CN113493420A - EGFR tyrosine kinase inhibitor and application thereof - Google Patents

EGFR tyrosine kinase inhibitor and application thereof Download PDF

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CN113493420A
CN113493420A CN202010193366.0A CN202010193366A CN113493420A CN 113493420 A CN113493420 A CN 113493420A CN 202010193366 A CN202010193366 A CN 202010193366A CN 113493420 A CN113493420 A CN 113493420A
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cancer
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amino
independently selected
hydrogen
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胡佳琪
陈弘道
郑杨
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Nanjing Maisheng Technology Co.,Ltd.
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Pharmablock Sciences Nanjing Inc
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Abstract

The invention provides a compound shown in formula (I), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, and an application thereof in preparing a medicament for preventing or treating related diseases caused by EGFR mutation.

Description

EGFR tyrosine kinase inhibitor and application thereof
Technical Field
The invention belongs to the field of chemical medicine, and particularly relates to an EGFR tyrosine kinase inhibitor and application thereof.
Background
Lung cancer is one of the most common malignancies in the world, and is classified into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), including squamous cell carcinoma (squamous carcinoma), adenocarcinoma, and large cell carcinoma. NSCLC has cancer cells that grow and divide slower and spread metastases are relatively late compared to SCLC. NSCLC accounts for approximately 80% of all lung cancers, has a poor prognosis, and is found in the middle and advanced stages in approximately 75% of patients. Whereas overexpression and mutations of the Epidermal Growth Factor Receptor (EGFR) have been clearly demonstrated to lead to uncontrolled cell growth, associated with the progression of most cancer diseases, especially NSCLC (oncotarget.2016Jul 5; 7(27): 41691-.
EGFR is one of the epidermal growth factor receptor (HER) family members, which consists of EGFR (Erb-Bl), Erb-B2(HER-2/neu), Erb-B3, and Erb-B4. EGFR is a glycoprotein, a receptor of cell proliferation and signaling of Epidermal Growth Factor (EGF), belonging to the tyrosine kinase type, which is penetrated through the cell membrane and located on the surface of the cell membrane (Clin Cancer Res; 21 (3); February 1,2015, 526-.
In NSCLC patients, EGFR is not only overexpressed, but also has an aberrantly activating mutation in its tyrosine kinase domain for kinase activity independent of ligand binding. The most common kinase activity mutations are EGFR (Exon 19del E746-A750) and EGFR (Exon 21L858R), the first generation of the marketed EGFR tyrosine kinase inhibitors (EGFR-TKI) gefitinib (gefitinib) and erlotinib (erlotinib) have been approved for the treatment of both, but the first generation of EGFR-TKI develops resistance during treatment, which is associated with a secondary mutation in which threonine at position 790 of EGFR is replaced with methionine (T790M). Second generation non-reversible covalent inhibitors such as Afatinib (Afatinib) are more effective in treating EGFR (delE746-a750) and EGFR (L858R) but are less effective in treating drug-resistant T790M mutant because they show dose-dependent toxicity (Biologics,2014,8: 183-. Third generation EGFR-TKIs have therefore been developed, such as AZD9291, CO-1686 and HM 61713. The third generation EGFR-TKI is a tyrosine kinase inhibitor with specific selectivity. Compared with the first generation and the second generation EGFR-TKI, the third generation EGFR-TKI reduces the inhibition of wild type EGFR, reduces the clinical toxic and side effects, and can obtain better curative effect by using higher clinical dose. (J Clin Oncol 2014; 32: abstr 8009; J Clin Oncol 2014; 32: abstr 8010).
Another EGFR mutation that is aberrantly activated to induce carcinogenesis is the exon20 insertion mutation (EGFR exon20ins), and previous studies have shown that this type of mutation accounts for 4% -10% of all EGFR mutant lung cancers (PLoS ONE 201510 (7): e 0133859). At present, no medicine can be clinically used for treatment (Mol Cancer ther.2013,12,220), such mutant patients are not sensitive to EGFR inhibitor medicines on the market at present, the current standard treatment scheme is cytotoxic chemotherapy, the prognosis effect is poor, the side effect is strong, and no targeted medicine is available at present. Candidate drugs Poziotinib and Ref-1 targeting EGFR exon20 insertion mutation have entered clinical research, but have very strong inhibition capability on wild type EGFR, and therefore, can bring about large toxic and side effects, such as skin toxicity and the like. The improvement of clinical dosage and clinical efficacy are severely limited. In addition, there are also the compounds CLN-081 and DZ9008, which have recently entered the clinic, and although their in vitro activity has been shown to reduce toxicity to wild type, their clinical effects have not been further demonstrated. In conclusion, compounds with higher activity and low toxicity have yet to be developed.
Disclosure of Invention
The invention discloses a compound capable of being used as an EGFR protein kinase inhibitor and application thereof in preparing a medicament for preventing or treating EGFR related diseases.
In one aspect, the present invention provides a compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
Figure BDA0002416713450000021
wherein Z is1is-NH-, -N (CH)3) -, -O-or-S-;
Z2、Z3、Z4、Z5each independently is selected from C or N;
each R1Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy; two adjacent R1May form, together with the atoms to which they are attached, a five to six membered saturated or unsaturated aromatic or heteroaromatic ring;
each R2Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, ammoniaA group, cyano or alkoxy;
R3selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;
R4selected from hydrogen, C1-C6Alkyl or-NR5R6；
R5And R6Each independently selected from hydrogen, C1-C6Alkyl, -R7N(R8)(R9)；
R7、R8、R9Each independently selected from hydrogen or C1-C6An alkyl group;
m and n are respectively and independently selected from 1,2 or 3;
the dotted line is a single or double bond.
