Thioaryl Naphthylmethanone Oxime Ether Analogs as Novel

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Thioaryl naphthylmethanone oxime ether analogs as novel anticancer agents Atul Kumar, Bandana Chakravarti, Tahseen Akhtar, Byanju Rai, Manisha Yadav, Jawed Akhtar Siddiqui, Shailendra Kumar Dhar Dwivedi, Ravi Thakur, Anup Kumar Singh, Abhishek Kumar Singh, Harish Kumar, Kainat Khan, Subhashis Pal, Srikanta Kumar Rath, Jawahar Lal, Rituraj Konwar, Arun Kumar Trivedi, Dipak Datta, Durga Prasad Mishra, Madan Madhav Godbole, Sabyasachi Sanyal, and Naibedya Chattopadhyay J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm500873e • Publication Date (Web): 08 Sep 2014 Downloaded from http://pubs.acs.org on September 9, 2014

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Journal of Medicinal Chemistry

Thioaryl naphthylmethanone oxime ether analogs as novel anticancer agents Bandana Chakravartib, Tahseen Akhtara,ByanjuRaia, Manisha Yadavc, Jawed Akhtar Siddiquib, Shailendra Kumar Dhar Dwivedib, Ravi Thakurb, Anup Kumar Singhc, Abhishek Kumar Singhc, Harish Kumarc, Kainat Khanb, Subhashis Palb, Srikanta Kumar Rathd, Jawahar Lale, Rituraj Konwarb, Arun Kumar Trivedic, Dipak Dattac, Durga Prasad Mishrab, Madan Madhav Godbolef, Sabyasachi Sanyalc, Naibedya Chattopadhyayb,*, Atul Kumara,* a

Medicinal and Process Chemistry Division; bEndocrinology Division; cBiochemistry Division; d

Toxicology Division; ePharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow 226031, India. fDepartment of Molecular medicine and

Biotechnology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India.

RECEIVED DATE

1

ACS Paragon Plus Environment


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 Corresponding authors: Atul Kumara: Tel.: +91-522-26 1241 1; fax: +91-522-2623 4050; email: [email protected] and Naibedya Chattopadhyayb Tel.: +91-522-26 4967 1; fax: +91522-2623 4050; email: [email protected] Abbreviations: MND 4-(methylthio)phenyl)(naphthalen-1-

TGF-β1 transforming growth factor b1

yl)methanone-O-2-(diethylamino)

MTA1

ethyl

oxime

Metastasis-associated

protein

MMP-9 Matrix metaloprotease-9

TK

Tyrosine kinase

ALDH1 Aldehyde dehydrogenase 1

GPCR

G-protein coupled receptor

EGF

Epidermal growth factor;

RTK

Receptor tyrosine kinase

IGF-1

Insulin-like growth factor-1

NRTK Non-receptor tyrosine kinase

INS

Insulin,

EGFR

Epidermal Growth Factor Receptor

PAI-1

Plasminogen activator inhibitor-1

Her2

Human EGF receptor 2

MCL-1 Myeloid cell leukemia-1

AML

Acute myelogenous leukemia

FAK

Focal adhesion kinase

CML

Chronic myelogenous leukemia

BCSC

Breast cancer stem cells

ABC

ATP binding cassette

PTX

Pertussis toxin

OH-TAM Hydroxy-tamoxifen

CTX

Cholera toxin

PTEN

1

Phosphatase and tensin homologue

VEGF-A

Vascular

endothelial

growth

factor-A

2


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Abstract Employing a rational design of Thioaryl naphthylmethanone oxime ether analogs containing functional properties of various anti-cancer drugs, a series of compounds were identified that displayed

potent

cytotoxicity

towards

various

cancer

cells,

out

of

which,

4-

(methylthio)phenyl)(naphthalen-1-yl)methanoneO-2-(diethylamino)ethyl oxime (MND) exhibited best safety profile. MND induced apoptosis, inhibited migration and invasion, strongly inhibited cancer stem cell population on a par with salinomycin, and demonstrated orally potent tumor regression in mouse MCF-7 xenografts. Mechanistic studies revealed that MND strongly abrogated EGF-induced proliferation, migration and tyrosine kinase (TK) signaling in breast cancer cells. However, MND failed to directly inhibit EGFR or other related receptor TKs in a cell-free system. Systematic investigation of putative target upstream of EGFR revealed that the biological effects of MND could be abrogated by pertussis toxin. Together, MND represents a new nonquinazoline potential drug candidate having a promising anti-proliferative activity with good safety index.

KEYWORDS: Thioaryl naphthylmethanone oxime ether analogs, Receptor tyrosine kinase; Epidermal Growth Factor Receptor; Cancer stem cell; Pertussis toxin; G protein-coupled receptor. 3



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Introduction Tyrosine kinases (TK) are important mediators of signal transduction and cellular communication and has been well studied pertaining to their roles in cancer1. TK inhibitors targeting both receptor and non-receptor tyrosine kinases (RTK and NRTK respectively) have been widely investigated and a number of such inhibitors have been approved for treatment against various cancers, expressing their target receptors1.

A detailed analysis of the published information regarding the efficacies and mechanisms of action of these kinase inhibitors, however, raised some interesting points that remain yet unexplained. Firstly, some of these inhibitors have been reported to inhibit cell proliferation and induce apoptosis in cells lacking their cognate targets, for example inhibitors of epidermal growth factor receptor (EGFR) and human EGF receptor 2 (Her2) including gefitinib (Iressa), lapatinib and erlotinib, induce apoptosis and differentiation in acute and chronic myelogenous leukemia (AML and CML respectively) cell lines and patient samples that are devoid of EGFR or Her2 expression2-5. Here, it is important to note that kinase interaction profile for lapatinib exhibits extreme specificity, where this compound inhibits only 4 out of 133 kinases tested 6. These findings, together with available kinase profiling data for gefitinib and erlotinib6 and the ATP binding cassette (ABC) transporter inhibitory activities of these drugs7-8 indicate that at least the anti-leukemic activities of these drugs could be kinase-independent. Secondly, despite 4


