CN108569990A - Polysubstituted pyrrole alkane derivatives and its application as Rho kinase inhibitors in cancer - Google Patents
Polysubstituted pyrrole alkane derivatives and its application as Rho kinase inhibitors in cancer Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
- C07D207/08—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by hetero atoms, attached to ring carbon atoms
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Abstract
The invention discloses a kind of polysubstituted pyrrole alkane derivatives, and structure is as shown in formula I：Wherein：R1、R2、R3、R4It is independently selected from H, F or OCH3.The present invention confirms that I compound represented of formula has the inhibitory activity of ROCK I by the enzyme inhibition activity experiment of ROCK I and cancer cells in vitro Inhibition test, inhibitory activity with a variety of cancer cells has an opportunity as the kind new medicine in the chemicals registration classification for the treatment of cancer.
Description
Technical field
A kind of application the present invention relates to polysubstituted pyrrole alkane derivatives and its as Rho kinase inhibitors in cancer.
Background technology
Rho kinases (Rho-associated kinases, ROCK) is to participate in cell mitogen adherency, cytoskeleton tune
A series of important enzyme of cell life phenomenons such as whole, muscle cell contraction, tumor cell invasion.Including ROCK I and ROCK II,
The former is primarily present in the cells such as non-nervous tissue such as heart, lung, skeletal muscle, and the latter is primarily present in central nervous system, such as
Hippocampal pyramidal neurons, cerebral cortex, Cerebellar Cortex Purkinje Cell etc..
Numerous studies show that Rho/ROCK signal paths take part in kinds cancer disease, such as breast cancer, lung cancer, colon
Cancer, liver cancer, the cancer of the brain, incidence cancer, carcinoma of testis and carcinoma of urinary bladder.The unconventionality expression of ROCK can cause the invasion of several types cancer cell
And transfer, it is found in model in vivo and in vitro, the Rho/ROCK accesses inhibition caused by ROCK inhibitor can effectively inhibit swollen
Tumor is grown.K.Itoh etc. has found that Y27632 can inhibit the activation that the Rho of actomyosin is mediated, and can also inhibit MM1 liver cancer
The invasion activity of cell, in addition, the continuous Y27632 that gives can reduce dissipating for the MM1 cells being implanted in homogenic mouse peritoneal
It broadcasts.S.Liu etc. observes that in metastatic human mammary tumor and breast tumor cell, ROCK is expressed by superelevation, it is interesting that
The overexpression of ROCK assigns a kind of transfer sex expression of locality MCF-7 breast cancer cells.In addition, the ROCK caused by Y27632 presses down
System is migrated and is spread with the cell in vitro that ROCK is targeting, can also in vivo be prevented in cell migration to the bone of the mankind.
RKI1447 is a kind of selective ROCK inhibitor found in 2012, and in transgenic mouse model, RKI1447 shows ten
Divide anti-aggression and the antitumor activity of important anti-breast cancer.These are the experiment proves that ROCK inhibitor is preventing tumors invading
Attack, migrate and proliferation in terms for the treatment of potential.
ROCK inhibitor used at present is roughly divisible into following a few classes：Iloquinoline derivative, benzopyrazoles class, urea class, amino
Miazines.The Recent Progresses In The Development of novel ROCK inhibitor is slow, and the research and development of novel ROCK inhibitor are of great significance.