In some embodiments, formula (i) is formula (ia):
Figure BDA0002416713450000031
Z1is-NH-, -N (CH)3) -, -O-or-S-;
Z2、Z3、Z4、Z5each independently is selected from C or N;
each R1Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy; two adjacent R1May form, together with the atoms to which they are attached, a five to six membered saturated or unsaturated aromatic or heteroaromatic ring;
each R2Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;
m and n are respectively and independently selected from 1,2 or 3;
the dotted line is a single or double bond.
In yet other embodiments of the present invention, the substrate is,Z1is-NH-, -N (CH)3) -or-O-;
Z2、Z3、Z4、Z5each independently selected from C or N, and Z3And Z4Not N at the same time;
each R1Independently selected from hydrogen, C1-C3Alkyl or halogen; two adjacent R1May form a benzene ring together with the atoms to which they are attached;
each R2Independently selected from hydrogen, C1-C3Alkyl, halo C1-C3Alkyl, halogen, cyano or methoxy;
m and n are respectively and independently selected from 1 or 2;
the dotted line is a single or double bond.
In yet other embodiments of the present invention, the substrate is,
Figure BDA0002416713450000041
selected from:
Figure BDA0002416713450000042
in yet other embodiments of the present invention, the substrate is,
Figure BDA0002416713450000043
selected from:
Figure BDA0002416713450000044
the aforementioned aryl groups are all-carbon monocyclic or fused-ring polycyclic aromatic groups having a conjugated pi-electron system. The aryl group may have 6 to 10 carbon atoms in one or more rings. Most commonly, the aryl group has 6 carbon atoms in the ring. For example, a C6-10 aryl group is an aromatic radical containing 6 to 10 carbon atoms, such as phenyl or naphthyl.
The aforementioned heteroaryl groups, i.e., monocyclic or fused ring polycyclic aromatic heterocyclic groups having in at least one ring one or more heteroatom ring members (ring-forming atoms) independently selected from O, S and N. Heteroaryl groups have 5 to 14 ring-forming atoms, including 1 to 13 carbon atoms and 1 to 8 heteroatoms selected from O, S and N. In some embodiments, heteroaryl groups have 5 to 10 ring-forming atoms, including one to four heteroatoms. Heteroaryl groups may also contain one to three oxo or thiono (i.e., ═ S) groups. In some embodiments, heteroaryl groups have 5 to 8 ring-forming atoms, including one, two, or three heteroatoms. For example, a 5-membered heteroaryl group is a monocyclic heteroaryl group as defined above, having 5 ring atoms in the monocyclic heteroaryl ring; a 6-membered heteroaryl is a monocyclic heteroaryl group as defined above, having 6 ring atoms in the monocyclic heteroaryl ring; a5-10 membered heteroaryl is a monocyclic or bicyclic heteroaryl group as defined above having 5, 6, 7, 8, 9 or 10 ring atoms in the monocyclic or bicyclic heteroaryl ring.
In other embodiments, the compound is selected from the group consisting of:
Figure BDA0002416713450000051
Figure BDA0002416713450000061
the compound can be used for preparing medicaments for preventing or treating receptor tyrosine kinase mutation, particularly EGFR mutation related diseases; the EGFR mutation-related diseases are cancers, particularly cancers related to EGFR mutation in exon20 domain, such as non-small cell lung cancer, breast cancer, brain cancer, ovarian cancer, pancreatic cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, bladder cancer, liver cancer, gastrointestinal tissue cancer, esophageal cancer, thyroid cancer, leukemia, lymphoma, multiple myeloma and the like.
The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, isomer thereof; a pharmaceutically acceptable carrier or excipient. The pharmaceutically acceptable carrier or excipient may comprise any conventional pharmaceutical carrier or excipient. Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents such as hydrates and solvents. The pharmaceutical composition may contain additional ingredients such as flavoring agents, binders, excipients, and the like, if desired.
The pharmaceutical composition can also comprise other one or more anticancer drugs, and the anticancer drugs are small molecule drugs, monoclonal antibodies or fusion protein drugs.
The compound with EGFR tyrosine kinase inhibitory activity provided by the application has strong inhibitory capacity on EGFR (D770_ N771insNPG) insertion mutation kinase activity. The inhibition on the proliferation capacity of a wild type EGFR high expression mouse pro-B cell (BaF3) is greatly lower than that of Poziotinib and Ref-1 in the clinical research stage at present, and meanwhile, the inhibition on the proliferation of an EGFR exon20 insertion mutation high expression BaF3 cell can be effectively realized. The selectivity of the inhibitory activity of mutant cells relative to wild-type cells is a more important and critical indicator as a clinical therapeutic window compared to the inhibitory activity of single, highly expressed tumor cells of mutant EGFR. Therefore, compared with the current clinical compounds, the compound provided by the application can be used in higher dose without obvious toxicity in clinical application, so that the curative effect is greatly improved, and a better prognostic effect is obtained. And has good inhibitory effect on the abnormal proliferation of common EGFR mutation, EGFR (delE746-A750), Her2 insertional mutation high expression tumor cells.
