CN106565685A - Tubulin inhibitor - Google Patents
Tubulin inhibitor Download PDFInfo
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- CN106565685A CN106565685A CN201610889855.3A CN201610889855A CN106565685A CN 106565685 A CN106565685 A CN 106565685A CN 201610889855 A CN201610889855 A CN 201610889855A CN 106565685 A CN106565685 A CN 106565685A
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- propylene
- compound
- piperazine
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- bases
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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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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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
The invention provides a novel tubulin inhibitor and an application thereof. The novel tubulin inhibitor is a series of substituted heterocyclic skeleton-based compounds with colchicine binding sites in tubulins as targets. The compounds have the following structure as shown in the specification, wherein the formula is as shown in the specification; n independently represents an integer of 0-5 under the condition that n is less than or equal to 5; A represents a monosubstituted or polysubstituted group; and the group is selected from H, C1-C20 acylamino, C1-C20 acyloxy, C1-C20 alkanoyl, C1-C20 alkoxycarbonyl, C1-C20 alkoxy, C1-C20 alkylamino, C1-C20 alkylcarboxy amino, aroyl, arylalkanoyl, carboxyl, cyano, halogen, hydroxyl, nitro and methylthienyl.
Description
Technical field
The present invention relates to drug world, relate in particular to as Antitubulin new series compound and its
Using.
The present invention also relates to prepare the midbody compound of the compound.
The invention further relates to the pharmaceutical composition containing the compound.
Background technology
Used as the Main Means of oncotherapy, antitumor drug is to extend the life span of patient and improve its life matter
Amount is made that suitable contribution.Wherein act on the medicine (microtubule inhibitors) of micro-pipe, and have in tumour medicine and quite weigh
The status wanted.But current clinical medicine, is affected by following bad problem：Poor water solublity, be unfavorable for administration and
Anaphylaxiss, serious toxic and side effects and the acquired resistance for easily causing, causes curative effect reduction, chemical constitution complexity to be difficult to
Synthesize and cause source rare, limit their further uses.Thus, it is badly in need of finding the new tubulin suppression of design
The micromolecular inhibitor of agent, especially simple structure.
Micro-pipe (microtubule) is the important component of eukaryotic cell, is also important antitumor drug action target.It is micro-
Pipe is the key component of cytoskeleton, is made up of alpha-tubulin and 'beta '-tubulin heterodimer, with hollow tubular
The characteristics of structure.In addition, 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 dynamicss of polymerization and depolymerization, conveys in cellular morphology, cell division, signal transduction and material
Play an important role Deng during.Micro-pipe becomes spindle in prophase of cell division polymerization, and spindle is led in mitosiss
Draw chromosome to move in two daughter cells to the two poles of the earth, complete cell propagation.As micro-pipe has extremely in cell division
Important effect, has become one of important target spot of antitumor drug research.The tubulin for acting on microtubule system suppresses
Agent has also become a class effective antitumour medicine.
Antitubulin has two kinds of sorting techniques.A kind of is to be divided into two types according to the difference of mechanism of action:①
Suppress the tubulin depolymerizing agent of tubulin polymerization；2. promote the Tubulin polymerization agent of tubulin polymerization.Another kind of point
Class method is to be divided into 3 classes according to Antitubulin is different from tubulin action site:1. act on Colchicine position
The Antitubulin of point；2. act on the Antitubulin in vinblastine site；3. act on the micro- of paclitaxel site
Tubulin inhibitor.
Now there are some researches show in tubulin (tubulin), there are three main drug binding sites：Paclitaxel is combined
Site (Taxol site), vinblastine binding site (Vinblastine sitc) and Colchicine binding site
(Colchieinesite).But in these three sites, Colchicine 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), which is cell cycle inhibitor, used as tumor growth inhibitors or fungistat.
Chinese patent application CN1934101A is related to the purposes of above-claimed cpd, close for reducing blood vessel hyperplasia and blood vessel
Degree, acts on tumor vessel, but plinabulin exclusive uses is weaker to the inhibitory action of tumor, dry to immune function
Disturb larger.
The content of the invention
It is an object of the present invention to provide a kind of new Antitubulin, it is a series of based on substituted heterocycle
The compound of skeleton, with the Colchicine binding site in tubulin as target.
