CN110128411A - A kind of bis- target spot inhibitor of c-Met/HDAC and its synthetic method and application - Google Patents
A kind of bis- target spot inhibitor of c-Met/HDAC and its synthetic method and application Download PDFInfo
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- CN110128411A CN110128411A CN201910428401.XA CN201910428401A CN110128411A CN 110128411 A CN110128411 A CN 110128411A CN 201910428401 A CN201910428401 A CN 201910428401A CN 110128411 A CN110128411 A CN 110128411A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
Abstract
The invention discloses a kind of bis- target spot inhibitor of c-Met/HDAC and its synthetic method and applications.Inhibitor of the invention replaces Buddhist nun and hdac inhibitor Vorinostat as guide structure using c-Met inhibitor gram azoles, a kind of double target spot inhibitor for the single structure designed, the cap-like structure for using gram azoles to replace the structure of Buddhist nun as hdac inhibitor, instead of the aniline acyl group in Vorinostat structure, retain chelation group of the hydroximic acid structure in Vorinostat as metal zinc ion, pass through the linking group of introducing different structure therebetween, utilize the principle of pharmacophore split, design the double target spot inhibitor of single structure, obtained double target spot inhibitor all have good inhibiting effect to c-Met and HDAC, and it can cooperate with and inhibit c-Met and HDAC；Preparation method of the invention is simple, mild condition, and yield is high.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to a kind of bis- target spot inhibitor of c-Met/HDAC and its synthetic method
With application.
Background technique
In HGF/MET signal path, HGF activates c-Met, activates RAS-RAF-MEK-MAPK and the PI3K-AKT in downstream
Signal path is expressed proto-oncogene (c-Myc) and G1/S- specificity cyclin-D1 (cyclin D1) and is increased, cell week
Phase checks that albumen p21 and p27 activity reduces, and promotes cell cycle progression and existence.Histon deacetylase (HDAC) (HDAC) be also
The key modulator of cell cycle progression inhibits HDAC, and the cell cycle can be promoted to check the expression of albumen p21 and p27, retardance
Cell cycle reduces the transcription of cyclin D1, accelerates the degradation of c-Myc.Therefore, c-Met inhibitor and hdac inhibitor are right
There is certain synergistic effect on the mechanism of action of cancer.
C-Met and gram azoles replace complex crystal structure such as Fig. 1 of Buddhist nun, it can be seen from the figure that compound structure occupies ATP
Binding pocket, 2,6- bis- chloro- 3- fluoro-phenyls and the residue Tyr1230 activated on ring form stronger π-π interaction, pyridine
Ring and pyrazole ring, which are incorporated in, to be formed by residue Lys1110, Leu1157, Val1092, Ala1108, Ile1084 and Tyr1159
In hydrophobic pocket, amino and Pro1158 on pyridine ring form hydrogen bond, and the piperidine ring being connected on pyrazole ring is then stretched over hydrophobic
The outside of pocket.Vorinostat is a kind of hdac inhibitor, clinical treatment T-cell lymphoma,cutaneous, but Vorinostat is to reality
Body tumor is without significant curative effect.The complex crystal structure of HDAC2 and Vorinostat such as Fig. 2, it can be seen from the figure that the hydroxyl oxime of chain
Acid groups go deep into the binding cavity of HDAC2, with Zn2+Chelate and with residue A sp268, His182, His142, His143 and Tyr312
Hydrogen bond is formed, and aniline acyl group is then exposed to except binding cavity.
Shown in the molecular formula such as formula (II) of gram azoles for Buddhist nun, shown in the structural formula of Vorinostat such as formula (III).
The transduction of multiple target point inhibitor tumor signal is the new direction of oncotherapy and drug development, studies have shown that multiple target point
The efficacy result of single entities drug is better than single target drug and has smaller side reaction.Have c-Met and HDAC bis- simultaneously
The inhibitor of target spot has the function of collaboration to anticancer, and there is no for the individual event with both target spots in the prior art
The research of drug.