Invention content
Compound provided by the invention is to be set on the basis of the spatial structural form of existing inhibitor information and ROCK
The compound of a kind of new structure type of meter.ROCK fine structures are that the pocket knot combined with ATP is collectively formed in the area A, F and D
Structure.Specially such as I compound represented of formula or its pharmaceutically acceptable salt, ester, stereoisomer, solvated compounds,
Wherein：R1、R2、R3、R4It is independently selected from-H ,-F or-OCH3。
The present invention also provides the synthetic method such as I compound represented of formula,
1) in a suitable solvent, to 1,1,1- tri- fluoro- 5- methyl hexanes -2,4- diketone and (E)-(2- nitro propyl-s 1-
Alkene -1- bases) benzene solution in be added DBU after 1,1,1- tri- fluoro- 6- methyl -4- (2- nitro -1- phenyl propyls) heptan is obtained by the reaction
Alkane -3,5- diketone (intermediate 1)；
2) using ethyl alcohol as solvent, Raney nickel is that catalyst under suitable pressure hydrogenates intermediate 1, through follow-up
2- methyl-1s-(2- methyl -3- phenyl -5- (trifluoromethyl) -3,4- dihydro-2 h-pyrrole -4- bases) propyl- 1- ketone is obtained after processing
(intermediate 2)；
3) using THF and the mixed solution of ethyl alcohol as solvent, into intermediate 2, sodium cyanoborohydride and bromocresol green is added.
After reaction intermediate 3 is obtained through silica gel chromatography；
4) intermediate 3 and corresponding bromine substituted compound heat 1 hour in the mixture of DIPEA and acetonitrile at 50 DEG C,
Corresponding pyrrolidin derivatives can be obtained after subsequent processing.
Further, the solvent in the step 1) can be 2- propyl alcohol, dichloromethane, DCM, THF etc., preferably THF and
The solution of 2- propyl alcohol.
Further, the pressure limit in the step 2) is 2-10atm, preferably 4atm.
Further, the eluant, eluent in the step 3) silica gel chromatograph is EtOAc- hexanes, and proportional region can be
65%:35%~95%:5%, preferably 85%EtOAc-15% hexanes.
The present invention also provides described if I compound represented of formula or its pharmaceutically acceptable salt are as Rho kinases
The application of inhibitor.
The present invention also provides described if I compound represented of formula or its pharmaceutically acceptable salt are in treating cancer medicine
Application in object.
Further, the cancer is selected from liver cancer, oophoroma, lung cancer, breast cancer, carcinoma of urinary bladder.
The present invention also provides a kind of pharmaceutical compositions, including such as I compound represented of formula and/or its is pharmaceutically acceptable
Salt and one or more pharmaceutically acceptable carriers.
Medicine group can be made with pharmaceutically various typical additives (such as diluent and excipient) in the compound of the present invention
Close object.According to therapeutic purposes, pharmaceutical composition can be made to various types of administration unit dosage forms, as tablet, pill, pulvis,
Liquid, suspension, lotion, granule, capsule, suppository and injection (solution and suspension) etc..
In order to make the pharmaceutical composition of tablet form shape, it can be used this field any of and widely used figuration
Agent.For example, carrier, such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, avicel cellulose and silicon
Acid etc.；Adhesive, such as water, ethyl alcohol, propyl alcohol, common syrup, glucose solution, starch solution, penetrating judgment solution, carboxymethyl cellulose
Element, lac, methylcellulose and potassium phosphate, polyvinylpyrrolidone etc.；Disintegrant, such as dried starch, mosanom, agar powder and sea
Band powder, sodium bicarbonate, calcium carbonate, the aliphatic ester of polyethylene sorbitan, lauryl sodium sulfate, stearic acid monoglycerides,
Starch and lactose etc.；Disintegration inhibitor, such as white sugar, glycerol tristearate, coconut oil and hydrogenated oil and fat；Adsorption enhancer, such as season
Amine base and lauryl sodium sulfate etc.；Wetting agent, such as glycerine, starch；Adsorbent, such as starch, lactose, kaolin, bentonite
With colloid silicic acid etc.；And lubricant, such as pure talcum, stearate, boric acid powder and polyethylene glycol etc..If necessary
Words can also use common coated material to make tablet as sugar coated tablet, painting gelatin film tablet, enteric coated tablets, film coated tablets, double
Tunic tablet and multilayer tablet.
In order to make the pharmaceutical composition of pill shape, it can be used this field any of and widely used inborn nature
Agent, for example, carrier, such as lactose, starch, coconut oil, hardened vegetable oils, kaolin and talcum etc.；Adhesive, such as gum arabic
Powder, Huang write rubber powder, gelatin and ethyl alcohol etc.；Disintegrant, such as agar and Kelp Powder.
