Bioorganic & Medicinal Chemistry Letters 24 (2014) 884–887
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs Xiangfeng Chen, Youguo Du, Huanliang Sun, Feidong Wang, Lingsheng Kong, Min Sun ⇑ Nanjing Haiguang Applied Chemistry Institute, Jiangsu Aosaikang Pharmaceutical Co. Ltd, Nanjing 211112, PR China
a r t i c l e
i n f o
Article history: Received 24 September 2013 Revised 16 December 2013 Accepted 19 December 2013 Available online 25 December 2013 Keywords: Tricyclic fused quinazolines Antitumor activity EGFR HER2
a b s t r a c t Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. Ó 2014 Published by Elsevier Ltd.
Lung cancer is the number one cause of cancer mortality in men globally, with an estimated 13% (1.6 million) of total cases and accounting for 18% (1.4 million) of total deaths worldwide in 2008.1 Although chemotherapy is the mainstay of cancer therapy, the use of available chemotherapeutics is often limited mainly due to undesirable side effects and a limited choice of available anticancer drugs. This clearly underlies the urgent need of developing novel chemotherapeutic agents with more potent antitumor activities.2 EGFR is a member of a family of closely related receptors, including EGFR (ErbB1), human epidermal growth factor receptor-2 (HER2)/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR is overexpressed in the majority of NSCLCs and its expression is inversely related to survival outcome.3 The two main signaling pathways activated by EGFR are the RAS/RAF/MEK/ERK pathway and the PI3K/AKT pathway, which lead to evasion of apoptosis (cell survival) and cell proliferation.4,5 Based on the critical role of the ErbB family of receptors in the growth and metastases of NSCLC and other human malignancies, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed as targeted antitumor agents.6 Currently gefitinib (Iressa™, AstraZeneca) and erlotinib (Tarceva™, Genentech) (Fig. 1), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with non–small cell lung cancer (NSCLC) in May 2003 and November 2004, respectively.7,8 Both are reversible competitive inhibitors at the adenosine triphosphate (ATP) binding site of the EGFR TK domain. ⇑ Corresponding author. Tel.: +86 25 51198557; fax: +86 25 52162777. E-mail address: [email protected] (M. Sun). 0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmcl.2013.12.079
Despite the benefits of reversible EGFR TKIs, the efficacy of these agents has been limited by the development of resistance in most, if not all, initially responsive patients, which leads to tumor progression and relapse after a median time of 12 months.9 Recently, second generation inhibitors, designed to address resistance, are currently under investigation in clinical trials. The two most advanced compounds are dacomitinib (PF-00299804, Pfizer) and afatinib (BIBW-2992, Boehringer–Ingelheim) (Fig. 1) which are currently approved and in phase III clinical trials.10 Both of these compounds are structurally very similar to gefitinib and erlotinib with the exception that they harbor Michael acceptors in the 6-position side chain of the quinazoline core. This leads to dacomitinib and afatinib to be irreversible inhibitors of EGFR.11 The results of several phase III clinical trials for dacomitinib and afatinib are expected to be completed in 2013. Moreover, many studies have been targeted at finding new structures based on quinazolines that are potent EGFR inhibitors.12–14 Based on erlotinib as the leading compound, we have devised and synthesized two series of novel tricyclic oxazine and oxazepine fused quinazolines through intramolecular cyclization which possessed a functional Michael acceptor group, with the aim of obtaining agents displaying more potent antitumor activities. The antitumor effect of all the newly synthesized compounds on the in vitro growth of five cell lines, namely human gastric carcinoma cell line NCI-N87 (HER2 overexpression), human epidermoid carcinoma cell line A431 (EGFR overexpression), human adenocarcinoma cell line NCI-H1975 (EGFR L858R/T790M mutation), human breast cancer cell line BT-474 (HER2 overexpression) and human adenocarcinoma cell line Calu-3(HER2 overexpression), was evaluated. Apparent growth inhibition was observed for most of the
X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
885
Figure 1. Selected 1st, and 2nd generation EGFR inhibitors for NSCLC.
