retracted: synthesis and cytotoxic activity of 2-anilinopyridine-3-acrylamides as tubulin...

DOI: 10.1002/cmdc.201400036

Synthesis and Cytotoxic Activity of 2-Anilinopyridine-3-Acrylamides as Tubulin Polymerization InhibitorsAhmed Kamal,*[a, b] Md. Ashraf,[a] M. Naseer Ahmed Khan,[a] Vijaykumar D. Nimbarte,[b]

Shaikh Faazil,[a] N. V. Subba Reddy,[a] and Shaik Taj[a]

Introduction

Cancer is a class of diseases characterized by uncontrolled cellgrowth, which can spread to other parts of the body throughthe blood and lymph systems. Cancer is a major cause ofdeath throughout the world,and chemotherapy is one thetreatments for cancer. The ulti-mate goal of cancer chemother-apy is to produce a drug thatcan specifically destroy cancercells without having any signifi-cant effects on normal cells.

Tubulin, the major componentof microtubules, is well estab-lished as a molecular target forantitumor drugs that disrupt mi-tosis.[1] The microtubule-target-ing antimitotic drugs consist oftwo groups: microtubule-desta-bilizing and microtubule-stabiliz-ing agents. The destabilizingagents inhibit microtubule poly-merization when present at highconcentrations and bind in oneof two domains on tubulin: the

vinca and colchicine domains. The microtubule-stabilizingagents enhance microtubule polymerization at high drug con-centrations and bind to the same, or an overlapping, taxoid

binding site on b-tubulin, which is located on the inside sur-face of the microtubule.[2] Many natural products such as com-bretastatin A-4 (CA-4, 1, Figure 1)[3] as well as some syntheticmolecules including sulfonamide E7010 (2), are known to me-diate their cytotoxic effects through a binding interaction withtubulin. E7010 reversibly binds to the colchicine binding site ofa-tubulin and causes cell-cycle arrest and apoptosis in the Mphase.[4, 5] This compound exhibits good in vivo antitumor ac-

In an attempt to develop potent anticancer agents, a series of2-anilinonicotinyl-linked acrylamide conjugates were designed,synthesized, and evaluated for cytotoxic activity against vari-ous human cancer cell lines, anti-tubulin activity and cell-cycleeffects. Among the series, compounds 6 d [(E)-N-(6-fluoroben-zo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)acrylamide] and 6 p [(E)-3-(2-((4-methoxyphenyl)amino)pyri-din-3-yl)-N-(6-nitrobenzo[d]thiazol-2-yl)acrylamide] showedpromising cytotoxicity, specifically against the A549 human

lung adenocarcinoma epithelial cell line, with GI50 values of0.6�0.23 and 1.8�0.22 mm, respectively. Furthermore, cell-cycle perturbation studies by flow cytometry analysis indicateddrastic cell-cycle effects in the G2/M phase in this cell line fol-lowed by caspase-3 activation and apoptotic cell death. Molec-ular docking studies of the most potent compound, 6 d, re-vealed that this compound interacts with and binds efficientlyin the active site of tubulin.

Figure 1. Structures of tubulin polymerization inhibitors as anticancer agents: combretastatin A-4 1, E7010 2, 2-anilinonicotinyl aminobenzothiazole conjugate 3, phenylcinnamide derivative 4, and 2-anilinopyridine acrylamideconjugates 5 a–h, 6 a–t.

[a] Dr. A. Kamal, M. Ashraf, M. N. A. Khan, S. Faazil, N. V. Subba Reddy, S. TajMedicinal Chemistry and PharmacologyCSIR – Indian Institute of Chemical TechnologyHyderabad 500 007 (India)E-mail : [email protected]

[b] Dr. A. Kamal, V. D. NimbarteDepartment of Medicinal ChemistryNational Institute of Pharmaceutical Education and Research (NIPER)Hyderabad 500 037 (India)

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tivity against several rodent and human tumors, andis presently in phase II clinical trials.[6] Based on theE7010 scaffold, we previously designed and synthe-sized a series of 2-anilinonicotinyl-linked aminoben-zothiazoles 3, which showed potent cytotoxicity, cell-cycle arrest, and DNA fragmentation.[7] Similarly, 2-anilinonicotinyl sulfonylhydrazide conjugates alsoshowed promising cytotoxic activity;[8] however, 2-anilinonicotinyl-linked oxadiazole derivatives provedto be very good inhibitors of tubulin polymeri-zation.[9] Benzothiazoles are well known to exhibitvarious biological properties including antimicrobial,anticancer, anti-amyloid, antirheumatic, and antiglu-tamate activities.[10–13] Various modified arylbenzothia-zoles and substituted 2-aminobenzothiazoles are alsoknown to possess significant anticancer activity bothin vitro and in vivo.[14–18] Phenylcinnamide derivative4[19] induced G2/M-phase cell-cycle arrest and celldeath in cancer cell lines at low micromolar concen-trations. The cytotoxic effect of this compound wasfound to be exerted through the disruption of micro-tubule dynamics.

In recent years, numerous efforts have been made in cancerchemotherapy research to identify potent and selective anti-cancer drugs through the discovery of a variety of novel drugs.As part of our ongoing effort to discover novel anticanceragents, we previously reported 2-anilino-linked sulfonylhydra-zides,[8] 2-aminobenzothiazole, triazolobenzothiadiazine,[7] and1,3,4-oxadiazole[9] conjugates as potent anticancer agents. Onthe basis of these findings, an attempt was made in the pres-ent study to incorporate a 2-anilinopyridyl ring in phenyl acryl-amides 5 a–h and benzothiazolyl acrylamides 6 a–t. These syn-thesized compounds were evaluated for their cytotoxic poten-tial, as well as effects toward tubulin polymerization, cell-cycleand apoptosis.