In order to make the pharmaceutical composition of suppository form shape, it can be used this field any known and widely used inborn nature
Agent, for example, polyethylene glycol, coconut oil, higher alcohol, the ester of higher alcohol, gelatin and semi-synthetic glyceride etc..
, can be by solution and suspension liquid disinfectant in order to prepare the pharmaceutical composition of injection form, and it is preferably added suitable chlorine
Change sodium, glucose or glycerine etc. are made and the isotonic injection of blood.When preparing injection, it is possible to use in the art any
Common carrier.For example, water, ethyl alcohol, propylene glycol, the isooctadecanol of ethoxylation, the isooctadecanol and polyethylene of polyoxy
The aliphatic ester etc. of anhydro sorbitol.In addition, common lytic agent, buffer and analgesic etc. can also be added.As needed, exist
During treating schizophrenia, colorant, preservative, fragrance, flavoring agent, sweetening agent and other medicines etc. can also be added.
In the present invention, the medication of the pharmaceutical composition is not particularly limited.Can according to patient age, gender and
Other conditions and symptom select the preparation of various dosage forms to be administered.For example, tablet, pill, solution, suspension, lotion, granule
It is oral medication with capsule；Injection can be administered alone, or (such as glucose solution and amino acid are molten with injection conveying liquid
Liquid) it is mixed into row vein injection, muscle can be carried out with injection, inject in intradermal, subcutaneous or abdomen merely if necessary；Suppository is
It is administered into rectum.
In the present invention, use can be properly selected according to method of administration, patient age, gender and other conditions and symptom
Pharmaceutical quantities.
Specific implementation mode
Intermediate 1：The synthesis of 1,1,1- tri- fluoro- 6- methyl -4- (2- nitro -1- phenyl propyls) heptane -3,5- diketone：
By 1,1,1- tri- fluoro- 5- methyl hexanes -2,4- diketone (35.0g, 0.192mol) and (E)-(2- nitro propyl- 1- alkene -
1- yls) benzene (37.9g, the 0.232mol) THF and 35mL that are dissolved in 210mL 2- propanol solutions in.It is double that 1g1,8- diazas are added
- ten one carbon -7- alkene (DBU) of ring [5.4.0], then reaction mixture is stirred at room temperature 2 hours, during churning, every 20
The DBU that 0.65g is added after minute, adds the DBU of 0.25g after 1 hour.After 2 hours, TLC (95%CH2Cl2- 5%
EtOAc most of disappearance of starting material) is shown.Solvent is concentrated in a vacuum and 500mL toluene is added.Organic phase dilute hydrochloric acid
Washing, is then washed with NaCl solution again.Use Na2SO4After drying, concentrate solution, residue by silica gel chromatography, with 3:1 hexane-
EtOAc is eluted, and obtains isomer mixture 3,1,1,1- tri- fluoro- 6- methyl -4- (2- nitro -1- phenyl propyls) heptane -3,5-
Diketone, yield are 53.82g, yield 78%.1H-NMR(400MHz,CDCl3)δ:1.03(s,3H),1.05(s,3H),1.79
(d,3H),2.39(m,1H),3.16(m,1H),3.46(dd,1H),3.97(d,1H),5.74(m,1H),7.21(t,1H),
7.28(m,4H).13C-NMR(125MHz,CDCl3)δ:17.41,18.56,38.70,42.88,48.41,64.03,80.87,
123.34,126.16,128.14,130.12,140.22,176.69,204.04.LC-MS(ESI,pos,ion)m/z:360[M+
H].