For these reasons, the present invention is specifically proposed.
Summary of the invention
In order to solve problem above of the existing technology, the present invention provides a kind of bis- target spot inhibitor of c-Met/HDAC
And its synthetic method and application, inhibitor of the invention use the principle of pharmacophore split, have synthesized the double target spots of single structure
Inhibitor can inhibit c-Met and HDAC simultaneously.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of bis- target spot inhibitor of c-Met/HDAC, shown in the structural formula of the inhibitor such as formula (I) or formula (I) is in medicine
Acceptable salt on:
Wherein, linker is linear chain structure, branched structure or aromatic ring structure.
Further, the linear chain structure isWherein n is any natural number of 1-7.
Further, the branched structure is
Further, the aromatic ring structure is
A kind of synthetic method of the bis- target spot inhibitor of c-Met/HDAC, the method include the following steps:
(1) synthetic intermediate (a) reaction equation first is as follows:
(2) the synthesis equation of intermediate (b) is as follows:
(3) the synthesis equation of intermediate (c) is as follows:
(4) target product synthesis equation is as follows:
Raw material in of the invention is all made of commercially available raw material or is formed using prior art preparation.
Further, step (1) is stirred to react 20-24 hours in room temperature, add water, extraction, washing, drying, filtering,
Concentration, obtains intermediate (a), and the yield of intermediate (a) is 70-75%.
Further, step (2) is reacted under alkaline condition, and reaction 3.5-4.5 hours is stirred at room temperature, and concentration adjusts pH
=5~6, filtering, drying obtain intermediate (b), and the yield of intermediate (b) is 85-95%.
Further, intermediate (b) is dissolved in DMF in step (3), and HATU and DIPEA is then added, is stirred to react 10-
20 minutes, O- (tetrahydro -2H- pyrans -2- base) azanol is added, reaction 3.5-4.5 hours is stirred at room temperature, layering, is done washing
Dry, filtering, concentration, obtain intermediate (c), and intermediate (c) yield is 65-75%.
Further, step (4) adjusts pH to neutrality, obtains target product, institute in room temperature reaction 25-35 minutes, concentration
The yield for the target product stated is 60-64%.
A kind of bis- target spot inhibitor application in preparations of anti-tumor drugs of c-Met/HDAC.
Compared with prior art, beneficial effects of the present invention are as follows:
(1) inhibitor of the invention replaces Buddhist nun and hdac inhibitor Vorinostat as guide structure using c-Met inhibitor gram azoles,
A kind of double target spot inhibitor for the single structure designed, the cap-like structure for using gram azoles to replace the structure of Buddhist nun as hdac inhibitor,
Instead of the aniline acyl group in Vorinostat structure, retain chelate group of the hydroximic acid structure in Vorinostat as metal zinc ion
Group, using the principle of pharmacophore split, designs the double targets of single structure therebetween by introducing the linking group of different structure
Point inhibitor, obtained double target spot inhibitor all have good inhibiting effect to c-Met and HDAC, and can cooperate with and inhibit c-
Met and HDAC；
(2) preparation method of the invention is simple, mild condition, and yield is high, provides a kind of new medicament research and development thinking, mentions
The high utilization efficiency and therapeutic effect of the drug for the treatment of cancer, has significant effect for the inhibition of solid tumor.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 is the complex crystal structure that c-Met and gram azoles replace Buddhist nun；
Fig. 2 is the complex crystal structure of HDAC2 and Vorinostat；
Fig. 3 is the synthesis path schematic diagram of double target spot inhibitor provided by the invention.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
Embodiment 1
The structural formula of the bis- target spot inhibitor of c-Met/HDAC in the present embodiment is as follows:
Wherein, linker isN=4.
