CN105037399A - Bcr-Abl amphiploid inhibitor, preparation method and application thereof - Google Patents
Bcr-Abl amphiploid inhibitor, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a Bcr-Abl amphiploid inhibitor, a preparation method and an application thereof. A compound or pharmaceutically acceptable salts thereof provided in the invention can be used as the Bcr-Abl amphiploid inhibitor which can effectively inhibit the activity of tyrosine kinase. The Bcr-Abl amphiploid inhibitor can be effectively used in treatment of diseases related to abnormal activation of the tyrosine kinase and has excellent treatment effect on malignant tumors. The inhibitor is easy to prepare, is low in cost and is excellent in application prospect.
Description
Technical field
The present invention relates to Bcr-Abl amphiploid inhibitor and its production and use.
Background technology
Chronic myelocytic leukemia is that karyomit(e) produces transgenation [t (9:22) (q34; Q11)] transposition mutually, the abl gene that karyomit(e) is 9 and the BCR gene of chromosome 22 position merge generation BCR-ABL mutually, a kind of 210kDa Tyrosylprotein kinase of this genes encoding merged Bcr-Abl, this albumen is the major cause causing chronic myelocytic leukemia.C-Abl is the monoploid Tyrosylprotein kinase be present in endochylema in normal cell, and after merging with Bcr, form changes, and become the tetramer by monoploid, this kinases is in the state of being activated all the time thus, leads oncogenic generation.Existing in Bcr protein sequence to cause the aminoacid sequence of multimerization to cause Bcr-Abl tetra-dimerization.Because c-Abl plays an important role in the normal cells such as cardiac muscle, if exploitation Selective depression carinogenicity Bcr-Abl but not the inhibitor of Abl, be then expected to greatly to reduce the side effects widely such as the cardiac toxic of this type of inhibitor.
Calendar year 2001, FDA ratifies the listing of first tyrosine kinase inhibitor (TKI) imatinib (Imatinib) 1, for the treatment of chronic myelocytic leukemia (CML).As first-generation Bcr-Abl inhibitor, imatinib has become the first-line drug for the treatment of CML.But finally all there is resistance to imatinib in Most patients.Research subsequently shows, the generation of IM resistance may be relevant with the transgenation in the Abl kinase activity districts such as G250E, Q252H, Y253F, Y253H, E255K, E355G, E255V, T315A, T315I, F317L, F317V, M351T, F359V, H396P, M244V, and transgenation causes imatinib and the kinase whose affinity of Abl to decline.Most transgenation makes the affinity decline 5-30 of imatinib doubly, and this is the major cause producing resistance.Wherein T315I is more special, reduces the most obvious, IC
50be down to about 6400nM, that is not only effective to T315I for Buddhist nun on the slope of recently exploitation, to wild-type and most mutant strain all effective.
From imatinib and slope, that is for the x-Ray eutectic structure of Buddhist nun with the kinases part of Abl albumen, and in a solvent, the slant range between two these nitrogen-atoms of inhibitor is about 24 peaces in the n-formyl sarcolysine base section exposure of the first class piperazine moiety in both structures.Bcr-Abl is owing to being the tetramer, can combine with 4 inhibitor simultaneously, if two inhibitor are coupled together by chain, then be expected to the affinity of the Bcr-Abl greatly improved four dimerizations, thus play the effect of Selective depression Bcr-Abl, and Abl is owing to being monoploid, can only combine with an inhibitor, amphiploid inhibitor does not have large impact to the affinity of enzyme.
Summary of the invention
The object of the present invention is to provide Bcr-Abl amphiploid inhibitor and its production and use.
Compound shown in formula I provided by the invention or its pharmacy acceptable salt, its structural formula is as follows:
Further, Linker is (Z
1)
a– (Z
2)
b– (Z
3)
c;
Wherein, Z
1, Z
2, Z
3separately be selected from
C(O)(CH
2)
dNH、CO(CH
2)
eC(O)、
(CH
2CH
2)
fNHC(O)、(CH
2CH
2)
g-C(O)NH、
(CH
2)
hC(O)(OCH
2CH
2)
iNHC(O)(CH
2)
jC(O)、
NH(CH
2)
K(OCH
2CH
2)
l-O(CH
2)
mNH、
(CH
2)
nC(O)(OCH
2CH
2)
ONH(C(O)(CH
2)
pNH)
qC(O)-alkyl、
C(O)NH(CH
2)
rNHC(O)-(CH
2)
sC(O)(NHCH
2CH
2)
tNH(C(O)(CH
2)
uNH)
v-
C(O)-alkyl；
A ~ v is separately selected from arbitrary numerical value in 1 ~ 20.