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exhibiting distinct kinase inhibitory profile, most of the kinase inhibitor drugs including, EGFR inhibitors that are named before, BCR-ABL inhibitors (Imatinib, Dasatinib and Nilotinib), FLT3 inhibitors (Sorafenib/BAY43-9006, Sunitinib/SU11248) and CDK inhibitors (Roscovitine and Flavopiridol) induce mitochondria-mediated apoptosis2, 9-17, however, the mechanism behind the mitochondrial damage and consequent rapid induction of apoptosis by these drugs that are expected to arrest cell growth remains elusive. Based on above observations we first asked if there exists a commonality in the structure of kinase inhibitors, which could be responsible for their apoptosis-inducing functions. Here we report the chemical synthesis of a new variety of nonquinazoline /nonpyrimidine compounds that were based on the commonality across some of the major chemotherapeutic pharmacophores in the area of TK inhibitors and detailed characterization of one of these compounds, referred here as MND, that displayed robust cancer cell-specific cytotoxicity. Figure 1

Rationale for design Structurally numerous TK inhibitors can be divided into having three major regions - “Base” (A), “Crown” (B) and a “Hinge” (C), with an aminoalkyl side chain as an additional arm. The base region that is mostly used in TKIs is aminoquinazoline (gefitinib, vandetanib, erlotinib, lapatinib and afatinib)18-22, aminopyrimidine (nilotinib, imatinib, bafetinib,dasatinib, taundutinib and saracatinib)23-28 or aminoquinoline (bosutinib)29 and aminopurines (roscovitine)30. We replaced the base moiety with naphthalene because a number of scaffolds with a naphthalene base such as resveratrol analog31, adamantyl phenyl naphthalenes32, furyl naphthalene derivatives33 and doramapimod34 exhibit promising anticancer activity. The crowns in all the 5



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TKIs are aryl moieties and in our design we kept the same aryl system intact. Structural analyses of all the TKIs revealed that the hinge region is highly conserved and all the active TKIs have amino functionality35-37. To explore if alterations of this conserved hinge region amino functionality could improve cytotoxic potency, we modified this region with oxime. It is important to note that a number of anticancer agents such as (hydroxyimino) naphthofuranones, methoxyphenyl oximes and Piperazinyl indenoquinolinone have oxime functionality as pharmacophores (Fig. 1)38-40. Together, the newly designed scaffold consists of three main components, i.e. naphthalene as nucleus, oxime and its alkyl amino derivatives as functional group in the hinge region and an aryl moiety constituting the crown. The attachment of substituted phenyl ring over naphthalene has been done at C-1 position of naphthalene ring, keeping in mind the attachments in gefitinib and vandetanib. The synthesized compounds together with intermediate oxime yielded a novel phenyl naphthalen-1-yl methanone oxime series that were then evaluated for their anti-proliferative and cytotoxic activities in various human cancer cells (Table 1). One such derivative, MND(4-(methylthio)phenyl)(naphthalen-1yl)methanone O-2-(diethylamino)ethyl oxime stood out in these screens (Table 1) and was selected as lead molecules for detailed mechanism of action.

Results Chemistry Ketones 3a-3b were synthesized via Friedel-Craft acylation of 1-napthoic acid 1 with thioanisole/anisole 2 according to the published procedure41. Ketone 3 on reaction with hydroxylamine hydrochloride yielded oxime 4. Intermediate 4 on Williamson O-alkylation with haloalkyl amines or dihaloalkanes afforded compound 5a-k and 6a-i. Compounds 7a-e and 8a-d 6


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were synthesized from 5j and 5k respectively on reaction with excess of corresponding amines. Oxime 4 on reaction with ethylbromoacetate gave 5l which on amidation in microwave yielded 5m. Subsequent treatment of compounds 5, 6 and 7 with organic acids in methanol lead to the formation of corresponding salts (Scheme 1). These compounds were tested against several cancer cell lines including MCF-7, MDA-MB231, DU-145, Ishikawa, and Hela. Scheme 1

Biological evaluation Antiproliferative effect of novel Thioaryl naphthylmethanone oxime ether analogs in cancer and non-cancer cells The synthesized compounds were first screened for their antiproliferative activity in various human endocrine cancer cell lines including MCF-7 (breast, ER positive), MDA-MB-231(breast, ER negative), DU-145 (prostate, androgen receptor positive), Ishikawa (endometrial adenocarcinoma, ER positive) and Hela (cervical cancer) using MTT assay. The EC50 values (effective concentration or drug concentration at which 50% inhibition of cell proliferation occurs) in various cell lines are presented in Table 1. Out of these, five compounds (5b, 5d, 5i, 7a and 8a) caused loss of viability of breast cancer cells at concentrations that were comparable to either Raloxifene or Hydroxy-tamoxifen (OH-TAM) and Gefitinib (Table 1). Table 1,

Because specific cytotoxicity towards the cancer cells without affecting normal cell growth is a key safety feature of cancer chemotherapy, assessments of growth inhibition by 5b, 5d, 5i, 7a and 8a were made in mammary epithelial cell line (MCF-10A). We next determined the safety 7



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indices [the ratio between the concentration of an agent causing cytotoxicity of normal breast cells and the concentration causing cytotoxicity of cancer cell lines; IC 50/EC50] of 5b, 5d, 5i, 7a, 8a, OH-TAM and gefitinib at 72 h. Table 2 show much greater safety index of 5d and 5i over OH-TAM and gefitinib in MCF-7 and MDA-MDA-231 cells. Out of the compounds tested, 5d exhibited much higher IC50 values in the normal cells compared to breast cancer cells (Table 2), and thus was selected as the lead compound (further abbreviated and addressed as MND henceforth). MND also displayed cytotoxicity in colon cancer cells with a much higher efficacy and greater safety index as compared to a human non-cancerous epithelial cell line (HEK-293) than the chemotherapeutic drug 5-Fluorouracil (5-FU) except in DLD1 cell line (Table 3). Table 2, 3

Effects of MND on reversibility of cell growth, apoptosis and cell cycle of breast cancer cells First we determined if the anti-proliferative effect of MND in MCF-7 and MDA-MB-231 was reversible. To this end, cells were first treated with different concentrations of MND for 24h and then were maintained in drug free medium for a further 72h, and as shown in Fig. 2a, at MND concentrations up to 5μM, the growth recovery was~50% in MCF-7 and ~20% in MDA-MB231, however, beyond 5μM, no growth recovery in either cell type was noted (Fig. 2a). This antiproliferative effect of MND was associated with apoptosis as determined by Annexin V/ PI staining followed by flow cytometric analysis (Fig. 2b). Consistent with the lower IC50 of MND for MDA-MB-231 in MTT assay, the apoptosis-inducing effect of MND was stronger in MDAMB-231 than in MCF-7 cells (Fig. 2b). Figure 2 8


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MND induced both caspase-8 and caspase-9 activities in MCF-7 and MDA-MB-231 cells (Fig. 2c), indicating that both extrinsic and intrinsic pathways of apoptosis are activated by MND. In addition, MND activated caspase-3 in MDA-MB-231 but not in MCF-7 cells (Fig.2c). Consistent with the increase in caspases, pan caspase inhibitor (z-VAD-FMK), caspase-8 inhibitor (z-IETD-FMK) and caspase 9 inhibitor (z-LEHD-FMK) protected against MNDinduced loss of viable cells (Fig.2d). MND activation of the mitochondrial death pathway was illustrated by MND-induced loss of mitochondrial membrane potential (indicating mitochondrial damage) (Fig. 2e) and increased in Bax-to-Bcl2 ratio in both MCF-7 and MDA-MB-231 cells (Fig. 2f). From these findings, it appears that treatment with MND leads to initiation of the both Type I and type II apoptotic programs via the disruption of mitochondrial function42.