compounds, with 1a–1h demonstrating more potent activities against all five cell lines as compared to gefitinib and erlotinib, respectively. Furthermore, all the synthesized compounds were assessed the in vitro inhibition of EGF-induced receptor autophosphorylation in the KB nasopharyngeal carcinoma cell line. Finally, compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. The synthetic route to tricyclic oxazine and oxazepine fused quinazolines (1a–1p) was illustrated in Scheme 1. Conversion of the fluoro group of 2 to the corresponding hydroxylethoxy (hydroxylpropoxy) compound 3. Treatment of 3 with phosphoryl chloride and thionyl chloride gave the dichloride 4. Coupling of 4 with 3-aminophenylacetylene 9 gave nitro compound 5. Reduction of 5 with iron/ammonium chloride gave the corresponding amine 6. Intramolecular cyclization of 6 with potassium carbonate gave 7. Acylation of 7 with the bromocrotonic acid chloride 10 generated from bromocrotonic acid and oxalyl chloride gave the bromocrotonamide 8, which was finally submitted to bromide displacement with different aliphatic amines to yield the corresponding target compounds 1a–1p. All the 16 newly synthesized erlotinib derivatives (1a–1p) were tested for cytoxicity15 toward cancer cell lines either with EGFR (A431) or HER2 (N87, BT474, Calu-3) overexpression. The data are summarized in Table 1. The tricyclic oxazine compounds
(1a–1h) demonstrated more remarkable inhibition activity against the four cell lines (IC50: 0.046–2.06 lM) compared with erlotinib (IC50: 0.75–>10 lM) and gefitinib (IC50: 0.36–1.00 lM). The activity of compound 1h16 (IC50: 0.046–0.24 lM) was found to be 3 to >210 fold and 1.5 to 22 fold more potent than erlotinib (IC50: 0.75–>10 lM) and gefitinib (IC50: 0.36–1.00 lM) anaginst the four cell lines, respectively. The in vitro sensitivity of NSCLC cell lines to gefitinib was most closely associated with the presence of activating mutations in EGFR. However, some EGFR mutations, such as T790M or exon 20 insertion mutations, were associated with gefitinib resistance in vitro and in vivo.17 Hence the efficacy of target compounds (1a–1p) to gefitinib and erlotinib in H1975 cell line (EGFR L858R/ T790M mutation) was compared (Table 1). The tricyclic oxazine compounds (1a–1h) demonstrated more remarkable inhibition activity against the H1975 cell line (IC50: 0.30–1.46 lM) compared with gefitinib (IC50: >10 lM) and erlotinib (IC50: 5.51 lM). The activity of compound 1h was found to be 33 fold and 18 fold more potent than gefitinib and erlotinib, respectively. To determine the potency of target compounds against EGFR autophosphorylation in intact cells, we performed ELISA tests18 with EGFR-specific antibodies and measured levels of receptor phosphorylation with increasing drug concentrations (Table 1). Compounds 1a–1h demonstrated potent cellular effects on EGFR
Scheme 1. Synthetic route for the preparation of the target compounds 1a1p. Reagents and conditions: (a) ethylene glycol, NaH, THF, 75 °C, 20 h, 99% of 3a; (a’) propylene glycol, NaH, THF, 75 °C, 20 h, 99% of 3b; (b) POCl3, SOCl2, 90 °C, 3 h, 83% of 4a and 4b; (c) 3-aminophenylacetylene (9), isopropanol, 50 °C, 2 h, 99% of 5a and 5b; (d) Fe/NH4Cl, DMF/H2O, 80 °C, 1 h, 80% of 6a and 76% of 6b; (e) K2CO3, KI, DMF, 110 °C, 24 h, 27% of 7a and 23% of 7b; (f) bromocrotonic acid chloride (10), Et3N, DCM, 35 °C, 24 h, 76% of 8a and 70% of 8b; (g) aliphatic amines, DMF, r.t, 1 h.