Further, described compound is:
Present invention also offers the method preparing above-claimed cpd BDB.
The preparation method of above-claimed cpd BDB, is characterized in that: it comprises following operation steps:
Further, it comprises following operation steps:
A, prepare compound 9:
Vinyl chloroformate, compound 8, diisopropylethylamine, in tetrahydrofuran (THF), in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 9;
B, prepare compound 11:
Compound 9, diisopropylethylamine, 4-(chloromethyl) Benzoyl chloride, in methylene dichloride, in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 11;
C, prepare compound 12:
Compound 11, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 2h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 12;
D, prepare compound 14:
Compound 12, compound 13,1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, I-hydroxybenzotriazole, diisopropylethylamine, in methylene dichloride, in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 14;
E, prepare compd B DB:
Compound 7, compound 14, salt of wormwood, in DMF, in 60 DEG C of stirring reaction 5h, obtain reaction solution; Reaction solution is separated, purifying, obtain compd B DB.
Further,
In step a, the mol ratio of described Vinyl chloroformate, compound 8, diisopropylethylamine is 46.5:44.5:264; Described Vinyl chloroformate is 46.5:350mmol/ml with the molecular volume ratio of tetrahydrofuran (THF);
In step b, the mol ratio of described compound 9, diisopropylethylamine, 4-(chloromethyl) Benzoyl chloride is 0.67:0.8:0.8; Described compound 9 is 0.67:50mmol/ml with the molecular volume ratio of methylene dichloride;
In step c, described compound 11 is 0.445:20mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M;
In steps d, the mol ratio of described compound 12, compound 13,1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, I-hydroxybenzotriazole, diisopropylethylamine is 0.445:0.445:0.534:0.534:0.534; Described compound 12 is 0.445:25mmol/ml with the molecular volume ratio of methylene dichloride;
In step e, the mol ratio of described compound 7, compound 14, salt of wormwood is 0.4:1:4.8; Described compound 7 is 0.4:50mmol/ml with the molecular volume ratio of DMF.
Further, in step e, described compound 7 is prepared according to following operation steps:
Further, described compound 7 is prepared according to following operation steps:
I, prepare compound 2:
Compound 1, BOC acid anhydrides react completely in methylene dichloride, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 2;
Ii, prepare compound 3:
Compound 2, triethylamine, glutaryl chlorine, in methylene dichloride, in stirred at ambient temperature reaction 3h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 3;
Iii, prepare compound 4
Compound 3, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 3h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 4;
Iv, prepare compound 5
Compound 4, diisopropylethylamine, 3-chlorpromazine chloride, in methylene dichloride, in stirred at ambient temperature reaction 3h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 5;
V, prepare compound 6
Compound 5, piperazine-1-t-butyl formate, diisopropylethylamine are in dioxane, and back flow reaction 18h, obtains reaction solution; Reaction solution is separated, purifying, obtain compound 6;
Vi, prepare compound 7:
Compound 6, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 2h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 7.
Further,
In step I, the mol ratio of described compound 1, BOC acid anhydrides is 45.5:43.18; Described compound 1 is 45.5:600mmol/ml with the molecular volume ratio of methylene dichloride;
In step I i, the mol ratio of described compound 2, triethylamine, glutaryl chlorine is 8.42:14.85:4.17; Described compound 2 is 8.42:120mmol/ml with the molecular volume ratio of methylene dichloride;
In step I ii, described compound 3 is 3.8:100mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M;
In step I v, the mol ratio of described compound 4, diisopropylethylamine, 3-chlorpromazine chloride is 0.9:6.15:1.81; The molecular volume of described compound 4 methylene dichloride is than being 0.9:50mmol/ml;
In step v, the mol ratio of described compound 5, piperazine-1-t-butyl formate, diisopropylethylamine is 0.14:2.68:0.77; Described compound 5 is 0.14:20mmol/ml with the molecular volume ratio of dioxane;
In step vi, described compound 3 is 0.1:20mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M.
Present invention also offers above-claimed cpd and the purposes of pharmacy acceptable salt in the medicine of preparation treatment cell breeding disease thereof.
Above-claimed cpd or its pharmacy acceptable salt purposes in the medicine of preparation treatment cell breeding disease.
Further, described medicine is tyrosine kinase inhibitor.
Further, described medicine is ABL inhibitor class medicine.
Further, described medicine is BCR-ABL or its mutant strain inhibitor class medicine.
Further, described BCR-ABL mutant strain is T315I mutant strain.