Results and Discussion

Chemistry

The routes used for the synthesis of compounds 5 a–h and6 a–t are shown in Schemes 1–3. The preparation of key inter-mediates (acryl acids 14 a–d) is described in Scheme 1. 2-Chlor-onicotinic acid (7) was converted into ethyl 2-chloronicotinate(8) by holding at reflux with ethanol and sulfuric acid at 80 8Cfor 2 h. Treatment of ester 8 with various substituted anilinesin ethylene glycol at 160 8C for 8 h yielded substituted ethyl 2-anilinonicotinates 10 a–d.[7] Further, conversion of these esters10 a–d into Weinreb amides using trimethylaluminum and N,O-dimethylhydroxylamine hydrochloride in dichloromethane for6 h afforded compounds 11 a–d,[20] which were converted intosubstituted 2-anilinonicotinaldehydes 12 a–d by using diisobu-tylaluminum hydride (DIBAL-H) in dichloromethane at �78 8Cfor 30 min.[21] Compounds 12 a–d were treated with ethyl 2-(tri-phenylphosphoranylidene)acetate in water at 100 8C for 30 minto give 13 a–d,[22] which were hydrolyzed under alkaline condi-tions to provide the corresponding acids 14 a–d.[7] Preparation

of 5 a–h was carried out by coupling the respective 2-anilinoni-cotinic acids 14 a–e with substituted anilines 15 a–e in thepresence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDCI) and hydroxybenzotriazole (HOBt) (Scheme 2, Table 1).

Similarly, the other desired compounds 6 a–t were obtained byreaction of respective 2-anilinonicotinic acids 14 a–e with 6-substituted 2-aminobenzothiazoles 16 a–e using EDCI andHOBt in dry N,N-dimethylformamide (DMF) as solvent(Scheme 3, Table 2).

Scheme 1. Reagents and conditions : a) H2SO4, ethanol, reflux, 2 h; b) ethylene glycol,160 8C, 6 h; c) Al(CH3)3, N,O-dimethylhydroxylamine hydrochloride, CH2Cl2, 0–30 8C, 8 h;d) DIBAL-H, CH2Cl2, �78 8C, 45 min; e) PPh3CH2COOC2H5, H2O, 80 8C, 30 min; f) 2 n NaOH,80 8C, 3 h.

Scheme 2. Synthesis of compounds 5 a–h. Reagents and conditions : a) EDCI,HOBt, DMF, room temperature, overnight.

Table 1. 2-Anilinopyridyl phenylacryamides 5 a–h and their clog P values.

Compd R1 R2 R3 X Y Z clog P

5 a OMe OMe OMe OMe OMe OMe 3.205 b OMe OMe OMe OMe OMe H 3.595 c OMe OMe OMe H OMe H 3.915 d OMe OMe OMe H F H 4.245 e H F H OMe OMe OMe 4.225 f H F H OMe OMe H 4.615 g H F H H OMe H 4.945 h H F H H F H 5.26



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Biological evaluation

Cytotoxic activity

We initially evaluated compounds 5 a–h for their cytotoxicityagainst various human cancer cell lines by using the MTTassay: lung adenocarcinoma (A549), cervix (HeLa), prostate(DU-145), and liver (HepG2).[23] Growth inhibitory activities (GI50

values) are listed in Table 3, with E7010 used as reference stan-dard. Most of the compounds from this series displayed potentbroad-spectrum growth inhibitory activities against all celllines tested. Some of these compounds showed particularlystrong inhibitory effects against the HepG2 cell line, with GI50

values in the 2.8–12.8 mm range.On the basis of cytotoxicity data listed in Table 1, we focused

our efforts on replacing the phenyl ring of acrylamide witha benzothiazolyl moiety, aiming to find more potent anti-prolif-erative agents. We hypothesized that the benzothiazolylmoiety may undergo more interactions with the hydrophobicresidues in the active site of the target protein. The heteroa-toms of this scaffold may also establish hydrogen bonds withresidues in the binding site. Based on this hypothesis, we de-signed and synthesized several derivatives as conjugates of 2-

anilinopyridyl acrylic acid and aminobenzothiazoles,giving compounds 6 a–t. As listed in Table 1, thesecompounds exhibited substantial cytotoxicity withsub-micromolar GI50 values.

In our SAR studies of the 2-anilinopyridyl acrylam-ide–benzothiazole conjugates, we first sought to in-vestigate whether the presence of the 3,4,5-trime-thoxyphenyl group influences along with the othersubstituents at the benzothiazolyl ring. The 3,4,5-tri-methoxyphenyl group at the 2-amino position of the

pyridyl ring was initially examined with various substituents atthe 4-benzothiazolyl ring (compounds 6 a–e). The electron-do-nating or -withdrawing effect on the benzothiazolyl ring didnot have much effect on potency (6 a and 6 c) ; however,a chloro substituent decreased the inhibitory effect in lungand prostate cancer cell lines (compound 6 b). Surprisingly,a fluoro substituent on the benzothiazolyl ring (compound 6 d)substantially enhanced potency toward all tested cell lines,having particularly strong inhibitory effects in the lung cancercell line (GI50 : 0.6 mm). Replacement of all three methoxygroups on the 2-anilinopyridyl ring with various substituents

Scheme 3. Synthesis of compounds 6 a–t. Reagents and conditions : a) EDCI, HOBt, DMF,room temperature, overnight.

Table 2. 2-Anilinopyridyl benzothiazolylacryamides 6 a–t and their clog Pvalues.

Compd R R1 R2 R3 clog P

6 a NO2 OCH3 OCH3 OCH3 4.526 b Cl OCH3 OCH3 OCH3 5.396 c OCH3 OCH3 OCH3 OCH3 4.976 d F OCH3 OCH3 OCH3 4.826 e OEt OCH3 OCH3 OCH3 4.986 f NO2 H H H 5.296 g Cl H H H 6.166 h OCH3 H H H 5.746 i F H H H 5.596 j OC2H5 H H H 6.276 k NO2 H Cl H 6.116 l Cl H Cl H 6.98

6 m OCH3 H Cl H 6.566 n F H Cl H 6.416 o OC2H5 H Cl H 7.096 p NO2 H OCH3 H 5.286 q Cl H OCH3 H 6.156 r OCH3 H OCH3 H 5.736 s F H OCH3 H 5.586 t OC2H5 H OCH3 H 6.26

Table 3. GI50 values of compounds 5 a–h and 6 a–t against a panel of celllines.