By the structural property of binding site of analyzing and researching, and the combination of itself and inhibitor is simulated, determine each subprovince domain
Property, pivotal role residue and potential binding site；Total group from inhibitor structure is started with, and progressively expands total
The property of group, so as to assume that the stay in place form of inhibitor.Grind from activity conformation and with reference to existing structure activity relationship again
Study carefully, propose the pharmacophore model of inhibitor and affect active part-structure factor.On this basis, carry out new micro-
The research of the design of pipe inhibitor, synthesis, structure of modification and external activity.
It is a further object to provide a kind of intermediate for preparing above-claimed cpd.
The present invention also provides the pharmaceutical composition comprising above-mentioned Antitubulin.
According to an aspect of the present invention, a kind of Antitubulin, with formula (I) structure：
Wherein：
N independently represents 0～5 integer, and condition is n≤5, and A represents monosubstituted or polysubstituted group, and the group is selected from
H, C1-C20 alkyl, C1-C20 alkyl, C1-C20 acylamino-s, C1-C20 acyloxy, C1-C20 alkanoyls, C1-C20 alcoxyl carbonyls
Base, C1-C20 alkoxyls, C1-C20 alkylaminos, C1-C20 alkane carboxylic amino, aroyl, aralkanoyl, carboxyl, cyano group, halogen, hydroxyl
Base, nitro and methylthiophene base.
It is further preferred that in above-mentioned formula (I), n independently represents 0～2 integer, and condition is n≤2, and A represents monosubstituted or many
Substituted group, the group are selected from H, vinyl, methyl, trifluoromethoxy, methoxyl group, cyano group, halogen and benzoyl.
The representative compound of the present invention includes：
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- ethylene
Base phenyl) -2- propylene subunits) piperazine-2,5-dione (9)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- fluorobenzene
Base) -2- propylene subunits) piperazine-2,5-dione (10)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- phenyl third
Alkene subunit) piperazine-2,5-dione (11)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (2,3- bis-
Aminomethyl phenyl) -2- propylene subunits) piperazine-2,5-dione (12)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- trifluoros
Methoxyphenyl) -2- propylene subunits) piperazine-2,5-dione (13)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (2,5- bis-
Fluorophenyl) -2- propylene subunits) piperazine-2,5-dione (14)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- methoxies
Base phenyl) -2- propylene subunits) piperazine-2,5-dione (15)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3,5- bis-
Methoxyphenyl) -2- propylene subunits) piperazine-2,5-dione (16)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- chlorobenzenes
Base) -2- propylene subunits) piperazine-2,5-dione (17)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- itrile groups
Phenyl) -2- propylene subunits) piperazine-2,5-dione (18)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- benzene first
Aminosulfonylphenyl) -2- propylene subunits) piperazine-2,5-dione (19)；
According to a further aspect in the invention, there is provided prepare the intermediate of the compounds of this invention, with formula (B) structure：
The preparation method of the compounds of this invention is illustrated by taking the representative compound 9 of the present invention as an example hereafter.
Synthetic route of the present invention 1 is as follows：
According to the method for route 1, the present invention has synthesized following some representational compounds：
Table one：Structural formula of compound
According to another aspect of the invention, there is provided a kind of pharmaceutical composition, (I) compound of the formula containing therapeutically effective amount with
And pharmaceutically acceptable carrier and/or adjuvant, for treating various cancers, infection, inflammation and routine propagation disease, or control
Treat Other diseases such as psoriasises and other dermatosiss that rapid proliferative cell is characterized occur.The therapeutically effective amount is referred to
The amount for leading to formula (I) compound contained by Pharmaceutical composition be enough to produce clinically desirable response to treatment, for example, make the tumor of drug user
Size is reduced in clinically-acceptable scope.
In a specific embodiment, compound of the invention or pharmaceutical composition are used to treating sarcoma, cancer and/or white
Disorders of blood.The compound or compositionss can be included fiber individually or as the Exemplary conditions of the part application of therapeutic scheme
Sarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphatic vessel meat
Tumor, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing' s tumor, leiomyoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast
Adenocarcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, syringocarcinoma, sebaceous gland carcinoma, mastoid process cancer, papillary adenocarcinoma,
Cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of biliary duct, choriocarcinoma, spermocytoma, embryonal carcinoma,
Wilm'stumor, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, star are thin
Born of the same parents' tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, auditory nerve tumor, few prominent god
Jing gliomas, meningioma, melanoma, neuroblastoma and retinoblastoma cell cancer.