Further, described cell breeding disease is cancer.
Further, described cancer is chronic myelocytic leukemia, intestines mesenchymoma, gynaecology's knurl, dermatofibrosarcoma protuberans, Ph+ALL.
Compound provided by the invention or its pharmacy acceptable salt; as Bcr-Abl amphiploid inhibitor; effectively can suppress tyrosine kinase activity; the disease treatment that such kinases abnormal activation is relevant can be effective to; to malignant tumour, there is good therapeutic action; the preparation method of such inhibitor is easy, with low cost, has a good application prospect.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
The raw material used in the specific embodiment of the invention, equipment are known product, obtain by buying commercially available prod.
The Chinese of abbreviation: DCM: methylene dichloride; (Boc)
2o:BOC acid anhydrides;
The preparation of embodiment 1 the compounds of this invention BDB
Synthetic route is as follows:
Concrete synthesis step is as follows:
Prepare compound 2:
By compound 1 (10g, 45.5mmol; Manufacturer: Sigma-Aldrich) be dissolved in 500mlDCM, under room temperature, by (Boc)
2o (9.4g, 43.18mmol) is dissolved in the DCM of 100ml, and slowly drop in reaction flask, reaction is spent the night; Water 200ml is added in reaction solution, after adjusting pH to 3-4, extraction removes organic phase impurity, and aqueous phase adjusts pH to 10, add sodium-chlor more saturated, extract and product, organic phase uses saturated sodium-chloride water solution (10ml × 6) to wash away the complete raw material of unreacted, dried over sodium sulfate again, solvent evaporated, obtain colorless oil, be compound 2 (4.7g, yield 34.1%).
1HNMR(400MHz,CDCl3):δ5.11(bs,1H),3.63-3.45(m,12H),3.21(t,J＝6.0Hz,2H),2.81(t,J＝6.4Hz,2H),1.77-1.42(m,6H),1.42(s,9H)。
Prepare compound 3:
At room temperature, by compound 2 (2.7g, 8.42mmol), triethylamine (1.5g, 14.85mmol) be dissolved in methylene dichloride (100ml), by glutaryl chlorine (0.7g, 4.17mmol) be dissolved in methylene dichloride (20ml), drip in reaction solution, time for adding is more than 1h, at room temperature stirring reaction 3h after finishing, then saturated sodium-chloride water solution (100ml × 2) is used to wash organic phase, organic phase with sodium sulfate is dry, vacuum concentration solvent evaporated, residue over silica gel column chromatography purification (methylene chloride/methanol=20/1), obtain colorless oil, be compound 3 (3.1g, yield 100%).
ESI-MSm/z:737.4[M+H]
+；
1HNMR(400MHz,CDCl3):δ6.55(bs,2H),5.06(bs,2H),3.63-3.49(m,24H),3.34(m,4H),3.20(m,4H),2.22(t,J＝7.2Hz,4H),1.93(m,2H),1.78-1.70(m,8H),1.41(s,18H)。
Prepare compound 4:
By compound 3 (2.8g, 3.80mmol) join HCl/ ethyl acetate (2M, 100ml), stirred at ambient temperature reaction 3h, vacuum concentration solvent evaporated, obtains colourless oil liquid, be compound 4 (2.6g, yield: ~ 100%), need not be further purified, and is directly used in next step reaction;
Prepare compound 5:
At room temperature, by compound 4 (484mg, 0.90mmol), diisopropylethylamine (DIPEA) (800mg, 6.15mmol) be dissolved in methylene dichloride (40ml), by 3-chlorpromazine chloride (230mg, 1.81mmol) with after methylene dichloride (10ml) dilution, drip in reaction solution, time for adding is more than 1h, at room temperature stirring reaction 3h after finishing, then saturated sodium-chloride water solution (50ml × 2) is used to wash organic phase, organic phase with sodium sulfate is dry, concentrating under reduced pressure solvent evaporated, in resistates, add normal hexane stir, filter, obtain white solid, be compound 5 (330mg, yield 50.9%).
1HNMR(400MHz,CDCl3):δ6.85(bs,2H),6.58(bs,2H),3.81(t,J＝6.4Hz,4H),3.66-3.53(m,24H),3.39-3.31(m,8H),2.64(m,4H),2.24(t,J＝7.2Hz,4H),1.81(m,2H),1.55-1.47(m,8H)。
Prepare compound 6:
By compound 5 (100mg, 0.14mmol), piperazine-1-t-butyl formate (500mg, 2.68mmol), diisopropylethylamine (DIPEA) (100mg, 0.77mmol), dioxane (20ml) joins in round-bottomed flask, heating reacts 18h under reflux, then after reaction solution being cooled to room temperature, decompression steams solvent, residue from dichloromethane dissolves, organic phase is washed again with saturated sodium-chloride water solution, use dried over sodium sulfate organic phase, concentrating under reduced pressure solvent evaporated, residue over silica gel column chromatography purification (methylene chloride/methanol=10/1), obtain colourless oil liquid, be compound 6 (50mg, yield 35.2%).
