarcinoma cell, SK-ES-1 human osteosarcoma cell, MDA-MB-231 human breast carcinoma
Cell, MIAPaCa-2 human pancreatic cancer cell derive from ATCC.
Experimental method
First day plating cells
1. measuring cell density after being resuspended using culture medium under digesting cell from cell culture using pancreatin.
RPM1640 culture medium is for cultivating ags cell (INVITROGEN)
McCoys ' 5A culture medium is for cultivating SK-ES-1 cell (INVITROGEN)
EMEM culture medium is for cultivating HT-1080 Caki-1 and MDA-MB-231 cell (INVITROGEN)
Tire ox is clear (GIBCO#10099-141)
Trypsase (GIBCO#25200-072)
Luminescence method cell viability detects test box (promega#G7572)
96 orifice plates (from Coming#3365)
96 orifice plate black (Costar#3340)
Bottom white pad pasting (Per#6005199)
2. by the cell solution after adjustment density in the addition cell experiment plate of 90 microlitres of every hole.
3. the tissue culture plate completed is put into incubator, at 37 degrees Celsius, 5% CO2Wet condition under be incubated for it is 24 small
When.
Second day plus compound
1. preparing the reference compound and compound solution to be tested of 200 times of concentration according to experiment pattern.
2. 7 microlitres of compound solutions is taken to be diluted into 133 microlitres of culture mediums.
3. the compound solution after 10 microlitres of dilutions is taken to be added in the previous day ready tissue culture plate.
4. the tissue culture plate that compound is added places back in incubator, at 37 degrees Celsius, 5% CO2Wet condition under
It is incubated for 72 hours.
Result detection in 5th day
1. detection reagent:
Substrate freeze-dried powder and buffer are mixed, are pressed by luminescence method cell viability detection kit (promega#G7572)
The amount in the hole 30ul/ is added in 96 orifice plates, is placed in room temperature and is balanced within 30 minutes before experiment.
2. 30 microlitres of detection reagents, rocker 10 minutes, inducing cell lysis is added in the every hole of tissue culture plate.
Tissue culture plate is incubated at room temperature 2 minutes with stabilized illumination signal after 3.10 minutes.
4. using Envision read plate, the time is set as 0.5 second every hole when read plate.
In HT-1080, AGS, A375, Fadu, NCI-H1975, Caki-1, SK-ES-1, MDA-MB-231 and MIA
On 9 cell strains of PaCa-2, the suppression level of 4 compound on intracellular is detected with cell proliferation experiment.Compound is from 600nM
Starting, 3 times of dilutions, multiple holes detection.As a result as shown in the table:
The suppression level of four: 4 compound on intracellular of table
The result shows that above compound for each investigation tumor cell line have very strong inhibiting effect, and with reference
Drug [(-) phenylahistin] is (commercially available) to be compared, and compound inhibits the intensity of tumour much higher than reference in the embodiment
Drug.In conjunction with the embodiments in 12 to the influence of different enzymes as can be seen that compound plays function of tumor inhibition in the present embodiment
Concentration is significantly lower than the concentration for inhibiting enzyme, it can therefore be concluded that compound is removed when inhibiting tumor proliferation in the present embodiment out
Have outside inhibiting effect to the enzyme being mentioned in the application, the enzyme activity or intracellular molecules not yet investigate to other still have stronger
Inhibiting effect may influence target spot inside and outside various kinds of cell to some extent and play potent collaboration function of tumor inhibition.
Embodiment 14: to the inhibiting effect of the proliferation of tumour
Experimental method
By MES-SA people's sarcoma of uterus cell (source ATCC) long-term cultivation in the adriamycin of low concentration, keep sufficient
Cell viability, inducing cell express p-glycoprotein.After culture 10 months, inducing expression result is detected using WB.Induction
After success, after a week using no adriamycin culture medium culture, cell concentration is adjusted, by tumor cell inoculation in nude mice oxter, and
Different drugs is given after transplantation tumor proliferation to certain volume to be treated.Different pharmaceutical is given respectively using blank mouse
Various dose, which is found out, does not influence the dosage (Fig. 1) that the dosage that the weight of animals increases as gives transplantation tumor treatment of animals.
The result shows that different drugs presses down nude mice model tumour in the case where giving the dosage that will not reduce the weight of animals
Rate processed has certain difference.Wherein (-) phenylahistin is almost used alone without confrontation tumor proliferation under non-toxic
Effect.And docetaxel (commercially available) drug action under no overt toxicity dosage is poor.Compound 9, compound 11,
Compound 15, compound 18 can effectively play the work of antitumor proliferation under the dosage having no significant effect to the weight of animals proliferation
With.