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X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
Table 1 Structure–activity relationships for compounds 1a1p O
R N O
Compd
1a
1i~1p
BT474
Calu-3
0.60
0.77
0.53
1.39
1.01
0.64
0.12
0.32
0.11
0.33
0.71
0.060
0.25
0.89
0.40
1.38
1.67
0.082
0.27
1.23
0.69
1.15
0.95
0.067
0.15
0.68
0.19
0.55
0.35
0.042
0.16
0.87
0.34
0.87
0.55
0.042
0.22
1.46
0.48
1.33
2.06
0.78
0.046
0.30
0.23
0.24
0.14
0.046
>10
>10
>10
>10
>10
>10
N
>10
>10
>10
>10
>10
>10
N
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.59
>10
>10
>10
>10
N
N
1e
N
1f N
N
N N
O N
1k N
1l
N
1m
EGFR (cellular) IC50 (lM)
A431
N
1d
1j
N N
O
H1975
N
1c
1i
N
N87 N
O
1b
1h
1a~1h
HN
N
N
Cytotoxicity in different cell lines (IC50lM)
R
1g
O
R
HN
1n
N
>10
1.74
1.82
>10
>10
>10
1o
N
>10
>10
2.16
>10
>10
>10
>10
1.86
1.75
>10
>10
>10
>10 1.00
5.51 >10
0.75 0.55
>10 0.36
0.93 0.99
0.019 0.025
1p erlotinib gefitinib
N
phosphorylation in line with the in vitro kinase results, and their potency was comparable to the reference compound erlotinib and gefitinib. Perhaps because of lacking cell permeability, all the tricyclic oxazepine compounds (1i–1p) did not show noticeable inhibition to all five cell lines and EGF-induced receptor autophosphorylation in the KB nasopharyngeal carcinoma cell line even at 10 lM. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity19 (Table 2). Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. We have synthesized two series of novel tricyclic oxazine and oxazepine fused quinazolines and tested for their antitumor activities on N87, A431, H1975, BT474 and Calu-3 cell lines. Compounds 1a–1h demonstrated more potent antitumor activities when compared with erlotinib and gefitinib. Moreover, compounds 1b–1f, 1h effectively inhibited EGFR autophosphorylation in KB cell and the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. From the structure-activity relationships we may conclude that compounds containing oxazine ring demon-
Table 2 In vitro enzyme inhibition (IC50) Compd
EGFR (lM)
HER2 (lM)
1a 1b 1c 1d 1e 1f 1g 1h erlotinib gefitinib
0.012 0.003 0.005 0.005 0.005 0.004 0.016 0.003 0.001 0.001
0.93 0.23 0.33 0.29 0.25 0.29 1.11 0.25 0.37 0.28
strated significantly better cytotoxic activities than those with oxazepine ring. The results further supported investigation of these target compounds in vivo for cancer therapy with amplifications of ErbB family members.
X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