Intermediate 2：2- methyl-1s-(2- methyl -3- phenyl -5- (trifluoromethyl) -3,4- dihydro-2 h-pyrrole -4- bases)
The synthesis of propyl- 1- ketone：
The intermediate 1 (41.00g, 0.114mol) being dissolved in 500mL ethyl alcohol uses 81.00g under 4atm pressure
Raney nickel (washs three times) hydrogenation using preceding with ethyl alcohol.After filtration catalytic agent, concentrate solution, residue by silica gel chromatography is used
It is dissolved in CH2Cl2In 8.5%EtOAc solution elution, obtain 2- methyl-1s-(2- methyl -3- phenyl -5- (trifluoromethyl) -
3,4- dihydro-2 h-pyrrole -4- bases) propyl- 1- ketone (intermediate 2), 32.23g, yield 95%.1H-NMR(400MHz,CDCl3)δ:
1.03(s,3H),1.05(s,3H),1.14(d,3H),2.19-2.34(m,2H),3.71(d,1H),4.28(m,1H),7.21
(t,1H),7.28(m,4H).13C-NMR(125MHz,CDCl3)δ:18.56,20.06,38.70,54.46,58.81,75.60,
124.23,124.79,128.52,129.68,141.16,160.67,208.88.LC-MS(ESI,pos,ion)m/z:298[M+
H].
Intermediate 3：2- methyl-1s-((2R, 3R, 4S) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrrolidin-3-yl)
The synthesis of propyl- 1- ketone：
Intermediate 2 (21.89g, 0.074mol) is dissolved in 27mLTHF and 54mL ethyl alcohol.Sodium cyanoborohydride is added
(23.50g, 0.374mol) and 5mg bromocresol greens.The ethanol solution (1 of dense HCl is added dropwise into the blue solution:2), so that molten
Liquid color remains that the rate of chartreuse is added dropwise.When HCl is not added dropwise, solution colour persistently keeps yellow, then should
Solution is stirred for 20 minutes.Solution is concentrated in vacuo, CHCl is then used3Extraction, uses KHCO3Solution washs.By organic phase separation,
Use Na2SO4It is dried and concentrated.Residue is subjected to chromatographic isolation on silica gel, with 85%EtOAc-15% hexane elutions, is obtained
13.75g2- methyl-1s-((2R, 3R, 4S) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrrolidin-3-yl) propyl- 1- ketone is (intermediate
Body 3) and 7.75g2- methyl-1s-((2R, 4R) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrrolidin-3-yl) propyl- 1- ketone (in
Mesosome 3 ") mixture.It is further eluted with pure ethyl acetate, obtains 4.18g pure 2- methyl-1s-((2R, 3S, 4S) -5- first
Base -4- phenyl -2- (trifluoromethyl) pyrrolidin-3-yl) propyl- 1- ketone (intermediate 3 ').By above-mentioned pure intermediate 3 ' (4.18g)
It is dissolved in 10mL ethyl alcohol, the ethanol solution of 20 drop sodium ethoxides is added.By the solution at reflux overnight.TLC (EtOAc) is shown without starting
Substance.With in the HCl in ethyl alcohol and excessive NaOEt, and solution is concentrated in vacuo.Residue is dissolved in toluene and is used in combination
KHCO3Solution washs.By toluene solution Na2SO4It is dried and concentrated, obtains the intermediate 3 that 3.89g is purified through TLC (EtOAc)
。1H-NMR(400MHz,CDCl3)δ:1.03(s,3H),1.05(s,3H),1.06(d,3H),1.51(s,1H),2.34(m,
1H),2.44(m,1H),3.43(t,1H),3.63(m,1H),4.10(m,1H),7.21(t,1H),7.28(m,4H).13C-NMR
(125MHz,CDCl3)δ:17.94,18.56,38.52,54.97,58.49,64.08,71.00,124.96,128.50,
129.49,129.79,141.06,217.91.LC-MS(ESI,pos,ion)m/z:300[M+H].
Embodiment 1：1- ((2S, 3R, 4S) -1- (4- butyl phenyls) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrroles
Alkane -3- bases) -2- methyl-propyl -1- ketone synthesis：
By intermediate 3, intermediate 3 " (total 25.39g, 0.085mol), 4- butyl bromobenzene (0.086mol), N, N- diisopropyls
The mixture of base ethamine (DIPEA) (18.30g, 0.142mol) and acetonitrile (55mL) heats 1 hour at 50 DEG C.TLC
(EtOAc) it is displayed without starting material.Toluene, mixture KHCO is added3Solution washs, and then uses Na2SO4It is dry, concentration.