Detailed Description
Example 1N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (4-methoxyphenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000071
The method comprises the following steps:
Figure BDA0002416713450000072
compound 1.1(15.000g, 54.57mmol, 1eq.) was dissolved in DMSO (100mL), compound 1.2(6.67g, 60.03mmol, 1.1eq.) was added, K2CO3(15.08g, 109.14mmol, 2eq.), heated to 80 ℃ and reacted for 15 hours. The system was poured into water (200mL), allowed to stand for 30min, filtered, and the filter cake dried to give a brown solid (7.350g, yield 38.53%).
Step two:
Figure BDA0002416713450000081
compound 1.3(2.500g, 7.15mmol, 1eq.) was dissolved in dioxane (100mL) and compound 1.4(1.30g, 8.58mmol, 1.2eq.) Pd (dppf) Cl was added2(523.35mg，715.25umol，0.1eq.)，K3PO4(1.52g，7.15mmol，1eq.)，H2O (10mL), nitrogen substitution three times, nitrogen protection, heating to 60 ℃, reaction for 1.5 hours, pouring the system into water (150mL), EtOAC (300mL), liquid separation, washing the organic phase once with saturated aqueous NaCl solution, liquid separation, drying the organic phase with anhydrous magnesium sulfate, filtration, concentration, column chromatography (n-heptane: EtOAC ═ 15:1) to obtain a white solid (2.100g, yield 89.04%).
Step three:
Figure BDA0002416713450000082
dissolving compound 1.5(2.100g, 6.37mmol, 1eq.) in dioxane (100mL), adding compound 1.6(1.19g, 6.37mmol, 1eq.), Pd2(dba)3(583.16mg，636.84umol，0.1eq.)，x-phos(607.18mg，1.27mmol，0.2eq.)，Cs2CO3(4.15g, 12.74mmol, 2eq.), three times of nitrogen replacement, nitrogen protection, heating to 100 ℃, reacting for 4 hours, cooling the system, filtering, and directly putting the filtrate into the next step.
Step four:
Figure BDA0002416713450000083
the filtrate of the previous step was added with N, N' -trimethylethylenediamine (3.25g, 31.81mmol, 5eq.), heated to 80 ℃ for reaction for 15 hours, poured into water (120mL), extracted with EtOAC (250mL), separated, the organic phase was washed once with saturated aqueous NaCl solution, separated, dried over anhydrous magnesium sulfate, filtered, concentrated, and subjected to column chromatography (DCM: MeOH: 30:1) to obtain a red oil (2.940g, yield: 82.29%).
Step five:
Figure BDA0002416713450000091
compound 1.8(2.940g, 5.23mmol, 1eq.) was dissolved in EtOH (80mL), Fe (1.46g, 26.17mmol, 5eq.) and NH added4Cl(1.29g，26.17mmol，5eq.)，H2O (20mL), heated to 80 ℃ and reacted for 5 hours. The system was filtered, washed with EtOAC (200mL), water (100mL) was added to the filtrate, the mixture was separated, the organic phase was washed once with saturated aqueous NaCl solution, the mixture was separated, the organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to give a brown solid (2.140g, yield: 76.89%).
Step six:
Figure BDA0002416713450000092
dissolving compound 1.9(2.140g, 4.03mmol, 1eq.) in DCM (20mL), cooling to-20 ℃ with a dry ice ethanol bath, adding triethylamine (814.66mg, 8.05mmol, 2eq.), compound 1.10(364.33mg, 4.03mmol, 1eq.), reacting for 10min, adding water (20mL) to the system, extracting with DCM (100mL), separating, washing the organic phase once with saturated aqueous NaCl solution, separating, drying the organic phase with anhydrous magnesium sulfate, filtering, concentrating, column chromatography (DCM: MeOH: 30:1), and slurrying with EtOAC to obtain an off-white solid (0.800g, 33.93%).1H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),8.53(s,1H),7.96-8.00(t,1H),7.90(m,2H),7.61(s,1H),7.41-7.44(d,2H),6.97-7.17(m,6H),6.36-6.43(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.79-3.81(d,6H),2.84-2.87(t,2H),2.70(s,3H),2.28-2.31(t,2H),2.20(s,6H)。LC-MS(m/z):587.00[M+H]+。
Example 2N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- (p-tolylamino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000101
Using a method similar to example 1, compound 1.2 of step one was replaced with p-toluidine and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.13(s,1H),8.60(s,1H),7.96(s,1H),7.86(s,1H)，7.84(s,1H),7.47-7.50(m,2H),7.42-7.45(d,2H),7.28-7.32(t,2H),6.94-6.98(m,3H),6.40-6.44(m,1H),6.18-6.22(dd,1H),5.73-5.76(dd,1H),3.80(s,3H),2.85-2.88(t,2H),2.71(s,3H),2.23-2.31(t,2H),2.18-2.22(m,9H)。LC-MS(m/z):569.69[M+H]+。
Example 3N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- ((2-methylpyrimidin-5-yl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000102