Reference 1. Ou, S. H. Crit. Rev. Oncol. Hematol. 2012, 83, 407. 2. Sun, M.; Wu, X.; Chen, J.; Cai, J.; Cao, M.; Ji, M. Eur. J. Med. Chem. 2010, 45, 2299. 3. Brabender, J.; Danenberg, K. D.; Metzger, R.; Schneider, P. M.; Park, J.; Salonga, D.; Hölscher, A. H.; Danenberg, P. V. Clin. Cancer Res. 1850, 2001, 7. 4. Yarden, Y.; Pines, G. Nat. Rev. Cancer 2012, 12, 553. 5. Hynes, N. E.; Lane, H. A. Nat. Rev. Cancer 2005, 5, 341. 6. Govindan, R. Clin. Lung Cancer 2010, 11, 8. 7. Cohen, M. H.; Williams, G. A.; Sridhara, R.; Chen, G.; Pazdur, R. Oncologist 2003, 8, 303. 8. Cohen, M. H.; Johnson, J. R.; Chen, Y. F.; Sridhara, R.; Pazdur, R. Oncologist 2005, 10, 461. 9. Riely, G. J.; Politi, K. A.; Miller, V. A.; Pao, W. Clin. Cancer Res. 2006, 12, 7232. 10. Giroux, S. Bioorg. Med. Chem. Lett. 2013, 23, 394. 11. Barf, T.; Kaptein, A. J. Med. Chem. 2012, 55, 6243. 12. Wu, J.; Chen, W.; Xia, G.; Zhang, J.; Shao, J.; Tan, B.; Zhang, C.; Yu, W.; Weng, Q.; Liu, H.; Hu, M.; Deng, H.; Hao, Y.; Shen, J.; Yu, Y. ACS Med. Chem. Lett. 2013, 4, 974. 13. Li, D. D.; Fang, F.; Li, J. R.; Du, Q. R.; Sun, J.; Gong, H. B.; Zhu, H. L. Bioorg. Med. Chem. Lett. 2012, 22, 5870. 14. Hu, S.; Xie, G.; Zhang, D. X.; Davis, C.; Long, W.; Hu, Y.; Wang, F.; Kang, X.; Tan, F.; Ding, L.; Wang, Y. Bioorg. Med. Chem. Lett. 2012, 22, 6301. 15. Cytotoxicity assay: A standard MTT assay was used to measure cell growth. Tumor cell lines in corresponding growth media recommended by American Type Culture Collection (ATCC) with 10% fetal bovine serum were plated in 96well cell culture plates (5.0 103 cells/well), and allowed to adhere at 37 °C, 5% CO2 for overnight. Test compound was added, cells were incubated at 37 °C, 5% CO2 for 72 h. Cell growth medium was removed afterwards, MTT was added to each well, and incubation at 37 °C for 4 h. Absorbance was measured at 550 nm with correction at 690 nm. IC50 values were determined by nonlinear regression analysis of the concentration response data. 16. Analytic data of potent inhibitor 1h: 1H NMR (DMSO-d6, 500MHz) d(ppm): 9.62(s, 1H, Ar-H), 8.52(s, 1H, Ar-H), 8.50(br s, 1H, NH), 7.99(s, 1H, Ar-H), 7.87(d, J = 8.2Hz, 1H, Ar-H), 7.38(t, J = 7.9Hz, 1H, Ar-H), 7.19–7.21(m, 2H, Ar-H), 6.86(td, J = 5.8, 15.2Hz, 1H, CH=CH), 6.61(d, J = 15.2Hz, 1H, CH=CH), 4.43(t, J = 4.6Hz, 2H, CH2O), 4.17(s, 1H, ethynyl-H), 4.06–4.08(m, 2H, CH2N), 3.06(d, J = 4.8Hz, 2H, CH2N) , 2.12(s, 6H, 2CH3); 13C NMR (DMSO-d6, 125MHz) d(ppm): 163.45, 157.51, 154.72, 151.70, 149.20, 140.15, 134.98, 129.92, 129.30, 126.98, 126.88, 125.16, 122.94, 122.16, 118.58, 112.22, 109.62, 84.03, 80.99, 67.41, 57.22, 45.82, 42.33; MS [ESI]+: m/z: 414[M+H]+; HRMS [ESI]+: m/z 414.1918 [M+H]+ calcd for C24H23N5O2: found 414.1852. 17. Engelman, J. A.; Zejnullahu, K.; Gale, C. M.; Lifshits, E.; Gonzales, A. J.; Shimamura, T.; Zhao, F.; Vincent, P. W.; Naumov, G. N.; Bradner, J. E.; Althaus, I.
