CN109265469A - Pyrimido thiazole heterocyclic compounds, composition and its purposes for treating lymphocytic leukemia - Google Patents
Pyrimido thiazole heterocyclic compounds, composition and its purposes for treating lymphocytic leukemia Download PDFInfo
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- CN109265469A CN109265469A CN201811340443.XA CN201811340443A CN109265469A CN 109265469 A CN109265469 A CN 109265469A CN 201811340443 A CN201811340443 A CN 201811340443A CN 109265469 A CN109265469 A CN 109265469A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
- C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
- C07D495/04—Ortho-condensed systems
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
Abstract
The present invention relates to pyrimido thiazole heterocyclic compounds, composition and its purposes for treating lymphocytic leukemia, the pyrimido thiazole heterocyclic compounds are specially logical formula (I) compound represented, and each substituent group of logical formula (I) is defined in the description.The invention further relates to the logical formula (I) compound represented or its pharmaceutically acceptable salts, or containing its pharmaceutical composition by inhibit JAK3 tyrosine kinase, and then tumor disease is treated, especially for treating the purposes of Burkitt's lymphoma, diffusivity large B cell lymphoid tumor, follicular lymphoma or chronic lymphocytic leukemia.
Description
Technical field
The present invention relates to pyrimido thiazole heterocyclic compounds, composition and its purposes for treating lymphocytic leukemia,
Belong to pharmaceutical technology field.
Background technique
Protein tyrosine kinase (protein tyrosine kinase, PTKs) is logical by the signal transduction of control cell
Road adjusts a series of physiological and biochemical procedures such as growth, differentiation, apoptosis of cell, be anti-tumor drug research and development important target spot it
One.Receptor type tyrosine kinase is a kind of kinases across cell membrane, the extracellular domain, transmembrane domain with ligand binding
In the specific tyrosine residue of phosphorylation and thus the intracellular domain of cell Proliferation is influenced with zymogenesis-is played.Numerous
(such as lung cancer, breast cancer, gastric cancer, oophoroma, lymthoma) is it has been found that the unconventionality expression of the kinases in cancer.
Recently researches show that: all there is JAK3 gene mutation in lymphocytic leukemia substantially, JAK-STAT inhibitor is
The effective way for treating Lymphoma.Carrying out a kind of JAK suppression for treating rheumatism of three phases clinical research assessment
Preparation also can induce Apoptosis, promote lymphoma cell strain dead (Cancer Discov.2012,2,591-597), this is
JAK3 inhibitor provides an important evidence and new thinking for treating lymphocytic leukemia.
In view for the treatment of leukaemia, there is an urgent need to it is necessary to developmental function mechanism, this field unique, the anti-leaching of structure novel
Bar chronic myeloid leukemia drug.
Summary of the invention
One of the objects of the present invention is to provide a kind of pyrimido thiazole heterocyclic compounds or its is pharmaceutically acceptable
Salt, such compound have good antilymphocyte leukocythemia liveness.
Another object of the present invention is to provide containing the pyrimido thiazole heterocyclic compounds or its is pharmaceutically acceptable
Salt pharmaceutical composition.
A further object of the present invention is to provide the pyrimido thiazole heterocyclic compounds or its is pharmaceutically acceptable
The purposes of salt or 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 NH or O；
R1Selected from hydrogen, chlorine, methyl, fluorine or methoxyl group；
R is selected from
In some embodiments, the logical formula (I) compound represented has structure shown in I-1~I-9:
Preferably, the logical formula (I) compound represented is I-3.
Structural compounds as shown above are pyrimido thiazole heterocyclic compounds, the anti-tumor activity screening display present invention
In compound have stronger inhibition lymphocyte oncocyte (Ramos cell and Raji cell) proliferative capacity, part chemical combination
Object is shown than the more excellent anti-JAK3 kinase activity unexpected referring to drug Spebrutinib and HM71224.As
The molecule of a kind of structure novel, the present invention in compound have exploitation at new and effective JAK3 inhibitor potentiality, to treatment
Relevant tumor disease especially lymthoma, follicular lymphoma or chronic lymphocytic leukemia have biggish application value.