In some specific embodiments, the present invention compound or compositionss be used for treatment such as breast, prostate,
The disease of the cancer that kidney, bladder or colon are formed.
In other specific embodiments, the compound or pharmaceutical composition of the present invention occur in being used to treat fatty tissue
Proliferative or tumor disease, such as adipose cell tumors be lipoma, fibrolipoma, into lipoblastoma, lipomatosises, palm fibre
Color lipoma (hibemomas), hemangioma and/or liposarcoma.
In other specific embodiments, it is also possible to the compositionss and compound control infective agent and parasite (such as
Antibacterial, trypanosoma, funguses etc.).
The Pharmaceutical composition of the present invention can be administered by approach such as intravenouss, Intradermal, intramuscular, subcutaneous, oral or suctions,
The dosage form of its Pharmaceutical composition can be gastrointestinal agents preparation such as tablet, capsule, pill etc., or parenteral administration
Preparation such as injection, external preparation etc..
Additionally, the present invention also provides the method for treating antitumor and relevant disease, the method is included to cancer or phase
Compound of the patient for closing the patient's condition using the described logical formula (I) of therapeutically effective amount.
The present invention adjusts microtubule polymerization method in also providing for patient, the method include drug treatment effective dose at least
A kind of the compounds of this invention or its pharmaceutically acceptable prodrug, salt, hydrate, solvate, crystal form or diastereomeric are different
Structure body.
The compound of the present invention can be tested and show the compounds of this invention and chemotherapy with chemotherapeutic agent application for the treatment of tumor
Agent can increase tumor inhibitory effect when being used in combination, while reducing the toxicity of chemotherapeutics.Most preferably compound is compound
(9) the conventional chemotherapy agent that, chemotherapeutics can be used by general therapeutic tumor, 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 be with reference to tubulin, can be with the growth of anticancer and propagation, to corresponding white thin
Born of the same parents almost without impact, when tumor proliferation is resisted, disturb less to function of immune system, considerably improve antitumor with granulocyte
The drawbacks of clinical drug is used.This compound can be additionally used in treating other diseases related to hyper-proliferative.
Description of the drawings
Fig. 1 is the tumor proliferation inhibitory action of the compound of the present invention；
Antitumor action and safety experiment result of the Fig. 2 for compound (9), in figure：
A：Compound suppresses the inhibitory action of tumor proliferation for nude mouse；B：When compound suppresses nude mouse tumor to breed
Impact to the weight of animals；C：Compound (9) and with docetaxel combined continuous administration after for SD Polymorphonuclear Leukocytes
The impact of counting；
Fig. 3 is the compound of the present invention for the impact that Polymorphonuclear Leukocyte is counted.
Specific embodiment
The present invention compound structure transformation be on the basis of with natural product Phenylahistin as lead compound,
And the structure activity relationship that combination has been reported carries out corresponding structure of modification.According to vinylogy principle, it is inserted between piperazine and phenyl ring
Different substituent groups, on Electrical distribution, just as piperazine diene and phenyl ring are joined directly together, then is incorporated into phenyl ring by one double bond
On, active similar or active higher compound may be obtained；And between piperazine ring and pyrazoles, it is inserted into a diene key
Make compound more stable, it is not easy to go bad at normal temperatures, therefore the stability of the compounds of this invention is more preferably, safety is higher.
The present invention is further illustrated with specific embodiment hereafter, but any restriction is not formed to the scope of the present invention.
Universal method：Fusing point is measured on RT-1 melting point apparatus (Tianjin analytical tool factory) equipment, temperature is calibrated.Bruker
1HNMR spectrum are recorded in AV400 (400MHz) spectrogrph and which is carried out with parts per million (ppm) downfield of TMS
Explanation.Pay in Nicolet Magna 550FT-IR infrared spectrum is recorded on vertical leaf spectrophotometer.With
HP1100Esquire2000 liquid chromatography/mass spectrometries combined instrument records mass spectrum.UV is recorded in Shimadzu UV2410 ultraviolet spectrophotometer
Spectrum.TLC is carried out on the efficient plate of silica GF254 (Zhifu silica gel development experiments factory of Yantai City).WZZ-1S polariscopy instrument (on
Extra large precision scientific instrument company limited) on carry out polariscopy.