887
W.; Gandhi, L.; Shapiro, G. I.; Nelson, J. M.; Heymach, J. V.; Meyerson, M.; Wong, K. K.; Jänne, P. A. Cancer Res. 2007, 67, 11924. 18. EGFR-mediated intracellular tyrosine phosphorylation assay: KB cells stimulated with EGF were used to quantify inhibition of EGFR phosphorylation. Cell were seeded at 1 105 cells/ml in 100 ll grow medium per well in 96-well plates. After 4 h, the growth medium was replaced with 100 ll serum-depleted medium overnight. Test compound was diluted in 100% DMSO, added to cells at a final DMSO of 0.1% v/v, and incubated at 37 °C for 1 h. KB cells were stimulated with EGF and incubated for another 6 min. Un-stimulated cells were used as blank and untreated but stimulated cells as control. The medium was removed, the cells were washed once with PBS, and then lysed with 100 ll lysis buffer. The diluted cell lysates were thoroughly mixed, 100 ll supernatant was transferred to ELISA capture plate and incubated at 37 °C for 2 h. ELISA capture plates were prepared by coating 96-well plates with 100 ll/well of 0.2–0.4 lg/ml EGFR capture antibody overnight at 4 °C. After 2 h incubation, the plates were washed with PBST for 3 times, then incubated with anti-phosphotyrosine antibody (1:2000) at 37 °C for 1 h. The plates were washed again and then incubated with HRP goat antimouse IgG (1:4000) at 37 °C for 1 h. The plates were washed a final time and then incubated with TMB and evaluated. IC50 values were calculated as percent inhibition of control. 19. In vitro kinase assay: A Caliper motility shift assay was used to measure the potency of title compounds against HER2 and EGFR. Compounds were prepared in DMSO at 10 mM and serially diluted in 96-well plates to obtain 50 compound solution in 100% DMSO of 10 concentrations ranging from 10 lM to 1 nM. 100 lL of 100% DMSO was transferred for DMSO control. Staurosporine was used as the reference compound. Kinase reaction: Add kinase (HER2 or EGFR) into 1 kinase base buffer (EGFR: 50 mM HEPES, pH 7.5; 0.0015% Brij-35; 10 mM MgCl2; 2.5 mM DTT. HER2: 20 mM HEPES, pH 7.5; 0.01% Triton X-100, 5 mM MnCl2; 2 mM DTT) to prepare 2.5 enzyme solution. Transfer 5 lL of each 5 compound solution in 10% DMSO to the 384-well assay plate in duplicate. Transfer 10 lL of 2.5 enzyme solution to each well of the 384-well assay plate. Incubate at room temperature for 10 min. Transfer 10 lL of 2.5 peptide solution (prepared by adding FAM-labeled peptide and ATP into the 1 kinase base buffer) to each well of the 384-well assay plate. Incubate at 28 °C for 40 min. Add 25 lL of stop buffer (100 mM HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTA) to stop the reaction. Collect conversion data on Caliper EZ Reader II. Convert conversion values to inhibition values according to percent inhibition = (max-conversion)/(maxmin) 100%, in which ‘max’ stands for DMSO control, ‘min’ stands for low control. Fit the data in XLfit to obtain IC50 values. The equation used is: Y = Bottom + (Top-Bottom)/(1 + 10^((log IC50 X) HillSlope)).
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs Xiangfeng Chen, Youguo Du, Huanliang Sun, Feidong Wang, Lingsheng Kong, Min Sun ⇑ Nanjing Haiguang Applied Chemistry Institute, Jiangsu Aosaikang Pharmaceutical Co. Ltd, Nanjing 211112, PR China
a r t i c l e
i n f o
Article history: Received 24 September 2013 Revised 16 December 2013 Accepted 19 December 2013 Available online 25 December 2013 Keywords: Tricyclic fused quinazolines Antitumor activity EGFR HER2
a b s t r a c t Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. Ó 2014 Published by Elsevier Ltd.