The synthetic method of double target spot inhibitor of the present embodiment is as follows:
(1) synthetic intermediate (a) reaction equation first is as follows:
At room temperature, gram azoles is replaced into Buddhist nun (900.6mg, 2mmol), 5- bromo pentane acid A ester (468.0mg,
It 2.4mmol) is added with potassium carbonate (830.0mg, 6mmol) into 30ml DMF, is stirred at room temperature for 24 hours, TLC (thin-layer chromatography) detection
(the volume ratio 10:1 of methylene chloride and methanol) raw material fully reacting.The addition 30ml water into reaction solution, stirring to clear liquid, then
With ethyl acetate (100ml) average mark 2 times extractions, extract liquor is with saturated salt solution (100ml) average mark 2 times washings, anhydrous sulphur
Sour sodium is dry, filters, filtrate decompression concentration, column chromatographic purifying (the volume ratio 30:1 of methylene chloride and methanol), obtains off-white color
Intermediate (a) 824.0mg, yield 73%.
(2) the synthesis equation of intermediate (b) is as follows:
Intermediate (a) (800mg, 1.42mmol) is dissolved in 10.0ml methanol, the sodium hydroxide of 2molL-1 is added dropwise
Aqueous solution (5.0ml) is stirred at room temperature 4 hours, and TLC detects (the volume ratio 20:1 of methylene chloride and methanol) fully reacting.Decompression
Concentration, residue are filtered with hydrochloric acid regulation system pH=5~6 of 1molL-1 with a large amount of white solids clearly with water-soluble,
And solid is washed with ice water, and it is dry, obtain (b) 694.0mg among white, yield 90%.
(3) the synthesis equation of intermediate (c) is as follows:
Intermediate (b) (550.5mg, 1.0mmol) is dissolved in 20ml DMF, then be added HATU (456.3mg,
1.2mmol) and n,N-diisopropylethylamine (DIPEA) (258.5mg, 2.0mmol), 15 minutes are stirred under condition of ice bath, so
O- (tetrahydro -2H- pyrans -2- base) azanol (NH2-O-THP) (140.6mg, 1.2mmol) is added afterwards, stirs at room temperature
4h, TLC monitor (the volume ratio 10:1 of methylene chloride and methanol) fully reacting.20ml water and 60ml ethyl acetate, layering is added
Organic phase, saturated common salt water washing are taken, anhydrous sodium sulfate dries, filters, and is concentrated under reduced pressure.Residue column chromatography (methylene chloride with
The volume ratio 20:1 of methanol) obtain off-white color intermediate (c) 454.7mg, yield 70%.
(4) target product synthesis equation is as follows:
Intermediate (c) (325.0mg, 0.5mmol) is dissolved in 10ml Isosorbide-5-Nitrae-dioxane and 3ml methanol, at 5 DEG C -10 DEG C
Condition of ice bath under, be added dropwise the HCl containing 1Nml/L Isosorbide-5-Nitrae-dioxane (2ml) solution, be added dropwise, react at room temperature 30 points
Clock.TLC monitors (the volume ratio 10:1 of methylene chloride and methanol) fully reacting.Reaction solution is concentrated under reduced pressure, residue is dissolved in water extremely
Clearly, PH is adjusted to neutrality with 0.5N NaOH aqueous solution, obtain solid crude product.Column chromatography (volume ratio 10 of methylene chloride and methanol:
1) target compound (I-1) 175.3mg, yield 62% are obtained.