Analysis of cell cycle phase distribution by flow cytometry revealed that MND caused greater cell accumulation in the G1 phase and decrease in the S phase (Fig.2g-i). Consistent with G1 arrest, MND decreased cyclin D1 and increased p27 protein levels (Fig. 2j, k).

Effects of MND on migration and invasion We next investigated the effect of MND on migration and invasion of MDA-MB-231 cells. As shown in Fig. 3a, MND caused a significant decrease (p50% increase in the percentage of TUNEL-positive cells in tumors from MND treated mice compared with that in tumors from control mice (Fig 4d). In addition, a significant reduction in the levels of PCNA (marker of proliferation) was observed in tumors from MND treated mice compared to control (Fig 4e). Consistent with increased apoptosis, we observed significantly elevated Bax-to-Bcl-2 ratio and down-regulation of cyclin D1 (corroborating our in vitro data indicating G1 arrest by MND) in the tumors of MND treated mice compared to control (Fig 4f).

Effect of MND on cancer stem cells Cancer stem cells (CSCs) are implicated in disease relapse and drug resistance47-48 and thus the ability to target this subpopulation could represent an extremely attractive property in an anticancer therapeutic. Since one of the properties of CSCs in anchorage-independent growth, leading to colony formation48, the effect of MND was first evaluated in a mammosphere formation assay and as demonstrated in Fig.5a MND strongly inhibited mammosphere formation in MDA-MB-231 cells.

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Since aldehyde dehydrogenase 1 (ALDH1) is an established CSC marker in breast cancer cells4950

, we also evaluated the effect of MND on ALDH1 +ve cells by flow cytometry and consistent

with its mammosphere inhibiting property MND remarkably reduced ALDH1 +ve MDA-MB231 cells (Fig. 5b). Figure 5

Cluster of differentiation 133 (also known as Prominin 1) is an established marker for colon cancer stem cells51. Since, MND exhibited cytotoxicity towards colon cancer cells (Supplementary Table 4), we tested if it reduced CSC subpopulation in colon cancer cells as well and as evident in Fig.5c, MND remarkably reduced CD133 +ve colon CSCs in DLD-1 cells. The efficacy of MND in these assays was comparable to an established CSC inhibitor, salinomycin52.

Effect of MND on EGFR signaling Given that MND was designed based on TKIs, to define its mechanism of action, first its ability to prevent enhancement of viability by a panel of tumorigenic growth factors/cytokines such as epidermal growth factor (EGF), VEGF, insulin like growth factor-1 (IGF-1), insulin (INS) and TGF-β were tested. In absence of MND, EGF, VEGF, IGF-1, increased viability of MCF-7 cells, whereas TGFinhibited it and INS had no significant effect (Fig. 6a). In MDA-MB-231 cells, EGF, VEGF, and TGF increased cell viability but IGF-1 and INS had no effect (Fig. 6a). Treatment with MND completely abolished the effect of EGF in both MCF-7 and MDA-MB231cells but it failed to affect cell viability induced by other growth factors/cytokines (Fig. 6a).

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We thus investigated if MND could inhibit TK activity of the EGF receptor (EGFR), since its over-expression and activation do correlate with neoplastic progression53-54. Treatment with increasing concentration of MND (0.1-1μM) inhibited EGF-induced phosphorylation of EGFR at different tyrosine residues; Tyr1068 at 0.5µM, Tyr1173 at 0.1µM and Tyr845 at 1µM (Fig.6b). MND also inhibited EGF-induced EGFR phosphorylation at Tyr1148, Tyr1045 and Tyr992 at 1µM, without affecting the level of total EGFR (Fig. 6c). Further, MND also inhibited EGF-stimulated phosphorylation of human EGFR receptor 2 (HER2) at 1M in MDA-MB-231cells (Fig. 6d) and its efficacy against p-EGFR and p-HER2 appeared to be higher than AG1478 and gefitinib (Fig. 6b, d). We also attempted to evaluate the effect of MND on phosphorylations of ErbB3 and ErbB4, however, we failed to detect these proteins inMDA-MB-231cells by ELISA (data not shown).

Consistent with its inhibition of EGFR phosphorylation, MND inhibited EGF-induced phosphorylation of ERK1/2, AKT, STAT-3 but had no effect on STAT-1 (Fig. 6e). MND also inhibited the basal level of p-ERK1/2, however, it failed to affect basal levels of p-AKT, pSTAT-1 and p-STAT-3 (Fig. 6e).

Effect of MND on EGF-induced migration and invasion Since EGF induction of breast cancer cell invasion and migration is well known55, we evaluated the effect of MND on EGF-induced invasion and migration ofMDA-MB-231cells using various assays. MND significantly inhibited EGF-induced invasion of MDA-MB-231 cells through matrigel (Fig. 6f). EGF is known to activate transcription factor ELK-1 and through it induces the expression of plasminogen activator inhibitor-1 (PAI-1) (a chemo attractant involved in 13



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breast cancer invasion and migration56) and myeloid cell leukemia-1 (MCL-1)57. The latter is an anti-apoptotic member of the BCL2 family, that has been linked to high tumor grade and poor patient survival57 and along with enhancing tumor survival is also known to influence cell migration, invasion and colony forming ability58-59. We therefore evaluated the ability of MND to influence these factors and MND at 1µM inhibited both basal and EGF- induced levels of pELK-1, Mcl-1 and PAI-1 in MDA-MB-231cells (Fig. 6g-h). Figure 6

Consistent with Figure 3, MND inhibited both basal and EGF-induced migration of MDA-MB231 cells (Fig. 6i). Focal adhesion kinase (FAK) activation has been found to be a key component of cellular migration, especially in context of growth factor-induced migration60-61, and MND inhibited both basal and EGF-induced FAK phosphorylation at Tyr397 (Fig. 6j). Since MND inhibited EGFR phosphorylation and relevant downstream signaling, we next assessed if MND is a direct inhibitor of EGFR or other related RTKs. However, cell free RTK assays revealed that MND had no significant effect on any of these kinases tested, indicating that the effect of MND on EGFR phosphorylation might result from modulation of factors acting upstream of EGFR (Fig. 6k).