Structure shown in aforementioned I-1~I-9 is respectively provided with following title:
(I-1) N- [3- [[2- [[3-4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D] pyrimidine-
4- yl] oxygroup] phenyl] -2- acrylamide；
(I-2) N- [3- [[2- [[3- methyl -4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D]
Pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
(I-3) N- [3- [[2- [[3- methyl -4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D]
Pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
(I-4) [[[2- [[the chloro- 4- of 3- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D] is phonetic by 3- by N-
Pyridine -4- base] amino] phenyl] -2- acrylamide；
(I-5) N- [3- [[2- [[4- (2- [[4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thieno [3,
2-D] pyrimidine-4-yl] oxygroup] phenyl] -2- acrylamide；
(I-6) N- [3- [[2- [[4- (2- [[4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thieno [3,
2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
(I-7) N- [3- [[2- [[4- (2- [[3- methyl -4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
Pheno simultaneously [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
(I-8) N- [3- [[2- [[4- (2- [[the chloro- 4- of 3- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
And [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
(I-9) N- [3- [[2- [[4- (2- [[2- methyl -4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
Pheno simultaneously [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide；
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.
For compound of the present invention due to their possibility purposes in drug, the salt preferred agents of formula (I) compound can
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 with mineral acid treatment free alkali, described inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.；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 (pyranosidy1acid), such as glucuronic acid or galacturonic acid,
'alpha '-hydroxy acids, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid or meat
Cinnamic acid, sulfonic acid, such as p- toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid etc..The embodiment of pharmaceutically acceptable salt include sulfate,
Pyrosulfate, disulfate, sulphite, bisulfites, phosphate, chloride, bromide, iodide, acetate, propionic acid
Salt, caprate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, propionate (propiolates),
Oxalates, malonate, benzoate, chloro benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxy benzoate,
Methoxy benzoic acid salt, phthalate, phenyl acetate salt, phenylpropionic acid salt, phenylbutyrate (phenylbutrates),
Citrate, lactate, gamma hydroxybutyrate, hydroxyl acetate, tartrate, amygdalate and sulfonate, such as diformazan
Benzene sulfonate, mesylate, propane sulfonic acid salt, naphthalene -1- sulfonate and naphthalene-2-sulfonic acid salt.
Pharmaceutical composition of the 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, pharmaceutical composition of the invention can also optionally include
One or more other pharmaceutically active compounds.
The present invention also provides the pyrimido thiazole heterocyclic compounds or its pharmaceutically acceptable carrier, the drugs
Composition inhibits the purposes of tumor proliferation by inhibiting JAK3 tyrosine kinase.Specifically, which predominantly prepares and uses
In the medicine for the treatment of Burkitt's lymphoma, diffusivity large B cell lymphoid tumor, follicular lymphoma or chronic lymphocytic leukemia
Purposes in object.
The present invention provides compound represented or its pharmaceutically acceptable salt or pharmaceutical composition of the present invention exists
Prepare the application in JAK3 tyrosine kinase inhibitor.
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 of preparation treatment tumour.Preferably, the tumour is selected from diffusivity large B cell lymph
One of tumor, follicular lymphoma and chronic lymphocytic leukemia are a variety of, the further preferred white blood of chronic lymphocytic
Disease.It is highly preferred that the purposes, which mainly passes through, inhibits the realization of JAK3 tyrosine kinase.
Detailed description of the invention
Fig. 1 is the anti-Ramos lymphocyte proliferation activity experimental result picture of compound I-3.
Fig. 2 is the lymphopoietic morphological observations figure of the anti-Ramos of compound I-3.
Fig. 3 is compound I-3 to PBMC cytotoxicity experiment result figure.
Specific embodiment
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 HSGF254 or Qingdao GF254 silica gel plate, and thin-layered chromatography (TLC) makes
The specification that silicon amine plate uses is 0.15mm-0.2mm, and 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, it is 20 DEG C -30 DEG C that the temperature of reaction, which is room temperature,.
The technical solution adopted by the invention is as follows:
Synthetic route, reagent and the condition of compound I- (1-9): (condition 1) diisopropyl ethyl amine, dioxane, 60
℃,2h；(condition 2) trifluoroacetic acid, isopropanol, 12h, 100 DEG C.
The synthesis of M-2
Take 2,4- dichloro-thiophene simultaneously [3,2-D] pyrimidine (7.24mmol) and intermediate M-1 (7.24mmol), DIPEA
Isosorbide-5-Nitrae-dioxane is added in flask in (10.86mmol), and 60 DEG C are reacted 2 hours, reaction solution evaporated under reduced pressure, adds elutriation to go out solid
Body filters, and drying obtains solid M-2.