Toluene is added, is concentrated in a vacuum again.Smell disappearance of the operation up to n,N-diisopropylethylamine (DIPEA) is repeated,
Obtain final product 1- ((2S, 3R, 4S) -1- (4- butyl phenyls) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrrolidines -3-
Base) -2- methyl-propyl -1- ketone, yield 30.81g, yield 84%.1H-NMR(400MHz,CDCl3)δ:0.89(t,3H),
1.04(d,6H),1.19-1.33(m,5H),1.56(m,2H),2.23-2.36(m,2H),2.55(t,2H),3.13(t,1H),
4.24(m,1H),4.45(m,1H),6.68(dt,2H),6.89(dt,2H),7.21-7.28(m,5H).13C-NMR(125MHz,
CDCl3)δ:14.00,16.42,18.56,21.71,31.87,35.81,38.52,53.65,59.02,63.1,64.07,
116.68,125.38,127.18,128.58,128.87,130.23,130.52,140.84,146.25,218.3.LC-MS
(ESI,pos,ion)m/z:432[M+H].
Embodiment 2：：1- ((2S, 3R, 4S) -1- (the fluoro- 4- butyl phenyls of 2-) -5- methyl 4-phenyls -2- (trifluoromethyl)
Pyrrolidin-3-yl) -2- methyl-propyl -1- ketone synthesis
By intermediate 3, intermediate 3 " (total 25.39g, 0.085mol), the fluoro- 4- butyl bromobenzenes (0.086mol) of 2-, N, N-
The mixture of diisopropylethylamine (DIPEA) (18.30g, 0.142mol) and acetonitrile (55mL) heats 1 hour at 50 DEG C.TLC
(EtOAc) it is displayed without starting material.Toluene, mixture KHCO is added3Solution washs, and then uses Na2SO4It is dry, concentration.
Toluene is added, is concentrated in a vacuum again.Smell disappearance of the operation up to n,N-diisopropylethylamine (DIPEA) is repeated,
Obtain final product 1- ((2S, 3R, 4S) -1- (4- butyl phenyls) -5- methyl 4-phenyls -2- (trifluoromethyl) pyrrolidines -3-
Base) -2- methyl-propyl -1- ketone, yield 30.95g, yield 81%.LC-MS(ESI,pos,ion)m/z:450[M+H].
Embodiment:3：1- ((2S, 3R, 4S) -1- (2- methoxyl group -4- butyl phenyls) -5- methyl 4-phenyl -2- (trifluoros
Methyl) pyrrolidin-3-yl) -2- methyl-propyl -1- ketone synthesis
By intermediate 3, intermediate 3 " (total 25.39g, 0.085mol), 2- methoxyl group -4- butyl bromobenzenes (0.086mol),
It is small that the mixture of n,N-diisopropylethylamine (DIPEA) (18.30g, 0.142mol) and acetonitrile (55mL) heats 1 at 50 DEG C
When.TLC (EtOAc) is displayed without starting material.Toluene, mixture KHCO is added3Solution washs, and then uses Na2SO4It is dry,
Concentration.Toluene is added, is concentrated in a vacuum again.The operation is repeated until N, the smell of N- diisopropylethylamine (DIPEA)
It disappears, obtains final product 1- ((2S, 3R, 4S) -1- (2- methoxyl group -4- butyl phenyls) -5- methyl 4-phenyl -2- (trifluoros
Methyl) pyrrolidin-3-yl) -2- methyl-propyl -1- ketone, yield 30.21g, yield 77%.LC-MS(ESI,pos,ion)m/
z:462[M+H].