Effect of MND on GPCR-mediated cell death GPCRs have been reported to influence EGFR signaling (reviewed in62-63), we therefore evaluated if MND could regulate primary GPCR signaling events. While MND enhanced cellular accumulation of cAMP both in absence or presence of adenylyl cyclase activator forskolin (Fig. 6l), it failed to influence cellular Ca2+ level (Fig. 6m). This enhancement in cAMP 14


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was not associated with inhibition of cellular phosphodiesterase activity (Fig. 6n). Finally to conclude that the effect of MND was indeed GPCR-dependent, we analyzed MND-induced cell death in presence of Gαi/o inhibitor, Pertussis toxin (PTX) and Gαs activator, Cholera toxin (CTX). As shown in Fig. 6o, PTX significantly blocked MND induced cell death but CTX failed to do so. Taken together, MND-induced cell death appears to result from modulation of a PTXsensitive GPCR, which in turn regulates the activity of EGFR and its downstream signaling events.

Discussion and conclusion We synthesized a series of phenyl naphthalen-1-yl methanone oxime analogs based on a rational design taking inputs from various scaffolds with anti-cancer effects. The series contains naphthalene as nucleus, oxime and its alkyl amino derivatives as a functional group and a substituted phenyl moiety. The aim was to create a novel pharmacophore against breast cancer and define its mechanism of action. The SAR of the series revealed a very distinct feature that appeared essential for the cytotoxic activity of compounds was S-alkyl substitutions in the aryl ring but no other substitutions viz., O-alkyl or OH in this ring. In addition, whereas both alkyl amine (5a) and alicyclic amines (5b) having two carbon alkyl chain had exhibited activities however, best activity was observed in aminoalkyl basic chain having N, N-diethylamino substitutions i.e. MND. The better safety profile of MND over 5b, 5i, 7a and 8a in normal cell line could also be attributed to the structural differences noted before, i.e. the amino alkyl linker at C1 position of naphthalene ring via a ketoxime hinge tie in MND as opposed to cyclic amine as amino alkyl linker in other compounds.

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4-anilinoquinazoline class of compounds that includes gefitinib, erlotinib and lapatinib targets the ATP binding site of EGFR to suppress TK activity of the receptor64. This class of compounds is clinically used in various cancers as reversible blockers of EGFR signaling. However, the contended specificity of their action could be disputed by the reports which showed induction of apoptosis and differentiation by a number of these compounds in AML and CML cells that do not express EGFR or Her2. Moreover, a variety of clinically used RTK and NRTK inhibitors exhibit mitochondrial damage and consequent induction of apoptosis while it’s still unclear as to how the inhibition of TKs and mitochondrial death programs are connected. Based on these observations highlighting a limited degree of specificity of RTK and NRTK compounds and yet exhibiting effective cancer chemotherapeutic activity, we embarked on synthesizing phenyl naphthyl methanone oxime, a novel scaffold taking cues from the major chemotherapeutic agents. MND, the compound with the best combination of strong cytotoxicity to a variety of cancer (breast, prostate and endometrial) cells but least so in case of normal cells, induced cancer cell apoptosis via mitochondrial damage in concordance with reports from other groups showing that gefitinib or erlotinib induce cell cycle arrest and apoptosis in cancer cells65. Proof-of-concept of anti-cancer activity of MND in vivo has been obtained by daily oral administration of this compound which resulted in substantial regression in the xenograft tumor volumes in mice. MND dose (16mg/kg) is less than the reported effective dose of gefitinib (50-200mg/kg/day) in similar preclinical chemotherapeutic model66-67, thus suggesting that MND possesses substantial anti-tumor efficacy in vivo.

CSCs represent a subset of cancer cells that possess normal stem cell-like properties, that is, selfrenewal and generation of diverse cells comprising a tumor through differentiation. CSCs are 16


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refractory to conventional cancer chemotherapeutic agents and are often responsible for drugresistance and disease relapse in a number of cancers, including breast and colon cancers. Salinomycin, initially used as a veterinary anti-protozoal agent, was identified as an anti-breast cancer CSC agent by Gupta et al, in a large screen of 16000 compounds for their anti-CSC activities52. However, salinomycin displays neural and reproductive toxicity in mice68-69 and therefore it is difficult to envisage its future as an anti-CSC agent against human malignancies. Intense search thus for new compounds with robust anti-CSC activities are currently the focus of cancer research. Given that MND inhibited growth and expansion of CSC subpopulation not only in breast but also in colon cancer cells as compared with salinomycin therefore is exciting indeed.

Given that MND was synthesized based on the commonality of TKI structures, our first attempt to identify its mechanism of action was to evaluate its potential to abrogate the downstream signaling of TKIs. Since MND action was blocked by administration of EGF, first the EGFR phosphorylation status was determined in presence of MND. MND indeed inhibited p-EGFR at all seven Tyr residues phosphorylated by EGF more potently than AG1478 and also inhibited EGF-induced p-HER2 more potently than gefitinib. Consistent with the abrogation of EGFR signaling by MND, post-receptor signaling elicited by EGF that are associated with proliferation, survival and migration of cancer cells such as phosphorylation of ERK, AKT, STAT-1, STAT-3 and Elk were suppressed by MND. Furthermore, EGF-induced invasion and migration of cancer cells were both suppressed by MND as well as inhibition of p-FAK and PAI-1 secretion, suggesting that MND could mitigate EGF-induced cancer metastasis.

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Interestingly, however, although MND activated EFGR signaling in breast cancer cells, it failed to affect the kinase activities of EGFR or any of the RTKs tested in a cell free RTK profiling which suggested that the effect of MND on EGFR phosphorylation might result from modulation of factors acting upstream of EGFR. GPCRs have been shown to modulate activities of EGFR or other TKs and we thus evaluated if MND could modulate GPCR-related signaling pathways. MND robustly induced accumulation of cellular cAMP, but had little effect on cellular Ca2+ levels. MND-induced cellular cAMP level was not associated with inhibition of phosphodiesterases as MND failed to inhibit any of the 11 phosphodiesterases tested, thus indicating that its action might be routed through a GPCR. We thus evaluated MND-mediated loss of cell survival in presence of CTX and PTX, that are modulators for Gs and Gi/0 respectively and MND action was significantly blocked by PTX but not CTX indicating that MND action could indeed by mediated through modulation of a PTX-sensitive GPCR70.