Lung cancer is the number one cause of cancer mortality in men globally, with an estimated 13% (1.6 million) of total cases and accounting for 18% (1.4 million) of total deaths worldwide in 2008.1 Although chemotherapy is the mainstay of cancer therapy, the use of available chemotherapeutics is often limited mainly due to undesirable side effects and a limited choice of available anticancer drugs. This clearly underlies the urgent need of developing novel chemotherapeutic agents with more potent antitumor activities.2 EGFR is a member of a family of closely related receptors, including EGFR (ErbB1), human epidermal growth factor receptor-2 (HER2)/neu (ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR is overexpressed in the majority of NSCLCs and its expression is inversely related to survival outcome.3 The two main signaling pathways activated by EGFR are the RAS/RAF/MEK/ERK pathway and the PI3K/AKT pathway, which lead to evasion of apoptosis (cell survival) and cell proliferation.4,5 Based on the critical role of the ErbB family of receptors in the growth and metastases of NSCLC and other human malignancies, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been developed as targeted antitumor agents.6 Currently gefitinib (Iressa™, AstraZeneca) and erlotinib (Tarceva™, Genentech) (Fig. 1), were approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with non–small cell lung cancer (NSCLC) in May 2003 and November 2004, respectively.7,8 Both are reversible competitive inhibitors at the adenosine triphosphate (ATP) binding site of the EGFR TK domain. ⇑ Corresponding author. Tel.: +86 25 51198557; fax: +86 25 52162777. E-mail address: [email protected] (M. Sun). 0960-894X/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.bmcl.2013.12.079
Despite the benefits of reversible EGFR TKIs, the efficacy of these agents has been limited by the development of resistance in most, if not all, initially responsive patients, which leads to tumor progression and relapse after a median time of 12 months.9 Recently, second generation inhibitors, designed to address resistance, are currently under investigation in clinical trials. The two most advanced compounds are dacomitinib (PF-00299804, Pfizer) and afatinib (BIBW-2992, Boehringer–Ingelheim) (Fig. 1) which are currently approved and in phase III clinical trials.10 Both of these compounds are structurally very similar to gefitinib and erlotinib with the exception that they harbor Michael acceptors in the 6-position side chain of the quinazoline core. This leads to dacomitinib and afatinib to be irreversible inhibitors of EGFR.11 The results of several phase III clinical trials for dacomitinib and afatinib are expected to be completed in 2013. Moreover, many studies have been targeted at finding new structures based on quinazolines that are potent EGFR inhibitors.12–14 Based on erlotinib as the leading compound, we have devised and synthesized two series of novel tricyclic oxazine and oxazepine fused quinazolines through intramolecular cyclization which possessed a functional Michael acceptor group, with the aim of obtaining agents displaying more potent antitumor activities. The antitumor effect of all the newly synthesized compounds on the in vitro growth of five cell lines, namely human gastric carcinoma cell line NCI-N87 (HER2 overexpression), human epidermoid carcinoma cell line A431 (EGFR overexpression), human adenocarcinoma cell line NCI-H1975 (EGFR L858R/T790M mutation), human breast cancer cell line BT-474 (HER2 overexpression) and human adenocarcinoma cell line Calu-3(HER2 overexpression), was evaluated. Apparent growth inhibition was observed for most of the
X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
885
Figure 1. Selected 1st, and 2nd generation EGFR inhibitors for NSCLC.
compounds, with 1a–1h demonstrating more potent activities against all five cell lines as compared to gefitinib and erlotinib, respectively. Furthermore, all the synthesized compounds were assessed the in vitro inhibition of EGF-induced receptor autophosphorylation in the KB nasopharyngeal carcinoma cell line. Finally, compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. The synthetic route to tricyclic oxazine and oxazepine fused quinazolines (1a–1p) was illustrated in Scheme 1. Conversion of the fluoro group of 2 to the corresponding hydroxylethoxy (hydroxylpropoxy) compound 3. Treatment of 3 with phosphoryl chloride and thionyl chloride gave the dichloride 4. Coupling of 4 with 3-aminophenylacetylene 9 gave nitro compound 5. Reduction of 5 with iron/ammonium chloride gave the corresponding amine 6. Intramolecular cyclization of 6 with potassium carbonate gave 7. Acylation of 7 with the bromocrotonic acid chloride 10 generated from bromocrotonic acid and oxalyl chloride gave the bromocrotonamide 8, which was finally submitted to bromide displacement with different aliphatic amines to yield the corresponding target compounds 1a–1p. All the 16 newly synthesized erlotinib derivatives (1a–1p) were tested for cytoxicity15 toward cancer cell lines either with EGFR (A431) or HER2 (N87, BT474, Calu-3) overexpression. The data are summarized in Table 1. The tricyclic oxazine compounds