The synthesis of object I
It takes amino-compound M-3 or M-4 (1.0mmol) to react respectively with pyrimidine intermediate M-2 (1.0mmol), is dissolved in
In 10mL 2-BuOH, trifluoroacetic acid (1.5mmol) slowly is added dropwise, heats up 100 DEG C and reacts 12 hours, drain after completion of the reaction molten
Agent, adds MeOH 10mL, then plus saturated sodium bicarbonate solid is precipitated, thin-layer chromatography isolates and purifies to obtain target compound I- (1-9).
Target molecule is synthesized according to above method, the physicochemical data of synthesized target molecule is as follows:
(I-1) N- [3- [[2- [[3-4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D] pyrimidine-
4- yl] oxygroup] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.44(s,1H),9.36(s,1H),8.29
(d, J=5.4Hz, 1H), 7.73 (t, J=2.0Hz, 1H), 7.62 (d, J=8.3Hz, 1H), 7.55 (d, J=7.6Hz, 2H),
7.46 (s, 1H), 7.38-7.29 (m, 1H), 7.08 (dd, J=7.9,1.9Hz, 1H), 6.76 (dd, J=30.0,8.7Hz,
2H), 6.46 (dd, J=16.9,10.1Hz, 1H), 6.29 (d, J=1.8Hz, 1H), 5.79 (dt, J=10.2,5.0Hz, 1H),
4.24 (s, 2H), 3.80 (s, 4H), 3.15 (d, J=50.8Hz, 6H)；13C NMR(101MHz,DMSO-d6)δ166.11(s),
165.01(s),164.77(s),159.19(s),153.59(s),141.73(s),138.24(s),135.90(s),133.01
(s),131.29(s),128.71(s),126.53(s),124.54(s),121.55(s),118.43(s),118.04(s),
115.86(s),114.30(s),64.92(s),56.70(s),53.26(s)；HRMS(ESI)for C28H29N5O4S,[M+H]+
Theoretical calculation: 532.2013, actual measurement: 532.21043；
(I-2) N- [3- [[2- [[3- methyl -4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D]
Pyrimidine-4-yl] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.41(s,1H),9.32(s,
1H), 8.18 (d, J=5.2Hz, 1H), 7.68 (t, J=2.0Hz, 1H), 7.58 (d, J=8.3Hz, 1H), 7.48 (d, J=
7.6Hz, 2H), 7.41 (s, 1H), 7.38-7.20 (m, 2H), 7.00 (dd, J=8.0,2.0Hz, 1H), 6.70 (dd, J=
30.0,8.8Hz, 2H), 6.40 (dd, J=16.0,10.0Hz, 1H), 6.20 (d, J=1.8Hz, 1H), 5.70 (dt, J=
10.0,5.0Hz, 1H), 4.20 (s, 2H), 3.80 (s, 4H), 3.10 (d, J=50.8Hz, 6H)；13C NMR(101MHz,DMSO-
d6)δ166.11(s),165.00(s),164.70(s),159.10(s),153.50(s),141.68(s),138.20(s),
135.90(s),133.00(s),131.20(s),128.70(s),126.50(s),124.48(s),120.11(s),118.33
(s),118.00(s),115.02(s),114.20(s),64.90(s),56.52(s),53.20(s)；HRMS(ESI)for
C28H30N6O3S,[M+H]+Theoretical calculation: 531.2173, actual measurement: 531.2199；
(I-3) N- [3- [[2- [[3- methyl -4- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D]
Pyrimidine-4-yl] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.32(s,1H),8.49(s,
1H), 8.19 (d, J=5.4Hz, 1H), 7.70 (t, J=2.0Hz, 1H), 7.53 (d, J=0.9Hz, 1H), 7.40 (t, J=
8.1Hz, 1H), 7.19 (dd, J=11.7,7.0Hz, 2H), 7.05 (dd, J=8.1,1.7Hz, 1H), 6.72 (d, J=2.7Hz,
1H), 6.59 (dd, J=8.7,2.6Hz, 1H), 6.45 (dd, J=17.0,10.1Hz, 1H), 6.28 (dd, J=16.9,
1.9Hz, 1H), 5.78 (dd, J=10.1,2.0Hz, 1H), 3.94 (t, J=6.3Hz, 2H), 3.64-3.50 (m, 4H), 2.40
(dd, J=15.6,8.0Hz, 6H), 2.12 (s, 3H), 1.90-1.79 (m, 2H)；13C NMR(101MHz,DMSO-d6)δ