Using a method similar to example 1, compound 1.2 of step one was replaced with 2-methylpyrimidin-5-amine and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.08(brs,1H),8.84(s,2H),8.55(brs,1H),8.37(s,1H),8.12(s,1H),7.93(s,1H),7.50-7.54(m,2H),7.29-7.34(t,2H),6.97(s,1H),6.34(s,1H),6.14-6.18(d,1H),5.70-5.73(m,1H),3.79(s,3H),2.91-2.93(m,2H),2.71(s,3H),2.28-2.50(m,9H)。LC-MS(m/z):571.66[M+H]+。
Example 4N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- ((5-methylpyrazin-2-yl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000111
Using a method similar to example 1, compound 1.2 of step one was replaced with 5-methylpyrazin-2-amine and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.17(s,1H),9.24(s,1H),8.57(s,1H),8.41(s,1H),8.09(s,1H),8.03(s,1H),7.88(s,1H),7.51-7.54(m,2H),7.32-7.37(m,2H),7.02(s,1H),6.32-6.39(m,1H),6.13-6.17(dd,1H),5.70-5.72(dd,1H),3.80(s,3H),2.92(m,2H),2.74(m,3H),2.35(m,5H),2.24(m5H)。LC-MS(m/z):571.28[M+H]+。
Example 5N- (5- ((5- (3-chloro-4-fluorophenyl) -4- (p-tolylamino) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000112
Using a method similar to example 1, compound 1.2 of step one was replaced with p-toluidine and compound 1.4 of step two was replaced with (3-chloro-4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.10(s,1H),8.57(s,1H),8.11(s,1H),7.88(s,1H),7.86(s,1H),7.63-7.66(dd,1H),7.41-7.51(m,4H),6.94-7.0(m,3H),6.38-6.44(m,1H),6.18-6.23(dd,1H),5.73-5.74(d,1H),3.80(s,3H),2.88(m,2H),2.71(s,3H),2.31(m,2H),2.19-2.22(m,9H)。LC-MS(m/z):603.25[M+H]+。
Example 6N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- (methyl (p-tolyl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000121
Using a method similar to example 1, compound 1.2 of step one was replaced with N, 4-dimethylaniline and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),9.06(s,1H),7.86(s,1H),7.78(s,1H),7.01-7.03(m,3H),6.71-6.83(m,6H),6.35-6.41(dd,1H),6.18-6.23(dd,1H),5.70-5.73(dd,1H),3.89(s,3H),3.44(s,3H),2.87-2.88(m,2H),2.70(s,3H)，2.20-2.30(m,8H),2.08(s,3H)。LC-MS(m/z):583.77[M+H]+。
Example 7N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- (p-tolyloxy) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000122
Using a method similar to example 1, compound 1.2 of step one was replaced with p-cresol and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.02(s,1H),8.41(s,1H),8.37(s,1H),8.16(s,1H),7.67-7.72(m,2H),7.10-7.15(m,4H),6.91(s,1H),6.39-6.43(dd,1H),6.22-6.27(dd,1H),5.75-5.78(dd,1H),3.76(s,3H),2.85(m,2H),2.67(s,3H),2.23-2.34(m,10H)。LC-MS(m/z):570.75[M+H]+。
Example 8N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- (naphthalen-1-yloxy) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000131
Using a method similar to example 1, compound 1.2 of step one was replaced with naphthalen-1-ol and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid, giving the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.0(brs,1H),8.45(s,1H),8.28(brs,1H),8.09(s,1H),7.96-7.98(m,1H),7.77-7.85(m,4H),7.43-7.57(m,4H),7.30-7.35(m,2H),6.83(s,1H),6.40(m,1H),6.22-6.26(d,1H),5.76-5.79(d,1H),3.66(s,3H),2.82(m,2H),2.64(s,3H),2.23-2.34(m,8H)。LC-MS(m/z):606.71[M+H]+。
Example 9N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluorophenyl) -4- ((2-fluorophenyl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000132
Using a method similar to example 1, compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.07(s,1H),8.50(s,1H),7.93(s,1H),7.78-7.88(m,3H),7.52-7.56(m,2H),7.31-7.36(t,2H),7.12-7.14(m,1H),6.95-7.05(m,3H),6.36-6.40(dd,1H),6.18-6.22(dd,1H),5.72-5.75(dd,1H),3.78(s,3H),2.83-2.86(t,3H),2.70(s,2H),2.27-2.30(t,2H),2.20(s,6H)。LC-MS(m/z):574.95[M+H]+。
Example 10N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluoro-5-methylphenyl) amino) -5- (4-fluorophenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000141
Using a method similar to example 1, compound 1.2 of step one was replaced with 2-fluoro-5-methylaniline and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):9.92(brs,1H),8.45(brs,1H),8.11(s,1H),7.92(m,2H),7.50-7.58(m,3H),7.36-7.30(m,2H),7.0-7.04(m,1H),6.94(s,1H),6.85-6.86(m,1H),6.19-6.23(d,1H)5.73-5.76(d,1H),3.81(s,3H),2.93(m,2H),2.66(s,3H),2.18-2.37(m,9H)。LC-MS(m/z):589.04[M+H]+。
Example 11N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluoro-4-methylphenyl) amino) -5- (4-fluorophenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000142
Using a method similar to example 1, compound 1.2 of step one was replaced with 2-fluoro-4-methylaniline and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.04(s,1H),8.57(s,1H),7.91(s,1H),7.78(s,1H),7.65-7.70(t,3H),7.51-7.55(m,2H),7.31-7.36(m,2H),6.96-6.99(m,2H),6.37-6.43(m,1H),6.18-6.23(dd,1H),5.73-5.76(dd,1H),3.79(s,3H),2.86(m,2H),2.70(s,3H),2.10-2.40(m,11H)。LC-MS(m/z):589.01[M+H]+。