Embodiment synthesizes formula (I) compound according to route 1
Embodiment 1
Step 1：
Magnesium chips (14.4g, 0.60mol) and the tetrahydrofuran (1L) being dried are added in the there-necked flask that 5000ml is dried,
Deca 1ml1,2- Bromofume initiation reactions.Then it is slowly added dropwise the tetrahydrochysene furan of bromoacetaldehyde condensed ethandiol (100g, 0.60mol)
Mutter solution (500ml).After completion of dropping, stirring disappears for two hours to magnesium chips.Then by the 5- tert-butyl group -1H- imidazoles -4- formaldehyde
The tetrahydrofuran solution (500ml) of (30g, 0.20mol) is slowly dropped in above-mentioned solution, is stirred overnight.Then use concentrated hydrochloric acid
Adjust to acid (pH=1), and be heated to 60 DEG C and stir 30 minutes.Revolving removes tetrahydrofuran, adds ethyl acetate extraction.Have
Machine mutually merges and washes with water twice, and saturated common salt is washed once, finally with anhydrous sodium sulfate drying, concentration.Crude by column chromatography
Purification (the volume ratio of petrol ether/ethyl acetate:2/1) yellow solid (17.8g, yield are obtained: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：
Addition DMF (100ml) in the there-necked flask that 250ml is dried, Isosorbide-5-Nitrae-diacetyl-piperazine -2,5- diketone (2.5g,
17.6mmol), 3- (the 5- tert-butyl group -1H- imidazoles -4-) acrylic aldehyde (2.09g, 11.7mmol) and cesium carbonate (5.74g,
17.6mmol).Mixture under nitrogen protection, is stirred at room temperature 16 hours.It is by reactant liquor diluted ethyl acetate to 1.5L, organic
Washed 3 times with Sal, anhydrous sodium sulfate drying, filtered, concentration.Crude by column chromatography purification be (eluant DCM/MeOH's
Volume ratio:200/1 to 30/1) bright yellow solid (1.89g, yield are obtained: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- vinylbenzaldehydes (13.2g, 0.10mol), formoxyl methylene triphen are added in the single-necked flask that 1L is dried
Base phosphine (33.5g, 0.11mmol) and toluene (200ml).Reaction system flows back 17 hours and then concentrates.Crude by column chromatography
Purification (eluant：The volume ratio of petrol ether/ethyl acetate:100/1 to 80/20) yellow solid (9.6g, yield are obtained:
60%) .ESI-MS:M/z=159.2 (M+H).
Step 4：
DMF (100ml), intermediate B (1.58g, 5mmol), 3- (3- vinyls are added in the single-necked flask that 250ml is dried
Phenyl) acrylic aldehyde (1.59g, 10mmol) and cesium carbonate (3.26g, 10mmol).Reaction system is stirred 2 hours at 80 DEG C, cooling
To room temperature, then to entering in frozen water.Ethyl acetate is extracted, and organic faciess are washed three times with Sal, anhydrous sodium sulfate drying, is filtered,
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 prepare intermediate B, with step 1～2 in embodiment 1.Other intermediate and finished product prepare such as following table
It is shown：
Table two：Other compounds process for production thereof
Embodiment 12：
Experimental technique：Using HTRF kinEASE TK kit detect 11 compounds to Lyn α, AKT2, KDR, IKK- β,
The inhibitory activity of c-RAF enzymes, by testing compound from 100 μM to 0.1 μM 10 concentration dilutions of grade, control DMSO concentration for 1%,
Each concentration is multiple holes.
In Lyn α, AKT2, KDR, IKK- β, c-RAF enzyme carries out inhibitory activity sieve to 11 compounds with 10 μM of concentration
Look into.As a result it is as shown in the table：
Table three：IC50 value of 11 compounds to 5 kinds of kinases
As a result show that the suppression different degrees of for Lyn α, AKT2, KDR, IKK- β, c-RAF have of these compounds is made
With.