Compd GI50 [mm][a]

A549[b] DU-145[c] HeLa[d] HepG2[e]

5 a 14.2�0.22 9.4�0.23 3.3�0.49 12.4�0.115 b –[f] –[f] 4.3�0.14 12.8�0.155 c 5.6�0.14 6�0.04 –[f] 4.4�0.035 d 14.6�0.21 16.6�0.07 –[f] 2.8�0.155 e 3.9�0.32 6.8�0.16 9.8�0.04 3.3�0.145 f 5.4�0.31 3.8�0.22 –[f] 2.8�0.155 g 13.8�0.35 4.7�0.15 –[f] 3.6�0.225 h 8.9�0.21 –[f] 6.9�0.31 –[f]

6 a 4.2�0.22 9.4�0.23 3.3�0.49 12.4�0.116 b –[f] –[f] 4.3�0.14 12.8�0.156 c 5.6�0.14 6�0.04 –[f] 4.4�0.036 d 0.6�0.33 2.4�0.22 1.2�0.03 3.9�0.326 e 3.9�0.32 6.8�0.16 9.8�0.04 3.3�0.146 f 14.6�0.21 16.6�0.07 –[f] 2.8�0.156 g 13.8�0.35 4.7�0.15 –[f] 3.6�0.226 h –[f] –[f] 8.8�0.01 4.8�0.026 i 9.2�0.20 –[f] 6.3�0.22 6.1�0.266 j 4.5 12.3�0.26 4.1�0.04 4.8�0.226 k –[f] 11.2�0.08 21�0.18 5.1�0.126 l 9.2�0.20 –[f] –[f] 6.5�0.14

6 m 4.5 5.8�0.12 3.9�0.16 5�0.126 n –[f] 8.2�0.06 5.9�0.03 1.8�0.256 o 44.7�0.06 4.8�0.22 22.5�0.53 9.4�0.236 p 1.8�0.22 2.4�0.22 2.3�0.22 5.8�0.226 q 8.7�0.06 4.8�0.22 11.5�0.53 9.4�0.236 r 4.8�0.22 9.4�0.23 3.3�0.49 12.4�0.116 s 4.3�0.22 4.1�0.22 4.3�0.14 8.8�0.156 t –[f] –[f] 8.8�0.01 4.8�0.02

E7010 0.51�0.03 0.16�0.03 0.81�0.03 0.6�0.03

[a] Values are the mean �SD from three separate experiments performedin triplicate. [b] A549: human lung adenocarcinoma epithelial cell line.[c] DU-145: human prostate cancer cell line. [d] HeLa: human cervixcancer cell line. [e] HepG2: human liver cancer cell line. [f] GI50 value notattained at the concentration used in the assay.




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on the benozothiazolyl ring (6 f–j) did not affect potencyagainst the liver cancer cell lines; however, the potency wasdecreased toward other cell lines. Notably, fluorinated ana-logue 6 i, which lacks a 3,4,5-trimethoxyphenyl pattern, exhibit-ed mild or no inhibitory effect in the cancer cell lines whencompared with its counterpart 6 d.

Removal of the 3,4,5-trimethoxy substituents from the anili-no moiety attached at the 2-pyridyl position retained potencyagainst the HepG2 cell line and led to decreased potencytoward other cell lines for compounds 6 f–i. Replacement of3,4,5-trimethoxy substituents from the aniline moiety linked tothe 2-pyridyl position did not affect the inhibitory potency ofcompounds 6 h–o, and interestingly, compound 6 o, with anethoxy substituent on the benzothiazolyl ring, exhibited selec-tive potency against HepG2 cells, with a GI50 value of 1.8 mm.In another set of compounds, 6 p–t, replacement of 3,4,5-tri-methoxy substituents with 4-methoxy group on the 2-anilinoring showed broad-spectrum and similar potency. In the caseof compound 6 p, a nitro group at the 4-position of the benzo-thiazolyl ring significantly enhanced cytotoxicity toward all celllines (GI50 : 1.8–5.8 mm). These results indicate the requirementfor a 3,4,5-trimethoxy substitution at the 2-anilino ring for opti-mum anti-proliferative activity in such conjugates.

Inhibition of tubulin polymerization

The inhibition of tubulin polymerization is one of the goals forcancer therapeutics.[1, 24] The dynamic aspect of tubulin mono-mers to heterodimerize and self-assemble to form microtu-bules is a time-dependent process. Therefore, effects on theprogression of tubulin polymerization by these conjugates wasmonitored by the increase in fluorescence emission at wave-lengths of 360 nm (excitation) and 420 nm (emission) in 384-well plates for 1 h at 37 8C.[25, 26] We examined the effect of themost potent cytotoxic compounds 6 d and 6 p for their abilityto inhibit tubulin polymerization at 2 mm, and E7010 was usedas a positive control. The results indicate that 6 d and 6 p havesubstantial activity in suppressing tubulin polymerization(Figure 2). These conjugates showed potent inhibition of tubu-

lin polymerization, with IC50 values of 2.98�0.63 and 3.13�0.15 mm, respectively, compared with E7010, with an IC50 valueof 2.32�0.27 mm (Table 4). The inhibition of tubulin assemblyby these compounds correlates well with their strong anti-pro-liferative effects.

Cell-cycle analysis and apoptosis

It is known that compounds that affect tubulin assembly cancause alteration of cell-cycle parameters, with preferential G2/M blockade. Therefore, we were interested in examining theeffect of 6 d and 6 p on cell-cycle progression by FACS analy-sis.[27] A549 cells were treated with conjugates 6 d or 6 p at2 mm, along with the reference standard E7010 at 2 mm, and in-cubated for 48 h. The data obtained shows that these com-pounds caused greater accumulation of cells in the G2/Mphase than control.

Conjugates 6 d and 6 p showed 58.1 and 55.3 % accumula-tion respectively, whereas control and standard showed 23 and68.5 % accumulation, as illustrated in Figure 3 and Table 5.

Figure 2. Effect of compounds 6 d and 6 p on tubulin polymerization. An invitro tubulin polymerization assay was carried out with compounds 6 d, 6 p,and E7010 at 2 mm. Tubulin polymerization was monitored by the increasein fluorescence (lex : 360 nm, lem : 420 nm) for 1 h at 37 8C.

Table 4. Effect of compounds 5 e, 5 f, 6 d, 6 e, 6 j, 6 m, 6 p and E7010 onin vitro tubulin polymerization (TP).[a]

Compd TP [% inhib.] Compd TP [% inhib.]

E7010 65 (2.32)[b] 6 e 445 e 43 6 j 375 f 46 6 m 486 d 61 (2.98)[b] 6 p 55 (3.13)[b]

[a] Compound concentration in each case was 2 mm. [b] IC50 values (mm)in brackets.

Figure 3. FACS analysis of compounds 6 d and 6 p. A549 cells were treatedwith a) no compound (negative control), b) E7010 (2 mm) as positive control,and c) 6 d and d) 6 p, each at 2 mm.




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Thus, the effect of compounds 6 d and 6 p on cell-cycle pro-gression correlates well with their anti-proliferative activity andtubulin inhibition.