Example 12N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluoro-3-methylphenyl) amino) -5- (4-fluorophenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000151
Using a method similar to example 1, compound 1.2 of step one was replaced with 2-fluoro-3-methylaniline and compound 1.4 of step two was replaced with (4-fluorophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.04(s,1H),8.52(s,1H),7.94(s,2H),7.76-7.80(m,1H),7.60(s,1H),7.52-7.57(m,2H),7.32-7.36(m,2H),6.97(s,1H),6.85-6.90(m,2H),6.36-6.42(dd,1H),6.17-6.21(dd,1H),5.72-5.75(dd,1H),3.79(s,3H),2.85-2.88(t,2H),2.70(s,3H),2.17-2.40(m,11H)。LC-MS(m/z):588.50[M+H]+。
Example 13N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (4- (trifluoromethyl) phenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000152
Using a method similar to example 1, compound 1.4 of step two was replaced with (4- (trifluoromethyl) phenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.46(s,1H),8.10(s,1H),8.01(s,1H),7.89(s,1H),7.82-7.84(d,2H),7.73-7.75(d,2H),7.65-7.69(m,1H),7.00-7.16(m,3H),6.94(s,1H),6.36-6.43(dd,1H),6.18-6.23(dd,1H),5.73-5.76(dd,1H),3.78(s,3H),2.83-2.86(t,2H),2.69(s,3H),2.21-2.30(m,8H)。LC-MS(m/z):625.16[M+H]+。
Example 14N- (5- ((5- (4-cyanophenyl) -4- ((2-fluorophenyl) amino) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000161
Using a method similar to example 1, compound 1.4 of step two was replaced with (4-cyanophenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.04(s,1H),8.46(s,1H),8.15(s,1H),8.02(s,1H),7.92-7.95(m,3H),7.73(d,2H),7.63-7.67(m,1H),7.0-7.16(m,3H),6.94(s,1H),6.37-6.43(dd,1H),6.19-6.23(dd,1H),5.73-5.76(dd,1H),3.78(s,3H),2.85(m,2H),2.68(s,3H),2.21-2.40(m,8H)。LC-MS(m/z):582.10[M+H]+。
Example 15N- (5- ((5- (4, 4-Difluorocyclohex-1-en-1-yl) -4- ((2-fluorophenyl) amino) pyrimidin-2-yl) amino) -2- ((2- (dimethylamino) ethyl) (methyl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000162
Using a method similar to example 1, compound 1.4 of step two was replaced with (4, 4-difluorocyclohex-1-en-1-yl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.49(s,1H),7.94(s,2H),7.71-7.85(m,2H),7.16-7.21(m.3H),6.93(s,1H),6.35-6.42(m,1H),6.17-6.22(m,1H),5.72-5.75(dd,1H),3.78(s,3H),2.82-2.85(t,2H),2.68-2.71(m,5H),2.20-2.28(m,9H)。LC-MS(m/z):596.48[M+H]+。
Example 16N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (4-propylphenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000171
Using a method similar to example 1, compound 1.4 of step two was replaced with (4-propylphenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.51(s,1H),7.93-7.98(m,3H),7.66(s,1H),7.41-7.43(d,2H),7.33-7.35(d,2H),7.11-7.16(m,1H),6.97-7.02(m,3H),6.37-6.43(dd,1H),6.18-6.22(dd,1H),5.72-5.75(dd,1H),3.79(s,3H),2.87(m,2H),2.70(s,3H),2.60-2.64(t,2H),2.22-2.31(m,8H),1.62-1.67(m,2H),0.92-0.96(t,3H)。LC-MS(m/z):599.27[M+H]+。
Example 17N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluoro-2-methylphenyl) -4- ((2-fluorophenyl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000172
Using a method similar to example 1, compound 1.4 of step two was replaced with (4-fluoro-2-methylphenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.52(s,1H),7.77(s,1H),7.72-7.75(m,2H),7.41(s,1H),7.29-7.32(m,1H),7.21-7.24(m,1H),7.0-7.17(m,4H),6.95(s,1H),6.36-6.43(dd,1H),6.18-6.22(dd,1H),5.73-5.76(dd,1H),3.80(s,3H),2.83-2.86(t,3H),2.69(s,3H),2.21-2.30(m,11H)。L C-MS(m/z):589.04[M+H]+。
Example 18N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((5- (4-fluoro-3-methylphenyl) -4- ((2-fluorophenyl) amino) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000181
Using a method similar to example 1, compound 1.4 of step two was replaced with (4-fluoro-3-methylphenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.05(s,1H),8.52(s,1H),7.93(s,1H),7.86-7.93(m,2H),7.73(s,1H),7.41-7.43(m,1H)7.33-7.34(m,1H),7.24-7.28(t,1H),7.14(m,1H),7.0-7.04(m,2H),6.96(s,1H),6.36-6.38(m,1H),6.18-6.22(d,1H),5.72-5.75(d,1H)3.79(s,3H),2.84-2.87(t,2H),2.70(s,3H),2.28-2.30(m,5H),2.21(s,6H)。LC-MS(m/z):589.04[M+H]+。
Example 19N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (pyridin-4-yl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000182
Using a method similar to example 1, compound 1.4 of step two was replaced with pyridin-4-ylboronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.03(s,1H),8.63-8.64(d,2H),8.45(s,1H),8.18(s,1H),8.06(s,1H),7.97(s,1H),7.67-7.70(t,1H),7.55-7.56(d,2H),7.01-7.17(m,3H),6.94(s,1H),6.37-6.43(dd,1H),6.19-6.23(d,1H),5.73-5.76(d,1H),3.78(s,3H),2.85(m,2H),2.69(s,3H),2.21-2.30(m,8H)。LC-MS(m/z):557.78[M+H]+。