Tumor control rate (%)=(1- treatment group tumor weight/control group tumor weight) * 100%, it is according to Fig. 1, mostly western
The tumor control rate that he matches is higher than compound 9, compound 11, compound 15, compound 18 close to 80%.
It is tested under same effective dose simultaneously in the present embodiment, the shadow that drug is distributed different tissues organ blood flow
It rings.2 hours after administration, comprising including tumor tissues, the distribution of blood flow is similar with blank solvent group in docetaxel group.And chemical combination
Object 9, compound 11, compound 15, compound 18 and (-) phenylahistin do not send out its hetero-organization in addition to tumor tissues
The existing apparent influence for intervening tissue blood distribution.For tumor tissues, compound 9, compound 11, compound 15, chemical combination
Object 18 and (-) phenylahistin group can significantly reduce blood distribution in tumour compared with blank solvent group.
Embodiment 15: the antitumor action of compound and safety
To inoculation tumour cell nude mouse give respectively blank solvent, docetaxel, compound 9 and docetaxel with
9 combination medicine of compound the volume of different point in time measurement tumours and detects tumor volume change process after administration, simultaneously
The changes of weight of nude mouse is detected, to evaluate the antitumous effect and toxic reaction degree of drug.Separately to normal SD rats, press
According to the above administration and packet mode, detection blood neutrophil count is horizontal before administration and after the last administration, to evaluate medicine
Influence degree of the object for neutrophil count.
The experimental results showed that compound 9 can effectively inhibit the proliferation of tumour in vivo, while combining with docetaxel and make
Anti-tumor activity can be dramatically increased after, simultaneously effective reduce whole adverse reaction when docetaxel is used alone, energy
Significantly reducing the weight of animals caused by docetaxel toxicity reduces.Docetaxel and compound is used in combination in normal rat body
After 9, compared with docetaxel is used alone, neutrophil count is significantly increased, caused by restrained effectively docetaxel
The adverse reaction that neutrophil count reduces.It is comprehensively compared, under the dosage for not influencing animal integrality, compound 9 is right
Antitumor significant effect be better than docetaxel, i.e., the oncotherapy window of compound 9 significantly be better than docetaxel, while for into
One step increases antitumous effect, and docetaxel and 9 use in conjunction of compound can be effectively reduced docetaxel toxic reaction, and
Increase antitumor action (Fig. 2).
Embodiment 16: the influence to neutrophil count
Experimental method
It gives different drugs respectively using multi-dose to investigate the inhibition level of nude mice model tumour, finds out each medicine
The equivalent dose of object, and by giving rat different pharmaceutical after dosage conversions between species.Giving rat relatively same drug effect
It learns under dosage, investigates the influence that drug counts Polymorphonuclear Leukocyte.After continuously giving Wista rat 2 weeks, rat is adopted
Blood simultaneously weighs each administration group weight (Fig. 3).
The result shows that under same effective dose, it is used continuously after each compound gives rat, conventional anticancer drugs
Docetaxel can significantly reduce neutrophil count in rat body, while observe that animal integrality is bad in experiment.(-)
Phenylahistin compared with docetaxel, under same pharmacodynamics dosage to neutrophil leucocyte reduction effect be lighter than more west he
Match, but neutrophil count is still significantly lower than blank solvent group and compound group.And compound 9, compound 11, compound
15,18 groups of compound under the same effective dose of other drugs, does not make significant difference to neutrophil leucocyte.Therefore, compound 9,
Compound 11, compound 15, compound 18 can effectively avoid reducing not neutrophil leucocyte when effectively antagonizing tumor proliferation
Good reaction.The variation tendency of the weight of animals is positively correlated with neutrophil count percentage change.
Embodiment 17: the effect to micro-pipe function
Human umbilical vein endothelial cells (HuVEC from Cambrex) is used in our current research, by carrying out to α-tubulin
Dyeing, compares colchicin and taxol has rated compound 9,11,15,18 and tert-butyl-phenyl ahistins to tubulin
Effect.
In the HuVEC cell, it is exposed to compound 9,11,15,18, tert-butyl-phenyl ahistins or colchicin
(being 2 μM) 30 minutes (shows as lacking complete induction of the opposite microtubule depolymerization effect of observed result in compareing with DMSO
Micro-tubular structure) and cell membrane blister (blebbing) (distinguishing mark of Apoptosis), and taxol under these conditions cannot
Induce microtubule depolymerization effect.Colchicin is known microtubule depolymerization reagent and taxol is tubulin stabilizer.By CCD-
27sk cell is exposed to chemical combination 9,11,15,18 or similar result can be observed in colchicin.
