CN108658874A - Thiopyrimidine heterocycle anti-tumor compounds and preparation method thereof and purposes - Google Patents
Thiopyrimidine heterocycle anti-tumor compounds and preparation method thereof and purposes Download PDFInfo
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- CN108658874A CN108658874A CN201810785653.3A CN201810785653A CN108658874A CN 108658874 A CN108658874 A CN 108658874A CN 201810785653 A CN201810785653 A CN 201810785653A CN 108658874 A CN108658874 A CN 108658874A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/48—Two nitrogen atoms
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- A—HUMAN NECESSITIES
- 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
- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
- C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
- C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
- C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
- C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
Abstract
The present invention relates to thiopyrimidine heterocycle anti-tumor compounds and preparation method thereof and purposes, which is specially structure shown in formula (I).The invention further relates to the formula (I) compound represented or its pharmaceutically acceptable salts, or tumor disease is treated by inhibiting Wild type EGFR, mutant egf R T790M skin factor receptor protein tyrosine kinases containing its pharmaceutical composition, it is especially used to treat Small Cell Lung Cancer, the purposes of non-small cell lung cancer, EGFR T790M saltant type non-small cell lung cancers.
Description
Technical field
The present invention relates to antitumoral compounds and preparation method thereof and purposes, specifically, the antitumoral compounds are sulphur
For pyrimidine heterocyclic class compound, belong to pharmaceutical technology field.
Background technology
The targeted therapy of malignant tumour substantially prolongs tumor patient life cycle, significantly improves the quality of life of patient.
The core of " molecular targeted " treatment is to aim at accurately molecule " target spot ", and then being applied can be with these target spot specificity knot
The small-molecule drug of conjunction.Protein tyrosine kinase (protein tyrosine kinase, PTKs) is that cell activities are important
Signal envoy, can be catalyzed and γ-phosphate group of the ends ATP is transferred on substrate, a variety of substrate protein white matter junket ammonia can be catalyzed
Sour residue phosphorylation has important work to transmit signal in cell Proliferation, survival, apoptosis, metabolism, transcription and differentiation
With.PTKs is ideal " target spot " in current antineoplastic target medicament research and development, is more than 50% proto-oncogene and oncogene
Product is all tyrosine kinase.Epidermal growth factor recipient tyrosine kinase (epidermal growth factor
Receptor tyrosine kinase, EGFR) it is one of the protein tyrosine kinase found earliest, the intracellular region of EGFR has
ATP-binding site, EGFR inhibitor competitive can be combined with ATP-binding site, to inhibit the Phosphorylation events of EGFR,
The conduction of downstream signal is blocked, and then inhibits growth, differentiation and transfer (Yun, et the al.Cancer Cell of tumour cell
2007,11,217-227).EGFR has been elucidated with as the biochemical process of anti-tumor target, crystal structure and active site
It is clearer, it has been applied to clinic as the drug Gefitinib, Erlotinib, Afatinib etc. of target spot.However this
A little drugs are inevitably present the problem of anti-drug resistance difference.Research shows that：The threonine in 790 site of EGFR kinase proteins arrives
The mutation (EGFR-T790M) of methionine is the main inducing of such drug resistant.Clinical case data show, greatly
There are about 60% patient's acquired resistances to all originate from caused by the mutation in the sites T790M.Therefore exploitation anti-drug resistance is stronger, toxicity more
Small, the stronger new E GFR inhibitor of activity has extremely important realistic price.
Ao Si is to take orally irreversible, T790M Catastrophic selection EGFR inhibitors, to exon in LoVo cells for Buddhist nun
The EGFR of 19 missingsL858R/T790MAnd EGFRWTIC50Respectively 11.44 and 493.8nM, in December, 2015 by U.S. FDA
Approval listing (WO2013014448) is for treating EGFR-T790M drug-resistant type patients with lung cancer.Sieve department is another irreversible for Buddhist nun
, Catastrophic selection EGFR-T790M inhibitor acts on EGFR-T790M and EGFR in Cell free assayWTIC50Respectively
For 21.5nM and 303.3nM, being currently in the clinical III phases studies (WO 2012061299).Other such as HM61713, EGF816,
PF-06747775, Ai Wei replace Buddhist nun, ASP8273 be also into clinical research EGFR-T790M inhibitor (Ma et al.,
J.Med.Chem., 2016,59,6580-6594), the patent being related to is such as：US20120157426、US8563568B2、
CN102740847、CN102083800.The discovery of said medicine meets EGFR-T790M drug resistance patients' to a certain extent
Medication demand, but said medicine belongs to pyrimidine derivatives, and drug effect and pharmacological mechanism are similar, and there are certain toxic side effect, answer
With being limited in scope.