In summary, the present study provides the proof-of-concept in preclinical cellular, molecular and animal settings toward MND being a low-molecular mass and orally potent anti-cancer agent. MND also has a great potential for further preclinical evaluation of its therapeutic efficacy in drug-resistant forms of breast cancers as well as other types of cancers. Finally, MND could contribute to an enhanced understanding of structure-based drug design to facilitate drug discovery and development of thioaryl naphthylmethanone oxime as GPCR inhibitors and nontoxic cancer chemotherapeutic agents. However, detailed mechanistic studies including identification of its GPCR target is necessary before it can be taken to the clinics.

Experimental section 18


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General methods: Chemistry All the chemical reagents were purchased from Sigma–Aldrich Chemical Company and were used directly without further any purification. NMR spectra were recorded on a Bruker DRX 200 MHz, 300 MHz or 400 MHz spectrometer. Chemical shifts (δ) are given in ppm relative to tetramethylsilane (TMS, δ=0.0 ppm) as internal reference. Coupling constants (J) are given in hertz (Hz). Mass spectrometry was performed by fast atom bombardment (FAB) or electrospray ionization (ESI) using JEOL SX-102 instrument. IR spectra were taken on a VARIAN FT-IR spectrometer as KBr pellets or neat. Elemental analysis was performed at a Perkin Elmer Autosystem XL Analyzer. Melting points were measured on a COMPLAB melting point apparatus and all the melting points were uncorrected. Unless otherwise mentioned all the analytical and spectral data (1H NMR, 13CNMR, Mass, IR elemental analysis and melting points) are given for the free base of the compounds. All the Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25 mm silica gel plates visualized with UV light. The purification of compounds was carried out by column chromatography using silica gel (100-200 mesh) or basic alumina and ethyl acetate/hexane or Methanol/Chloroform as eluent as per requirement. Microwave irradiation was carried out with Initiator 2.5 Microwave Synthesizers from Biotage. All the reactions were performed in special 5 mL glass vessels under an atmosphere of nitrogen. The temperature was fixed and maintained to 120 °C. The purity of all the compounds was determined by using reversed phase HPLC method on a Discovery HS C-18 (Supelco, Bellefonte, USA) column (5 μm, 100 x 4.6 mm id) preceded with a C-18 guard column (5 µm, 20 x 4.0 mm, id) packed with the same material under isocratic condition at ambient temperature using mobile phase [acetonitrile:methanol:phosphate buffer (10 mM, pH 3.5), 30:35:35, v/v] at a flow rate of 0.6 mL/min. The HPLC system consisted of a pump (LC-10AT 19



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VP with FCV-10AL VP), degasser (DGU-14A) and auto-injector (SIL-HTc, fixed with a 100 μl loop) (Shimadzu, Japan). Eluents were monitored at 294 nm with UV-Vis multiple wavelength detector and chromatograms were integrated using Class-VP (version 6.12 SP5) software (Shimadzu, Japan). The Purity of the tested compounds was reported ≥95% on the basis of HPLC analysis. Compound synthesis and characterization: The complete Synthetic procedures and compound characterization details for all the synthesized compounds are given in supplementary information.

Chemicals and reagents: biology Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA). Unless otherwise mentioned, all the chemicals used in the present study were from Sigma–Aldrich, USA. All inhibitors/antagonist used in this study were from Tocris Biosciences (Ellisville, MO) unless otherwise indicated. HTRF cAMP femto kit was from Cisbio bioassays (Parc Marcel Boiteux, France).

Antibodies and ELISA kits: Bcl-2 (sc-492), Bax (sc-493), PCNA (sc-56) and mouse β-Actin (sc-47778) antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). p27 (ab7961) and cyclin D1 (ab16663) were from Abcam. p-ERK (4370), ERK (9102), p-AKT (4060), AKT (9272), p-STAT1 (9171), STAT1 (9172), p-STAT3 (9131), STAT3 (9132), p-ELK1 (9181), ELK1 (9182), p-FAK (3283), FAK (3285), Mcl-1 (5453), p-EGFRTyr1086 (2220), p-EGFRTyr1148 (4404), p-EGFRTyr1045 (2237), p-EGFRTyr992 (2235), Phospho-HER4/ErbB4Tyr984 (3790), HER4/EbbB4 (4795) and rabbit β-Actin (4970) antibodies were from Cell Signaling Technology 20


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(Boston, MA). Phospho-EGF ReceptorTyr1068 (7240), Phospho-EGF ReceptorTyr1173 (7187), Phospho-EGF ReceptorTyr845 (7189), Total EGF Receptor (7250), phospho-HER2/ErbB2panTyr (7968), total HER2/ErbB2 (7310), Phospho-HER3/ErbB3panTyr (7890), and total HER3/ErbB3 (7888) Sandwich ELISA kits were from Cell Signaling Technology (Boston, MA). Human Plasminogen activator inhibitor-1 (PAI-1) ELISA kit (KHC3071) was from Invitrogen. CDC42 G-LISA kit (BK127) was from Cytoskeleton, Denver, CO, USA.

Cells and cell culture condition: MCF-7, MDA-MB-231(breast cancer cell lines), DU-145 (prostate cancer cell line), Ishikawa (endometrial cancer cell line), MCF-10A (non-tumorigenic mammary epithelial cell line), and colon cancer cells (SW620, DLD1, HCT116, and Colo205) were obtained from ATCC, USA and grown according to the manufacturer’s recommendations.

MTT Cytotoxicity Assay: Cells (1×104 cells/well) were seeded in 96-well plates in DMEM medium containing 10% FBS and allowed to adhere overnight and synchronized by serum starvation for 4 h. Cells were then treated with different doses of compounds (4a, 5a-m, 6a-i, 7ae and 8a-d), various growth factors (EGF - 100ng/ml, VEGF- 10ng/ml, IGF-1 - 10ng/ml, insulin – 10ng/ml, TGF-β – 5ng/ml) and caspase inhibitors (pan-capase inhibitor Z-VAD-FMK-10µM, caspase-8 inhibitor Z-IETD-FMK-10µM and caspase-9 inhibitor Z-LEHD-FMK-10µM ) or vehicle (0.1% DMSO] as control for different time period, as indicated in result section. For caspase inhibition assay, cells were pre-incubated with zVAD-FMK (pan caspase inhibitor), zIETD-FMK (caspase-8 inhibitor) and z-LEHD-FMK (caspase-9 inhibitor) for 2 h. After 2h cells were treated with 5μM MND for additional 24 h and cell viability was determined71.