The 1HNMR data of the target product formula (I-1) of the present embodiment are as follows:
1HNMR (400MHz, DMSO-d6): ppm:10.46 (1H, s), 7.96 (1H, s), 7.75 (d, J=1.6Hz, 1H),
7.56-7.59 (dd, J=4.76Hz, 5.04Hz, 1H), 7.52 (s, 1H), 7.45 (t, J=8.68Hz, 8.68Hz 1H) 6.89
(d, J=1.68,1H), 6.06-6.11 (q, J=6.72Hz, 1H), 5.67 (s, 2H), 4.06-4.10 (m, 1H), 2.93 (d, J
=11.48Hz, 2H), 2.3 (t, J=8Hz, 8Hz, 2H), 1.88-2.06 (m, 8H), 1.79 (d, J=6.72Hz, 3H), 1.22-
1.52(m,4H)。
Embodiment 2
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isN=5 is pressed
It is synthesized according to the method for embodiment 1,5- bromo pentane acid A ester is replaced with into 6- bromocaproic acid methyl esters, test parameters carries out conventional tune
It is whole, target product (I-2) is obtained, 1HNMR data are as follows:
1H NMR (400MHz, DMSO-d6): ppm:10.44 (s, 1H, N-OH), 7.96 (s, 1H), 7.75 (d, J=
1.68Hz, 1H), 7.56-7.59 (dd, J=4.76Hz, 5.04Hz, 1H), 7.52 (s, 1H), 7.44 (t, J=8.68Hz,
8.68Hz, 1H), 6.89 (d, J=1.4Hz, 1H), 6.06-6.11 (q, J=6.48Hz, 6.72Hz, 6.48Hz, 1H), 5.67
(s, 2H), 4.06-4.13 (m, 1H), 2.95 (d, J=10.96Hz, 2H), 2.28-2.31 (t, J=6.72Hz, 7.56Hz,
2H), 1.89-2.08 (m, 8H), 1.80 (d, J=6.72Hz, 3H), 1.22-1.54 (m, 6H).
Embodiment 3
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isN=6 is pressed
It is synthesized according to the method for embodiment 1,5- bromo pentane acid A ester is replaced with into 7- bromine methyl heptanoate, test parameters carries out conventional tune
It is whole, target product (I-3) is obtained, 1HNMR data are as follows:
1HNMR (400MHz, DMSO-d6): ppm:10.37 (s, 1H), 8.70 (s, 1H), 7.96 (s, 1H), 7.75 (d, J
=1.68Hz, 1H), 7.57 (dd, J=4.76Hz, 5.04Hz, 1H), 7.52 (s, 1H), 7.44 (t, J=8.68Hz, 1H),
6.90 (d, J=1.4Hz, 1H), 6.06-6.11 (q, J=6.44Hz, 6.72Hz, 6.44Hz, 1H), 5.66 (s, 2H), 4.05-
4.11 (m, 1H), 2.94 (d, J=11.48Hz, 2H), 2.29 (t, J=7Hz, 2H), 1.89-2.06 (m, 8H), 1.80 (d, J=
6.72Hz,3H),1.23-1.50(m,8H)。
Embodiment 4
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isN=7 is pressed
It is synthesized according to the method for embodiment 1,5- bromo pentane acid A ester is replaced with into 8- bromine methyl caprylate, test parameters carries out conventional tune
It is whole, target product (I-4) is obtained, 1HNMR data are as follows:
1HNMR (400MHz, DMSO-d6): ppm:10.37 (s, 1H), 7.96 (s, 1H), 7.75 (d, J=1.68Hz,
1H), 7.57 (dd, J=5.04Hz, 4.76Hz, 1H), 7.52 (s, 1H), 7.44 (t, J=8.68Hz, 8.68Hz, 1H), 6.89
(d, J=1.68Hz, 1H), 6.06-6.11 (q, J=7.04Hz, 6.72Hz, 6.72Hz, 1H), 5.65 (s, 2H), 4.04-4.10
(m, 1H), 2.92 (d, J=11.24Hz, 2H), 2.27 (t, J=7.28Hz, 2H), 1.91-2.03 (m, 8H), 1.80 (d, J=
6.44Hz,3H),1.23-1.51(m,10H)。
Embodiment 5
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isAccording to implementation
The method of example 1 is synthesized, and 5- bromo pentane acid A ester is replacedLinker isTest parameters carries out general adjustment, obtains target product (I-5), 1HNMR data are as follows:
1HNMR (400MHz, DMSO-d6): ppm:11.24 (s, 1H), 9.06 (s, 1H), 7.98 (s, 1H), 7.75 (d, J
=1.68Hz, 1H), 7.72 (s, 1H), 7.64 (d, J=7.6Hz, 1H), 7.57 (dd, J=5.04Hz, 1H), 7.53 (s, 1H),
7.39-7.47 (m, 3H), 6.90 (d, J=1.68Hz, 1H), 6.09-6.11 (q, J=6.72Hz, 6.76Hz, 6.72Hz, 1H),
5.67 (s, 2H), 4.08-4.16 (m, 1H), 3.54 (s, 2H), 2.88 (d, J=19.2Hz, 2H), 1.90-2.15 (m, 6H),
1.79 (d, J=6.72Hz, 3H).