1HNMR(400MHz,CDCl3):δ7.98(bs,2H),7.07(bs,2H),3.66-3.53(m,24H),3.46(m,8H),3.36-3.31(m,8H),2.71(m,4H),2.47(m,12H),2.27(t,J＝6.8Hz,4H),1.95(m,2H),1.79-1.75(m,8H),1.45(s,18H)。
Prepare compound 7
Joined by compound 6 (100mg, 0.10mmol) in HCl/ ethyl acetate (2M, 20ml), stirred at ambient temperature reaction 2h, then vacuum concentration solvent evaporated, obtains colourless oil liquid, is compound 7 (100mg; Yield ~ 100%).
1HNMR(400MHz,D2O)δ3.62(m,30H),3.53(m,14H),3.21(q,J＝7,2Hz,8H),2.77(t,J＝6.8Hz,4H),2.21(t,J＝7.6Hz,4H),1.82(m,2H),1.74(m,8H)。
Prepare compound 9:
By Vinyl chloroformate (4.5ml, 46.5mmol) be dissolved in tetrahydrofuran (THF) (100ml), ice bath cools, by compound 8 (10g, 44.5mmol) with diisopropylethylamine (DIPEA) (46ml, 264mmol) be dissolved in tetrahydrofuran (THF) (250ml), drip in reaction solution lentamente at 0 ~ 5 DEG C, finish the rear stirring at room temperature that naturally rises to and react 16h, then by reaction solution vacuum concentration solvent evaporated, residue with ethyl acetate dissolves, and wash organic phase with water, use dried over sodium sulfate organic phase, vacuum decompression solvent evaporated, residue over silica gel column chromatography purification (ethyl acetate: sherwood oil=1:3) obtains white solid, be compound 9 (8.0g, yield 60.7%).
1HNMR(400MHz,CD3OD)δ4.72(s,2H),4.40(q,J＝7.2Hz,2H),4.12(d,J＝7.2Hz,2H),1.53(s,9H),1.40(t,J＝6.8Hz,3H)。
Prepare compound 11:
Be dissolved in methylene dichloride (25ml) by compound 9 (200mg, 0.67mmol), diisopropylethylamine (DIPEA) (103mg, 0.8mmol), frozen water cools; By 4-(chloromethyl) Benzoyl chloride (compound 10) (151mg, 0.8mmol) be dissolved in methylene dichloride (25ml), drip in 0 ~ 5 DEG C of downhill reaction liquid, finish the rear stirring at room temperature that naturally rises to and react 16h, by reaction solution solvent evaporated under vacuum decompression, residue with ethyl acetate dissolves, and wash organic phase with water, organic phase with sodium sulfate is dry, evaporated under reduced pressure solvent, residue over silica gel column chromatography purification (ethyl acetate: sherwood oil=1:2), obtains yellow solid, be compound 11 (220mg, yield 73%).
1HNMR(400MHz,CD3OD)δ7.98(d,J＝8.4Hz,2H).7.60(d,J＝7.6Hz,2H).4.74(s,2H),4.69(s,4H),4.50(q,J＝7.2Hz,2H),1.55(s,9H),1.45(t,J＝7.2Hz,3H)。
Prepare compound 12:
Joined by compound 11 (200mg, 0.445mmol) in HCl/ ethyl acetate (2M, 20ml), stirred at ambient temperature reaction 2h, vacuum concentration solvent evaporated, obtains white solid, is compound 12 (172mg; Yield ~ 100%); Need not be further purified, be directly used in next step reaction;
1HNMR(400MHz,CD3OD)δ7.98(d,J＝8.0Hz,2H),7.60(d,J＝8.0Hz,2H),4.74(s,2H),4.71(s,4H),4.52(q,J＝7.2Hz,2H),1.46(t,J＝7.2Hz,3H)。
Prepare compound 14:
At room temperature, by compound 12 (153mg, 0.445mmol), compound 13 (74mg, 0.445mmol), 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride (EDCI) (91mg, 0.534mmol) with I-hydroxybenzotriazole (HOBT) (72mg, 0.534mmol) be dissolved in methylene dichloride (25ml), then in reaction solution, diisopropylethylamine (DIPEA) (115mg is slowly dripped, 0.89mmol), finish rear stirring reaction 16h. in reaction solution, then add water dilution, and by methylene dichloride (100ml × 2) aqueous phase extracted, merge organic phase, and wash organic phase with saturated sodium-chloride water solution, organic phase with sodium sulfate is dry, evaporated under reduced pressure solvent, residue over silica gel column chromatography purification (ethyl acetate: sherwood oil=1:1), obtain yellow solid, be compound 14 (100mg, yield 45%).
