This invention relates to utilize a new compound to treat human non-small cell lung carcinoma, more particularly, to utilize the compound for overcoming drug resistance of EGFR mutation and cancerous stemness in the above mentioned disease.
There are two main types of lung cancer; one is small cell lung carcinoma (SCLC) (15%˜25% of cases), the other is non-small cell lung carcinoma (NSCLC) (75%˜80% of cases). The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor which belongs to the ErbB family, EGFR overexpresses in many types of cancers, including 25% of lung cancer and occurs in 40%˜80% of patients with NSCLC which is a promising therapeutic target.
Abnormal activation of tyrosin kinase increases cell proliferation, survival, and cytotoxic drug resistance of malignant cells. Approximately 90% of EGFR mutations are exon 19 deletions and exon 21 L858R point mutations which are associated with sensitivity to the small-molecule kinase inhibitors in treating cancer. L858R point mutation of EGFR seemed to cause the repositioning of crucial residues that surround the ATP-binding cleft of the EGFR tyrosine kinase domain to result in small molecule tyrosine kinase inhibitors (TKIs) such as Gefitinib inhibiting EGFR mutation by competing with ATP binding within the catalytic kinase domain of tyrosine kinase receptor.
Although EGFR TKI has revolutionized treatment of EGFR-mutant NSCLC, most patients acquired resistance after several months' treatment. EGFR mutation such as T790M mutation correlates resistance to EGFR TKI treatment which can be detected approximately 50%˜60% after resistance develops. T790M the most common secondary point mutation in which methionine is to substitute threonine at amino acid position 790 is one of the mutations identified from the NSCLC patients acquiring resistance to Gefitinib. T790M mutation increases the affinity of the receptor for ATP, thereby reducing the effectiveness of TKI, Gefitinib.
Development of novel drugs for overcoming drug resistance in NSCLC therapy is highly desired. This invention provides a Gefitinib derivative, Dodecyl-4-(4-(3-(4-(3-chloro-4-fluorophenylamino)-7-methoxyquinazolin-6-yloxy)propyl)piperazin-1-yl)-4-oxobutanoate, hereinafter referred to as SP101. The chemical structure of SP101 is as follows:
SP101 can bind the ATP-binding pocket of EGFR to inhibit its EGFR kinase activity. The EGFR mutation at T790M and cancerous stemness are associated with drug resistance in NSCLC therapy. In this invention, SP101 shows the ability to reduce drug resistances of EGFR (T790M) and cancerous stemness in NSCLC. SP101 displays the anticancer ability in inducing cell death and apoptosis in the Gefitinib-resistant EGFR (T790M) of H1975 cells. SP101 inhibits the phosphorylated EGFR and its downstream surviving protein expression and induced the caspase 3 activation for apoptosis induction.
Moreover, SP101 is more potent on the reduction of cell viability than Gefitinib and also induced more apoptosis than Gefitinib in the A549-ON which expressed Oct4 and Nanog proteins in lung cancer cells displaying cancerous stemness and drug resistance. In addition, SP101 is more effective in inducing cell death and apoptosis than Gefitinib in the PLC26 cells which were from clinical lung cancer patients having Gefitinib-drug resistance. More importantly, SP101 significantly inhibits drug-resistant tumor growth in the xenograft human lung tumors in nude mice.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
A detailed description of this invention is illustrated by the following specific embodiments. Person skilled in the art can conceive other advantages and effects of this invention based on the disclosure contained in the specification of this invention. This invention can be executed or applied by the other methods. Without affecting the purpose of this invention, any detail in the description can be modified or changed based on different viewpoints and applications and it shall still be covered within the scope of this invention.
I. Preparation of SP101
The detailed synthesis process of SP101 is disclosed in U.S. Pat. No. 9,512,086 filed on Jan. 14, 2015, the entire content of which is incorporated herein by reference.
A mixture of succinic anhydride (1.00 g, 10.00 mmol) and dry methanol (20 mL 500 mmol) was stirred vigorously while heated at reflux 2.5 hours. The excess of methanol was removed under reduced pressure and the residue was taken up in water, and the solution was extracted with dichloromethane (hereinafter referred to DCM), dried over MgSO4, and evaporated to obtain Compound-1 (0.90 g, 6.81 mmol) in 68% yield.