Embodiment 6
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isAccording to implementation
The method of example 1 is synthesized, and 5- bromo pentane acid A ester is replacedLinker isTest parameters carries out general adjustment, obtains target product (I-6), 1HNMR data are as follows:
1HNMR(400MHz,DMSO-d6): ppm:11.19 (s, 1H), 9.05 (s, 1H), 7.97 (s, 1H), 7.71-7.75
(m, 3H), 7.57 (dd, J=4.76Hz, 5.04Hz, 1H), 7.52 (s, 1H), 7.39-7.46 (m, 3H), 6.89 (d, J=
1.4Hz, 1H), 6.06-6.11 (q, J=6.72Hz, 1H, CH-CH3),5.67(s,2H),4.07-4.15(m,1H),3.55(s,
2H), 2.87 (d, J=11.2Hz, 2H), 1.92-2.15 (m, 6H), 1.79 (d, J=6.44Hz, 3H).
Embodiment 7
In the bis- target spot inhibitor structure formulas of the c-Met/HDAC of the present embodiment, linker isAccording to
The method of embodiment 1 is synthesized, and 5- bromo pentane acid A ester is replacedLinker isTest parameters carries out general adjustment, obtains target product (I-7), 1HNMR data are as follows:
1HNMR (400MHz, DMSO-d6): ppm:10.8 (s, 1H), 9.09 (s, 1H), 7.97 (s, 1H), 7.76 (s,
1H), 7.35-7.59 (m, 8H), 6.90 (s, 1H), 6.46 (d, J=16.04Hz, 1Hz, 1H), 6.06-6.11 (q, J=
6.16Hz, 1H), 5.68 (s, 2H), 4.03-4.11 (m, 1H), 3.52 (s, 2H), 2.88 (d, J=10.08Hz, 2H), 1.92-
2.14 (m, 6H), 1.80 (d, J=6.44Hz, 3H, CH-CH3).
Embodiment 8
Compound (I-1) 565mg (1mmol) is dissolved in the mixed solution (10:1) of 10ml methylene chloride and methanol, in ice
0.5N HCl/1,4- dioxane solution 4ml are added dropwise under conditions of water-bath, there is solid precipitation, stirs 4h, TLC is detected, and raw material is anti-
Should reaction be stopped, crude product is filtered to obtain, under condition of ice bath, 3h is stirred in acetonitrile, filter to obtain I-A 341mg, yield completely
60%.1HNMR (400MHz, DMSO-d6): ppm:10.39 (1H, s), 7.96 (1H, s), 7.75 (d, J=1.6Hz, 1H),
7.56-7.59 (m, 1H), 7.52 (s, 1H), 7.45 (t, J=8.68Hz, 8.68Hz 1H) 6.89 (d, J=1.68,1H),
6.06-6.11 (q, J=6.72Hz, 1H), 4.06-4.10 (m, 1H), 2.93 (d, J=11.48Hz, 2H), 2.3 (t, J=8Hz,
8Hz, 2H), 1.88-2.06 (m, 8H), 1.79 (d, J=6.72Hz, 3H), 1.22-1.52 (m, 4H).