Distribution of soluble versus polymerized tubulin in cells

Microtubules constantly undergo the dynamic process of poly-merization and depolymerization, which are mediated mainlyby a- and b-tubulins. This dynamic balance is a potentialtarget for drug development against cancer, as in the case ofthe colchicine and paclitaxel compound classes. Therefore, tounderstand the effect of these conjugates on tubulin polymeri-zation, the ratio of free to polymerized tubulin was calculatedby western blot analysis. This study was carried out by treatingA549 cells with 2 mm each of 6 d and 6 p along with 1 mm no-codazole (a microtubule-destabilizing agent) as positive con-trol, and paclitaxel (a microtubule-stabilizing agent) as a nega-tive control[28] for 24 h. By western blot analysis, we calculatedthe percentage of supernatant to pellet fractions. There wasa nearly 5- to 10-fold increase in tubulin content of the super-natant fraction of 6 d and 6 p at 2 mm, respectively, relative tocontrol (Figure 4), whereas nocodazole-treated cells demon-

strated more tubulin content in the soluble fraction than poly-merized fraction. In contrast, the tubulin protein was more inthe polymerized fraction than the soluble portion of paclitaxel-treated cells. Therefore, these results suggest that the conju-gates 6 d and 6 p act as microtubule-destabilizing agentsunder both in vitro and intracellular conditions.

Effect on caspase-3

It has been reported that cell-cycle arrest at G2/M phase leadsto induction of apoptosis. Our interest was therefore focusedon examination of conjugates 6 d and 6 p to determine wheth-er their cytotoxicity is brought about by apoptotic cell death.Caspases, a family of conserved cysteine proteases, play a vital

role in the execution of apoptosis by initiating apoptotic sig-nals.[29] Caspase-3,[30] one of the primary effector caspases, hasbeen considered, as it cleaves and activates caspases-6 and -7,and the protein itself is processed and activated by caspases-8,-9, and -10. Therefore, the activation of caspase-3 was studiedby Annexin V flow cytometric assays; A549 cells were treatedwith 6 d and 6 p at both 2 and 4 mm for 24 h, and E7010 wasused as a positive control. A significant three- to fivefold in-crease was observed in the induction of caspase-3 in cellstreated with these conjugates relative to the control, as shownin Figure 5. This result indicates that 6 d and 6 p induced apop-tosis in A549 cells by activation of caspase-3.

Hoechst staining for morphological analysis of apoptosis

Apoptosis is one of the major pathways that lead to the pro-cess of cell death. Chromatin condensation and fragmentednuclei are the classic characteristics of apoptosis.[31] We there-fore investigated the apoptosis-inducing effect of compounds6 d and 6 p by Hoechst staining (H 33258) method in the A549human lung adenocarcinoma epithelial cell line. Cells weretreated with compounds at 1 mm for 24 h, and E7010 was usedas a reference compound. Based on chromatin condensation,fragmented nuclei, presence of apoptotic bodies, and relativefluorescence of the tested compounds (6 d, 6 p, and E7010) re-vealed a significant increase in the percentage of apoptoticcells relative to that of untreated control cells (Figure 6).

Immunocytochemistry

To validate the effect of compounds on cellular tubulin immu-nohistochemistry, studies were carried out to examine the insitu effects of compounds 6 d and 6 p on cellular microtubulesin A549 cancer cells. A549 cells seeded on a sterile cover slipswere treated with the compounds at 2 mm, and E7010 wasused as a reference standard at 1 mm for 48 h. In this study, un-treated human lung cancer cells displayed the normal distribu-tion of microtubules (Figure 7). However, cells treated withcompounds 6 d and 6 p showed disrupted microtubule organi-zation, as shown in Figure 3, thus demonstrating the inhibitionof tubulin polymerization. However, the standard E7010 also

Table 5. Effect of compounds 6 d, 6 p, and E7010 on cell-cycle phase dis-tribution in A549 cells.

Compd[a] G0/G1 [%] S [%] G2/M [%]

control 65.6 11.4 236 d 33.2 8.7 58.16 p 36.8 7.9 55.3E7010 21.7 9.8 68.5

[a] Concentration: 2 mm.

Figure 4. Western blot analysis of a-tubulin in the soluble (S) and polymer-ized (P) fractions of A549 cells treated with compounds 6 d and 6 p at 2 mm.Nocodazole and paclitaxel were used at 1 mm for 24 h.

Figure 5. Effect of compounds 6 d, 6 p, and E7010 on caspase-3 activity.A549 cells were treated for 24 or 48 h with compounds 6 d, 6 p (each at 2 or4 mm), and E7010 (1 mm, positive control).




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showed disrupted microtubule organization. This immuno-fluorescence study showed that the level of tubulin polymeri-zation inhibition is similar to that of E7010 for compounds 6 dand 6 p.

Molecular docking studies

To rationalize the potential binding modes of these acryla-mides in tubulin, docking studies were performed with the

most potent compound, (E)-N-(6-fluorobenzo[d]thia-zol-2-yl)-3-(2-(3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)acrylamide (6 d) in this series. Using X-ray crystalstructure information for tubulin (PDB ID: 3E22),[32]

possible binding interactions with 6 d were probedwith the AutoDock 4.2 software package.[33]

In-depth analysis of docking conformation was car-ried out to evaluate the binding sites of 6 d in the ab

interface of colchicine binding sites in tubulin. Dock-ing analysis of 6 d supports inhibition of tubulin poly-merization at the molecular level, by showing the3,4,5-trimethoxyphenyl group binding in the colchi-cine binding site of b-tubulin. Some important hydro-gen binding interactions were observed betweenaSer178, aTyr224, and bLys224 with the methoxygroup O atoms at positions 4 and 5 of the phenylring, which are positioned 1.9–2.2 � away (Figure 8).The NH group of the amide linkage is involved in hy-drogen bonding interactions with aAsn10 andaThr179 residues of tubulin. Some electrostatic inter-

Figure 6. Hoechst staining in the A549 human lung adenocarcinoma epithelial cell line.a) A549 control cells, b) E7010 (1 mm), c) 6 d (2 mm), and d) 6 p (2 mm).

Figure 7. A549 cells were treated with compounds 6 d and 6 p at 2 mm for48 h, followed by staining with a-tubulin antibody. Microtubule organizationwas clearly observed by green tubulin network-like structures in controlcells, and was found to be disrupted in cells treated with compounds 6 dand 6 p. E7010 was used as a positive control.