166.47(s),164.94(s),164.69(s),161.04(s),156.92(s),153.48(s),141.59(s),137.82
(s),133.02(s),132.24(s),131.18(s),128.69(s),128.33(s),124.45(s),118.28(s),
117.79(s),117.17(s),114.09(s),112.88(s),108.21(s),67.55(s),67.10(s),56.24(s),
54.74(s),27.28(s),19.65(s)；HRMS(ESI)for C29H31N5O4S,[M+H]+Theoretical calculation: 546.2170, it is real
It surveys: 546.2155；
(I-4) [[[2- [[the chloro- 4- of 3- (2- (4- morpholinyl) propoxyl group) phenyl] amino] thieno [3,2-D] is phonetic by 3- by N-
Pyridine -4- base] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.41(s,1H),10.12(s,1H),
9.51 (s, 1H), 8.31 (d, J=5.2Hz, 1H), 7.76 (d, J=18.1Hz, 2H), 7.68-7.22 (m, 4H), 7.02 (dd, J
=46.5,8.3Hz, 2H), 6.64-6.02 (m, 2H), 5.77 (d, J=10.1Hz, 1H), 4.01 (d, J=24.6Hz, 4H),
3.67 (s, 2H), 3.19 (d, J=63.8Hz, 4H), 2.50 (s, 2H), 2.13 (s, 2H)；13C NMR(101MHz,DMSO-d6)δ
165.94(s),165.06(s),164.75(s),158.88(s),153.49(s),149.32(s),141.83(s),138.53
(s),136.42(s),133.04(s),131.41(s),128.68(s),124.55(s),122.65(s),121.29(s),
119.72 (s), 118.22 (d, J=6.7Hz), 115.87 (s), 114.08 (s), 109.23 (s), 67.83 (s), 64.87 (s),
55.12(s),52.67(s),24.65(s)；HRMS(ESI)for C28H28ClN5O4S,[M+H]+Theoretical calculation: 566.1623,
Actual measurement: 566.1620；
(I-5) N- [3- [[2- [[4- (2- [[4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thieno [3,
2-D] pyrimidine-4-yl] oxygroup] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.35(s,1H),9.30
(s, 1H), 8.27 (d, J=5.4Hz, 1H), 7.72 (t, J=2.0Hz, 1H), 7.62-7.58 (m, 1H), 7.47 (dd, J=
16.8,8.6Hz, 3H), 7.34 (d, J=5.4Hz, 1H), 7.10-7.06 (m, 1H), 6.72 (d, J=8.8Hz, 2H), 6.44
(dd, J=17.0,10.1Hz, 1H), 6.27 (dd, J=17.0,1.9Hz, 1H), 5.78 (dd, J=10.1,1.9Hz, 1H),
3.64–3.54(m,4H),3.46(s,4H),3.33(s,2H)；13C NMR(101MHz,DMSO-d6)δ167.57(s),166.15
(s),165.00(s),164.73(s),159.24(s),154.01(s),153.59(s),141.69(s),138.15(s),
135.48(s),132.99(s),131.32(s),128.73(s),124.58(s),121.58(s),118.45(s),118.02
(s), 115.65 (s), 114.30 (s), 108.60 (s), 67.53 (d, J=17.6Hz), 46.21 (s), 42.97 (s)；HRMS
(ESI)for C27H25N5O5S,[M+H]+Theoretical calculation: 532.1649, actual measurement: 532.1635；
(I-6) N- [3- [[2- [[4- (2- [[4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thieno [3,
2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.28(s,1H),9.13
(s, 1H), 8.20 (d, J=5.4Hz, 1H), 7.62 (t, J=2.0Hz, 1H), 7.62-7.60 (m, 1H), 7.40 (dd, J=
16.8,8.6Hz, 3H), 7.30 (d, J=5.4Hz, 1H), 7.10-7.00 (m, 2H), 6.70 (d, J=8.8Hz, 2H), 6.40
(dd, J=16.0,10.0Hz, 1H), 6.20 (dd, J=17.0,1.9Hz, 1H), 5.70 (dd, J=10.1,1.9Hz, 1H),
3.64–3.50(m,4H),3.40(s,4H),3.30(s,2H)；13C NMR(101MHz,DMSO-d6)δ167.50(s),166.10
(s),165.00(s),164.70(s),159.20(s),154.00(s),153.50(s),141.60(s),138.10(s),
135.40(s),132.90(s),131.30(s),128.70(s),124.50(s),121.50(s),118.40(s),118.00
(s), 115.60 (s), 114.28 (s), 108.45 (s), 67.50 (d, J=16.0Hz), 46.20 (s), 42.97 (s)；HRMS