Example 20N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (2-methoxyphenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000191
Using a method similar to example 1, compound 1.4 of step two was replaced with (2-methoxyphenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.06(s,1H),8.53(s,1H),8.05(m,1H),7.95(s,1H),7.90(s,1H),7.43-7.45(m,1H),7.30-7.32(m.2H),7.09-7.19(m,3H),6.96-6.99(m,3H),6.36-6.39(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.80-3.83(d,6H),2.85-2.88(m,2H),2.71(s,3H),2.29-2.31(m,2H),2.21(s,6H)。LC-MS(m/z):587.18[M+H]+。
Example 21N- (2- ((2- (dimethylamino) ethyl) (methyl) amino) -5- ((4- ((2-fluorophenyl) amino) -5- (3-methoxyphenyl) pyrimidin-2-yl) amino) -4-methoxyphenyl) acrylamide
Figure BDA0002416713450000192
Using a method similar to example 1, compound 1.4 of step two was replaced with (3-methoxyphenyl) boronic acid to give the title compound.1H NMR(400MHz,DMSO-d6)(ppm):10.06(s,1H),8.51(s,1H),7.96-8.0(m,3H),7.75(s,1H),7.41-7.45(m,1H),6.97-7.18(m,7H),6.46-6.43(dd,1H),6.17-6.22(dd,1H),5.72-5.75(dd,1H),3.80-3.85(d,6H),2.85-2,88(t,2H),2.70(s,3H),2.20-2.40(m,8H)。LC-MS(m/z):587.07[M+H]+。
Experimental example 1 EGFR D770-N771 insNPG kinase Activity inhibition assay
EGFR D770-N771 insNPG kinase expressed in baculovirus expression system was purchased from Shanghai Yongwei (univ Biological Inc.). TK kinase HTRF Detection kit (#62TK0PEC) containing a biotin-labeled polypeptide Substrate TK Substrate-biotin, a Eu-labeled specific phosphorylated polypeptide Antibody TK Antibody-Cryptate, a HTRF fluorescence acceptor reagent Streptavidin-XL665, and a 5 Xkinase reaction buffer, Detection buffer was purchased from Cisbio Bioassays (Codolet, France). Dithiothreitol (DTT), magnesium chloride, manganese chloride, Adenosine Triphosphate (ATP), dimethyl sulfoxide (DMSO), and HEPES buffer were obtained from Sigma at the highest purity levels available.
General methods for EGFR kinase activity inhibition assay: the phosphorylation reaction buffer was composed of 1M HEPES (pH 7.0), 5mM MgCl2、1mM MnCl2Composition, 1mM DTT was added to the buffer immediately before the start of the experiment.Preparing a test compound DMSO storage mother liquor, and performing three-time concentration gradient dilution by using DMSO according to the experiment requirement. The obtained gradient diluted solution is further diluted by phosphorylation reaction buffer solution to obtain a working solution of the compound to be detected dissolved in the reaction buffer solution containing 5% DMSO. mu.L of the compound working solution and 4. mu.L of EGFR kinase solution diluted in reaction buffer were added to a white low volume 384-well microtiter plate, and 4. mu.L of a mixed solution of ATP and biotin-labeled polypeptide substrate in reaction buffer was added to start the phosphorylation reaction. In the WT EGFR assay, the final concentrations of WT kinase, polypeptide substrate, ATP and DMSO were 0.01ng/ul, 500nM, 4. mu.M and 1%, respectively, and in the EGFR D770_ N771insNPG assay, the final concentrations of kinase, polypeptide substrate, ATP and DMSO were 0.01ng/ul, 200nM, 4. mu.M and 1%, respectively. The reaction was carried out at room temperature for 60 minutes under protection from light. mu.L each of TK Antibody-Cryptate Antibody and Streptavidin-XL665 diluted in detection buffer was added thereto, and incubated at room temperature for 60 minutes. The final concentration of Streptavidin-XL665 in the WT EGFR kinase reaction system was 15.61nM, the final concentration of Streptavidin-XL665 in the EGFR D770_ N771insNPG kinase reaction system was 31.25nM, and the antibodies were diluted to the final concentrations provided by the supplier. Use of Tecan (
Figure BDA0002416713450000201
Figure BDA0002416713450000202
Switzerland) multifunctional microplate reader Spark to read the plate, and detect two groups of homogeneous time-resolved fluorescence intensities, wherein the excitation wavelength is 320nm, and the emission wavelengths are 665nm and 620nm respectively. Using Prism 7(La Jolla,15CA) to plot the ratio of 665nm/620nm fluorescence intensity against the inhibitor concentration, the resulting inhibition curves were normalized by a sigmoidal dose-dependent curve model, and the IC of the inhibitor was obtained50The value is obtained. EGFRD 770-N771 insNPG kinase inhibitory Activity IC of a fraction of the Compound prepared according to the preceding examples50The results are shown in Table 1.