Figure 8. a) Binding conformation of 6 d (in green) in the colchicine bindingsite in ab-tubulin. b) Interactions of compound 6 d with aSer178, aTyr224,aGln11, aAsn101, bLeu247. c) Docking conformation of compound 6 d inthe colchicine binding site of tubulin.




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actions were observed between the pyridine ring N atom andbGln247, in the distance range of 2.9–3.2 �. Some hydrophobicand electrostatic interactions were observed between the Sand N benzothiazole ring atoms of 6 d and aSer178, aThr179,and aAsn101. Some electrostatic interactions were predictedto occur between the F atom on the benzothiazole ring andaGln11 (Figure 8). In addition to these interactions, some hy-drophobic interactions were observed between the pyridinering and bAsn288, aVal181, and bLys352. Several hydrogenbonding, hydrophobic, and electrostatic contacts were ob-served between the 3,4,5-trimethoxy group O atoms andbLeu255, bThr353, bCys241, bLeu298, and bAla250, a patternthat resembles the colchicine interaction with tubulin (Fig-ure 8 c).

Some decisive hydrophobic interactions were observed be-tween aSer178, aTyr224, aGln11, aAsn101 and the 3,4,5-trime-thoxyphenyl group, and a few between the pyridine ring andbAsn258, aVal181, bMet259, and bLys352. Apart from these,some additional hydrophobic interactions were observed withthe carbonyl oxygen of the amide group present between thetrimethoxyphenyl group and pyridine, with aAla180 andaAsn101. For the benzothiazole ring, some hydrophobic inter-actions were observed with bLeu255, bLeu248, bAla250,bVal318. The 3- and 4-methoxy groups were specifically in-volved in hydrophobic interactions with bAla316, bAla354,bThr353, bLeu255, bVal318, and bAla250. In this case, few hy-drophobic interactions were the as same as those of colchicine(bVal318, bCys241, bLeu248, bAla250, bLeu255, and bAla316,respectively). All compounds were expected to show a posesimilar to that of compound 6 d.

Conclusions

In this study, a new series of 2-anilinopyridine-3-acrylamides5 a–h and 6 a–t were designed, synthesized, and evaluated fortheir anti-proliferative activities against four human cancer celllines (A549, HeLa, DU-145, and HepG2). These compounds ex-hibited potent cytotoxicity, with GI50 values ranging from 0.6to 44.7 mm. Among these compounds, 6 d and 6 p exhibitedpotent activity in the A549 human lung adenocarcinoma epi-thelial cell line, with GI50 values in the range of 0.6–1.8 mm. Thespecificity of compounds 6 d and 6 p in inhibiting tubulin poly-merization by binding to the colchicine binding site was ob-served from the increased ratio of soluble to polymerized tu-bulin. Thus, these functions as microtubule-destabilizingagents are supported both by in vitro tubulin polymerizationassays and intracellular models. Both compounds showedpotent inhibition of tubulin polymerization, with IC50 values of2.98 and 3.13 mm, compared with E7010 (IC50 : 2.02 mm). Fur-thermore, cell-cycle analysis in A549 cells revealed compounds6 d and 6 p to cause cell-cycle arrest at the G2/M phase fol-lowed by three- and fivefold increases in induction of caspase-3 activity relative to control at concentrations of 2 and 4 mm.These mechanistic studies can provide more insight for furtherdevelopment of tubulin inhibitors as potential anticanceragents.

Experimental Section

Chemistry

All chemicals and reagents were obtained from Sigma–Aldrich(St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Co.,Ward Hill, MA, USA), and Spectrochem Pvt. Ltd. (Mumbai, India),and were used without further purification unless indicated other-wise. THF and CH2Cl2 were distilled from NaH and CaH2, respective-ly, immediately prior to use. Reactions were monitored by TLC per-formed on glass plates coated with silica gel 60 GF254, visualized byUV light and iodine indicator, as well as by charring. Column chro-matography was performed with Merck 60–120 mesh silica gel.1H NMR spectra were recorded on Bruker UXNMR/XWIN-NMR(300 MHz) or Inova Varian VXR Unity (400, 500 MHz) instruments.Chemical shifts (d) are reported in ppm downfield from an internalTMS standard. ESIMS spectra were recorded on a Micromass Quat-tro LC instrument using ESI + software at a capillary voltage of3.98 kV and ESI mode positive ion trap detector. High-resolutionmass spectra (HRMS) were recorded on a QSTAR XL Hybrid MS–MSmass spectrometer. Melting points were determined with an Elec-trothermal melting point apparatus, and are uncorrected.

General method for the synthesis of substituted ethyl 2-(phe-nylamino)nicotinates 10 a–d

A mixture of ethyl 2-chloronicotinate 8 (2 g, 0.01 mol) and corre-sponding anilines (0.01 mol) in ethylene glycol was stirred at160 8C for 8 h. The reaction mixture was cooled to room tempera-ture and poured into water. The aqueous layer was extracted withEtOAc (3 � 30 mL). The combined organic layer was washed withbrine, dried over MgSO4, and concentrated in vacuo. The crudeproduct was purified by column chromatography eluting withEtOAc/hexane to afford substituted ethyl 2-(phenylamino)nicoti-nates 10 a–d.

Ethyl 2-((3,4,5-trimethoxyphenyl)amino)nicotinate (10 a): Com-pound 10 a was prepared according to the method describedabove by using ethyl 2-chloronicotinate 8 (1.5 g,1 mmol) and 3,4,5-trimethoxyaniline (147 mg, 1 mmol) to obtain the pure product10 a as a yellow solid. Yield: 236 mg, 71 %; mp: 143–145 8C;1H NMR (CDCl3, 500 MHz): d= 10.14 (bs, 1 H), 8.37 (dd, J = 5.3,2.3 Hz, 1 H), 8.25 (dd, J = 7.6, 2.3 Hz, 1 H), 7.00 (s, 2 H), 6.71 (dd, J =8.3, 5.3 Hz, 1 H), 4.38 (q, J = 7.5 Hz, 2 H), 3.88 (s, 6 H), 3.83 (s, 3 H),1.43 ppm (t, J = 7.5 Hz, 33 H); EIMS: m/z 332 [M]+ .