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To study the reversibility of the anti-proliferative effect of MND, cells were pre-incubated in 96well plates to approximately 70% of confluence and exposed to MND for 24 h, after which they were allowed to recover for 72 h in drug free medium and cell viability was determined by MTT assay.

Apoptosis detection by flow cytometry: MCF-7 and MDA-MB231 cells (1×106) were seeded in 6 well plates and allowed to grow overnight. The medium was then, replaced with fresh complete medium containing 2.5μM, 5μM and 7.5μM final concentration of compound MND for 24 h or with vehicle (0.001% DMSO) for control. Annexin V-FITC apoptosis detection kit was used to detect early and late apoptosis by flow cytometry71.

Cell cycle analysis: MCF-7 and MDA-MB-231cells were treated with compound MND, at the end of the incubation adherent cells were trypsinized and combined with any floating cells present and then washed with cold PBS. Cells were fixed in 70% ethanol and incubated with RNase A and stained with 50μg/ml propidium iodide (PI) for 30 min before acquiring the flow cytometry reading (FACScan, BD Biosciences, USA)71.

Measurement of mitochondrial membrane potential (MMP): In brief, MCF-7 and MDAMB-231cells were treated with MND (5μM) for 24 h and harvested by trypsinization. Cells were incubated with 2 ml of medium containing JC-1(1μg/ml) for 15 min at 37°C. Stained cells were washed with PBS and subjected to flow cytometry analyses as per standard protocol using FL1, and FL2 channel71.

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qPCR: qPCR was performed for assessing the expression of metastatic gene in MND (1µM) treated MDA-MB-231 breast cancer cells, following our optimized protocol. The house keeping gene GAPDH was used as the internal control. Primers were designed using the Universal Probe Library (Roche Applied sciences) for different genes (Supplementary table S1). cDNA was synthesized with a RevertAid cDNA synthesis kit (Fermentas, Austin, USA) using 2.0μg of total RNA. SYBR green chemistry was used to perform quantitative determination of relative expression of transcripts for all genes. All genes were analyzed using the Light Cycler 480 (Roche Molecular Biochemicals, Indianapolis, Indiana, USA) real time PCR machine72.

Receptor Enzyme-linked immunosorbant assay (ELISA): MDA-MB-231 cells were seeded in 6 well plates and treated with MND at indicated conc. in Fig.5c, d for 1 h in serum free medium prior to stimulation with EGF for 15 min. After drug treatment, cells were washed with ice-cold PBS and lysed in ice-cold cell lysis buffer (20mM Tris pH 7.5, 150mM NaCl, 10mM EDTA, 1% w/v NP-40, 20mM sodium fluoride, 5mM sodium pyrophosphate, 1mM sodium vanadate, 10% v/v glycerol, 1× protease inhibitor cocktail). Protein concentration was determined using the Bradford method. Equal amount of cell lysates were used for determination of p-EGFR (EGFR phosphorylation at different tyrosine residues including pTyr1068, pTyr845, and pTyr1173), total EGFR, p-Her2 and total Her2 using ELISA kits according to manufacturer’s instruction. Values for receptor phosphorylation were determined by measuring absorbance at 450 nm using ELISA plate reader (μQuant, BioTek) and normalized with total EGFR and HER2 protein73.

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Plasminogen activator inhibitor-1 (PAI-1) and CDC42 ELISA: MDA-MB-231 cells were seeded in 6 well plates and treated with MND (1µM) for 24 h with or without EGF. After that conditioned medium was collected and PAI-1 protein concentration was determined using a PAI1 ELISA kit according to the manufacturer’s instructions. CDC42 protein level was determined in MND (1µM) treated MDA-MB-231 cells for 24 h using an ELISA kit according to the manufacturer’s protocols74.

Migration assay: MDA-MB-231cells were seeded in a six-well plate and grown overnight to confluence. The monolayers were scratched with a 200-µL pipette tip to create a wound, and then washed twice with serum-free DMEM to remove floating cells. Cells were treated with MND at 1μM in serum-free medium for 24 h with or without EGF (100ng/ml) and the wound was photographed 24 h later using light microscope in six random fields at 10× magnification. The rate of wound closure was assessed by measuring distances from six randomly selected fields using image analysis software (Image-Pro Plus 6.1.0)75.

Invasion assay: Transwell invasion assays were performed using BD BioCoat matrigel invasion chambers in 24-well tissue culture plates according to the manufacturer’s protocol (BD Biosciences). In brief, MCF-7 and MDA-MB-231cells (1x105cells/mL) were plated in medium containing 2% FBS to the upper chamber of an 8-µM-pore matrigel coated invasion chamber (BD Bioscience). In lower chamber, medium was supplemented with MND (1µM) and EGF (100ng/ml). After 24 h, cells that had migrated through the matrigel to the underside of the transwell filters of an 8-µm pore size membrane were fixed, stained (crystal violet stain), and counted under a light microscope in six random fields at 20× magnification. Stained cells were 24


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quantified by measuring the absorbance of dissolved crystals in 0.1% SDS at 540nm. All assays were done in triplicate chambers76.

Mammosphere formation assay: Human breast epithelial single cells were plated in ultralow attachment plates (Corning Inc, Corning NY) at a density of 100,000 viable cells/ml in primary culture and 5,000 cells/ml in passages. Primary mammospheres were allowed to form for 7–10 days in serum-free mammary epithelial basal medium (MEBM) with MND and salinomycin. Primary mammospheres were centrifuged (1,000 rpm), dissociated with trypsin and used for serial passage. Sphere number was counted manually and verified by two independent observers blinded to treatment. Breast stem cells were identified using Aldefluor (Stem Cell Technologies, Newark, NJ) and antibodies against stem cell surface marker according to the method of Dontu et al 77.

Cyclic AMP measurement: Cellular cAMP was estimated using a homogenous time-resolved fluorescence (HTRF) - based assay kit from Cisbio International using manufacturer’s protocol. Briefly, MDA-MB-231 cells seeded onto 96 well plates were serum starved for 4h followed by treatment with 500μM 3- isobutyl-1-methylxanthine (IBMX) (Sigma Aldrich) for 30 min and the cells were then treated with the indicated compounds for additional 30 min. After completion of incubation with compounds, cells were lysed and the lysates were used for estimation of cAMP using a fluorimeter (Fluostar Omega, BMG Labtech; Ortenberg, Germany)78.