Experimental example 1
Double target spot inhibitor compound (I-1)~(I-7) of embodiment 1-7 preparation are evaluated as follows respectively:
1, biological evaluation:
According to the kit operation instructions of Cisbio company,Kinases TM- tyrosine kinase assay (KinEASETM-TK ASSAY)、The test of HDAC2 DNA methylase inhibitor (
HDAC2HISTONE H3K4DEACETYLATION ASSAY) andThe test of HDAC1 DNA methylase inhibitor (HDAC1HISTONE H3K9 DEACETYLATION ASSAY) use homogeneous phase time discrimination fluorescence (HTRF) technology
Depression effect of the detection untested compound to c-Met kinase activity and the deacetylated effect of HDAC2 and HDAC1 respectively.It tested
Journey is divided into three steps: enzyme reaction step, detecting step and data processing.
(1) enzyme reaction step
It is sequentially added into compound, substrate-biotin (TK Substrate-biotin), enzyme respectively in 384 orifice plates
Deng, by plank sealing be placed in 37 DEG C of incubators be incubated for carry out enzyme reaction.
(2) detecting step
Detection reagent Streptavidin-XL665 (Streptavidin-XL665) is added and the corresponding antibody of enzyme, room temperature are put
620nm and 665nm fluorescent value is detected with microplate reader after setting 1 hour.
(3) data processing
The detected value in every hole: Ratio=(665nm/620nm) × 104, the specific signals of each sample well are sample well
Detected value and negative control hole detected value difference: Delta Ratio=Ratio (Sample)-Ratio (Negative
Control), the inhibiting rate of each compound is calculated according to specific signals: (Delta Ratio positive control-Delta
Ratio compound)/Delta Ratio positive control.According to the concentration of compound and corresponding inhibiting rate, compound is sought
IC50。
2, to the screening of c-Met inhibitory activity
(1) material prepares
Positive reference compound is that gram azoles replaces Buddhist nun.The compound for preparing 2 × 104uM is mother liquor, is stored in -80 DEG C of refrigerator
In it is spare.The concentration that diluted chemical compound is 200nM is subjected to primary dcreening operation.According to saying for HTRF kinEASETM-TK assay kit
The concentration of bright reagent preparation simultaneously carries out being diluted to working solution concentration: streptavidin-XL665 (66.68 ×), TK
Substrate-biotin(10×)、TK Antibody-Cryptate(1×)、Detection buffer(1×)、c-Met
Kinases (0.4ng/ul), DTT (100mM), ATP (5mM) and MgCl2 (1M), Enzymatic buffer (5 × Enzymatic
Buffer, 200 × MgCl2,100 × DTT, DDH2O).
(2) enzyme reaction step
1 × kinase of 6ul buffer is added in negative control hole, 1 × kinase of 4ul is added in Positive control wells
4ul compound (200nM) is added in buffer, sample well；Then 2ul TK Substrate-biotin (10 ×) are added in every hole；
Other than negative control hole, every hole adds the c-Met kinases (0.4ng/ul) of 2ul；Last every hole adds the ATP of 2ul
(5mM) is incubated for a hour under conditions of 37 DEG C.
(3) detecting step
Every hole sequentially adds the Streptavidin-XL665 (66.68 ×) of the 5ul and TK Anti-body- of 5ul
Cryptate (1 ×), after being placed at room temperature for one hour.The fluorescent value of microplate reader detection 665nm and 620nm.
According to the method for data processing, inhibiting rate is calculated, the results are shown in Table 1.
Table 1
According to primary dcreening operation as a result, the comparable compound of inhibiting rate of selection and positive compound, further to be screened.Secondary screening
Compound selects six concentration (200nM, 100nM, 20nM, 10nM, 2nM, 0.2nM) respectively, and experimentation is same as above, secondary screening chemical combination
The IC50 value of object is shown in Table 2.