In view for the treatment of cancer, especially drug-resistant type cancer there is an urgent need to, this field it is necessary to develop novel molecular skeleton,
Mechanism of action and better good antitumor drug.
Invention content
One of the objects of the present invention is to provide new antitumoral compounds or its pharmaceutically acceptable salts, this is antitumor
Compound is specially thiopyrimidine heterocycle compound, such compound has good antitumor activity.
Another object of the present invention is to provide the preparation methods of aforementioned antitumoral compounds.
Another object of the present invention is to provide containing the thiopyrimidine heterocycle compound or its is pharmaceutically acceptable
The pharmaceutical composition of salt.
It is still another object of the present invention to provide the thiopyrimidine heterocycle compound or its pharmaceutically acceptable salt,
Or the purposes of the composition.
On the one hand, the present invention provides a kind of logical formula (I) compound represented or its pharmaceutically acceptable salt, the general formula
(I) compound represented has the following structure：
Wherein,
X is selected from O, NH；
M is selected from S (O)n, n=0,1；
R is selected from
Preferably, the logical formula (I) compound represented has structure shown in (I-1)~(I-6)：
Preferably, the logical formula (I) compound represented is I-1, I-2, I-4, I-5.
Structural compounds as shown above are thiopyrimidine heterocycle compound, during antitumor activity screening display is of the invention
Compound all have unexpected anti-lung cancer cell (A431 phosphorus cell, HCC827 adenocarcinoma cells and A549 adenocarcinoma cells) and
The proliferative capacity of EGFR T790M drug-resistant types lung carcinoma cell (H1975 cells)；Part of compounds also shows more lucky than with reference to drug
The non-more excellent anti-EGFR-T790M kinase activities for replacing Buddhist nun unexpected for Buddhist nun and Luo Si.Point as a kind of structure novel
Son, the present invention in compound have exploitation at new and effective EGFR-T790M saltant types kinase inhibitor potentiality, to treatment
Have in relevant tumor disease especially Small Cell Lung Cancer, non-small cell lung cancer, EGFR-T790M drug-resistant type non-small cell lung cancers
Larger application value.
Structure is respectively provided with following title shown in aforementioned (I-1)~(I-6)：
(I-1) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) rosickyite base) aniline] -4- pyrimidines] amino] phenyl] -2- third
Acrylamide；
(I-2) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) rosickyite base) aniline] -4- pyrimidines] oxygroup] phenyl] -2- third
Acrylamide；
(I-3) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) propyl thionyl) aniline] -4- pyrimidines] amino] phenyl] -
2- acrylamides；
(I-4) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetylthio) aniline] -4- pyrimidines] amino] phenyl] -2-
Acrylamide；
(I-5) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetylthio) aniline] -4- pyrimidines] oxygroup] phenyl] -2-
Acrylamide；
(I-6) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetyl group thionyl) aniline] -4- pyrimidines] amino] benzene
Base] -2- acrylamides.
On the other hand, the present invention provides the preparation method of aforementioned antitumoral compounds, which presses following road
It is prepared by line：
On the other hand, the present invention provides a kind of pharmaceutical composition, the logical formula (I) institute of the present invention containing effective dose
The compound shown or its pharmaceutically acceptable salt and pharmaceutical carrier.
Compound of the present invention is due to their purposes in drug, and the salt preferred agents of compound can shown in formula (I)
The salt of receiving.The compound of the present invention is alkali, wherein required salt form can be prepared by appropriate method known in the art, is wrapped
It includes and uses mineral acid treatment free alkali, the inorganic acid is such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid；Or at organic acid
Manage free alkali, the organic acids such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, acetone
Acid, oxalic acid, hydroxyacetic acid, salicylic acid, pyranose thuja acid, such as glucuronic acid or galacturonic acid, 'alpha '-hydroxy acids, such as lemon
Acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or cinnamic acid, sulfonic acid, such as p-
Toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid etc..The embodiment of pharmaceutically acceptable salt includes sulfate, pyrosulfate, hydrogen sulfate
Salt, sulphite, bisulfites, phosphate, chloride, bromide, iodide, acetate, propionate, caprate, octanoic acid
Salt, acrylates, formates, isobutyrate, caproate, enanthate, propionate, oxalates, malonate, benzoate, chlorine
Benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxy benzoate, methoxy benzoic acid salt, phthalic acid
Salt, phenyl acetate salt, phenylpropionic acid salt, phenylbutyrate, citrate, lactate, gamma hydroxybutyrate, hydroxyl acetate,
Tartrate, amygdalate and sulfonate, such as xylenesulfonate, mesylate, propane sulfonic acid salt, naphthalene -1- sulfonate and
Naphthalene-2-sulfonic acid salt.