Ethyl 2-(phenylamino)nicotinate (10 b): Compound 10 b was pre-pared according to the method described above by using ethyl 2-choronicotinate 8 (2 g, 0.010 mol) and aniline (1 g, 0.1 mol) to give3.20 g of pure ethyl 2-(phenylamino)nicotinate (10 b) as a pale-yellow solid. Yield: 75 %; mp: 54–56 8C; 1H NMR (200 MHz, CDCl3 +[D6]DMSO): d= 10.22 (bs, 1 H), 8.34 (dd, J = 4.7, 1.5 Hz, 1 H), 8.20(dd, J = 7.9, 2.3 Hz, 1 H), 7.68 (m, 2 H), 7.30 (m, 2 H), 7.00 (dt, J = 7.1,1.6 Hz, 1 H), 6.66 (dd, J = 7.8, 4.7 Hz, 1 H), 4.38 (q, J = 7.0 Hz, 2 H),1.43 ppm (t, J = 7.0 Hz, 3 H); EIMS: m/z 242 [M]+ .

Ethyl 2-((4-chlorophenyl)amino)nicotinate (10 c): Compound 10 cwas prepared according to the method described above, usingethyl 2-chloronicotinate 8 (1.5 g, 1 mmol) and 4-chloroaniline(147 mg, 1 mmol) to obtain the pure product 10 c as a yellowsolid. Yield: 195 mg, 78 %; mp: 67–69 8C; 1H NMR (200 MHz,CDCl3 + [D6]DMSO): d= 10.19 (bs, 1 H), 8.32 (dd, J = 4.6, 2.0 Hz, 1 H),8.22 (dd, J = 8.0, 2.0 Hz, 1 H), 7.65 (m, 2 H), 7.00 (m, 2 H), 6.70 (dd,J = 7.3, 4.6 Hz, 1 H), 4.40 (q, J = 7.3 Hz, 2 H), 1.44 ppm (t, J = 7.3 Hz,3 H); EIMS m/z 260 [M]+ .




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Ethyl 2-((4-methoxyphenyl)amino)nicotinate (10 d): Compound10 d was prepared according to the method described above,using ethyl 2-chloronicotinate 8 (1.5 g, 1 mmol) and 4-methoxyani-line (147 mg, 1 mmol) to obtain the pure product 10 d as a yellowsolid. Yield: 218 mg, 80 %; mp: 88–90 8C; 1H NMR (200 MHz,CDCl3 + [D6]DMSO): d= 10.00 (s, 1 H), 8.32 (dd, J = 4.6, 2.3 Hz, 1 H),8.20 (dd, J = 7.8, 2.3 Hz, 1 H), 7.52 (dd, J = 7.0, 2.3 Hz, 2 H), 6.88 (dd,J = 7.0, 2.3 Hz, 2 H), 6,64 (dd, J = 7.8, 4.6 Hz, 1 H), 4.38 (q, J = 7.0 Hz,2 H), 3.82 (s, 3 H), 1.43 ppm (t, J = 7.0 Hz, 3 H); EIMS m/z 272 [M]+ .

General method for the synthesis of substituted compounds11 a–d

A flame-dried flask (two-neck 100 mL) fitted with one additionfunnel and an N2 inlet tube was charged with N,O-dimethylhydrox-ylamine hydrochloride (2.03 g, 20.65 mmol) in CH2Cl2 (10 mL), andthe reaction mixture was stirred at �5 8C. A solution of trimethyla-luminum (10 mL, 20.65 mmol) in toluene was added dropwise over30 min to the N,O-dimethylhydroxyamine·HCl solution. To this solu-tion was added the substituted ethyl 2-(phenylamino)nicotinate(1 g, 4.13 mmol) as a solution in CH2Cl2 (5 mL) over 10 min. Themixture was allowed to warm to room temperature for 8 h. The re-action mixture was hydrolyzed with aqueous NH4Cl, and extractedwith CHCl3. The combined organic layers were dried over Na2SO4.Evaporation of the solvent left a foamy material, which was puri-fied by column chromatography on silica gel with EtOAc/hexaneto provide 11 a–d as a yellow liquid. Yield: 71 % in each case.

N-Methoxy-N-methyl-2-((3,4,5-trimethoxyphenyl)amino)nicotina-mide (11 a): Compound 11 a was prepared according to themethod described above, using N,O-dimethylhydroxylamine hydro-chloride (2.03 g, 18.38 mmol), trimethylaluminum (9.19 mL,18.38 mmol) and 2-(3,4,5-trimethoxyphenylamino)nicotinate (1 g,3.67 mmol) to obtain the pure product 11 a as a yellow liquid.Yield: 71 %; 1H NMR (300 MHz, CDCl3): d= 8.77 (s, 1 H), 8.26 (dd, J =3.0, 2.2 Hz 1 H), 7.86 (dd, J = 6.0, 1.5 Hz, 1 H), 7.22 (s, 2 H), 6.87 (dd,J = 6.0, 2.2 Hz 1 H), 3.89 (s, 6 H), 3.86 (s, 3 H), 3.59 (s, 3 H), 3.37 ppm(s, 3 H); MS (ESI): m/z 347 [M]+ .

N-Methoxy-N-methyl-2-(phenylamino)nicotinamide (11 b): Com-pound 11 b was prepared according to the method describedabove, using N,O-dimethylhydroxylamine hydrochloride (2.03 g,20.65 mmol), trimethylaluminum (10 mL, 20.65 mmol) and ethyl 2-(phenylamino)nicotinate (1 g, 4.13 mmol) to obtain the pure prod-uct 11 b as a yellow liquid. Yield: 71 %; 1H NMR (300 MHz, CDCl3):d= 8.92 (s, 1 H), 8.29 (dd, J = 3,0, 1.5 Hz, 1 H), 7.87 (dd, J = 5.2,2.2 Hz, 1 H), 7.60 (d, J = 7.5 Hz, 2 H), 7.31 (t, J = 8.3 Hz, 2 H), 7.00 (t,J = 7.5 Hz, 2 H) 6.72 (dd, J = 4.5, 3.0 Hz, 1 H), 3.57 (s, 3 H), 3.38 ppm(s, 3 H); MS (ESI): m/z 257 [M]+ .

2-((4-Chlorophenyl)amino)-N-methoxy-N-methylnicotinamide(11 c): Compound 11 c was prepared according to the method de-scribed above, using N,O-dimethylhydroxylamine hydrochloride(2.03 g, 18.38 mmol), trimethylaluminum (9.19 mL, 18.38 mmol)and 2-(4-chlrophenylamino)nicotinate (1 g, 3.67 mmol) to obtainthe pure product 11 c as a yellow liquid. Yield: 71 %; 1H NMR(300 MHz, CDCl3): d= 9.01 (s, 1 H), 8.28 (dd, J = 3.0, 2.2 Hz, 1 H), 7.91(dd, J = 5.28, 2.2 Hz, 1 H), 7.60–7.55 (m, 2 H), 7.29–7.22 (m, 2 H), 6.73(dd, J = 4.5, 3.0 Hz, 1 H) 3.58 (s, 3 H), 3.39 ppm (s, 3 H); MS (ESI): m/z291 [M]+ .