(ESI)for C27H26N6O4S,[M+H]+Theoretical calculation: 531.1809, actual measurement: 531.1756；
(I-7) N- [3- [[2- [[4- (2- [[3- methyl -4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
Pheno simultaneously [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide；1H NMR(400MHz,DMSO-d6)δ10.32(s,
1H), 8.51 (s, 1H), 8.19 (d, J=5.4Hz, 1H), 7.70 (t, J=2.1Hz, 1H), 7.56-7.50 (m, 1H), 7.41
(t, J=8.1Hz, 1H), 7.23 (d, J=5.4Hz, 1H), 7.19 (d, J=8.7Hz, 1H), 7.09-7.03 (m, 1H), 6.75
(d, J=2.8Hz, 1H), 6.62 (dd, J=8.7,2.7Hz, 1H), 6.44 (dd, J=17.0,10.1Hz, 1H), 6.27 (dd, J
=17.0,2.0Hz, 1H), 5.78 (dd, J=10.1,2.0Hz, 1H), 3.66-3.54 (m, 4H), 3.46 (d, J=3.3Hz,
4H),3.34(s,2H),2.12(s,3H)；13C NMR(101MHz,DMSO-d6)δ167.53(s),166.45(s),164.96
(s),164.70(s),161.01(s),156.27(s),153.48(s),141.59(s),137.80(s),135.69(s),
133.01(s),132.70(s),131.20(s),128.71(s),128.32(s),124.51(s),118.34(s),117.84
(s),117.31(s),114.08(s),113.07(s),108.28(s),67.45(s),46.20(s),42.98(s),19.70
(s)；HRMS(ESI)for C28H27N5O5S,[M+H]+Theoretical calculation: 546.1806, actual measurement: 546.1790；
(I-8) N- [3- [[2- [[4- (2- [[the chloro- 4- of 3- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
And [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.34(s,1H),
9.47 (s, 1H), 8.30 (d, J=5.4Hz, 1H), 7.73 (dd, J=8.7,6.7Hz, 2H), 7.64-7.57 (m, 1H), 7.56-
7.25 (m, 3H), 7.13-7.03 (m, 1H), 6.85 (d, J=9.1Hz, 1H), 6.43 (dd, J=17.0,10.1Hz, 1H),
6.26 (dd, J=17.0,2.0Hz, 1H), 5.79-5.75 (m, 1H), 4.83 (s, 2H), 3.71-3.50 (m, 4H), 3.46 (s,
4H)；13C NMR(101MHz,DMSO)δ16-d67.05(s),165.94(s),165.04(s),164.71(s),158.90(s),
153.47(s),149.17(s),141.79(s),138.45(s),136.17(s),133.01(s),131.41(s),128.69
(s), 124.58 (s), 122.12 (s), 121.39 (s), 119.61 (s), 118.23 (d, J=10.7Hz), 115.32 (s),
114.04(s),109.19(s),68.24(s),67.44(s),46.24(s),43.02(s)；HRMS(ESI)for
C27H24ClN5O5S,[M+H]+Theoretical calculation: 566.1259, actual measurement: 566.1265；
(I-9) N- [3- [[2- [[4- (2- [[2- methyl -4- [((1- morpholine) acetyl group) oxygroup] phenyl] amino] thiophene
Pheno simultaneously [3,2-D] pyrimidine-4-yl] amino] phenyl] -2- acrylamide,1H NMR(400MHz,DMSO-d6)δ10.18(s,
1H), 8.50 (s, 1H), 8.10 (d, J=5.6Hz, 1H), 7.78 (t, J=2.0Hz, 1H), 7.52-7.50 (m, 2H), 7.40
(t, J=8.0Hz, 1H), 7.20 (d, J=5.4Hz, 1H), 7.10 (d, J=8.6Hz, 1H), 7.08-7.00 (m, 1H), 6.58
(d, J=2.8Hz, 1H), 6.60 (dd, J=8.7,2.7Hz, 1H), 6.40 (dd, J=17.0,10.0Hz, 1H), 6.26 (dd, J
=17.0,2.0Hz, 1H), 5.70 (dd, J=10.6,2.0Hz, 1H), 3.66-3.48 (m, 4H), 3.40 (d, J=3.3Hz,
4H),3.30(s,2H),2.10(s,3H)；13C NMR(101MHz,DMSO-d6)δ167.18(s),166.42(s),164.90
(s),164.60(s),161.00(s),156.20(s),153.40(s),141.50(s),137.70(s),135.60(s),
133.00(s),132.25(s),131.29(s),128.66(s),128.24(s),124.50(s),118.30(s),117.80
(s),117.30(s),114.00(s),113.12(s),108.20(s),67.40(s),46.10(s),42.50(s),19.59
(s)；HRMS(ESI)for C28H28N6O4S,[M+H]+Theoretical calculation: 545.1966, actual measurement: 545.1906.