The pharmaceutical composition of the present invention usually contains a kind of the compounds of this invention.However, in some embodiments, this hair
Bright pharmaceutical composition, which contains, has more than a kind of the compound of the present invention.In addition, the pharmaceutical composition of the present invention can also optionally include
One or more other pharmaceutically active compounds.
The present invention also provides the thiopyrimidine heterocycle compound or its pharmaceutically acceptable salt, the pharmaceutical compositions
Object inhibits the purposes of tumor proliferation by inhibiting EGFR-T790M kinases.Specifically, which predominantly prepares for controlling
Treat Small Cell Lung Cancer, non-small cell lung cancer, EGFR-T790M drug-resistant type non-small cell lung cancers drug in purposes.
The present invention provides compound represented or its pharmaceutically acceptable salt or pharmaceutical composition of the present invention exists
Prepare the application in EGFR-T790M kinase inhibitors.
The present invention provides the logical formula (I) compound represented or its pharmaceutically acceptable salt or of the present invention
Purposes of the pharmaceutical composition in the drug for preparing treatment tumour.Preferably, the tumour is selected from Small Cell Lung Cancer, non-small cell
Lung cancer, EGFR-T790M drug-resistant type non-small cell lung cancers it is one or more, further preferred EGFR-T790M drug-resistant types are non-small
Cell lung cancer.It is highly preferred that the purposes is mainly by inhibiting EGFR-T790M kinases to realize.
Description of the drawings
Fig. 1 shows compound I-1 different disposals groups to change over time situation to the inhibiting effect of H1975 cells.
Fig. 2 indicates the influence result figure of Apoptosis in the H1975 cells that compound I-1 is mutated EGFR-T790M.
Specific implementation mode
The explanation present invention is further described below in conjunction with specific embodiment, but these embodiments are not meant as limiting this hair
Bright range.
Test method without specific conditions in the embodiment of the present invention, usually according to normal condition, or according to raw material or
Condition proposed by commodity manufacturer.The reagent in specific source is not specified, for the conventional reagent of market purchase.
The preparation of 1 target molecule of embodiment
Tlc silica gel plate uses Yantai Huanghai Sea GF254 or Qingdao GF254 silica gel plates, thin-layered chromatography (TLC) to use
The specification that uses of silicon amine plate be 0.15mm-0.2mm, the specification that thin-layer chromatography isolates and purifies product use is 0.4mm-0.5mm.
The raw material that the present invention uses mainly is purchased from commercially available from Sinopharm Chemical Reagent Co., Ltd., Beijing coupling science and technology
Co., Ltd, reaches the companies such as auspicious chemicals at Aladdin chemical reagent Co., Ltd.
Without specified otherwise in embodiment, solution refers to aqueous solution.
Without specified otherwise in embodiment, the temperature of reaction is room temperature, is 20 DEG C -30 DEG C.
The technical solution adopted by the present invention is as follows：
Compound (I-1), (I-2), the synthetic route reagents of (I-3) and condition:(a) acryloyl chloride, NaHCO3, CH3CN,
Room temperature, 0.5 hour, 60-80%；(b) when X=OH, K2CO3, DMF, 60 DEG C；X=NH2When, DIPEA, Isosorbide-5-Nitrae-dioxanes, room temperature,
2 hours, 91%；(c) the chloro- 3- N-Propyl Bromides of 1-, K2CO3, CH3CN, 70 DEG C, 12 hours, 95%；(d) morpholine, K2CO3, KI, DMF,
80 DEG C, 12 hours, 81%；(e)Fe-NH4Cl, MeOH-H2O, 2 hours, 60 DEG C, 72%；(f) TFA, 2-BuOH, 100 DEG C, 12 is small
When, 22-37%；(g) m-CPBA, CH2Cl2, room temperature, 31%.
Compound (I-4), (I-5), the synthetic route reagents of (I-6) and condition:(a) 2- ethyl thioglycolates, K2CO3,
KI, DMF, 100 DEG C, 12 hours, 95%；(b) KOH, H2O, 50 DEG C, 24 hours, 76%；(c)SOCl2, 80 DEG C, 2 hours；(d)
Quinoline, NaHCO3, CH3CN, room temperature, 0.5 hour, 95%；(e)Fe-NH4Cl, MeOH-H2O, 2 hours, 60 DEG C, 72%；(f) pyrimidine
3a-b, TFA, 2-BuOH, 100 DEG C, 12h, 16-19%；(g)m-CPBA,CH2Cl2, room temperature, 25%.