1H NMR (400 MHz, CDCl3, δ): 10.89 (b, 1H), 3.70 (s, 3H), 2.71-2.61 (m, 4H); 13C NMR (100.6 MHz, CDCl3, δ): 178.1, 172.6, 51.9, 28.8, 28.6; IR (KBr): 3028, 2957, 1736, 1690, 1175, 1003 cm−1; MS m/z: 132.0 (M+, 0.1), 114.1 (11.2), 101.0 (100.0), 73.1 (20.9), 59.1 (17.2), 55.0 (41.8); HRMS-EI (m/z): [M]+ calculated for C5H8O4, 132.0423; found, 132.0424.
A solution of Compound-1 (0.35 g. 2.65 mmol) and thionyl chloride (0.22 mL, 2.9 mmol) in benzene (5 mL) was refluxed for 1.5 hours. Subsequently, the majority of the thionyl chloride and benzene were removed by distillation. The mixture was cooled down to room temperature and dried under a vacuum to give a crude 3-chlorocarbonyl-propionic acid methyl ester. A solution of 3-chlorocarbonyl-propionic acid methyl ester (0.5 g, 2 mmol) in dichloromethane (5 mL) was added to a round flask containing 1-benzylpiperazin (0.50 g, 2.84 mmol) in dichloromethane through cannula, and subsequently pyridine (0.65 mL, 8.00 mmol) was added. The resulting solution was stirred at room temperature overnight, and quenched by adding water. The pH of the solution was made basic (pH 9), by adding 2 M NaOH solution. The solution was extracted with dichloromethane, dried over MgSO4, and evaporated to give a crude residue which was purified by column chromatography, eluting by ethyl acetate/hexane (1:2.3, 1:1, 3:1) to provide Compound-2 (0.26 g, 0.90 mmol) in 34% yield.
1H NMR (400 MHz, CDCl3, δ): 7.32-7.29 (m, 4H), 7.28-7.27 (m, 1H), 3.69 (s, 3H), 3.63-3.60 (t, J=5.1 Hz, 2H), 3.51 (s, 2H), 3.49-3.47 (t, J=5.1 Hz, 2H), 2.68-2.59 (m, 4H), 2.45-2.39 (m, 4H); 13C NMR (100.6 MHz. CDCl3, δ): 173.7, 169.5, 137.6, 129.1, 128.3, 127.3, 62.9, 52.9, 52.7, 51.8, 45.3, 41.8, 29.1, 27.9; IR (KBr): 2949, 1736, 1646, 1438, 1226, 1165, 998, 744 cm−1; MS in/z: 290.1 (M+, 14.0), 259.1 (16.4), 146.1 (48.7), 134.1 (21.5), 91.1 (100.0); HRMS-EI (m/z): [M]+ calculated for C16H22N2O3, 290.1630; found, 290.1634.
A mixture of Compound 2 (0.21 g, 0.72 mmol) and 10% palladium on carbon (Pd/C) (22 mg. 10 wt %) in methanol (20 mL) in a Parr glass vessel and carefully flushed three times with hydrogen gas. The vessel was finally charged with hydrogen gas (60 psi) and shaken mechanically for 12 hours. After completion of the reaction, the reaction mixture was filtered through a pad of celite and washed with, excess methanol. The filtrate was concentrated under reduced pressure to obtain 4-oxo-4-piperazin-1-yl-butyric acid methyl ester (0.095 g, 0.48 mmol) in 66% yield. 4-oxo-4-piperazin-1-yl-butyric acid methyl ester was dissolved in tetrahydrofuran (hereinafter referred to THF) (10 mL), and subsequently added triethylamine (0.08 mL, 0.57 mmol) and 1-bromo-3-chloropropane (0.057 mL, 0.57 mmol). The solution was stirred at room temperature overnight. The reaction mixture was quenched by adding water. The resulting solution was extracted with ethyl acetate, dried over MgSO4, and evaporated to give a residue purified by column chromatography (Al2O3), eluting by ethyl acetate to provide Compound-3 (0.024 g, 0.087 mmol) in 18% yield.
1H NMR (400 MHz, CDCl3, δ): 3.67 (s, 3H), 3.60-3.57 (t, J=6.3 Hz, 4H), 3.48-3.45 (t, J=5.1 Hz, 2H), 2.66-2.58 (m, 4H), 2.49-2.46 (t, J=7.1 Hz, 2H), 2.43-2.41 (t, J=5.0 Hz, 2H), 2.39-2.36 (t. J=5.1 Hz, 2H), 1.95-1.88 (m, 2H); 1C NMR (100.6 MHz, CDCl3, δ): 173.6, 169.5, 55.1, 53.3, 52.8, 51.8, 45.2, 43.0, 41.7, 29.7, 29.0, 27.9; IR (KBr): 2950, 2814, 1736, 1647, 1438, 1369, 1227, 1168 cm−1; MS m/z: 276.1 (M+, 8.6), 245.1 (28.2), 213.1 (100.0), 132.1 (15.4); HRMS-EI (m/z): [M]+ calculated for C12H21ClN2O3, 276.1241; found, 276.1242.