Embodiment 13：Cellular level Inhibition test
Cell strain is originated：
HT-1080 CAKi-1 human fibrosarcoma cells, AGS human gastric adenocarcinomas, A375 Human skin melanoma cells,
Fadu people's pharynx squamous cell carcinoma, NCI-H1975 human lung adenocarcinoma cells, SK-ES-1 human osteosarcoma cells, MDA-MB-231 human breast carcinomas
Cell, MIAPaCa-2 human pancreatic cancer cells derive from ATCC.
Experimental technique
First day plating cells
1. under digested cell from cell culture, using the resuspended rear measure cell density of culture medium.
RPM1640 culture medium is used to cultivate ags cell (INVITROGEN)
McCoys ' 5A culture medium is used to cultivate SK-ES-1 cells (INVITROGEN)
EMEM culture medium is used to cultivate HT-1080 Caki-1 and MDA-MB-231 cells (INVITROGEN)
Tire cattle is clear (GIBCO#10099-141)
Trypsin GIBCO#25200-072)
Luminescence method cell viability detection test box (promega#G7572)
96 orifice plates (from Coming#3365)
96 orifice plate black (Costar#3340)
Bottom white pad pasting (Per#6005199)
2. the cell solution after adjustment density is added in cell experiment plate for 90 microlitres with every hole.
3. the Tissue Culture Plate completed is put into into incubator, at 37 degrees Celsius, 5% CO2Wet condition under incubation it is 24 little
When.
Second day plus compound
1. the reference compound and compound solution to be tested of 200 times of concentration are prepared according to experiment pattern.
2. take 7 microlitres of compound solutions to be diluted in 133 microlitres of culture medium.
3. take the compound solution after 10 microlitres of dilutions and add the previous day ready Tissue Culture Plate.
4. add the Tissue Culture Plate of compound to place back in incubator, at 37 degrees Celsius, 5% CO2Wet condition under
Incubation 72 hours.
Result detection in 5th day
1. detectable：
Luminescence method cell viability detection kit (promega#G7572), substrate lyophilized powder and buffer is mixed, is pressed
During the amount in 30ul/ holes adds 96 orifice plates, it is positioned over room temperature and is balanced within 30 minutes before experiment.
2. Tissue Culture Plate adds 30 microlitres of detectable, rocker 10 minutes per hole, inducing cell lysis.
Tissue Culture Plate is incubated 2 minutes at room temperature with stabilized illumination signal after 3.10 minutes.
4. Envision read plates are used, and during read plate, the time is set as 0.5 second per hole.
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 it is as shown in the table：
Table four：The suppression level of 4 compound on intracellular
As a result show, above-claimed cpd for it is each investigation tumor cell line have very strong inhibitory action, and with reference
(commercially available) contrast of medicine [(-) phenylahistin], in the embodiment, compound suppresses the intensity of tumor far above reference
Medicine.Impact in conjunction with the embodiments in 12 to different enzymes is as can be seen that compound plays function of tumor inhibition in the present embodiment
Concentration is significantly less than the concentration of inhibitory enzyme, it can therefore be concluded that go out compound in the present embodiment, when tumor proliferation is suppressed, removing
Enzyme to being mentioned in the application has outside inhibitory action, and the enzyme activity or intracellular molecules not yet investigate to other still has stronger
Inhibitory action, may affect target spot inside and outside various kinds of cell to play potent collaboration function of tumor inhibition to some extent.
Embodiment 14：Inhibitory action to the propagation of tumor
Experimental technique
MES-SA people's sarcoma of uterus cell (source ATCC) long-term cultivation, in the amycin of low concentration, is kept into sufficient
Cell viability, inducing cell expression p-glycoprotein.After culture 10 months, abduction delivering result is detected using WB.Induction
After success, after without amycin culture medium culturing one week, cell concentration is adjusted, by tumor cell inoculation in nude mice oxter, and
Different medicines are given to after certain volume after transplantation tumor propagation to be treated.Different pharmaceutical is given respectively using blank mice
Various dose is found out the dosage for not affecting the weight of animals to increase and is the dosage (Fig. 1) for giving transplantation tumor treatment of animals.
As a result show, in the case where the dosage that will not reduce the weight of animals is given, different medicines are for the suppression of nude mice model tumor
Rate processed has certain difference.Wherein (-) phenylahistin is almost used alone without antagonism tumor proliferation under non-toxic
Effect.And docetaxel (commercially available) is poor without drug action under the overt toxicity dosage.Compound 9, compound 11,
Compound 15, compound 18 can effectively play the work of antitumor propagation under the dosage having no significant effect to the weight of animals propagation
With.