N-Methoxy-2-((4-methoxyphenyl)amino)-N-methylnicotinamide(11 d): Compound 11 d was prepared according to the method de-scribed above, using N,O-dimethylhydroxylamine hydrochloride(2.03 g, 18.38 mmol), trimethylaluminum (9.19 mL, 18.38 mmol)

and 2-(4-methoxyphenylamino)nicotinate (1 g, 3.67 mmol) toobtain the pure product 11 d as a yellow liquid. Yield: 71 %;1H NMR (300 MHz, CDCl3): d= 9.02 (s, 1 H), 8.28 (dd, J = 3.0, 2.2 Hz,1 H), 7.91 (dd, J = 5.2, 2.2 Hz, 1 H), 7.59–7.55 (m, 2 H), 7.28–7.23 (m,2 H), 6.75 (dd, J = 4.5, 3.0 Hz, 1 H), 3.58 (s, 3 H), 3.39 ppm (s, 3 H); MS(ESI): m/z 275 [M]+ .

General method for the synthesis of compounds 12 a–d

To a stirred solution of N-methoxy-N-methyl-2-(phenylamino)nicoti-namide 11 a–d (1 g, 3.89 mmol) in CH2Cl2 was added diisobutylalu-minum hydride (8.75 mL) at �78 8C under N2 atmosphere. Afterstirring for 45 min at �78 8C, the solution was quenched withmethanol. The reaction mixture was extracted with CH2Cl2 anddried over MgSO4. After evaporation of the solvent, the residuewas purified by silica gel column chromatography to give the cor-responding aldehydes 12 a–d. Yields: 80–90 %.

2-((3,4,5-Trimethoxyphenyl)amino)nicotinaldehyde (12 a): Com-pound 12 a was prepared according to the general method, usingN-methoxy-2-((3,4,5-trimethoxyphenyl)amino)-N-methylnicotina-mide 11 a (1.5 g, 4.34 mmol) and diisobutylaluminum hydride(9.75 mL) to obtain the pure product 12 a as a yellow solid. Yield:80 %; mp: 130–132 8C; 1H NMR (300 MHz, CDCl3): d= 10.38 (s, 1 H),9.88 (s, 1 H), 8.41 (dd, J = 3.0 2.2 Hz, 1 H), 7.86 (dd, J = 5.2, 2.2 Hz,1 H), 7.04 (s, 2 H), 6.87–6.83 (m, 1 H), 3.89 (s, 6 H), 3.84 ppm (s, 3 H);MS (ESI): m/z 288 [M]+ .

2-(Phenylamino)nicotinaldehyde (12 b): Compound 12 b was pre-pared according to the general method, using N-methoxy-N-methyl-2-(phenylamino)nicotinamide 11 b (1 g 3.89 mmol) inCH2Cl2 ; to this was added diisobutylaluminum hydride (8.75 mL) toobtain the pure product 12 b as a yellow solid. Yield: 89 %; 1H NMR(300 MHz, CDCl3): d= 10.44 (S, 1 H), 9.80 (s, 1 H), 8.41 (dd, J = 2.6,1.8 Hz, 1 H), 7.86 (dd, J = 2.6, 1.8 Hz, 1 H), 7.74 (d, J = 7.9 Hz, 2 H),7.36 (t, J = 8.28, 7.5 Hz, 2 H), 7.09 (t, J = 7.5, 7.1 Hz, 1 H), 6.83 ppm(dd, J = 4.89, 2.6 Hz, 1 H); MS (ESI): m/z 198 [M]+ .

2-((4-Chlorophenyl)amino)nicotinaldehyde (12 c): Compound 12 cwas prepared according to the general method, using N-methoxy-2-((4-chlorophenyl)amino)-N-methylnicotinamide 11 c (1 g,3.58 mmol) and diisobutylaluminum hydride (8.00 mL) to obtainthe pure product 12 c as a yellow solid. Yield: 80 %; mp: 60–62 8C;1H NMR (300 MHz, CDCl3): d= 10.35 (s, 1 H), 9.86 (s, 1 H), 8.37 (dd,J = 2.2 Hz, 1 H), 7.85 (dd, J = 5.28, 2.2 Hz, 1 H), 7.70–7.64 (m, 2 H),7.09–700 (m, 2 H), 6.83 ppm (dd, J = 4.53, 3.0 Hz, 1 H); MS (ESI): m/z232 [M]+ .

2-((4-Methoxyphenyl)amino)nicotinaldehyde (12 d): Compound12 d was prepared according to the general method, using N-me-thoxy-2-((4-methoxyphenyl)amino)-N-methylnicotinamide (11 d,1 g, 3.63 mmol) and diisobutylaluminum hydride (8.15 mL) toobtain the pure product 12 d as a yellow solid. Yield: 80 %; mp:85–87 8C; 1H NMR (300 MHz, CDCl3): d= 10.26 (s, 1 H), 9.86 (s, 1 H),8.37 (dd, J = 3.0, 1.3 Hz, 1 H), 7.84 (dd, J = 5.6, 1.8 Hz, 1 H), 7.58 (d,J = 8.6 Hz, 2 H), 6.92 (d, J = 8.6 Hz, 2 H), 6.76(dd, J=4.8, 2.6 1 H)3.81 ppm (s, 3 H); MS (ESI): m/z 228 [M]+ .

General method for the synthesis of compounds 13 a–d

To a solution of ethyl 2-(triphenylphosphoranylidene)acetate(1.91 g, 5.5 mmol) in anhydrous THF was added sodium hydride(134 mg, 5.5 mmol) at 0 8C. After 10 min, substituted 2-(phenylami-no)nicotinaldehyde 12 a–d (1 g, 3.7 mmol) was added to the reac-tion mixture at 0 8C. The ice bath was removed, and the reaction




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mixture was stirred at room temperature under N2 atmosphere for3 h. Saturated NH4Cl (20–30 mL) was added to the mixture, andthe aqueous layer was extracted with EtOAc. The combined organ-ic layers were washed with saturated NaCl, dried over Na2SO4, andpurified by column chromatography using petroleum ether/EtOAc9:1 as eluent to yield yellow solid products 13 a–d. Yields: 80–90 %.