Intracellular Calcium measurement: Fluo-4 AM (Invitrogen) was used to determine changes in intracellular calcium upon stimulation with the test compounds. MDAMB-231 cells were 25



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trypsinized, washed with phosphate buffer saline (pH 7.4) followed by labeling with Fluo-4AM for 45 minutes in HBSS buffer. Cells were then washed and suspended in calcium free HBSS. A baseline flourescence was measured in a BD FACS Calibur instrument for 2 minutes followed by stimulation with MND for another 7 minutes and analyzed using Cell Quest Pro software78.

Western blot: Western blot was performed following previously described protocol and reprobed with actin71.

PDE profiling: MND’s ability to modulate phosphodiesterase isoforms were determined at SB Drug discovery (Glasgow, UK; http://www.sbdrugdiscovery.com/), using IMAP technology (except for PDE6AB), which is based on the high affinity binding of phosphate by immobilized metal coordination complexes on nanoparticles. The binding reagent complexes with phosphate groups on nucleotide monophosphate generated from cyclic nucleotides (cAMP/cGMP) through phosphodiesterases. With fluorescence polarisation detection, binding causes a change in the rate of the molecular motion of the phosphate bearing molecule, and results in an increase in the fluorescence polarization value observed for the fluorescent label attached to the substrate. For PDE6AB, radiometric assay was used, which is a modification of the two-step method of Thompson and Appleman79.

Standards used for each isoforms are; PDE1A3; 10µM IBMX, PDE2A3; 1nM BAY 60-7550, PDE3CAT; 1µM Cilostazol, PDE4CAT; 10µm Rolipram, PDE5CAT and PDE6AB; 0.1µM and 1µM Sildenafil respectively, PDE7A; 10mM BRL-50481, PDE8A1; 10µM BRL-50481, PDE9A1; 1µM SB 36216, PDE10A1; 1µM Papaverine, PDE11A1; 1µm Dipyridamole. 26


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Cell-free Kinase assays: MND’s ability to modulate EGFR or related kinases was assessed at Millipore (Kinase profiling services; Dundee, UK) using standard -33P-ATP-based in vitro kinase assay, where ATP concentration was kept within 15 μM of the apparent Km for ATP where determined80.

Development of MCF-7 xenograft tumor, morphological analyses and TUNEL staining: MCF-7 cells (5x106 cells in 100μl PBS) were injected into the peritoneal cavity of NIH-III strain of nude mice. After induction of tumor, two groups were recruited for each compound out of which one group (n=10) for control (only vehicle); and other group (n=10, each) for MND (8 and 16 mg.kg-1.day1). Animals were treated p.o. for 29 days with above mentioned doses in 1% carboxymethyl cellulose as vehicle. Tumor volumes were measured using vernier calipers every third day. Assuming that tumors formed in the animals were spherical, their volume was calculated using the formula [π/6×d3], where d is the mean diameter. Tumor samples from each animal were removed and fixed in 4% buffered formalin phosphate, sectioned to 5μm thickness, and stained with hematoxylin and eosin (H/E)71. Animal studies were approved by Institutional Animal Ethics Committee (IAEC) of Advanced Center for Treatment, Research & Education in Cancer (ACTREC), Navi Mumbai, India. This facility is accredited to AALAC and the registration number of ACTREC is 65/1999 granted by Committee for the Purpose of Control and Supervision of Experiments on Animals, Ministry of Environment and Forest, Government of India.

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Apoptotic DNA degradation in tumor tissue was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) method. In this study, In Situ Cell Death Detection Kit, Fluorescein (Roche) was used for this purpose. Tumor sections were observed under fluorescence microscope (Nikon 80i, Japan) at 40×, and images were captured digitally with NIS Elements F 3.0 camera (Nikon, Japan). Quantification of TUNEL positive cells was done using image analysis software (Image-Pro Plus 6.1.0)71.

Statistical analysis: Data are expressed as mean ± SEM with at least three independent experiments. The data were analyzed using one-way ANOVA followed by post hoc NewmanKeuls multiple comparison test of significance or unpaired, two-tailed student’s t-test, as appropriate. Qualitative observations have been represented following assessments made by three individuals blinded to the experimental designs.

Acknowledgements: Authors acknowledge funding received from CSIR network projects and DST (BSC0201 to NC; BSC0103 to SRK, AKT, DPM and SS, BSC0101 to RK, SB/FT/LS368/2012 to BC and BSC0108 to AK).

No author has Competing Financial Interest.

Supporting Information: The supporting information is available free of charge at http://pubs.acs.org and includes synthetic scheme, spectral data (1H-NMR and

13

C-NMR) for

characterization of compounds, and biological results.

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novel

analogue

of

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33



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FIGURE CAPTIONS: Fig 1.Designing strategy of Thioaryl naphthylmethanone oxime ether analogs The Base (A) of the designed pharmacophore is taken from natural product resveratrol, whereas the crown (B) part is kept as aryl system as in most TKIs. Major portion is contributed by the hinge (C) which is based on novel oxime and their corresponding ether alkyl amine. Fig 2.MND irreversibly inhibits cell growth, induces apoptosis and cell cycle arrest of MCF-7 and MDA-MB-231cells a. Cells were exposed to MND for 24 h (MCF-7-24h, MDA-MB-231-24h) after which they were allowed to recover for 72 h in drug free medium (MCF-7-W, MDA-MB-231-W), and cell viability was determined by MTT assay. b. Apoptosis was assessed at indicated concentration by flow cytometry using the FL1-H channel (Annexin-V) and FL2-H channel (PI) of a Becton Dickinson FACS Calibur. Shown are representative dot plots. c. Caspase-8, caspase-9 and caspase-3 activity was determined by Caspase-Glo® 8, 9 and 3 Assay kit. d. Cell viability was determined by MTT assay in presence of MND with or without caspase-inhibitors. e. Drops in 40


Page 41 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


membrane potential (ΔΨm) were assessed by JC-1 staining using flow cytometry analysis; f. Immunoblotting of Bax and Bcl-2 protein. Protein loading was controlled with an anti-actin. g. Cell cycle distribution was assessed by flow cytometry. Quantification of flow cytometry data has been shown as percent of cells in different phases; h. MCF-7 and i. MDA-MB-231. Cells were treated with MND (1μM), j.MCF-7 and k. MDA-MB-231 for the indicated length of time. Cell lysates were used for western blot to detect the protein levels of cyclin D1 and p27. Protein loading was controlled with an anti-actin. Data presented as mean ± SEM from three independent experiments; *P< 0.05,

**

P< 0.01, and

***

P< 0.001 compared to vehicle (N=3). As

indicated *a50

>50

>50

>50

11

5f

S

6

73

>50

>50

>50

>50

>50

12

5g

S

5

67

>50

>50

>50

>50

>50

13

5h

S

6

79

>50

>50

>50

>50

>50

14

5i

S

6

50

8

7.2

9.5

7

8

15

5j

S

8

69

Int.