Target molecule at salt method
The preparation method of inorganic acid salt: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly drips
The 5mL absolute methanol solution for adding inorganic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, then first is evaporated off in room temperature
Alcohol to get target molecule inorganic acid salt.
The preparation method of acylate: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly drips
The 5mL dry ether for adding organic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, and then solvent is evaporated off in room temperature,
Up to the acylate of target molecule.
The preparation of two target molecule mixtures
Above-mentioned two target molecule of equimolar amounts (1mmol) (5mL) in anhydrous methanol is taken, is stirred at room temperature 10 minutes,
Solvent is evaporated off to get the mixture of target molecule in room temperature.
2 target molecule biological evaluation of embodiment
1, in vitro to receptor tyrosine kinase inhibitory activity test method
(1) kinase assay buffer is prepared
1. melting kinase assay buffer (Kinase Detection Buffer) in room temperature, precipitating has been seen whether.
2. it is just shaken in 37 DEG C of incubations (Kinase Detection Buffer) 15 minutes and often if there is precipitating,
Dissolution precipitating.Alternatively, supernatant is carefully siphoned away, removal precipitating.
(2) kinase assay reagent is prepared
1. using preceding at equilibrium at room temperature kinase assay buffer (Kinase Detection Buffe) and kinase assay bottom
Object (Kinase Detection Substrate).
2. all pouring into kinase assay buffer (Kinase Detection Buffer) equipped with kinase assay substrate
In the brown bottle of (Kinase Detection Substrate), freeze-dried powder substrate is dissolved, kinase assay has thus been made
Reagent.
3. gently shaking, being vortexed or being mixed by inversion, 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, it is believed that the reagent prepared passes through
Cycle signal activity is not all lost after freeze thawing several times.
(3) production ATP is converted to the standard curve of ADP
1. the Ultra provided with 1 × kinase reaction buffer (kinase reaction buffer) dilution kit
50 μM of ADP of 900 μ L 50 μM of ATP and 500 μ L are made in Pure ATP and 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 in every holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
4. 10 μ L kinase assay reagents (Kinase Detection Reagent), which are added, in every hole is converted to ATP for ADP, and
Luciferase and luciferin are introduced to detect ATP.
5. measuring fluorescent in incubation at room temperature 30-60 minutes with multi-function microplate reader and recording fluorescent value.
6. drawing the standard curve that ATP is converted to ADP.
(4) IC of kinase inhibitor is determined50Value
1. preparing 1 × kinase reaction buffer (kinase reaction according to promega kit specification
Buffer), 2.5 × 50ng/ μ L kinases and 2.5 × 0.5 μ g/ μ L substrates and 125 μM of ATP.
2. 3 μ 1 × kinase reaction of L buffers (kinase reaction buffer), 2 μ are added in no enzyme control wells
The μ g/ μ L substrate of L2.5 × 0.5 and 125 μM of ATP.1 μ L 1 × kinase reaction buffer (kinase is added in negative control hole
Reaction buffer), 2 μ L 2.5 × 50ng/ μ L kinases, 2 μ L, 2.5 × 0.5 μ g/ μ L substrate and 125 μM of ATP.It is testing
1 μ L 5 × drug to be measured, 2 μ L2.5 × 50ng/ μ L kinases, 2 μ L, 2.5 × 0.5 μ g/ μ L substrate and 125 μM of ATP are added in hole.
3. mixing plate, it is incubated for 60 minutes.
4. the ADP-Glo of 5 μ L is added in every holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
5. 10 μ L kinase assay reagents (Kinase Detection Reagent), which are added, in every hole is converted to ATP for ADP, and
Luciferase and luciferin are introduced to detect ATP.In incubation at room temperature 30-60 minutes, fluorescent was measured with multi-function microplate reader and is remembered
Record fluorescent value.
6. interpretation of result, the results are shown in Table 2.
2, inhibit JAK3 high expressing cell growth experiment (CCK-8 detection method)
(1) cell type and selection: Ramos cell (people's Burkitt's lymphoma cell, the expression of JAK3 kinases height),
Raji cell (people's Burkitt's lymphoma cell, JAK3 kinases height expression), Raji cell (people's NAMAWAL lymphoma cell,
JAK3 kinases height expression).