1HNMR(400MHz，CD
3OD)δ7.96(d,J＝8.0Hz,2H),7.59(d,J＝8.0Hz,2H),7.37～7.48(m,4H),5.13(s,1H),4.69～4.79(m,6H),4.50(q,J＝7.2Hz,2H),3.44(s,3H),1.46(t,J＝7.2Hz,3H)。
Preparation BDB:
By compound 7 (327mg, 0.4mmol), compound 14 (500mg, 1mmol) and K
2cO
3(664mg, 4.8mmol), DMF (50ml) join in round-bottomed flask, stirring reaction 5h at 60 DEG C, after question response liquid is cooled to room temperature, pours in 200ml water by reaction solution, be extracted with ethyl acetate aqueous phase, merge organic phase, and wash organic phase with saturated sodium-chloride water solution, organic phase with sodium sulfate is dry, evaporated under reduced pressure solvent, obtain pale yellowish oil liquid, be compd B DB (522mg, yield 81%).
1HNMR(400MHz,D2O)δ7.77(d,J＝7.2Hz,2H),7.68(d,J＝8Hz,2H),7.47(d,J＝7.2Hz,2H),7.43(d,J＝7.6Hz,2H),7.35(bs,J＝7.2Hz,10H),5.01(s,1H),4.98(s,1H,s)，4.38(s,4H)，3.54～3.42(m,44H),3.27～3.26(bs,6H),3.11(t,J＝6.8Hz,4H),3.04(t,J＝6.8Hz,4H),2.66(bs,4H),2.05(m,4H),1.75～1.54(m,10H)。
The medical active of test example 1 the compounds of this invention
1, tyrosine kinase inhibitory activity
Reference: AmyCardetal., JournalofBiomolecularScreening14 (1) 2009; JudeDunneetal., Assay & DrugDevelopmentTechnologiesVol.2, No.2,2004.
Utilize the method for MobilityShiftAssay, vitro kinase Abl is carried out to the screening of the compounds of this invention BDB, compd B DB concentration is diluted to 0.005 μM successively by 100 μMs of three times of dilution method 100%DMSO, totally 10 concentration point, carry out detecting in (2 multiple hole), adopt compound staurosporine as standard control, if Vehicle controls (10%DMSO) and negative control (EDTA).
By enzyme linked immunoassay on 384 hole Sptting plates, detect the suppression situation of compd B DB to kinases Abl of each concentration, Caliper upper reading and converting rate data, and conversion is become inhibiting rate.
Percentinhibition＝(max-conversion)/(max-min)*100。Wherein max refers to that the transformation efficiency that DMSO contrasts, min refer to the transformation efficiency without enzyme contrast alive.The compounds of this invention BDB is calculated to the enzymic activity half-inhibition concentration IC of c-Abl Tyrosylprotein kinase according to the inhibiting rate of each concentration
50.
Suppress result as follows:
The IC of compd B DB
50: 0.05 μM;
Test-results illustrates, the compounds of this invention BDB has stronger inhibit activities to c-Abl Tyrosylprotein kinase.
2, the compounds of this invention BDB is to KBM5 (Bcr-Abl is positive) and KBM5R (T315I mutant strain) Proliferation of Tumor Cells In Vitro inhibition test
Measure the cytotoxicity of BDB series compound of the present invention by ATP method, KBM5 and KBM5R cell is adjusted suitable cell density, with every hole 140ul cell suspension inoculation 96 orifice plate, the inoculum density of often kind of cell is: 2000-6000; Above-mentioned Tissue Culture Plate is placed in incubator and within 24 hours, makes it be attached to completely on hole wall, adopt three times of dilution methods that each compound in the compounds of this invention BDB series is mixed with suitable different concns respectively, join and be vaccinated with in the 96 corresponding holes of orifice plate of cell in advance, every hole 10 μ L, makes its ultimate density be respectively: 100 μMs, 33.3 μMs, 11.1 μMs, 3.70 μMs, 1.23 μMs, 0.41 μM, 0.14 μM, 0.046 μM, 0.015 μM.Each concentration establishes three multiple holes respectively, and after hatching 72 hours in 37 DEG C of incubators, ATP method measures each porocyte proliferative conditions, calculates each drug level to the inhibiting rate of KBM5 and KBM5R cell proliferation and median lethal concentration (IC
50).