Embodiment 4：1- ((2S, 3R, 4S) -1- (bis- fluoro- 4- butyl phenyls of 2,6-) -5- methyl 4-phenyl -2- (fluoroforms
Base) pyrrolidin-3-yl) -2- methyl-propyl -1- ketone synthesis
By intermediate 3, intermediate 3 " (total 25.39g, 0.085mol), 2,6- bis- fluoro- 4- butyl bromobenzenes (0.086mol),
It is small that the mixture of n,N-diisopropylethylamine (DIPEA) (18.30g, 0.142mol) and acetonitrile (55mL) heats 1 at 50 DEG C
When.TLC (EtOAc) is displayed without starting material.Toluene, mixture KHCO is added3Solution washs, and then uses Na2SO4It is dry,
Concentration.Toluene is added, is concentrated in a vacuum again.The operation is repeated until N, the smell of N- diisopropylethylamine (DIPEA)
It disappears, obtains final product 1- ((2S, 3R, 4S) -1- (2,6- bis- fluoro- 4- butyl phenyls) -5- methyl 4-phenyl -2- (trifluoros
Methyl) pyrrolidin-3-yl) -2- methyl-propyl -1- ketone, yield 31.39g, yield 79%.LC-MS(ESI,pos,ion)m/
z:468[M+H].
5~embodiment of embodiment 9：
Test example 1：The enzyme inhibition activity of ROCK I is tested
One, experimental principle：
The measurement of ROCK I inhibitory activity uses the Z '-LYTE of Invitrogen companiesTMTechnology, the technology are based on fluorescence
Resonance energy transfer (FRET) principle, the sensitivity differences cut to proteolysis using phosphorylation and non-phosphorylated polypeptide is bases
Plinth.
Two, experimental method：
1, the preparation of reagent
Kinase buffer liquid：The 5X kinase buffer liquids of 2ml are diluted with water to 10ml；
Compound：Institute's compound to be tested is diluted with water to a concentration gradient；
The mixed solution of ROCK kinases/substrate：Prepare the mixed solution of 2250 μ LROCK/ substrates；
A concentration of 10ng/ml of enzyme, a concentration of 4 μM of substrate, solvent is kinase buffer liquid；
Phosphorylated polypeptide solution：7 peptide of serine/threonine phosphorylation of 2 μ L is added in the kinase buffer liquid of 498 μ L,
It mixes well；
ATP solution：The ATP solution of 1110 μ L is prepared, a concentration of 50 μM, solvent is kinase buffer liquid；
Color developing agent：By 1:32768 dilution proportion color developing agent A.
2, experimental procedure
(1) the 2.5 prepared compound solutions of μ L are added to 384 orifice plate of black；
(2) mixed solution of 5 μ L ROCK kinases/substrate is added；
(3) the ATP solution of 2.5 μ L is added；
(4) 384 orifice plates are shaken at room temperature and is cultivated 1 hour；
(5) 5 μ L color developing agents are added in orifice plate, the reaction was continued 1 hour；
(6) orifice plate is placed in microplate reader and is read.
Three, experimental result：
The inhibitory activity that ROCK I are carried out using the compounds of this invention of various concentration is tested, and finds the compounds of this invention tool
There are good inhibitory activity, 503nhibiting concentration IC50As shown in the table：
| Embodiment | IC50(μM) |
| Embodiment 1 | 0.32 |
| Embodiment 2 | 0.27 |
| Embodiment 3 | 0.20 |
| Embodiment 4 | 0.24 |
Test example 2：Cancer cells in vitro Inhibition test
One, experimental principle
MTT analytic approach is measured using microplate reader at 490nm based on living cells metabolin reducing agent MTT tetrazolium bromides
Optical density OD values, to reflect number of viable cells, to measure the fragmentation effect of compound on tumor cell.Two, experimental procedure
(1) cell of logarithmic growth phase, trypsin digestion, 1640 cell culture fluid tune concentration of cell suspension of RPMI
It is 6 × 104A/mL.Add 100 μ L of cell suspension per hole in 96 well culture plates, sets 37 DEG C, 5%CO2It is cultivated in incubator for 24 hours,
Cell is adherent.
(2) 1640 cell culture fluids of RPMI are removed, 1640 cell culture of RPMI of the drug to be measured of concentration gradient is added
100 μ L of liquid, each concentration set 6 parallel holes.96 orifice plates after dosing are placed in 37 DEG C, 5%CO248h is cultivated in incubator,
Set the function and effect of microscopically observation drug.