TABLE 1
Compound (I) IC50(nM)
Example 1 0.85
Example 2 0.83
Example 9 2.00
Example 10 5.67
Example 11 4.93
Example 12 8.94
The experimental results show that the compounds described in the present application can effectively inhibit the EGFR D770-N771 insNPG kinase activity, wherein the IC of examples 1 and 250Are all less than 1 nM.
Experimental example 2 cell proliferation inhibition experiment
H358, HCC827, NCI-H1781, Calu-3 cells were purchased from Chinese academy of sciences cell Bank (Shanghai), BaF3 EGFR WT, BaF3 EGFR D770_ N771insSVD, BaF3 EGFR V769_ D770insASV, BaF3 ERBB2A775_ G776insYVMA cells were purchased from Beijing Congyuan Bochu, MDA-MB-231, SK-BR-3, BT474 were purchased from Nanjing Kebai Biotech Co. MEM culture medium, RPMI1640 culture medium, penicillin-streptomycin double antibody, 0.5% pancreatin (10X) and epidermal cell growthThe long factor EGF was purchased from ThermoFisher (Waltham, MA, USA). Certified Fetal Bovine Serum (FBS) was purchased from Biological Industries (Israel). Corning 96 and 384-well cell culture plates were purchased from corning (usa). Cell-Titer
Figure BDA0002416713450000211
Available from Promega Corporation (Madison, Wis., USA).
In order to evaluate the ability of the synthesized compounds to inhibit the proliferation of lung cancer cells H358, HCC827, NCI-H1781, Calu-3 and BaF3 EGFRWT, BaF3 EGFR D770_ N771insSVD, BaF3 EGFR V769_ D770insASV, BaF3 ERBB2A775_ G776insYVMA cells, cells exponentially increased by Calu-3 were inoculated into MEM medium containing 10% bovine serum and 1% penicillin-streptomycin double antibody, cells exponentially increased by H358, HCC827, NCI-H1781, BaF3 EGFR D770_ N771 insD, BaF3 EGFR V769_ D insASV 770, BaF 4 ERBB 5A 775_ G776insYVMA were inoculated into RPMI medium containing 10% bovine serum and 1% penicillin-streptomycin double antibody, cells exponentially increased by BaF3 wt. 10% WT, 10% WT-10% penicillin-EGF double antibody, HCF 1640 medium containing 10% penicillin-EGF, 7550% HCF 1640-EGF double antibody, and VMA/102 mL RPMI-102/102 mL of RPMI-EGF medium, h358 density 100000 cells/mL, BaF3 EGFR D770_ N771insSVD, BaF 3V 769_ D770insASV density 50000 cells/mL, BaF3 EGFR WT density 125000 cells/mL, 384 well plates, 20. mu.L per well, placed at 37 ℃, 5% CO2Overnight in an incubator. Compounds were diluted to 12 points in DMSO, 3-fold gradient dilutions, starting at 2 mM. mu.L of DMSO solution from the compound stock plate was added to 99. mu.L of cell culture medium (final maximum concentration of compound in the assay was 10. mu.M, and final concentration of DMSO was 0.5%). mu.L of compound solution in culture medium was added to each well of the 384 plates according to a gradient. After addition of the compound solution, 384-well plates were placed at 37 ℃ in 5% CO2Incubate in incubator for 3 days. Cell viability was determined by quantifying the ATP present in the cell culture using the CellTiter-Glo assay kit from Promega (Madison, Wis., USA). After 20 minutes incubation, readings were taken under a chemiluminescent program using a SPARK multifunctional microplate reader from TECAN. In Prism 7 (LaJolla)CA) concentration of compound that inhibits cell viability by 50% (IC) was determined using a sigmoidal dose response model (variable slope, four parameters)50Value).
The drug-property control substances Poziotinib, Osimertinib and Pyrotinib used in the experiment are all obtained in a commercial mode, Ref-1 is prepared according to the preparation method recorded in WO2015195228A1, and the structures are as follows:
Figure BDA0002416713450000221
the results of the partial compounds prepared according to the preceding examples for the inhibition of BaF3 EGFR WT, BaF3 EGFR D770_ N771insSVD, BaF3 EGFR V769_ D770insASV cell proliferation are shown in table 2.
TABLE 2
Figure BDA0002416713450000222
Figure BDA0002416713450000231
The results show that the activity of the compound of the invention on the proliferation inhibition of WT EGFR high-expression BaF3 cells is far lower than that of two current candidate drugs in clinical research stages, and the toxicity caused by the activity is predicted to be greatly reduced. Compared with the selectivity between the cell proliferation inhibition activities of BaF3 highly expressed by two most extensive mutations D770_ N771insSVD and V769_ D770insASV in WT EGFR and EGFR Exon20 insertions, the two clinical compounds are 0.18/0.33 and 0.25/0.18 at present, respectively, which indicates that the clinical curative dose is far higher than the toxic dose, so the clinical application is greatly limited. The selectivity of most compounds of the invention is obviously higher than that of clinical compounds, for example, examples 9 and 13 are 5.5/5.0 and 1.3/3.3 respectively, which are 30/15, 7.2/10 and 22/28,5.2/18 times of Poziotinib and Ref-1 respectively, thus indicating that the clinical treatment space is greatly improved and the drug effect is more obvious.