Int.

Int.

Int.

Int.

16

5k

S

8

71

Int.

Int.

Int.

Int.

Int.

17

5l

S

6

65

30

35

>50

>50

18

18

5m

S

0.05c

54

28

30

>50

>50

25

19

6°

O 6

71

18

>50

>50

>50

15

20

6b

O 6

65

25

>50

>50

>50

20

21

6c

O 6

69

15

>50

>50

>50

23 46


Page 47 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


22

6d

O 6

59

20

>50

>50

>50

>50

23

6e

O 6

63

23

>50

>50

>50

>50

24

6f

O 6

51

>50

>50

>50

>50

>50

25

6g

O 6

63

25

30

35

>50

20.5

26

6h

O 4

79

20

25

>50

>50

>50

27

6i

O 6

66

20.5

>50

>50

>50

>50

28

6j

O 8

87

Int.

Int.

Int.

Int.

Int.

29

7°

S

6

91

5

20

15

12

6.5

30

7b

S

6

60

>50

>50

>50

>50

>50

31

7c

S

6

72

6.5

20

10

>50

>50

32

7d

S

6

68

>50

>50

>50

>50

8

33

7e

S

6

72

>50

>50

>50

>50

>50

34

8°

O 7

84

8

18

7.5

18

18

47



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

35

8b

36

8c

37

8d

38

Raloxifene

39 40

Page 48 of 57

O 7

69

>50

>50

>50

>50

>50

O 6

73

18

20

>50

>50

>50

O 7

72

>50

>50

>50

>50

10

-

-

-

84

23

30

>50

20

N.D.

OH-TAM

-

-

-

85

8.8

8

10

11.5

N.D.

Gefitinib

-

-

-

85

>50

50

N.D. N.D.

N.D.

N H

N

a

Reactions were conducted under anhydrous conditions, bisolated yields, cmicrowave assisted reaction 120 oC. The EC50 values of compounds were determined from dose response curve (doses 100nM-50µM) using GraphPad Prism 3.02 software. Compounds which were not tested listed as N.D. Abbreviations; N.D. (Not done), and Int. (Intermediate products).

48


Page 49 of 57

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Table 2 Safety indexes of compounds at 72h; EC50 and IC50 values are in μM Activity/Treatments

5b 5d 5i 7a 8a OH-TAM Gefitinib

MCF-7(EC50) MDA-MB231 (EC50)

MCF-10A (IC50)

5 3 1 5 8 7.4 35

5.5 50 30 10 10 10 30

3.8 1 5.6 20 18 7.8 25

Safety index IC50/EC50 MCF-7 1.1 16.6 30 2 1.2 13.5 0.857

MDA-MB231 1.4 50 5.3 0.5 0.5 1.28 1.2

Table 3 Safety indexes of compounds in colon cancer cell line, EC50 and IC50 values are in μM. Compound SW620 (EC50) MND 8.78 5-FU >50

DLD1 (EC50) 15.6 16.3

HCT116 (EC50) 7.7 >50

Colo205 (EC50) 4.2 38.06

Vero (IC50) >25 ND

NIH3T3 (IC50) >20 ND

Safety index (IC50/EC50) HEKSW620 DLD1 HCT116 Colo205 293(IC50) 20 2.2 1.2 2.5 4.7 48.7 0.97 2.98 0.97 1.2

49



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Page 50 of 57

Scheme 1: Chemical synthesis of Thioarylnaphthylmethanone oxime ether analogsa

a

Reagent and conditions: a) NH2OH.HCl, pyridine, ethanol, anhyd., reflux; b) haloalkylamines,

K2CO3, Acetone/DMF, anhyd., reflux; c) dihaloalkanes, K2CO3, Acetone/DMF, anhyd., reflux; d) Primary or secondary amines, MeOH/DMF, anhyd., reflux;e) organic acid, dry methanol, dry ether; f) Ethylbromoacetate, K2CO3, Acetone, anhyd., reflux; g) t-BuOK, diethylamine, microwave assisted reaction, 120 oC.

50


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 1

F H

O N

N

O

Gefitinib

H

Cl

N

N

O

N

F

NH

O

Afatinib

N O

HN

Cl

HN O EGFR inhibitors

O

O

Lapatinib

SO

F H N

Imatinib

N

N

N

O

N NC

RX

N O

N

O

Bosutinib

N

O N

O

N

N H

N N

HO

N

N

S

HN

O N H

H N

HN

Designed prototype

N

N

Scaffold -Naphthyl

N

Saracatinib O SRC

Cl

N

OH O OH

OMe O

N

N

resveratrol analogue

N

N

O O

O HN

OH

O

N

OH

FLT3/ CDK/ SRC Inhibitors

O

RO Piperazinyl indeno quinolinone

N H

N

Functional group Oxime

O N

N O

N BCR-ABL Inhibitors

(hydroxyimino) naphthofuranone

N

Roscovitine CDK

N

N

N H

N

N

N

O

Cl O

N

Dasatinib

N H3CO

Base Region A

N N H Bafetinib

N

N

N

CH3 N

F3C

Tandutinib FLT3

Hinge Region C

N N

NH Cl

O

O Crown Region B

HN

N

H N

N

N O

N

H N

Nilotinib

O NH

NH

N

F 3C

O O

Erlotinib

Cl

Cl

O

N Vandetanib

N

F

O

N N

O

N

O

N

Br

adamantyl phenyl naphthalene

HO

OH

OH

C2H5 O

O

N OH

methoxyphenyl oxime Cytotoxic scaffolds with oxime functionality

furyl naphthalene O

N OCH3

N

H N

H N O

O Dormapimod H3C COOCH3 Naphthalene scaffolds as cytotoxic agents

O N

Figure 1 Designing strategy of Thioaryl naphthylmethanone oxime ether analogs

51



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Page 52 of 57

Fig. 2

52


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Fig. 3

53



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Page 54 of 57

Fig. 4

54


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Fig. 5

55



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Page 56 of 57

Fig. 6

56


Page 57 of 57

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Table of Content Graphics Table of Content Graphics

57


Thioaryl Naphthylmethanone Oxime Ether Analogs as Novel

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