Target molecule is synthesized according to above method, the physicochemical data of synthesized target molecule is as follows：
(I-1) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) rosickyite base) aniline] -4- pyrimidines] amino] phenyl] -2- third
Acrylamide yields:36.52%, yellow solid1H NMR(400MHz,DMSO-d6):δ1.60-1.63(m,2H),2.28-2.32
(m, 6H), 2.82 (t, J=8.0Hz, 2H), 3.53 (t, J=8.0Hz, 4H), 5.76 (dd, J=4.0Hz, 8.0Hz, 1H),
6.26 (dd, J=4.0Hz, 16.0Hz, 1H), 6.44 (dd, J=8.0Hz, 16.0Hz, 1H), 7.11 (d, J=8.0Hz, 2H),
7.18-7.35(m,2H),7.52-7.57(m,3H),7.89(s,1H),8.15(s,1H),9.00(s,1H),9.41(s,1H),
10.22(s,1H)；13C NMR(100MHz,DMSO-d6):δ26.37,32.42,53.98(2C),57.39,66.86(2C),
104.60,116.17,116.24,120.03(2C),120.14,127.35,127.64,129.32,131.03(2C),
132.53,139.46,139.76,139.83,155.48,156.96,158.18,163.84；HRMS (ESI),
C26H29ClN6O4S, m/z, [M+H]+, theoretical calculation：525.1834, actual measurement:525.1843.
(I-2) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) rosickyite base) aniline] -4- pyrimidines] oxygroup] phenyl] -2- third
Acrylamide yields:22.14%；Yellow solid；1H NMR(400MHz,DMSO-d6):δ1.64-1.69(m,2H),2.32-2.36
(m, 6H), 2.87 (t, J=8.0Hz, 2H), 3.58 (t, J=8.0Hz, 4H), 5.83 (dd, J=4.0Hz, 8.0Hz, 1H),
6.32 (dd, J=4.0Hz, 16.0Hz, 1H), 6.49 (dd, J=12.0Hz, 16.0Hz, 1H), 7.06-7.10 (m, 3H), 7.40
(d, J=8.0Hz, 2H), 7.52 (d, J=8.0Hz, 2H), 7.76 (s, 1H), 8.54 (s, 1H), 9.89 (s, 1H), 10.46 (s,
1H)；13C NMR(100MHz,DMSO-d6):δ26.01,31.83,53.67(2C),57.04,66.56(2C),105.09,
113.30,117.02,117.39,119.75(2C),127.79,128.01,130.31(2C),130.39,131.97,
138.56,140.78,152.66,157.75,158.51,163.73,164.26；HRMS(ESI),C26H28ClN5O3S,m/z,[M
+H]+, theoretical calculation:526.1674, actual measurement:526.1678.
(I-3) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) propyl thionyl) aniline] -4- pyrimidines] amino] phenyl] -
2- acrylamide yields:31.0%；Brown solid；1H NMR(400MHz,DMSO-d6):δ1.56-1.68(m,2H),2.26-
2.32 (m, 6H), 2.74-2.86 (m, 2H), 3.52 (t, J=8.0Hz, 4H), 5.76 (dd, J=4.0Hz, 8.0Hz, 1H),
6.27 (dd, J=4.0Hz, 16.0Hz, 1H), 6.48 (dd, J=12.0Hz, 16.0Hz, 1H), 7.30-7.40 (m, 5H),
7.53-7.58 (m, 1H), 7.83 (d, J=8.0Hz, 2H), 8.22 (s, 1H), 9.10 (s, 1H), 9.73 (s, 1H), 10.28 (s,
1H)；13C NMR(100MHz,DMSO-d6):δ19.14,19.47,53.68(2C),57.13,66.78(2C),105.28,
114.31,116.17,116.35,119.33(2C),125.25(2C),126.64,127.63,129.53,132.56,
135.60,139.91,143.62,155.47,157.03,158.01,163.86；HRMS(ESI),C26H29ClN6O3S,m/z,[M
+H]+, theoretical calculation:541.1783, actual measurement:541.1788.