(1a–1h) demonstrated more remarkable inhibition activity against the four cell lines (IC50: 0.046–2.06 lM) compared with erlotinib (IC50: 0.75–>10 lM) and gefitinib (IC50: 0.36–1.00 lM). The activity of compound 1h16 (IC50: 0.046–0.24 lM) was found to be 3 to >210 fold and 1.5 to 22 fold more potent than erlotinib (IC50: 0.75–>10 lM) and gefitinib (IC50: 0.36–1.00 lM) anaginst the four cell lines, respectively. The in vitro sensitivity of NSCLC cell lines to gefitinib was most closely associated with the presence of activating mutations in EGFR. However, some EGFR mutations, such as T790M or exon 20 insertion mutations, were associated with gefitinib resistance in vitro and in vivo.17 Hence the efficacy of target compounds (1a–1p) to gefitinib and erlotinib in H1975 cell line (EGFR L858R/ T790M mutation) was compared (Table 1). The tricyclic oxazine compounds (1a–1h) demonstrated more remarkable inhibition activity against the H1975 cell line (IC50: 0.30–1.46 lM) compared with gefitinib (IC50: >10 lM) and erlotinib (IC50: 5.51 lM). The activity of compound 1h was found to be 33 fold and 18 fold more potent than gefitinib and erlotinib, respectively. To determine the potency of target compounds against EGFR autophosphorylation in intact cells, we performed ELISA tests18 with EGFR-specific antibodies and measured levels of receptor phosphorylation with increasing drug concentrations (Table 1). Compounds 1a–1h demonstrated potent cellular effects on EGFR
Scheme 1. Synthetic route for the preparation of the target compounds 1a1p. Reagents and conditions: (a) ethylene glycol, NaH, THF, 75 °C, 20 h, 99% of 3a; (a’) propylene glycol, NaH, THF, 75 °C, 20 h, 99% of 3b; (b) POCl3, SOCl2, 90 °C, 3 h, 83% of 4a and 4b; (c) 3-aminophenylacetylene (9), isopropanol, 50 °C, 2 h, 99% of 5a and 5b; (d) Fe/NH4Cl, DMF/H2O, 80 °C, 1 h, 80% of 6a and 76% of 6b; (e) K2CO3, KI, DMF, 110 °C, 24 h, 27% of 7a and 23% of 7b; (f) bromocrotonic acid chloride (10), Et3N, DCM, 35 °C, 24 h, 76% of 8a and 70% of 8b; (g) aliphatic amines, DMF, r.t, 1 h.
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X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
Table 1 Structure–activity relationships for compounds 1a1p O
R N O
Compd
1a
1i~1p
BT474
Calu-3
0.60
0.77
0.53
1.39
1.01
0.64
0.12
0.32
0.11
0.33
0.71
0.060
0.25
0.89
0.40
1.38
1.67
0.082
0.27
1.23
0.69
1.15
0.95
0.067
0.15
0.68
0.19
0.55
0.35
0.042
0.16
0.87
0.34
0.87
0.55
0.042
0.22
1.46
0.48
1.33
2.06
0.78
0.046
0.30
0.23
0.24
0.14
0.046
>10
>10
>10
>10
>10
>10
N
>10
>10
>10
>10
>10
>10
N
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.59
>10
>10
>10
>10
N
N
1e
N
1f N
N
N N
O N
1k N
1l
N
1m
EGFR (cellular) IC50 (lM)
A431
N
1d
1j
N N
O
H1975
N
1c
1i
N
N87 N
O
1b
1h
1a~1h
HN
N
N
Cytotoxicity in different cell lines (IC50lM)
R
1g
O
R
HN
1n
N
>10
1.74
1.82
>10
>10
>10
1o
N
>10
>10
2.16
>10
>10
>10
>10
1.86
1.75
>10
>10
>10
>10 1.00
5.51 >10
0.75 0.55
>10 0.36
0.93 0.99
0.019 0.025
1p erlotinib gefitinib
N
phosphorylation in line with the in vitro kinase results, and their potency was comparable to the reference compound erlotinib and gefitinib. Perhaps because of lacking cell permeability, all the tricyclic oxazepine compounds (1i–1p) did not show noticeable inhibition to all five cell lines and EGF-induced receptor autophosphorylation in the KB nasopharyngeal carcinoma cell line even at 10 lM. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity19 (Table 2). Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. We have synthesized two series of novel tricyclic oxazine and oxazepine fused quinazolines and tested for their antitumor activities on N87, A431, H1975, BT474 and Calu-3 cell lines. Compounds 1a–1h demonstrated more potent antitumor activities when compared with erlotinib and gefitinib. Moreover, compounds 1b–1f, 1h effectively inhibited EGFR autophosphorylation in KB cell and the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib. From the structure-activity relationships we may conclude that compounds containing oxazine ring demon-
Table 2 In vitro enzyme inhibition (IC50) Compd
EGFR (lM)
HER2 (lM)
1a 1b 1c 1d 1e 1f 1g 1h erlotinib gefitinib
0.012 0.003 0.005 0.005 0.005 0.004 0.016 0.003 0.001 0.001
0.93 0.23 0.33 0.29 0.25 0.29 1.11 0.25 0.37 0.28
strated significantly better cytotoxic activities than those with oxazepine ring. The results further supported investigation of these target compounds in vivo for cancer therapy with amplifications of ErbB family members.