Tumor control rate (%)=(1- treatment groups tumor weight/matched group tumor weight) * 100%, it is according to Fig. 1, more western
The tumor control rate that he matches is close to 80%, and is higher than compound 9, compound 11, compound 15, compound 18.
Tested under equal effective dose in the present embodiment simultaneously, medicine is for the shadow that different tissues organ blood flow is distributed
Ring.2 hours after administration, tumor tissues in docetaxel group, are included, the distribution of blood flow is similar with blank solvent group.And chemical combination
Thing 9, compound 11, compound 15, compound 18 and (-) phenylahistin are not sent out to its hetero-organization in addition to tumor tissues
The existing obvious impact for intervening tissue blood distribution.For tumor tissues, compound 9, compound 11, compound 15, chemical combination
Thing 18 and (-) phenylahistin group are compared with blank solvent group, can significantly reduce intra-tumor blood distribution.
Embodiment 15：The antitumor action of compound and safety
To be inoculated with tumor cell nude mouse give respectively blank solvent, docetaxel, compound 9 and docetaxel with
9 combination medicine of compound, the volume of point in time measurement tumor different after administration simultaneously detect tumor volume change process, while
The body weight change of detection nude mouse, to evaluate the antitumous effect and toxic reaction degree of medicine.Separately to normal SD rats, press
Administration and packet mode according to more than, detect blood neutrophil count level, before administration and after last dose to evaluate medicine
Thing is for the influence degree of neutrophil count.
Test result indicate that, compound 9 can effectively suppress the propagation of tumor in vivo, while combine with docetaxel making
Anti-tumor activity can be dramatically increased with after, overall untoward reaction when docetaxel is used alone, energy is simultaneously effective reduced
Significantly reduce the weight of animals reduction that docetaxel toxicity causes.Docetaxel and compound is used in combination in normal rat body
After 9, compare with docetaxel is used alone, neutrophil count is significantly raised, and restrained effectively what docetaxel caused
The untoward reaction that neutrophil count is reduced.Integrated comparative, under the dosage for not affecting animal integrality, compound 9 pairs
Antineoplastic effect is significant is significantly better than docetaxel better than the oncotherapy window of docetaxel, i.e. compound 9, at the same be 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 impact of centering granulocyte count
Experimental technique
Different medicines are given respectively using multiple dose to investigate the inhibition level of nude mice model tumor, find out each medicine
The dose,equivalent of thing, and rat different pharmaceutical is given after dosage conversions between species.Giving rat relatively equal drug effect
Learn under dosage, medicine is investigated for the impact that Polymorphonuclear Leukocyte is counted.After continuously giving Wista rats 2 weeks, rat is adopted
Blood simultaneously weighs each administration group body weight (Fig. 3).
As a result show, under equal effective dose, continuously given after rat using each compound, conventional anticancer drugs
Docetaxel can significantly reduce neutrophil count in rat body, while observing in experiment that animal integrality is not good.(-)
Phenylahistin compared with docetaxel, under equal pharmacodynamicss dosage centering granulocyte reduction effect be lighter than many west he
Match, but neutrophil count is still significantly lower than blank solvent group and compound group.And compound 9, compound 11, compound
15th, under with the equal effective dose of other drugs, centering granulocyte does not make significant difference 18 groups of compound.Therefore, compound 9,
, when tumor proliferation is effective against, can be prevented effectively from centering granulocyte reduces not for compound 11, compound 15, compound 18
Good reaction.The variation tendency of the weight of animals is proportionate with neutrophil count change percentage in arid.
Embodiment 17:Effect to micro-pipe function
Human umbilical vein endothelial cells (from the HuVEC of Cambrex) are used in our current research, by carrying out to α-tubulin
Dyeing, contrast Colchicine and paclitaxel have rated compound 9,11,15,18 and tert-butyl-phenyl ahistins to tubulin
Effect.
In the HuVEC cells, compound 9,11,15,18, tert-butyl-phenyl ahistins or Colchicine are exposed to
(being 2 μM) 30 minutes, induction of observed result in compareing with DMSO, relative microtubule depolymerization effect (shows as lacking complete
Micro-tubular structure) and cell membrane blister (blebbing) (apoptotic distinguishing mark), and paclitaxel under these conditions can not
Induction microtubule depolymerization effect.Colchicine is known microtubule depolymerization reagent and paclitaxel is tubulin stabilizer.By CCD-
27sk cells are exposed to chemical combination 9,11,15,18 or Colchicine can be observed similar result.