Table 2
| Group | IC50(nM) |
| Gram azoles replaces Buddhist nun | 36.3 |
| I-6 | 33.1 |
| I-7 | 150 |
3, to the screening of HDAC2 inhibitory activity
(1) material prepares
Positive reference compound is Vorinostat.The compound for preparing 2 × 104uM is mother liquor, is stored in -80 DEG C of refrigerator
In it is spare.It selects to carry out preliminary screening in the concentration of 20uM first.According to the concentration for illustrating reagent preparation and carry out being diluted to work
Make liquid concentration: H3K4me0-Eu (K) Ab (50 ×), streptavidin-XL665 (83.35 ×), Detection buffer (1
×), Histone H3 (1-21) lysine 4 acetylated biotinylated peptide (1000nM), HDAC2
(2ng/ul), 1 × Enzymatic buffer (50mM Tris-HCl PH 8.0,1mM DTT, 0.01%Tween20,
0.01%BSA).
(2) enzyme reaction step
1 × kinase of 6ul buffer is added in negative control hole, 1 × kinase of 4ul is added in Positive control wells
4ul compound (20uM) is added in buffer, detection hole；Then other than negative control hole, every hole adds the HDAC2 of 2ul
(2ng/ul)；4 acetylated of substrate Histone H3 (1-21) lysine of 4ul is added in last every hole
Biotinylated peptide (1000nM) is incubated for 2 hours under conditions of 37 DEG C.
(3) enzyme detecting step
Every hole all sequentially adds H3K4me0-Eu (K) Ab of the Streptavidin-XL665 (83.35 ×) and 5ul of 5ul
(50 ×), after being placed at room temperature for one hour.The fluorescent value of microplate reader detection 665nm and 620nm.
(4) data processing
According to the data processing method in experimental method, inhibiting rate is calculated, experimental data is shown in Table 3.
Table 3
4, to the screening of HDAC1 inhibitory activity
(1) material prepares
Positive reference compound is Vorinostat.The compound for preparing 2 × 104uM is mother liquor, is stored in -80 DEG C of refrigerator
In it is spare.It selects to carry out preliminary screening in the concentration of 2uM first.According to the concentration for illustrating reagent preparation and carry out being diluted to work
Liquid concentration: H3K9me0-Eu (K) Ab (50 ×), streptavidin-XL665 (416.5 ×), Detection buffer (1
×), Histone H3 (1-21) lysine 9 acetylated biotinylated peptide (200nM), HDAC1
(20ng/ul), 1 × Enzymatic buffer (50mM Tris-HCl PH 8.0,137mM NaCl, 2.7mM KCl, 1mM
MgCl2,0.01%Tween20).
(2) enzyme reaction step
1 × kinase of 6ul buffer is added in negative control hole, 1 × kinase of 4ul is added in Positive control wells
4ul compound (2uM) is added in buffer, detection hole；Then other than negative control hole, every hole adds the HDAC1 of 2ul
(20ng/ul)；9 acetylated of substrate Histone H3 (1-21) lysine of 4ul is added in last every hole
Biotinylated peptide (200nM) is incubated for 2 hours under conditions of 37 DEG C.
(3) detecting step
Every hole all sequentially adds H3K9me0-Eu (K) Ab of the Streptavidin-XL665 (83.35 ×) and 5ul of 5ul
(50 ×), after being placed at room temperature for one hour.The fluorescent value of microplate reader detection 665nm and 620nm.
(4) data processing
According to the data processing method in experimental method, inhibiting rate is calculated, experimental data is shown in Table 4.