X. Chen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 884–887
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887
W.; Gandhi, L.; Shapiro, G. I.; Nelson, J. M.; Heymach, J. V.; Meyerson, M.; Wong, K. K.; Jänne, P. A. Cancer Res. 2007, 67, 11924. 18. EGFR-mediated intracellular tyrosine phosphorylation assay: KB cells stimulated with EGF were used to quantify inhibition of EGFR phosphorylation. Cell were seeded at 1 105 cells/ml in 100 ll grow medium per well in 96-well plates. After 4 h, the growth medium was replaced with 100 ll serum-depleted medium overnight. Test compound was diluted in 100% DMSO, added to cells at a final DMSO of 0.1% v/v, and incubated at 37 °C for 1 h. KB cells were stimulated with EGF and incubated for another 6 min. Un-stimulated cells were used as blank and untreated but stimulated cells as control. The medium was removed, the cells were washed once with PBS, and then lysed with 100 ll lysis buffer. The diluted cell lysates were thoroughly mixed, 100 ll supernatant was transferred to ELISA capture plate and incubated at 37 °C for 2 h. ELISA capture plates were prepared by coating 96-well plates with 100 ll/well of 0.2–0.4 lg/ml EGFR capture antibody overnight at 4 °C. After 2 h incubation, the plates were washed with PBST for 3 times, then incubated with anti-phosphotyrosine antibody (1:2000) at 37 °C for 1 h. The plates were washed again and then incubated with HRP goat antimouse IgG (1:4000) at 37 °C for 1 h. The plates were washed a final time and then incubated with TMB and evaluated. IC50 values were calculated as percent inhibition of control. 19. In vitro kinase assay: A Caliper motility shift assay was used to measure the potency of title compounds against HER2 and EGFR. Compounds were prepared in DMSO at 10 mM and serially diluted in 96-well plates to obtain 50 compound solution in 100% DMSO of 10 concentrations ranging from 10 lM to 1 nM. 100 lL of 100% DMSO was transferred for DMSO control. Staurosporine was used as the reference compound. Kinase reaction: Add kinase (HER2 or EGFR) into 1 kinase base buffer (EGFR: 50 mM HEPES, pH 7.5; 0.0015% Brij-35; 10 mM MgCl2; 2.5 mM DTT. HER2: 20 mM HEPES, pH 7.5; 0.01% Triton X-100, 5 mM MnCl2; 2 mM DTT) to prepare 2.5 enzyme solution. Transfer 5 lL of each 5 compound solution in 10% DMSO to the 384-well assay plate in duplicate. Transfer 10 lL of 2.5 enzyme solution to each well of the 384-well assay plate. Incubate at room temperature for 10 min. Transfer 10 lL of 2.5 peptide solution (prepared by adding FAM-labeled peptide and ATP into the 1 kinase base buffer) to each well of the 384-well assay plate. Incubate at 28 °C for 40 min. Add 25 lL of stop buffer (100 mM HEPES, pH 7.5; 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTA) to stop the reaction. Collect conversion data on Caliper EZ Reader II. Convert conversion values to inhibition values according to percent inhibition = (max-conversion)/(maxmin) 100%, in which ‘max’ stands for DMSO control, ‘min’ stands for low control. Fit the data in XLfit to obtain IC50 values. The equation used is: Y = Bottom + (Top-Bottom)/(1 + 10^((log IC50 X) HillSlope)).