(I-4) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetylthio) aniline] -4- pyrimidines] amino] phenyl] -2-
Acrylamide yields:16.04%；White solid；1H NMR(400MHz,DMSO-d6):δ3.41-3.54(m,8H),3.80(s,
2H), 5.76-5.79 (m, 1H), 6.28 (dd, J=4.0Hz, 16.0Hz, 1H), 6.48 (dd, J=8.0Hz, 16.0Hz, 1H),
7.20 (d, J=8.0Hz, 2H), 7.36 (d, J=8.0Hz, 2H), 7.45-57 (m, 3H), 7.91 (s, 1H), 8.18 (s, 1H),
9.03(s,1H),9.46(s,1H),10.24(s,1H)；13C NMR(100MHz,DMSO-d6):δ37.70,42.74,47.14,
67.04(2C),105.06,116.42,116.59,120.18(2C),120.48,126.44,127.95,129.62,132.30
(2C),132.84,139.75,140.12,140.71,155.75,157.25,158.43,164.15,167.63；HRMS
(ESI),C25H25ClN6O3S,m/z,[M+H]+, theoretical calculation:525.1470, actual measurement:525.1484.
(I-5) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetylthio) aniline] -4- pyrimidines] oxygroup] phenyl] -2-
Acrylamide yields:19.01%；White solid；1H NMR(400MHz,DMSO-d6):δ 3.39 (t, J=4.0Hz, 4H),
3.50 (t, J=4.0Hz, 4H), 3.77 (s, 2H), 5.77 (dd, J=4.0Hz, 8.0Hz, 1H), 6.26 (dd, J=4.0Hz,
16.0Hz, 1H), 6.45 (dd, J=8.0Hz, 16.0Hz, 1H), 7.00-7.03 (m, 1H), 7.11 (d, J=8.0Hz, 2H),
7.35 (d, J=8.0Hz, 2H), 7.46 (d, J=8.0Hz, 1H), 7.57 (d, J=8.0Hz, 1H), 7.70 (s, 1H), 8.48
(s,1H),9.85(s,1H),10.39(s,1H)；13C NMR(100MHz,DMSO-d6):δ36.89,42.14,46.51,66.43
(2C),105.30,113.26,117.09,117.43,119.60(2C),126.84,127.81,130.40,131.36(2C),
131.98,139.24,140.78,152.67,157.74,158.50,163.77,164.28,166.95；HRMS(ESI),
C25H24ClN5O4S,m/z,[M+H]+, theoretical calculation：526.1310, actual measurement:526.1323.
(I-6) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholines) acetyl group thionyl) aniline] -4- pyrimidines] amino] benzene
Base] -2- acrylamide yields:25.0%；Brown solid；1H NMR(400MHz,DMSO-d6):δ3.37-3.52(m,8H),
3.93 (d, J=12.0Hz, 1H), 4.11 (d, J=12.0Hz, 1H), 5.77 (dd, J=4.0Hz, 8.0Hz, 1H), 6.27 (dd,
J=4.0Hz, 16.0Hz, 1H), 6.47 (dd, J=8.0Hz, 16.0Hz, 1H), 7.37 (d, J=8.0Hz, 2H), 7.49 (d, J
=8.0Hz, 2H), 7.82-7.94 (m, 4H), 8.22 (s, 1H), 9.11 (s, 1H), 9.72 (s, 1H), 10.25 (s, 1H)；13C
NMR(100MHz,DMSO-d6):δ42.39,46.71,61.09,66.61,66.78,105.36,116.23,116.43,
119.23(2C),120.43,125.78(2C),127.71,129.36,132.53,135.41,139.40,139.91,
143.96,155.48,157.09,158.01,163.89,163.91；HRMS(ESI),C25H25ClN6O4S,m/z,[M+H]+, reason
By calculating:541.1419, actual measurement:541.1441.
Target molecule at salt method
The preparation method of inorganic acid salt：It takes target molecule (1mmol) to be dissolved in 10mL absolute methanols, under ice bath, slowly drips
The 5mL absolute methanol solutions for adding inorganic acid (1mmol), are added dropwise, and are stirred 30 minutes at a temperature of this, then first is evaporated off in room temperature
Alcohol to get target molecule inorganic acid salt.Hydrochloride (I-1-1), the hydrobromate of compound I-1 are prepared for by this method
(I-1-2), sulfate (I-1-3) and phosphate (I-1-4)；
The preparation method of acylate：It takes target molecule (1mmol) to be dissolved in 10mL absolute methanols, under ice bath, slowly drips
The 5mL dry ethers for adding organic acid (1mmol), are added dropwise, and are stirred 30 minutes at a temperature of this, and then solvent is evaporated off in room temperature,
Up to the acylate of target molecule.Maleate (I-1-5), the succinate (I- of compound I-1 are prepared for by this method
1-6) and fumarate (I-1-7).