A mixture of 6-(benzyloxy)-N-(3-chloro-4-fluorophenyl)-7-methoxyquinazolin-4-amine (0.15 g, 0.37 mmol) and 10% Pd/C (25 mg, 10 wt %) in methanol (20 mL) in a Pan glass vessel and carefully flushed three times with hydrogen gas. The vessel was finally charged with hydrogen gas (60 psi) and shaken mechanically for 24 hours. After completion of the reaction, the reaction mixture was filtered through a pad of celite and washed with excess methanol. The filtrate was concentrated under reduced pressure to obtain Compound-4 (96 mg, 0.3 mmol) in 82% yield.
1-(3-chloropropyl)-4-(t-butyloxycarbonyl)-piperaxine (4.0 g, 15.2 mmol) was treated with hydrochloric acid in ethyl acetate to obtain Compound-5 (3.17 g, 13.5 mmol) in yield 89%.
1H NMR (400 MHz, D2O, δ): 3.63-3.54 (m, 9H), 3.39 (s, 1H), 3.40-3.35 (m, 2H), 2.21-2.14 (m, 2H). 13C NMR (100.6 MHz, D2O, δ): 54.8, 48.6, 41.1, 40.7, 26.2. IR (KBr): 3356, 3001, 1443, 1301, 1160, 1084 cm−1 MS nil: 162.1 (M+, 13.6), 120.1 (100.0). 99.1 (79.5), 70.1 (29.2), 56.1 (49.6). HRMS-EI (m/z): [M]+ calculated for, C7H15ClN2, 162.0924; found, 162.0930.
A mixture of succinic anhydride (1.0 eq) and dry alcohol (carbon number is from 2 to 20) (1 eq) in 4 mL toluene was heated at reflux 2.5 hours. The toluene was removed under reduced pressure and the residue was taken up in water, and the solution was extracted with dichloromethane, dried over MgSO4, and evaporated to obtain monoalkylsuccinic acid (carbon number is from 2 to 20).
A solution of monoalkyl succinic acid (carbon number of the alhyl is from 2 to 20) (1.2 eq) and thionyl chloride (1.4 eq) in benzene (5 mL) was refluxed for 3 hours. Subsequently, the majority of the thionyl chloride and benzene were removed by distillation. The mixture was cooled down to room temperature and dried under a vacuum to give a crude chlorocarbony-alkyl ester (carbon number of the alhyl is from 2 to 20). A solution of chlorocarbonyl-alkyl ester (carbon number of the alhyl is from 2 to 20) in 5 mL dichloromethane was added to a round flask containing Compound-5 (1 eq) by cannula, and subsequently added pyridine (1.4 eq). The resulting solution was stirred at room temperature overnight, and quenched by adding water. The solution was extracted with ethyl acetate, dried over MgSO4, and evaporated to give a residue which was purified by column chromatography (Al2O3), eluting by ethyl acetate/hexane (1:15) to provide Compound-21:
1H NMR (400 MHz, CDCl3, δ): 4.07-4.03 (t, J=6.8 Hz, 2H), 3.60-3.57 (m, 4H), 3.48-3.46 (t, J=4.9 Hz, 2H), 2.65-2.57 (m, 4H), 2.50-2.46 (t, J=6.9 Hz, 2H), 2.44-2.37 (m, 4H), 1.95-1.92 (m, 2H), 1.63-1.57 (m, 2H), 1.34-1.23 (b, 18H), 0.88-0.85 (t, J=6.6 Hz, 3H); 13C NMR (100.6 MHz, CDCl3, δ): 173.2, 169.5, 64.8, 55.1, 53.2, 52.7, 45.2, 42.9, 41.7, 31.9, 29.7, 29.6, 29.5, 29.5, 29.3, 29.2, 29.2, 28.5, 27.8, 25.8, 22.6, 14.0; IR (KBr): 2925, 2854, 1734, 1651, 1444, 1365, 1223, 1169 cm-1. MS m/z: 430.3 (M+, 62.5), 281.1 (11.7), 228.0 (11.7), 221.1 (11.1), 207.1 (18.7), 139.4 (41.3), 60.3 (100.0). HRFAB (m/z): [M]+ calculated for C23H43ClN2O3, 430.2963; found, 430.2954.