Table 4
In summary data are analyzed, and compound prepared by the present invention all has certain suppression to C-Met and HDAC1/HDAC2
Production is used, wherein compound I-1, I-2, I-3, I-6, I-7 replace Buddhist nun similar the inhibiting rate of C-Met with gram azoles, compound I-6
IC50 value and gram azoles for Buddhist nun on the same order of magnitude；The inhibiting rate and Fu Li of compound I-3, I-4, I-7 to HDAC1/HDAC2
He is similar for promise, therefore the compound of the present invention can play the role of while inhibit C-Met and HDAC1/HDAC2, antitumor
Mechanism on there are certain synergistic effect, the drug effect of the compound in vivo is expected to reduce medicine there may be the effect of superposition
The dosage and toxic side effect of object.
The applicant has also carried out above-mentioned test to compound prepared by other embodiments, as a result almost the same, due to
Length is limited, will not enumerate.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of bis- target spot inhibitor of c-Met/HDAC, which is characterized in that shown in the structural formula of the inhibitor such as formula (I) or
Formula (I) pharmaceutically acceptable salt:
Wherein, linker is linear chain structure, branched structure or aromatic ring structure.
2. the bis- target spot inhibitor of c-Met/HDAC according to claim 1, which is characterized in that the linear chain structure isWherein n is any natural number of 1-7.
3. the bis- target spot inhibitor of c-Met/HDAC according to claim 1, which is characterized in that the branched structure is
4. the bis- target spot inhibitor of c-Met/HDAC according to claim 1, which is characterized in that the aromatic ring structure is
5. a kind of synthetic method of the bis- target spot inhibitor of c-Met/HDAC described in claim 1-4 any one, feature exist
In the method includes the following steps:
(1) synthetic intermediate (a) reaction equation first is as follows:
(2) the synthesis equation of intermediate (b) is as follows:
(3) the synthesis equation of intermediate (c) is as follows:
(4) target product synthesis equation is as follows:
6. the synthetic method of the bis- target spot inhibitor of c-Met/HDAC according to claim 5, which is characterized in that step (1)
It is stirred to react 20-24 hours in room temperature, adds water, extraction, washing, drying, filtering, concentration, obtain intermediate (a), it is intermediate
The yield of body (a) is 70-75%.
7. according to the synthetic method of the bis- target spot inhibitor of claim 5 c-Met/HDAC according to claim 5, feature
It is, step (2) is reacted under alkaline condition, and reaction 3.5-4.5 hours is stirred at room temperature, and concentration adjusts pH=5~6, filter,
It is dry, intermediate (b) is obtained, the yield of intermediate (b) is 85-95%.
8. according to the synthetic method of the bis- target spot inhibitor of claim c-Met/HDAC according to claim 5, feature
It is, intermediate (b) is dissolved in DMF in step (3), and HATU and DIPEA is then added, is stirred to react 10-20 minutes, and O- is added
Reaction 3.5-4.5 hours is stirred at room temperature in (tetrahydro -2H- pyrans -2- base) azanol, and layering, drying, is filtered, concentration at washing,
Intermediate (c) is obtained, intermediate (c) yield is 65-75%.
9. according to the synthetic method of the bis- target spot inhibitor of claim c-Met/HDAC according to claim 5, feature
It is, step (4) adjusts pH to neutrality, obtain target product, the target product in room temperature reaction 25-35 minutes, concentration
Yield be 60-64%.
10. a kind of bis- target spot inhibitor of c-Met/HDAC described in claim 1-4 any one are in the preparation of antitumor drugs
Application.
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| CN113444069A (en) * | 2021-07-07 | 2021-09-28 | 新乡医学院 | 2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC double-target inhibitor |
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| CN106916101A (en) * | 2017-02-15 | 2017-07-04 | 聚缘（上海）生物科技有限公司 | Double target spot inhibitor of NAMPT/HDAC and preparation method thereof |
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| CN113444069A (en) * | 2021-07-07 | 2021-09-28 | 新乡医学院 | 2-aryl-4- (1H-pyrazol-3-yl) pyridine LSD1/HDAC double-target inhibitor |
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