Culture solution is discarded after the centrifugation of (3) 96 orifice plates, after carefully being rushed 2~3 times with PBS, adds the RPMI containing 0.5%MTT
1640 cell culture fluid, 100 μ L continue to cultivate 4h.
(4) supernatant is removed, 150 μ L dimethyl sulfoxide (DMSO)s are added per hole, sets low-speed oscillation 10min on shaking table, makes formazan
Crystallization fully dissolving.
(5) optical density (OD values) in each hole is measured at enzyme-linked immunosorbent assay instrument 490nm.
(6) parallel hole OD values are indicated with mean ± SD, calculate inhibiting rate formula：[(ODControl group-ODBlank group)-(ODDrug study group-
ODBlank group)]/(ODControl group-ODBlank group) * 100%.
Control group (drug to be measured that concentration gradient is added is changed to that 1640 cell culture fluids of RPMI of not drug containing are added)；
Blank group (compared with the control group, is not added with cell).
(7) 5 data processing softwares of GraphPad Prism are used, by drawing amount effect curve calculation of half inhibitory concentration
(IC50)。
Three, experimental result
In liver cancer cells, ovarian cancer cell, liver cancer cells, lung cancer cell line, breast carcinoma cell strain, bladder cells strain etc.
Research in find that the excessively high expression of ROCK, this experiment confirm that the compounds of this invention inhibiting tumor cell is lived using a variety of cancer cells
Property, the 4 kinds of cancer cells used are human lung cancer cell A549, human liver cancer cells Hep G2, human large cell lung cancer cell NCI-
H460, human breast cancer cell line Bcap-37.The IC of listed four kinds of cancer cells of embodiment compound pair in following table50Equal 10 μM of ＜, to part
The IC of cancer cell50Equal 5 μM of ＜.
In conclusion the inhibitory activity such as I compound represented of formula with ROCK I of the present invention, has a variety of cancers
The inhibitory activity of cell has an opportunity as the kind new medicine in the chemicals registration classification for the treatment of cancer.
Obviously, the above according to the present invention is not departing from this hair according to the ordinary technical knowledge and means of this field
Under the premise of bright above-mentioned basic fundamental thought, the modification, replacement or change of other diversified forms can also be made.
Claims (4)
1. such as I compound represented of formula or its pharmaceutically acceptable salt,
Wherein：R1、R2、R3、R4It is independently selected from-H ,-F or-OCH3。
2. as described in claim 1 such as I compound represented of formula or its pharmaceutically acceptable salt as ROCK inhibitor
Using.
3. as described in claim 1 if I compound represented of formula or its pharmaceutically acceptable salt are in treating cancer drug
Application.
4. application as claimed in claim 3, characterized in that the cancer is selected from liver cancer, oophoroma, lung cancer, breast cancer, wing
Guang cancer.
Priority Applications (1)
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265097A (en) * | 1997-06-17 | 2000-08-30 | 艾博特公司 | Pyrrolidine carboxylic acid derivatives as endothelin antagonists |
| CN101583361A (en) * | 2006-12-18 | 2009-11-18 | 印斯拜尔药品股份有限公司 | Cytoskeletal active RHO kinase inhibitor compounds, composition and use |
| TW201114741A (en) * | 2009-10-20 | 2011-05-01 | Takeda Pharmaceutical | Substituted pyrrolidine derivatives and use thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1265097A (en) * | 1997-06-17 | 2000-08-30 | 艾博特公司 | Pyrrolidine carboxylic acid derivatives as endothelin antagonists |
| CN101583361A (en) * | 2006-12-18 | 2009-11-18 | 印斯拜尔药品股份有限公司 | Cytoskeletal active RHO kinase inhibitor compounds, composition and use |
| TW201114741A (en) * | 2009-10-20 | 2011-05-01 | Takeda Pharmaceutical | Substituted pyrrolidine derivatives and use thereof |
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