Test-results:
The cytotoxicity of compd B DB to KBM5 (people source wild-type Bcr-Abl) tumour cell is as follows:
The IC of compd B DB
50: 7 μMs;
The cytotoxicity of compd B DB to KBM5R tumour cell is as follows:
The IC of compd B DB
50: 8.9 μMs;
Test-results illustrates, the compounds of this invention BDB in vitro cell levels has stronger inhibit activities.
Leukemia Subcutaneous Xenograft Proliferation Ability test in the body of 3, the compounds of this invention BDB
According to the result of in-vitro multiplication inhibition test, in BDB series compound of the present invention, select IC
50minimum inhibitor, have evaluated BDB at C57BL/6 (Es-1
c) growth inhibitory effect of nude mice by subcutaneous Transplanted Human source leukemia KBM5 and KBM5R cell.
The dosage of the compounds of this invention BDB is respectively: 80mg/Kg, 160mg/Kg, 350mg/Kg, establish positive controls (the imatinib Imatinib of 160mg/Kg) and blank group (physiological saline) simultaneously, after mice with tumor grouping daily once, continuous intraperitoneal injection, after two weeks, detects the volume of tumour.
Investigate tumor growth whether can suppressed, delay or cure.Twice or use vernier caliper measurement diameter of tumor every other day weekly.The calculation formula of gross tumor volume is: V=0.5a × b
2, a and b represents major diameter and the minor axis of tumour respectively.
Data analysis: T compares between checking and being used for two groups; Compare between three groups or many groups and use one-wayANOVA.If F value has significant difference, after ANOVA analyzes, multiple comparisons should be carried out again.Two-wayANOVA is used for the potential synergy of analysis joint administration group.All data analyses are carried out with SPSS17.0; P<0.05 thinks significant difference.
Test-results: for people source leukemia KBM5 (Bcr-Abl is positive) subcutaneous transplantation knurl model, the compounds of this invention BDB has antitumor drug effect, suitable with the inhibition of imatinib 160mg/kg under 80mg/kg dosage.For people source leukemia KBM5R (Bcr-AblT315I mutant strain) subcutaneous transplantation knurl model, the compounds of this invention BDB has and the inhibition suitable to KBM5 subcutaneous transplantation knurl, and imatinib has no obvious inhibition.
4, the Initial pharmacokinetic research of the compounds of this invention BDB
Female C57BL/6 (Es-1
c) after the tested the compounds of this invention BDB of nude mice Single-dose intravenous, by its concentration in Main Tissues of liquid chromatography tandem mass spectrometry quantitative assay and Plasma Concentration, investigate test medicine at female C57BL/6 (Es-1
c) distributional difference in nude mouse inner blood and Main Tissues; With imatinib as a comparison.
Experimental technique and process as follows: female C57BL/6 (Es-1
c) nude mice, body weight 18-25g, 6-8 week age, in addition, 3 female C57BL/6 (Es-1
c) nude mice for gathering blank sample, preparation analyze needed for typical curve.Intravenous administration dosage is 1mg/kg, and administration volume is 5mL/kg.Intravenous administration solvent: DMSO:SolutolHS15:Saline=5:5:90, v/v/v.Set up LC-MS/MS method, measure test medicine Plasma Concentration and tissue concentration with inner mark method ration, linearity range 1 ~ 1000ng/mL, lower limit of quantitation scope is generally 1ng/mL.Female C57BL/6 (Es-1
c) nude mice gathers whole blood about 20 μ L at 0.25h, 2h, 8h and 24h single from mouse tail vein after intravenous administration, uses K
2eDTA anti-freezing, the redistilled water adding 3 times according to volume ratio obtains the blood sample after diluting, and is stored in-70 DEG C until analyze.Gather the heart, liver, spleen, lung, kidney, stomach, small intestine, the tissue samples such as pancreas simultaneously, clean with physiological saline, weigh and record after filter paper blots, be then stored in-70 DEG C until analyze.After weighed organs and tissues is thawed, the heart, liver, spleen, lung, kidney, stomach, small intestine, pancreas adds 3 times of PBS buffering salt Beadbeater homogenate; Adopt the PBS buffering salt of 0.3mL to rinse when bone marrow specimens gathers, then 12000 leave the heart 5 minutes, remove 0.25mL supernatant liquor, remaining cell sample adds the homogenate of 150mLPBS buffering salt.