(2) cell inoculation: collecting logarithmic growth phase cell, concentration of cell suspension is adjusted, with every hole 4 × 103A cell, often
100 μ L of pore volume is inoculated into 96 orifice plates, and every group sets 3 multiple holes (edge hole is filled with sterile PBS)；
(3) cell culture: after cell inoculation, control group is cultivated with 10%FBS RPMI-1640, and experimental group uses 10 μ L respectively
Spebrutinib or Ibrutinib (1.25-40 μm of ol/L), the variant drug (1.25-40 μm of ol/L) of various concentration gradient
Intervene, 37 DEG C, 5%CO2Continue to cultivate (cultivating different time respectively by requirement of experiment) in incubator；
(4) colour generation: 10 μ L CCK-8 solution (5mg/ml) are added in two groups of cells after cultivating 48h, terminate training after 4h
It supports, in low-speed oscillation 10min on shaking table, makes to crystallize abundant dissolution；
(5) colorimetric: measuring each hole shading value (OD value) on enzyme-linked immunosorbent assay instrument, 450nm wavelength is selected, with cell-free
I.e. RPMl-1640 culture solution blank well zeroing, survey the absorbance value in each hole.Experiment is in triplicate；
(6) record result: inhibitory rate of cell growth=(control group absorbance value-experimental group absorbance value)/control group is inhaled
Shading value × 100%, cell proliferation rate=(experimental group absorbance value/control group absorbance value) × 100；
(7) draw cell growth curve: using the time as abscissa, inhibiting rate/proliferation rate is that ordinate draws cell growth
Curve.Figure is done for inhibitor concentration in the GraphPad Prism mapping software in GraphPad software, so as to by log
[inhibitor] estimates IC relative to reaction, variable slope model50。
Test result is as shown in table 2, and table 2 shows that compound obtained is inhibiting JAK3 kinases and anti-tumour cell proliferative
In active effect (A:1-10nM；B:10-50nM；C:1-10μM；D:10-20μM).
Table 2
Meanwhile this experiment discovery compound I-3 has very big pass to the proliferation activity of Ramos cell and time and concentration
System, as shown in Figure 1, with the increase of concentration, cell survival rate reduces, and the Ramos cell after especially 72h is in drug concentration up to 10
It when μm ol/L, can hardly survive, hence it is evident that be higher than 48h, thus provable drug belongs to concentration and time dependence drug.This
Outside, with compared with medicine Spebrutinib, compound I-3 has abrupt mention to the fragmentation effect of Ramos after 72h
It is high.In addition, the cellular morphology and quantity after the administration shown in Fig. 2 also further demonstrate that, compound I-3 compared with
The anti-Ramos cell activity of Spebrutinib, which has, to be remarkably reinforced.
3, active medicine toxicity test
Peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) include lymphocyte,
Monocyte (monocyte), Dendritic Cells and other a small amount of cells (candidate stem cell etc.).Carrying out toxicity test to it can
To prove whether drug of the invention has lethality to normal immunocyte.The common method of separation PBMC is both at home and abroad at present
Glucan-Hypaque density gradients centrifugal process, experimental procedure are as follows:
(1) take a blood sample and dilute: venous blood sampling 2ml is added in the test tube of the anticoagulant solution containing ACD, mixes, makes blood anticoagulant.
With PBS solution by 1 times of anticoagulant hemodilution；
(2) it is loaded: drawing 2ml lymphocytes separating solution (Tianjin TBD) and be placed in graduated centrifuge tube, then centrifuge tube inclines
Diluted whole blood is added slowly to above separating liquid by oblique 45° angle with capillary burette along tube wall, it should be noted that keeps the two interface clear
It is clear；
(3) it is centrifuged: at 18 DEG C~20 DEG C, 20min being centrifuged with 2000r/min with horizontal centrifuge.From tube bottom after centrifugation
To four layers of liquid level point, it is followed successively by red blood cell and granulocyte layer, layering liquid layer, mononuclearcell layer, plasma layer；
(4) it recycles: being gently inserted into muddy band with capillary syring, this confluent monolayer cells is gently sucked out along tube wall, move into another fragmented
In heart pipe.All mononuclearcells are drawn, avoid drawing excessive layering liquid or blood plasma again, in order to avoid it is mixed into other cells
Ingredient；
(5) it washs: being washed cell 3 times with PBS liquid.First time 2000r/min, 10min；2nd~3 1500r/min,
10min can remove the blood platelet largely mixed；
(6) sedimentation cell is suspended from spare in culture medium；
(7) bed board is counted: adjustment concentration of cell suspension, with every hole 2.5 × 105A cell, every 500 μ L of pore volume are inoculated into
24 orifice plates, every group sets 2 multiple holes；
(8) cell culture: after cell inoculation, control group is cultivated with 10%FBS RPMI-1640, and experimental group uses 50 μ L respectively
Spebrutinib (5-20 μm of ol/L), the I-3 (5-20 μm of ol/L) of various concentration gradient intervene, and 37 DEG C, 5%CO2In incubator
Continue to cultivate (cultivating different time respectively by requirement of experiment)；
(9) dye: 20 μ L, 1 μ g/ μ l AO (acridine orange), 20 μ L, 1 μ g/ μ l PI (propidium iodide) is added in culture afterwards for 24 hours
5min is dyed, observes and takes pictures under inverted fluorescence microscope.