2 target molecule biological evaluation of embodiment
1, in vitro to receptor tyrosine kinase inhibitory activity test method
Prepare kinase assay buffer
1. melting kinase assay buffer in room temperature, precipitation has been seen whether.
2. if there is precipitation, it is just incubated kinase assay buffer at 37 DEG C 15 minutes and often shakes, dissolving precipitation.Or
Person carefully siphons away supernatant, removal precipitation.
Prepare kinase assay reagent
1. using preceding in equilibrium at room temperature kinase assay buffer and kinase assay substrate.
2. all pouring into kinase assay buffer in the brown bottle equipped with kinase assay substrate, keep freeze-dried powder substrate molten
Solution, has thus been made kinase assay reagent.
3. gently concussion, vortex or reverse mixing, become homogeneous solution, substrate should dissolve in 1 minute.
4. kinase assay reagent should use immediately after preparing, or packing is stored in -20 DEG C, and the reagent prepared passes through freeze thawing several times
Cycle signal activity is not all lost afterwards.
Make the standard curve that ATP is converted to ADP
1. the ultrapure ATP and ADP that are provided with 1 × kinase reaction buffer solution dilution kit, are made 900 μ L, 50 μM of ATP
With 500 μ L, 50 μM of ADP.
2. by 50 μM of ATP and 50 μM of ADP solution that previous step prepares by being mixed in 384 orifice plate A1-A12 shown in table 1,
The concentration for simulating the ATP and ADP of each conversion percentages, mixes.
Table 1. prepares 50 μM of series A TP+ADP standard items
3. the ADP-Glo of 5 μ L is added per holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
4. 10 μ L kinase assays reagents are added per hole is converted to ATP by ADP, and introduces luciferase and luciferin to detect
ATP。
5. in incubation at room temperature 30-60 minutes, measures fluorescent with multi-function microplate reader and record fluorescent value.
6. drawing the standard curve that ATP is converted to ADP.
Determine the IC50 values of kinase inhibitor
1. preparing 1 × kinase reaction buffer solution, 2.5 × 50ng/ μ L kinases and 2.5 according to promega kit specifications
× 0.5 μ g/ μ L substrates and 125 μM of ATP.
2. 3 μ L 1 × kinase reaction buffer solutions are added in no enzyme control wells, 2 μ L, 2.5 × 0.5 μ g/ μ L substrates and 125 μ
M ATP.It is added 1 μ L 1 × kinase reaction buffer solutions in negative control hole, 2 μ L 2.5 × 50ng/ μ L kinases, 2 μ L 2.5 ×
0.5 μ g/ μ L substrates and 125 μM of ATP.1 μ L 5 × drug to be measured, 2 μ L 2.5 × 50ng/ μ L kinases, 2 μ are added in instrument connection
2.5 × 0.5 μ g/ μ L substrates of L and 125 μM of ATP.
3. mixing tablet, it is incubated 60 minutes.
4. the ADP-Glo of 5 μ L is added per holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
5. 10 μ L kinase assays reagents are added per hole is converted to ATP by ADP, and introduces luciferase and luciferin to detect
ATP.In incubation at room temperature 30-60 minutes, measures fluorescent with multi-function microplate reader and record fluorescent value.
6. interpretation of result, the results are shown in Table 2.
2, cell growth assay (MTT detection methods)
Cell inoculation：Exponential phase cell is collected, concentration of cell suspension is adjusted, with every hole 7x103A cell, per hole body
100 μ L of product are inoculated into 96 orifice plates, and every group sets 4 multiple holes (edge hole is filled with sterile PBS)；
Cell culture：After cell is adherent, 0%FBS RPMI-1640 starvation 8h, control group 10%FBS RPMI-1640
Culture, 37 DEG C, 5%CO2Continue to cultivate (empirically requiring to cultivate different time respectively) in incubator；
Colour generation：10 μ L MTT solution (5mg/mL) are added in three groups of cells after cultivating 72h, terminate culture after 4h, often
100 μ L, tri- liquid is added in hole makes crystallization fully dissolve in low-speed oscillation 10min on shaking table；
Colorimetric：Each hole shading value (OD values) is measured on enzyme-linked immunosorbent assay instrument, 570nm wavelength is selected, with acellular
I.e. RPMl-1640 culture solutions blank well returns to zero, and surveys the absorbance value in each hole.Experiment is in triplicate
Record result：Inhibitory rate of cell growth=(one experimental group absorbance value of control group absorbance value)/control group extinction
Angle value × 100%, cell proliferation rate=(experimental group absorbance value/control group absorbance value) × 100；
Draw cell growth curve：Using the time as abscissa, inhibiting rate/proliferation rate is that ordinate draws cell growth song
Line.