Embodiment 18：Pharmaceutical composition
Compound of the invention can be combined with physiology's acceptable carrier or medium to provide pharmaceutical composition,
Such as the lyophilized powder that form is tablet or capsule containing various fillers and binding agent.Similarly, compound can be with which
Its medicament co-administered, co-administered should be represented and at least provide two kind medicaments combinations beneficial using two kinds of medicaments to patient
Effect.For example, medicament can be used simultaneously or sequentially within a certain period of time.Widely can be selected by those skilled in the art
The effective dose of compound in empirically determined compositionss.In addition, according to indication and required therapeutic effect, the compound of the present invention
Can be used alone or the medicament extra with one or more is used in combination.By it is contemplated by the invention that therapeutic alliance include, for example,
The compounds of this invention and additional agent use and the compounds of this invention in single medicine formula and additional agent 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 accessory drugss such as crystallite
Dimension element is administered to newborn animal, preferred people together in the pharmaceutical composition of establishing criteria medicinal practice.Compound Orally-administrable
Or parenteral administration, including intravenouss, intramuscular, intraperitoneal, 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 is applied as aqueous solution or suspension.For oral tablet, being usually added into common carrier includes Lactose and corn starch, and
Lubricant such as magnesium stearate.For being applied with capsules per os, the diluent that can be used includes that Lactose and anhydrous Semen Maydiss form sediment
Powder, when the water slurry for needing to orally use, active component is mixed with emulsifying agent and suspending agent.If desired, may be used
Add some sweeting agents and/or correctivess.For intramuscular, intraperitoneal, 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
The reactive compound of the present invention, disintegrating agent and filler are taken in proportion crosses 60～100 mesh sieves, mix homogeneously, with 2～
The ethanol solution soft material of 20% Povidone K 30, crosses the granulation of 20～50 mesh sieves, and 40～90 DEG C of dryings, pellet moisture control exist
Within 3%, appropriate lubricant, mix homogeneously, tabletting after granulate, is added to obtain final product.
Specifically, the pharmaceutical composition of above-described embodiment can also be prepared via a method which：By 50 times of weighing of recipe quantity
Compound 9,18, filler and disintegrating agent cross 60,80 mesh sieves, mix homogeneously, with 50% ethanol of 2～20% Povidone K 30 successively
Solution soft material, the granulation of 30 mesh sieves, 60 DEG C of dryings, pellet moisture are controlled within 3%, add appropriate profit after 20 mesh sieve granulate
Lubrication prescription, mix homogeneously, tabletting obtain final product product.
19 study on the stability of embodiment
The compound 9 of acquisition is carried out into study on the stability (accelerated tests of 10 days), 40 DEG C, 60 DEG C, humidity 75%,
92.5%th, the moisture of compound, purity, maximum single miscellaneous and total miscellaneous data with 0 day are contrasted under illumination condition, is as a result shown
Show the stability of compounds of acquisition.And (-) phenylahistin is under the conditions of 60 DEG C, degraded is obvious, illustrate high temperature to (-) phenyl Ah
Sunset, the stability of this fourth had an impact.
Five compound of table, 9 influence factor's result of the test
Table six (-) phenylahistin influence factor's result of the test
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 deformation, without departing from the spirit of the present invention, all should belong to the scope of the claims in the present invention definition.
Claims (10)
1. the compound that below formula (I) is represented：
Wherein：
N independently represents 0～5 integer, and condition is n≤5, and A represents monosubstituted or polysubstituted group, the group selected from H,
C1-C20 alkyl, C1-C20 alkyl, C1-C20 acylamino-s, C1-C20 acyloxy, C1-C20 alkanoyls, C1-C20 alkoxy carbonyl groups,
C1-C20 alkoxyls, C1-C20 alkylaminos, C1-C20 alkane carboxylic amino, aroyl, aralkanoyl, carboxyl, cyano group, halogen, hydroxyl,
Nitro and methylthiophene base.