The results of partial compounds prepared according to the previous examples on the inhibition of HCC827, H358 WT cell proliferation are shown in table 3.
TABLE 3
Figure BDA0002416713450000232
Figure BDA0002416713450000241
The results show that the compound of the invention has far lower inhibition on the proliferation of H358 NSCLC cells expressing wild-type EGFR than Poziotinib, and is close to or even lower than the third-generation EGFR inhibitor Osimertinib with completely verified clinical safety.
The results of the partial compounds prepared according to the preceding examples for the inhibition of Calu-3, NCI-H1781 and BaF3 ERBB2A 775-G776 insYVMA cell proliferation are shown in Table 4.
TABLE 4
Figure BDA0002416713450000242
The results show that although the inhibition of example 2 on the proliferation of BaF3 ERBB2A775_ G776insYVMA and NCI-H1781 cells is lower than that of the Pyrotinib in the clinical research stage of the ERBB2A775_ G776insYVMA and ERBB 2G 776_ V777insV high-expression non-small cell lung cancer treatment, the inhibition of the proliferation of the WT ERBB2 high-expression Calu-3 is obviously lower than that of the Pyrotinib, and the selectivity and the clinical treatment space are more excellent.

Claims (13)

1. A compound of formula (I), a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:
Figure FDA0002416713440000011
wherein Z is1is-NH-, -N (CH)3) -, -O-or-S-;
Z2、Z3、Z4、Z5each independently is selected from C or N;
each R1Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy; two adjacent R1May form, together with the atoms to which they are attached, a five to six membered saturated or unsaturated aromatic or heteroaromatic ring;
each R2Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;
R3selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;
R4selected from hydrogen, C1-C6Alkyl or-NR5R6；
R5And R6Each independently selected from hydrogen, C1-C6Alkyl, -R7N(R8)(R9)；
R7、R8、R9Each independently selected from hydrogen or C1-C6An alkyl group;
m and n are respectively and independently selected from 1,2 or 3;
the dotted line is a single or double bond.
2. A compound according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein formula (i) is formula (la):
Figure FDA0002416713440000012
Z1is-NH-, -N (CH)3) -, -O-or-S-;
Z2、Z3、Z4、Z5each independently is selected from C or N;
each R1Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy; two adjacent R1May form, together with the atoms to which they are attached, a five to six membered saturated or unsaturated aromatic or heteroaromatic ring;
each R2Independently selected from hydrogen, C1-C6Alkyl, halo C1-C6Alkyl, halogen, hydroxy, nitro, amino, cyano or alkoxy;
m and n are respectively and independently selected from 1,2 or 3;
the dotted line is a single or double bond.
3. A compound according to claim 2, a stereoisomer, or a pharmaceutically acceptable salt thereof, wherein:
Z1is-NH-, -N (CH)3) -or-O-;
Z2、Z3、Z4、Z5each independently selected from C or N, and Z3And Z4Not N at the same time;
each R1Independently selected from hydrogen, C1-C3Alkyl or halogen; two adjacent R1May form a benzene ring together with the atoms to which they are attached;
each R2Independently selected from hydrogen, C1-C3Alkyl, halo C1-C3Alkyl, halogen, cyano or methoxy;
m and n are respectively and independently selected from 1 or 2;
the dotted line is a single or double bond.
4. A compound according to claim 1, a stereoisomer or pharmaceutically acceptable salt thereof, wherein
Figure FDA0002416713440000021
Selected from:
Figure FDA0002416713440000022
5. a compound according to claim 1, a stereoisomer or pharmaceutically acceptable salt thereof, wherein
Figure FDA0002416713440000023
Selected from:
Figure FDA0002416713440000031
6. the compound according to claim 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, which is selected from the following compounds:
Figure FDA0002416713440000032
Figure FDA0002416713440000041
7. use of a compound according to any one of claims 1 to 6 for the preparation of a medicament for the prophylaxis or treatment of a disease associated with mutation of a receptor tyrosine kinase.
8. The use of claim 7, wherein the receptor tyrosine kinase mutation is an EGFR mutation.
9. The use of claim 8, wherein the related disease caused by EGFR mutation is cancer.
10. The use of claim 9, wherein the cancer is non-small cell lung cancer, breast cancer, brain cancer, ovarian cancer, pancreatic cancer, uterine cancer, cervical cancer, skin cancer, prostate cancer, bladder cancer, liver cancer, cancer of gastrointestinal tissue, esophageal cancer, thyroid cancer, leukemia, lymphoma, or multiple myeloma.
11. The use of claim 7, wherein the related disease caused by EGFR mutation is related cancer caused by a mutation in exon20 domain of EGFR.
12. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-6 and a pharmaceutically acceptable carrier or excipient.
13. The pharmaceutical composition of claim 12, further comprising an additional anti-cancer drug or drugs, said anti-cancer drug or drugs being small molecule drugs, monoclonal antibodies or fusion protein drugs.
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