Do figure for inhibitor concentration in GraphPad Prism mapping softwares in GraphPad softwares, so as to by
Log [inhibitor] estimates IC relative to reaction, variable slope model50。
Test result such as table 2, table 3, shown in table 4, table 2 indicates that compound inhibits EGFR and EGFR-T790M kinase activities,
Table 3 indicates the proliferation activity of compound antitumor cell and human normal cell, and Fig. 1 shows compound I-1 different disposal groups pair
The inhibiting effect of H1975 cells changes over time situation.
Table 2
Using ADP-GloTMThe such molecule of kinase assays system evaluation is to Wild type EGFR and mutant egf R-T790M/
The inhibitory activity of L858R kinases replaces Buddhist nun as positive control using representative EGFR inhibitor drug Gefitinib and Luo Si.Table 2
In the test result that provides clearly illustrate that such compound has EGFR-T790M/L858R kinase inhibiting activities outstanding,
The inhibitory activity of middle compound I-1 and I-5 is most strong, IC50Value is respectively 27.5 and 9.1nM.With oxygenatedchemicals I-7 (IC50
=100.5nM) activity compare, sulfur-containing compound I-1 (IC50=27.5nM) improve nearly 10 times；With reference medicine phases ratio, activity
More advantage.It is particularly concerned with, the invalid (IC of such compounds against wild type EGFR50>980nM), show them to mutation
EGFR-T790M has highly selective inhibitory activity.Especially inhibitor I-1 (SI>36.4) and I-5 (SI>110.2) have than
Sieve department foretells (SI=25) much higher selectivity for Buddhist nun, also indicates their lower toxic side effects.
Table 3
aFour kinds of typical NSCLC cell lines：A431EGFR-WTCell, H1975EGFR-T790MMutant clone, A549EGFR - WT and k-ras mutationAnd HCC827EGFR del E746_A750 mutationCell strain and two kinds of normal cell systems (human bronchial epithelial cell (HBE)) and
Liver cell (LO-2)
In addition, evaluating antiproliferative activity of all compounds to NSCLC lung cancer cell lines using MTT measuring methods.Such as 3 institute of table
Show, compound I-1~I-5 has the anti-H1975 cells for being significantly higher than Gefitinib in 0.074 to 1.050 μM of concentration range
The activity of proliferation；It is suitable for Buddhist nun and Gefitinib with Ross to the A431 cellular potencies for carrying Wild type EGFR.The present invention
The selection index of compound I-1~I-5 is above Gefitinib, and the selection index of compound I-1, I-2, I-5 are replaced higher than Ross
Buddhist nun, especially compound I-1 have very strong effect at a concentration of 0.074 μM to H1975 cells.Compound I-1 also has
There is the medium activity (IC for inhibiting A431 cell Proliferations50=3.893 μM), show its highly selective (SI to mutation H1975 cells
=52.6).In addition, all these compounds are all mutated to carrying the highly sensitive EGFR del E746-A750 of HCC827 cells
It is highly effective, IC50Value is less than 0.308 μM.Compound I-4 most has strongest inhibition HCC827 cell activity, IC50Value is to have
0.050μM.What is more attracted people's attention is that this most of compound are non-toxic to normal cell system HBE and LO-2, especially inhibitor
I-1、I-4(IC50Value is all higher than 40 μM) show rather low cytotoxicity.
Fig. 1 shows most potent inhibitor I-1 with time (24,48 and 72 hours) and drug concentration (0.2,0.5 and 1.0 μM)
Increase and significantly block the effect of the proliferation of H1975 cells.As the I-1 using 1.0 μM of concentration, after being administered 72 hours, cell
Survival rate (26.8%) is about 2 times lower for Buddhist nun's non-(44.8%) than sieve department, shows anti-gefitinib resistant enhancing.These biology
Assessment shows that compound I-1 may be used potentially as the drug resistant inhibitor of EGFR-T790M for NSCLC.The present invention is also ground
The influence of Apoptosis in the H1975 cells that most effective inhibitor I-1 is mutated EGFR-T790M is studied carefully.Acquired results are as schemed
Shown in 2, compound I-1 induces H1975 Apoptosis in a manner of dosage and time dependence.With compound I-1 (0.2,0.5 Hes
1.0 μM) apoptosis rate of H1975 cells of processing 72 hours obviously increases to 96.8% from 45.6%.