Compound-4 (1 eq) was dissolved in DMF (1.0 mL), potassium carbonate (2 eq) and Compound-21 (1 eq) were added respectively and heated at 80° C. overnight. The reaction mixture was then cooled to room temperature and quenched by adding water. The resulting solution was extracted with ethyl acetate; the combined extracts were washed with water and brine, dried over MgSO4, and evaporated to give a crude residue which was purified by column chromatography to provide SP101:
1H NMR (400 MHz, CDCl3, δ): 8.65 (s, 1H), 7.86-7.84 (m, 1H), 7.54-7.51 (m, 2H), 7.24 (s, 1H), 7.17-7.13 (m, 2H), 4.18-4.15 (t, J=6.4 Hz, 2H), 4.07-4.03 (t, J=6.8 Hz, 2H), 3.98 (s, 3H), 3.61 (b, 2H), 3.48 (b, 2H), 2.65-2.56 (m, 6H), 2.48 (b, 2H), 2.44-2.42 (m, 2H), 2.11-2.08 (t, J=6.7 Hz, 2H), 1.61-1.58 (m, 2H), 1.32-1.25 (b, 18H), 0.89-0.85 (t, J=6.7 Hz, 3H). 13C NMR (100.6 MHz, CDCl3, δ): 173.3, 169.8, 156.3, 155.2, 153.5, 153.4, 149.0, 147.4, 135.5, 124.2, 121.8, 121.7, 121.0, 120.8, 116.6, 116.4, 109.0, 107.8, 101.2, 67.5, 64.9, 56.1, 54.7, 53.2, 52.7, 45.1, 41.6, 31.9, 29.6, 29.5, 29.3, 29.2, 28.6, 27.8, 26.2, 25.9, 22.7, 14.1. IR (KBr): 3672, 2925, 2854, 1732, 1625, 1578, 1500, 1470, 1429, 1217 cm-1. MS m/z: 714.3 (M+, 3.4), 551.4 (1.5), 71.7 (49.1), 52.8 (100). HRFAB (m/z): [M]+ calculated for C38H53ClFN5O5, 713.3719; found, 713.3717.
Materials—Antibodies and Cell Lines
Anti-EGFR, anti-p-EGFR (Tyr 1068), anti-cleaved Caspase 3 antibodies were purchased from Cell Signaling, Inc. Anti-Survivin antibody was purchased from Santa Cruz Biotechnology, Inc. Anti-actin antibody was purchased from Millipore. Anti-Nanog and anti-Oct4 antibodies were purchased from Abcam, Inc. Anti-GAPDH, goat anti-rabbit IgG horseradish peroxidase (HRP) and goat anti-mouse IgG horseradish peroxidase (HRP) antibodies were purchased from GeneTex Inc. Anti-rabbit IgG Dylight 488 antibody was purchased from Thermo, Forma. Scientific Inc.
A549 (ATCC number: CCL-185) was a non-small cell lung carcinoma cell line which expressed wild type EGFR and with K-Ras mutation (G12S). H1975 (ATCC number: CRL-5908) was a non-small cell lung carcinoma cell line which expressed EGFR L858R/T790M mutation. Oct4 and Nanog overexpression in A549-ON presented the properties of cancer stem-like cells, as well as epithelial-mesenchymal transdifferentiation. PLC26 was lung cancer cells that separated from pleural effusion of a clinical lung cancer patient (IRB number: 103-011-E). A549-ON cells were cultured in DMEM medium. A549 and H1975 cells were maintained in RPMI-1640 medium. PLC26 cells were maintained in LHC-9 medium. DMEM and RPMI medium were supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and sodium bicarbonate. LHC-9 medium was supplemented with 5% FBS and 100 units/ml penicillin. These cells were cultured at 37° C. and 5% CO2 in a humidified incubator.
III. SP101 is More Potent on the Inhibition of Cell Viability of NSCLC Cells with EGFR (T790M) Mutation
Please refer to Table 1, which indicates the genotype of EGFR and K-Ras in HFL1 normal lung fibroblast and various human lung cell lines, including A549, A549-ON, H358 and H1975.