Embodiment 18: pharmaceutical composition
Compound according to the present invention can in conjunction with the acceptable carrier of physiology or medium to provide pharmaceutical composition,
Such as the lyophilized powder that form is the tablet containing various fillers and binder or capsule.Similarly, compound can be with it
Its medicament co-administered, co-administered should indicate at least to patient using two kinds of medicaments with provide two kinds of medicaments combine it is beneficial
Effect.For example, medicament can be used simultaneously or sequentially within a certain period of time.It can widely be selected by those skilled in the art
The effective dose of compound in empirically determined composition.In addition, according to indication and required therapeutic effect, the compound of the present invention
It can be used alone or be used in combination with one or more additional medicaments.By it is contemplated by the invention that combination therapy include, for example,
The use and the compounds of this invention and additional agent of the compounds of this invention and additional agent in single medicine formula are separating
Use in pharmaceutical formulation.
The compounds of this invention can be independent, or with pharmaceutical acceptable carrier or diluent, it is optionally fine with known adjuvant such as crystallite
Dimension element is administered to newborn animal, preferably people in the pharmaceutical composition of establishing criteria medicinal practice together.Compound Orally-administrable
Or in parenteral administration, including intravenous, intramuscular, peritonaeum, subcutaneous, rectum and topical routes of administration.
For the oral application of chemotherapy compound of the present invention, selected compound can for example in the form of a tablet or capsule or
Person applies as aqueous solution or suspension.For oral tablet, being usually added into common carrier includes lactose and cornstarch, and
Lubricant such as magnesium stearate.For being applied with capsules per os, workable diluent includes that lactose and anhydrous corn form sediment
Powder mixes active component with emulsifier and suspending agent when the water slurry for needing to be administered orally.If necessary, may be used
Some sweeteners and/or corrigent is added.For in intramuscular, peritonaeum, subcutaneous or intravenous application, the total dense of solute should be controlled
Degree is so that preparation is isotonic.
18.1 pharmaceutical compositions containing compound 9
Prescription 1:
Prescription 2:
Prescription 3:
Prescription 4:
Prescription 5:
Prescription 6:
Prescription 7:
The preparation method of 18.2 preparations
It takes reactive compound of the invention, disintegrating agent and filler to cross 60~100 meshes in proportion, is uniformly mixed, with 2~
The ethanol solution softwood of 20% PVP K30, crosses the granulation of 20~50 meshes, 40~90 DEG C of dryings, and pellet moisture control exists
Suitable lubricant is added within 3%, after whole grain, be uniformly mixed, tabletting to get.
Specifically, the pharmaceutical composition of above-described embodiment can also be prepared via a method which: by 50 times of weighing of recipe quantity
Close object 9,18, filler and disintegrating agent successively cross 60,80 meshes, be uniformly mixed, with 50% ethyl alcohol of 2~20% PVP K30s
Solution softwood, the granulation of 30 meshes, 60 DEG C of dryings, pellet moisture control within 3%, and suitable profit is added after 20 mesh sieves
Lubrication prescription is uniformly mixed, and tabletting is to get product.
19 study on the stability of embodiment
The compound 9 of acquisition is subjected to study on the stability (10 days accelerated tests), 40 DEG C, 60 DEG C, humidity 75%,
92.5%, the moisture of compound, purity, largest single impurity and total miscellaneous data with 0 day are compared under illumination condition, is as a result shown
Show that the compound of acquisition is stablized.And (-) phenylahistin is under the conditions of 60 DEG C, degradation is obvious, illustrate high temperature to (-) phenyl Ah
The stability of this fourth of sunset has an impact.
Five compound of table, 9 influence factor test result
Table six (-) phenylahistin influence factor test result
The above detailed description of the present invention is not intended to limit the present invention, and those skilled in the art can make on this basis
Various changes and variants, as long as it does not depart from the spirit of the invention, should belong to the range that the claims in the present invention define.
Claims (9)
1. leading to the compound that formula (I) indicates as follows:
Wherein:
N independently indicates 0~5 integer, and condition is n≤5, and A indicates monosubstituted or polysubstituted group, the group be selected from H,
C1-C20 alkyl, C1-C20 acylamino-, C1-C20 acyloxy, C1-C20 alkanoyl, C1-C20 alkoxy carbonyl group, C1-C20 alcoxyl
Base, C1-C20 alkylamino, C1-C20 alkane carboxylic amino, aroyl, aralkanoyl, carboxyl, cyano, halogen, hydroxyl, nitro and methyl
Thienyl.