Data analysis: adopt WinNonlin (version 6.2) software, calculates pharmacokinetic parameters by non-compartment model.
Experimental result shows, the transformation period (t of the compounds of this invention BDB
1/2) be 3.8h, be far longer than the transformation period (t of imatinib
1/2for 1.5h); Compared with imatinib, the compounds of this invention BDB has obviously advantage on the pharmacokinetic properties after intravenous injection.
In sum; compound provided by the invention or its pharmacy acceptable salt; as Bcr-Abl amphiploid inhibitor; effectively can suppress tyrosine kinase activity; can be effective to the disease treatment that such kinases abnormal activation is relevant, have good therapeutic action to malignant tumour, the preparation method of such inhibitor is easy; with low cost, have a good application prospect.
Claims (16)
1. the compound shown in formula I or its pharmacy acceptable salt, its structural formula is as follows:
2. compound according to claim 1 or its pharmacy acceptable salt, is characterized in that: Linker is (Z
1)
a– (Z
2)
b– (Z
3)
c;
Wherein, Z
1, Z
2, Z
3separately be selected from
C(O)(CH
2)
dNH、CO(CH
2)
eC(O)、(CH
2CH
2)
fNHC(O)、(CH
2CH
2)
g-C(O)NH、(CH
2)
hC(O)(OCH
2CH
2)
iNHC(O)(CH
2)
jC(O)、NH(CH
2)
k(OCH
2CH
2)
l-O(CH
2)
mNH、(CH
2)
nC(O)(OCH
2CH
2)
oNH(C(O)(CH
2)
pNH)
qC(O)-alkyl、C(O)NH(CH
2)
rNHC(O)-(CH
2)
sC(O)(NHCH
2CH
2)
tNH(C(O)(CH
2)
uNH)
v-C(O)-alkyl；
A ~ v is separately selected from arbitrary numerical value in 1 ~ 20.
3. compound according to claim 1 and 2 or its pharmacy acceptable salt, is characterized in that: described compound is:
4. the preparation method of compd B DB described in claim 3, is characterized in that: it comprises following operation steps:
5. preparation method according to claim 4, is characterized in that: it comprises following operation steps:
A, prepare compound 9:
Vinyl chloroformate, compound 8, diisopropylethylamine, in tetrahydrofuran (THF), in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 9;
B, prepare compound 11:
Compound 9, diisopropylethylamine, 4-(chloromethyl) Benzoyl chloride, in methylene dichloride, in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 11;
C, prepare compound 12:
Compound 11, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 2h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 12;
D, prepare compound 14:
Compound 12, compound 13,1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, I-hydroxybenzotriazole, diisopropylethylamine, in methylene dichloride, in stirred at ambient temperature reaction 16h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 14;
E, prepare compd B DB:
Compound 7, compound 14, salt of wormwood, in DMF, in 60 DEG C of stirring reaction 5h, obtain reaction solution; Reaction solution is separated, obtains compd B DB.
6. preparation method according to claim 5, is characterized in that:
In step a, the mol ratio of described Vinyl chloroformate, compound 8, diisopropylethylamine is 46.5:44.5:264; Described Vinyl chloroformate is 46.5:350mmol/ml with the molecular volume ratio of tetrahydrofuran (THF);
In step b, the mol ratio of described compound 9, diisopropylethylamine, 4-(chloromethyl) Benzoyl chloride is 0.67:0.8:0.8; Described compound 9 is 0.67:50mmol/ml with the molecular volume ratio of methylene dichloride;
In step c, described compound 11 is 0.445:20mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M;
In steps d, the mol ratio of described compound 12, compound 13,1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, I-hydroxybenzotriazole, diisopropylethylamine is 0.445:0.445:0.534:0.534:0.534; Described compound 12 is 0.445:25mmol/ml with the molecular volume ratio of methylene dichloride;
In step e, the mol ratio of described compound 7, compound 14, salt of wormwood is 0.4:1:4.8; Described compound 7 is 0.4:50mmol/ml with the molecular volume ratio of DMF.