Table 1. The Genotype of EGFR and K-Ras in HFL1 Normal Lung Fibroblast and Various Human Lung Cell Lines
Please refer to
Please refer to
IV. SP101 Inhibits Protein Level of Phosphorylated EGFR in NSCLC Cells with EGFR (T7901M) Mutation
EGFR binding cognate ligands may lead to autophosphorylation of receptor tyrosine kinase and subsequent activation of signal transduction pathways of cell survival. Besides, aberrant tyrosine kinase activation increases proliferation and drug resistance of malignant cells. Please refer to
V. SP101 Reduces Protein Level of Survivin and Increases Active Caspase 3 Protein in NSCLC Cells with EGFR (T790M) Mutation
Survivin protein has been suggested to involve in tumorigenesis through diverse mechanisms, including prevent caspase-mediated cell death and regulate cell cycle. Reduction of Survivin protein expression has shown to inhibit tumor growth in NSCLC. Please refer to
Further, immunofluorescence staining and confocal microscopy were utilized to determine the expression and cell morphology of Survivin and cleaved-form Caspase 3 after treatment with SP101 in H1975 cells with EGFR (T790M) mutation. The cells were treated with or without 30 μM of SP101 for 24 hours. At the end of treatment, the cells were fixed with 4% paraformaldehyde at 37□C for 1 hour. The fixed cells were incubated with rabbit anti-Survivin antibody or rabbit anti-cleaved caspase-3 antibody at 4□C overnight. Then the cells were stained with goat anti-rabbit IgG Dylight 488 at 37□C for 1 hour. Please refer to
VI. SP101 Enhances Inhibition of Tumor Growth in the Xenografted Human NSCLC Model with EGFR (T790M) Mutation
To compare the antitumor activity of SP101 and Gefitinib, the xenografted human
NSCLC tumors in nude mice were established. Seven-week-old nude mice were subcutaneously injected with 3×106 H1975 cells. Please refer to
VII. SP101 Reduces Survivin Protein and Increases Active Caspase 3 Protein in the Tumor Tissues of Xenografted Human NSCLC in the Nude Mice with EGFR (T790M) Mutation
Please refer to
Please refer to
These findings demonstrated that SP101 decreases Survivin protein but conversely increases active Caspase 3 protein for apoptosis induction to block tumor growth in NSCLC.
VIII. SP101 is More Potent on the Reduction of Cell Viability than Gefitinib in NSCLC Cells with K-Ras Mutation
Please refer to
IX. SP101 Inhibits Protein Level of Phosphorylated EGFR in NSCLC Cells with K-Ras Mutation and Induces Apoptosis
Please refer to
X. SP101 Inhibits Protein Levels of Oct4 Nanog and Survivin its NSCLC Cells with Cancerous Stemness
Oct4 and Nanog have been served as markers of tumorigenesis. Furthermore, Oct4 played a pivotal role in Gefitinib resistance in lung cancer stem cells. Oct4 was demonstrated that regulated Survivin protein expression in cancers. Please refer to
XI. SP101 is More Potent on Apoptosis Induction than Gefitinib in NSCLC Cells with Cancerous Stemness
We compared the apoptosis levels of SP101 and Gefitinib in cancer stem-like A549-ON cells by Annexin V and PI staining. Please refer to
XII. SP101 is More Effective on the Reduction of Cell Viability than Gefitinib in the PLC26 Lung Cancer Cells Separated from the Pleural Effusion of a Clinical Lung Cancer Patient
We have further investigated the effect of SP101 in PLC26 lung cancer cells separated from the pleural effusion of a clinical lung cancer patient. To compare the cell viability of SP101 and Gefitinib in PLC26 cells, the cells were treated with 0˜40 μM SP101 or Gefitinib for 24 hours, and analyzed by MTT assays. Please refer to
Number | Name | Date | Kind |
---|---|---|---|
5770599 | Gibson | Jun 1998 | A |
8501413 | Varmus et al. | Aug 2013 | B2 |
8546107 | Freeman et al. | Oct 2013 | B2 |
9512086 | Chao et al. | Dec 2016 | B2 |
20050272083 | Seshagiri | Dec 2005 | A1 |
20160068495 | Chao et al. | Mar 2016 | A1 |
Number | Date | Country |
---|---|---|
105503746 | Apr 2016 | CN |
201609672 | Mar 2016 | TW |
Entry |
---|
Yin et al. Bioorganic & Medicinal Chemistry Letters, 2014, vol. 24, pp. 5247-5250. |
Jui-I Chao, Ya-Ping Hsu, Kuang-Kai Liu, Chien-Jen Chang, Chinpiao Chen; SP101, A Novel Synthetic Compound Reduces the Drug Resistance of EGFR (T790M) and Cancerous Sternness in Non-Small Cell Lung Carcinoma; Experimental Biology 2016, San Diego Convention Center, Sails Pavilion, National Chiao Tung University, Taiwan, R.O.C.; Apr. 6, 2016. |