2. compound as described in claim 1, it is characterised in that n independently indicates 0~2 integer, condition in the logical formula (I)
N≤2, A indicates monosubstituted or polysubstituted group, the group be selected from H, vinyl, methyl, trifluoromethoxy, methoxyl group,
Cyano, halogen and benzoyl.
3. compound as claimed in claim 1 or 2 is selected from following compounds:
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- vinyl benzene
Base) -2- propylene subunit) piperazine-2,5-dione (9)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- fluorophenyl) -
2- propylene subunit) piperazine-2,5-dione (10)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) ((E) -3- phenylpropen is sub- by -6-
Base) piperazine-2,5-dione (11)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (2,3- dimethyl
Phenyl) -2- propylene subunit) piperazine-2,5-dione (12)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- trifluoro methoxy
Base phenyl) -2- propylene subunit) piperazine-2,5-dione (13)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (2,5- difluorobenzene
Base) -2- propylene subunit) piperazine-2,5-dione (14)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- methoxybenzene
Base) -2- propylene subunit) piperazine-2,5-dione (15)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3,5- dimethoxy
Base phenyl) -2- propylene subunit) piperazine-2,5-dione (16)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- chlorphenyl) -
2- propylene subunit) piperazine-2,5-dione (17)；
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- itrile group benzene
Base) -2- propylene subunit) piperazine-2,5-dione (18)；With
(3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazole radicals -4- base) propylene subunit) -6- ((E) -3- (3- benzoyl
Phenyl) -2- propylene subunit) piperazine-2,5-dione (19).
4. a kind of pharmaceutical composition, the described in any item compounds of claims 1 to 3 including therapeutically effective amount.
5. pharmaceutical composition as claimed in claim 4, it is characterised in that further containing pharmaceutically acceptable carrier and/or
Auxiliary material.
6. a kind of intermediate for being used to prepare compound described in claim 1 has formula (B) structure:
7. application of the compound described in claim 1 in preparation tumor.
8. the application of compound described in claim 1 and chemotherapeutic agent in preparation tumor.
9. application according to any one of claims 8, wherein the compound is (3Z, 6Z) -3- ((E) -3- (5- tert-butyl) -1H- imidazoles
Base -4- base) propylene subunit) -6- ((E) -3- (3- ethenylphenyl) -2- propylene subunit) piperazine -2,5- diketone (9), describedization
Treatment agent is docetaxel.
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| CN201610889855.3A CN106565685B (en) | 2016-10-11 | 2016-10-11 | Antitubulin |
| PCT/CN2017/104307 WO2018068665A1 (en) | 2016-10-11 | 2017-09-29 | Microtubule protein inhibitor |
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| CN109580801B (en) * | 2018-06-26 | 2021-09-07 | 深圳海王医药科技研究院有限公司 | High performance liquid chromatography for detecting tubulin inhibitor and related impurities |
| CN109030642B (en) * | 2018-06-26 | 2021-03-09 | 深圳海王医药科技研究院有限公司 | Method for measuring dehydrophenylalsteine analogue and isomer thereof |
| CN111840291B (en) * | 2020-07-14 | 2021-12-21 | 深圳海王医药科技研究院有限公司 | Application of compound with synergistic effect in tumor treatment |
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| CN1684955A (en) * | 2002-08-02 | 2005-10-19 | 尼瑞斯药品公司 | Dehydrophenylahistins and analogs thereof and the synthesis of dehydrophenylahistins and analogs thereof |
| CN1890218A (en) * | 2003-12-03 | 2007-01-03 | 西托匹亚研究有限公司 | Tubulin inhibitors |
| WO2007035841A1 (en) * | 2005-09-21 | 2007-03-29 | Nereus Pharmaceuticals, Inc. | Analogs of dehydrophenylahistins and their therapeutic use |
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| WO2012035436A1 (en) * | 2010-09-15 | 2012-03-22 | Tokyo University Of Pharmacy And Life Sciences | Plinabulin prodrug analogs and therapeutic uses thereof |
| CN106565685B (en) * | 2016-10-11 | 2019-03-01 | 深圳海王医药科技研究院有限公司 | Antitubulin |
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| CN1684955A (en) * | 2002-08-02 | 2005-10-19 | 尼瑞斯药品公司 | Dehydrophenylahistins and analogs thereof and the synthesis of dehydrophenylahistins and analogs thereof |
| CN1890218A (en) * | 2003-12-03 | 2007-01-03 | 西托匹亚研究有限公司 | Tubulin inhibitors |
| WO2007035841A1 (en) * | 2005-09-21 | 2007-03-29 | Nereus Pharmaceuticals, Inc. | Analogs of dehydrophenylahistins and their therapeutic use |
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