(10) test results are shown in figure 3, and Fig. 3 is compound I-3 to PBMC cell toxicity data.
By the bioactivity of table 2, the result shows that, the molecule in the present invention has stronger inhibitory effect (IC to JAK3 kinases50
=1-10nM), reach the active rank of nanomolar range, has than the activity referring to medicine Ibrutinib and Spebrutinib bright
It is aobvious to improve.Antiproliferation result discloses, and majority of compounds has B lympha tumour cell Ramos and Raji and has very much
The inhibiting effect of effect, IC50Value is between 1-10 μM, better than referring to medicine Ibrutinib and Spebrutinib (10-20 μM).Simultaneously
Fig. 3 shows that the cytotoxicity of compound I-3 is also substantially reduced than Spebrutinib, mitigates medicine while keeping pharmaceutical activity
The toxicity of object has potential medical value.In conclusion the compound of the present invention is to caused by treatment JAK3 tyrosine kinase
Disease has very deep potential significance, especially for treating diffusivity large B cell lymphoid tumor, follicular lymphoma or chronic
Lymphocytic leukemia.Indicate the potentiality that such molecule has exploitation into new and effective JAK3 tyrosine kinase inhibitor.
The above is only preferred embodiments of the invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of logical formula (I) compound represented or its pharmaceutically acceptable salt, the logical formula (I) compound represented has
Such as flowering structure:
Wherein,
X is selected from NH or O；
R1Selected from hydrogen, chlorine, methyl, fluorine or methoxyl group；
R is selected from
2. logical formula (I) compound represented according to claim 1 or its pharmaceutically acceptable salt, wherein described logical
Formula (I) compound represented has structure shown in I-1~I-9:
3. logical formula (I) compound represented according to claim 1 or its pharmaceutically acceptable salt, wherein described logical
Formula (I) compound represented is I-3.
4. a kind of pharmaceutical composition, change shown in formula (I) is led to described in any one of claims 1 to 3 containing effective dose
Close object or its pharmaceutically acceptable salt and pharmaceutical carrier.
5. leading to formula (I) compound represented or its pharmaceutically acceptable salt or right described in any one of claims 1 to 3
It is required that pharmaceutical composition described in 4 is preparing the application in JAK3 tyrosine kinase inhibitor.
6. leading to formula (I) compound represented or its pharmaceutically acceptable salt or right described in any one of claims 1 to 3
It is required that purposes of the pharmaceutical composition described in 4 in the drug of preparation treatment tumour.
7. purposes according to claim 6, wherein the tumour is selected from diffusivity large B cell lymphoid tumor, follicularis lymph
One of tumor and chronic lymphocytic leukemia are a variety of.
8. purposes according to claim 7, wherein the tumour is chronic lymphocytic leukemia.
9. the purposes according to any one of claim 5~8, wherein the purposes, which mainly passes through, inhibits JAK3 tyrosine
What kinases was realized.
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| LT2975042T (en) * | 2010-06-23 | 2019-01-25 | Hanmi Science Co., Ltd. | Novel fused pyrimidine derivatives for inhibition of tyrosine kinase activity |
| CN106565614A (en) * | 2016-11-16 | 2017-04-19 | 大连医科大学 | Diphenylaminopyrimidine compound, composition and application |
| CN109265469A (en) * | 2018-11-12 | 2019-01-25 | 大连医科大学附属第医院 | Pyrimido thiazole heterocyclic compounds, composition and its purposes for treating lymphocytic leukemia |
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