The above bioactivity the result shows that, the present invention in molecule antitumous effect active effect it is notable, toxic side effect is low,
It is mainly manifested in the following aspects:1) compound I-1 and I-5 shows that anti-EGFR-T790M/L858R very outstanding lives
Property, IC50Value is respectively 27.5 and 9.1nM, and effect is than with reference to unexpected strong of medicine Gefitinib；2) compound I-1 and I-5
Two are also significantly better than with reference to medicine to the selective depression index SI of EGFR-T790M, it is unexpected to indicate that such molecule has
Lower toxic side effect；3) compound I-1 is less than 100nM to the inhibitory activity of drug-resistant type lung carcinoma cell H1975, is significantly better than Ji
It is non-to replace Buddhist nun, it is horizontal for Buddhist nun to reach sieve department；4) I-4, I-6 compound are less than 100nM to the inhibition of lung carcinoma cell HCC827, resist
NSCLC cell Proliferations effect is very prominent；4) majority of compounds to NSCLC squamous cell carcinomas A431 and adenocarcinoma cell A549 also all
There is very strong inhibitory activity, the EGFR inhibitor drug than having listed has more obvious progress and advantage；5) most of
Compound is to people upper respiratory tract normal cell HBE and people's normal cell lines of human liver LO-2 is not significantly interfered with and inhibiting effect, shows this
Class molecule has unexpected lower cytotoxicity.
As the molecule of a kind of structure novel, research compound has exploitation at new and effective EGFR-T790M in the present invention
The potentiality of kinase inhibitor, to treatment-related tumor disease especially Small Cell Lung Cancer, non-small cell lung cancer, EGFR-T790M
Drug-resistant type non-small cell lung cancer has larger application value.
Claims (9)
1. a kind of antitumoral compounds or its pharmaceutically acceptable salt, which has structure shown in formula (I)：
Wherein,
X is selected from O, NH；
M is selected from S (O)n, n=0,1；
R is selected from
2. antitumoral compounds according to claim 1 or its pharmaceutically acceptable salt, wherein the logical formula (I) institute
The compound shown has structure shown in (I-1)~(I-6)：
3. a kind of pharmaceutical composition, the antitumoral compounds as claimed in claim 1 or 2 containing effective dose or its pharmaceutically
Acceptable salt and pharmaceutical carrier.
4. the preparation method of antitumoral compounds as claimed in claim 1 or 2 or its pharmaceutically acceptable salt, described antitumor
Compound is prepared by following route：
5. the medicine described in antitumoral compounds as claimed in claim 1 or 2 or its pharmaceutically acceptable salt or claim 3
Application of the compositions in preparing EGFR-T790M saltant type skin factor receptor protein tyrosine kinase inhibitor.
6. the medicine described in antitumoral compounds as claimed in claim 1 or 2 or its pharmaceutically acceptable salt or claim 3
Purposes of the compositions in the drug for preparing treatment tumour.
7. purposes according to claim 6, wherein the tumour is selected from Small Cell Lung Cancer, one kind of non-small cell lung cancer
Or it is a variety of.
8. purposes according to claim 7, wherein the non-small cell lung cancer is EGFR-T790M saltant type non-small cells
Lung cancer.
9. the purposes according to any one of claim 6~8, wherein the purposes is mainly by inhibiting EGFRT790M prominent
What modification epidermis factor receptor proteins tyrosine kinase was realized.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102083800A (en) * | 2008-06-27 | 2011-06-01 | 阿维拉制药公司 | Heteroaryl compounds and uses thereof |
| CN105884695A (en) * | 2015-02-13 | 2016-08-24 | 山东轩竹医药科技有限公司 | Heterocyclic derivate tyrosine kinase inhibitor |
| CN105968056A (en) * | 2016-05-28 | 2016-09-28 | 大连医科大学 | Diarylpyrimidine compound, composition and application |
| CN106565614A (en) * | 2016-11-16 | 2017-04-19 | 大连医科大学 | Diphenylaminopyrimidine compound, composition and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102083800A (en) * | 2008-06-27 | 2011-06-01 | 阿维拉制药公司 | Heteroaryl compounds and uses thereof |
| CN105884695A (en) * | 2015-02-13 | 2016-08-24 | 山东轩竹医药科技有限公司 | Heterocyclic derivate tyrosine kinase inhibitor |
| CN105968056A (en) * | 2016-05-28 | 2016-09-28 | 大连医科大学 | Diarylpyrimidine compound, composition and application |
| CN106565614A (en) * | 2016-11-16 | 2017-04-19 | 大连医科大学 | Diphenylaminopyrimidine compound, composition and application |
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