2. compound as claimed in claim 1, it is characterised in that n independently represents 0～2 integer, condition in the logical formula (I)
N≤2, A represents monosubstituted or polysubstituted group, the group selected from H, vinyl, methyl, trifluoromethoxy, methoxyl group,
Cyano group, halogen and benzoyl.
3. compound as claimed in claim 1 or 2, selected from following compounds：
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- vinyl benzenes
Base) -2- propylene subunits) piperazine-2,5-dione (9)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- fluorophenyls) -
2- propylene subunits) piperazine-2,5-dione (10)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) ((E) -3- phenylpropens are sub- for -6-
Base) piperazine-2,5-dione (11)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (2,3- dimethyl
Phenyl) -2- propylene subunits) piperazine-2,5-dione (12)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- trifluoro methoxies
Base phenyl) -2- propylene subunits) piperazine-2,5-dione (13)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (2,5- difluorobenzenes
Base) -2- propylene subunits) piperazine-2,5-dione (14)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- methoxybenzenes
Base) -2- propylene subunits) piperazine-2,5-dione (15)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3,5- dimethoxies
Base phenyl) -2- propylene subunits) piperazine-2,5-dione (16)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- chlorphenyls) -
2- propylene subunits) piperazine-2,5-dione (17)；
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- itrile group benzene
Base) -2- propylene subunits) piperazine-2,5-dione (18)；With
(3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- imidazole radicals -4- bases) propylene subunit) -6- ((E) -3- (3- benzoyls
Phenyl) -2- propylene subunits) piperazine-2,5-dione (19).
4. the compound described in a kind of any one of claims 1 to 3 of pharmaceutical composition, including therapeutically effective amount.
5. pharmaceutical composition as claimed in claim 4, it is characterised in that further containing pharmaceutically acceptable carrier and/or
Adjuvant.
6. a kind of intermediate for preparing the compound described in claim 1, with formula (B) structure：
7. application of the compound described in claim 1 in tumor is prepared.
8. purposes of the compound described in claim 1 as Antitubulin.
9. application of the compound described in claim 1 with chemotherapeutic agent in tumor is prepared.
10. the application described in claim 9, wherein the compound is (3Z, 6Z) -3- ((E) -3- (the 5- tert-butyl groups) -1H- miaows
Oxazolyl -4- bases) propylene subunit) -6- ((E) -3- (3- ethenylphenyls) -2- propylene subunits) piperazine -2,5- diketone (9) is described
Chemotherapeutics are 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018068665A1 (en) * | 2016-10-11 | 2018-04-19 | 深圳海王医药科技研究院有限公司 | Microtubule protein inhibitor |
| CN109030642A (en) * | 2018-06-26 | 2018-12-18 | 深圳海王医药科技研究院有限公司 | A kind of measuring method of dehydrophenylahistin analog and its isomers |
| CN109580801A (en) * | 2018-06-26 | 2019-04-05 | 深圳海王医药科技研究院有限公司 | A kind of high performance liquid chromatography detecting Antitubulin and related impurities |
| CN111840291A (en) * | 2020-07-14 | 2020-10-30 | 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 |
| WO2012035436A1 (en) * | 2010-09-15 | 2012-03-22 | Tokyo University Of Pharmacy And Life Sciences | Plinabulin prodrug analogs and therapeutic uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| WO2012035436A1 (en) * | 2010-09-15 | 2012-03-22 | Tokyo University Of Pharmacy And Life Sciences | Plinabulin prodrug analogs and therapeutic uses thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018068665A1 (en) * | 2016-10-11 | 2018-04-19 | 深圳海王医药科技研究院有限公司 | Microtubule protein inhibitor |
| CN109030642A (en) * | 2018-06-26 | 2018-12-18 | 深圳海王医药科技研究院有限公司 | A kind of measuring method of dehydrophenylahistin analog and its isomers |
| CN109580801A (en) * | 2018-06-26 | 2019-04-05 | 深圳海王医药科技研究院有限公司 | A kind of high performance liquid chromatography detecting Antitubulin and related impurities |
| 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 |
| CN111840291A (en) * | 2020-07-14 | 2020-10-30 | 深圳海王医药科技研究院有限公司 | Application of compound with synergistic effect in tumor treatment |
| 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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| WO2018068665A1 (en) | 2018-04-19 |
| CN106565685B (en) | 2019-03-01 |
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