7. preparation method according to claim 5, is characterized in that: in step e, and described compound 7 is prepared according to following operation steps:
8. preparation method according to claim 7, is characterized in that: in step e, and described compound 7 is prepared according to following operation steps:
I, prepare compound 2:
Compound 1, BOC acid anhydrides react completely in methylene dichloride, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 2;
Ii, prepare compound 3:
Compound 2, triethylamine, glutaryl chlorine, in methylene dichloride, in stirred at ambient temperature reaction 3h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 3;
Iii, prepare compound 4
Compound 3, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 3h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 4;
Iv, prepare compound 5
Compound 4, diisopropylethylamine, 3-chlorpromazine chloride, in methylene dichloride, in stirred at ambient temperature reaction 3h, obtain reaction solution; Reaction solution is separated, purifying, obtain compound 5;
V, prepare compound 6
Compound 5, piperazine-1-t-butyl formate, diisopropylethylamine are in dioxane, and back flow reaction 18h, obtains reaction solution; Reaction solution is separated, purifying, obtain compound 6;
Vi, prepare compound 7:
Compound 6, in HCl/ ethyl acetate, in stirred at ambient temperature reaction 2h, obtains reaction solution; Reaction solution, except desolventizing, obtains compound 7.
9. preparation method according to claim 7, is characterized in that:
In step I, the mol ratio of described compound 1, BOC acid anhydrides is 45.5:43.18; Described compound 1 is 45.5:600mmol/ml with the molecular volume ratio of methylene dichloride;
In step I i, the mol ratio of described compound 2, triethylamine, glutaryl chlorine is 8.42:14.85:4.17; Described compound 2 is 8.42:120mmol/ml with the molecular volume ratio of methylene dichloride;
In step I ii, described compound 3 is 3.8:100mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M;
In step I v, the mol ratio of described compound 4, diisopropylethylamine, 3-chlorpromazine chloride is 0.9:6.15:1.81; Described compound 4 is 0.9:50mmol/ml with the molecular volume ratio of methylene dichloride;
In step v, the mol ratio of described compound 5, piperazine-1-t-butyl formate, diisopropylethylamine is 0.14:2.68:0.77; Described compound 5 is 0.14:20mmol/ml with the molecular volume ratio of dioxane;
In step vi, described compound 6 is 0.1:20mmol/ml with the molecular volume ratio of HCl/ ethyl acetate; Described HCl/ ethyl acetate, its concentration is 2M.
10. compound described in claims 1 to 3 any one or its pharmacy acceptable salt purposes in the medicine of preparation treatment cell breeding disease.
11. purposes according to claim 10, is characterized in that: described medicine is tyrosine kinase inhibitor.
12. purposes according to claim 10 or 11, is characterized in that: described medicine is ABL inhibitor class medicine.
13. the purposes according to claim 10 or 11, is characterized in that: described medicine is
BCR-ABL or its mutant strain inhibitor class medicine.
14. purposes according to claim 13, is characterized in that: described BCR-ABL mutant strain is T315I mutant strain.
15. the purposes according to claim 10 ~ 14 any one, is characterized in that: described cell breeding disease is cancer.
16. purposes according to claim 14, is characterized in that: described cancer is chronic myelocytic leukemia, intestines mesenchymoma, gynaecology's knurl, dermatofibrosarcoma protuberans, Ph+ALL.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225085A (en) * | 2007-01-17 | 2008-07-23 | 天津天士力集团有限公司 | Phenyl urazan nitrogen nitric oxide donor 2-aniline pyrimidine derivatives, preparation method, compound containing the same and use thereof |
| CN101410108A (en) * | 2006-03-30 | 2009-04-15 | 内尔维阿诺医学科学有限公司 | Use of a kinase inhibitor for the treatment of particular resistant tumors |
| CN101588800A (en) * | 2006-11-03 | 2009-11-25 | 内尔维阿诺医学科学有限公司 | A method of administering an antitumor compound |
| WO2012125379A1 (en) * | 2011-03-14 | 2012-09-20 | Cellworks Research India Private Limited | Compositions, process of preparation of said compositions and method of treating inflammatory diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101410108A (en) * | 2006-03-30 | 2009-04-15 | 内尔维阿诺医学科学有限公司 | Use of a kinase inhibitor for the treatment of particular resistant tumors |
| CN101588800A (en) * | 2006-11-03 | 2009-11-25 | 内尔维阿诺医学科学有限公司 | A method of administering an antitumor compound |
| CN101225085A (en) * | 2007-01-17 | 2008-07-23 | 天津天士力集团有限公司 | Phenyl urazan nitrogen nitric oxide donor 2-aniline pyrimidine derivatives, preparation method, compound containing the same and use thereof |
| WO2012125379A1 (en) * | 2011-03-14 | 2012-09-20 | Cellworks Research India Private Limited | Compositions, process of preparation of said compositions and method of treating inflammatory diseases |
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