(E)-Ethyl 3-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)acry-late (13 a): Compound 13 a was prepared according to the generalmethod, using ethyl 2-(triphenylphosphoranylidene)acetate (12 a,1.75 g, 5.03 mmol), 2-(3,4,5-trimethixyphenylamino)nicotinaldehyde(1 g, 3.3 mmol) and sodium hydride (120 mg, 5.03 mmol) to obtainthe pure product 13 a as a yellow solid. Yield: 75 %; mp: 230–232 8C; 1H NMR (300 MHz, CDCl3): d= 8.25 (dd, J = 3.0, 2.2 Hz, 1 H),7.81 (d, J = 15.8 Hz, 1 H), 7.70 (dd, J = 6.0, 1.5 Hz, 1 H), 6.85–6.80 (m,1 H), 6.77 (s, 2 H), 6.44 (d, J = 15.8 Hz, 2 H), 4.29 (q, J = 6.7 Hz, 2 H),3.86 (s, 6 H), 3.82 (s, 3 H), 1.34 ppm (t, J = 6.7 Hz, 3 H); MS (ESI): m/z358 [M]+ .

(E)-Ethyl 3-(2-(phenylamino)pyridin-3-yl)acrylate (13 b): Com-pound 13 b was prepared according to the general method, usingethyl 2-(triphenylphosphoranylidene)acetate (1.91 g, 5.5 mmol) and2-(phenylamino)nicotinaldehyde (12 b, 1 g, 3.7 mmol) to obtainpure product 13 b as a yellow solid. Yield: 78 %; 1H NMR (300 MHz,CDCl3): d= 8.25 (dd, J = 3.0, 1.6 Hz, 1 H), 7.86 (d, J = 15.8 Hz, 1 H),7.69 (dd, J = 6.1, 1.3 Hz, 1 H), 7.48 (d, J = 7.4 Hz, 2 H), 7.33 (t, J =8.3 Hz, 2 H), 7.04 (t, J = 7.4, 1 H), 6.82 (dd, J = 4.7, 2.7 Hz, 1 H), 6.48(s, 1 H), 6.41 (d, J = 15.7 Hz, 1 H), 4.28 (q, J = 7.0 Hz, 2 H), 1.34 ppm(t, J = 7.0, 3 H); MS (ESI): m/z 268 [M]+ .

(E)-Ethyl 3-(2-((4-chlorophenyl)amino)pyridin-3-yl)acrylate (13 c):Compound 13 c was prepared according to the general method,using ethyl 2-(triphenylphosphoranylidene)acetate (1.8 g,5.2 mmol), sodium hydride (125 mg, 5.2 mmol) and 2-(4-chlorophe-nylamino)nicotinaldehyde (1 g, 3.4 mmol) to obtain the pure prod-uct 13 c as a yellow solid. Yield: 80 %; mp: 192–194 8C; 1H NMR(300 MHz, CDCl3): d= 8.45 (dd, J = 3.1, 1.6 Hz, 1 H), 7.96 (d, J =15.6 Hz, 1 H), 7.68 (dd, J = 6.1, 1.5 Hz, 1 H), 7.51 (d, J = 7.6 Hz, 2 H),7.32 (t, J = 8.8 Hz, 2 H), 7.04 (t, J = 7.4, 1 H), 6.82 (dd, J = 4.7, 2.7 Hz,1 H), 6.38 (s, 1 H), 6.41 (d, J = 15.6 Hz, 1 H), 4.28 (q, J = 7.0 Hz, 2 H),1.34 ppm (t, J = 7.0, 3 H); MS (ESI): m/z 303 [M]+ .

(E)-Ethyl 3-(2-((4-methoxyphenyl)amino)pyridin-3-yl)acrylate(13 d): Compound 13 d was prepared according to the generalmethod, using ethyl 2-(triphenylphosphoranylidene)acetate (1.75 g,5.03 mmol), sodium hydride (120 mg, 5.03 mmol) and 2-(4-methox-yphenylamino)nicotinaldehyde (1 g, 3.3 mmol) to obtain the pureproduct 13 d as a yellow solid. Yield: 80 %; mp: 206–208 8C;1H NMR (300 MHz, CDCl3): d= 8.19 (dd, J = 4.9, 1.7 Hz, 1 H), 7.76 (d,J = 15.8 Hz, 1 H), 7.64 (dd, J = 5.8, 1.7 Hz, 1 H), 7.34 (d, J = 8.8 Hz,2 H), 6.90 (d, J = 15.8 Hz, 1 H), 6.36 (s, 1 H), 4.29 (q, J = 7.1 Hz, 2 H),1.34 ppm (t, J = 7.1 Hz, 3 H); MS (ESI): m/z 298 [M]+ .

General procedure for the synthesis of substituted (E)-3-(2-(phenylamino)pyridin-3-yl)acrylic acids 14 a–d

Substituted (E)-3-(2-(phenylamino)pyridin-3-yl)acrylic acids 13 a–dwere held at reflux with 2 n NaOH in ethanol for 2 h. The reactionmixture was cooled and left acidified with 2 n HCl to obtaina white solid, which was filtered and washed with water to givepure compounds 14 a–d.

General procedure for the synthesis of target compounds5 a–h and 6 a–t

To a stirred solution of substituted (E)-3-(2-(phenylamino)pyridin-3-yl)acrylic acid (14 a–d, 1 mmol) in dry DMF (5 mL), was added HOBt(1.2 mmol) at 0 8C. After 10 min, EDCI (1.2 mmol) was added, and fi-nally substituted 2-aminobenzothiazole/aniline (0.8 mmol) wasadded. The resulting mixture was then stirred at room temperaturefor 8–10 h. The reaction mixture was quenched with NaHCO3 andextracted with EtOAc from the ice-cold aqueous layer and driedover anhydrous Na2SO4. The resulting product was purified bycolumn chromatography using EtOAc/Hexane as eluent.

(E)-N-(3,4,5-Trimethoxyphenyl)-3-(2-((3,4,5-trimethoxyphenyl)-amino)pyridin-3-yl)acrylamide (5 a): Compound 5 a was preparedaccording to the general method, using (E)-3-(2-((3,4,5-trimethoxy-phenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and 3,4,5-trimethoxyaniline (146 mg, 0.8 mmol) to obtain pure product 5 a asa yellow solid. Yield: 83 %; mp: 241–243 8C; 1H NMR (300 MHz,[D6]DMSO): d= 10.21 (s, 1 H), 8.55 (s, 1 H), 8.17 (d, J = 3.7 Hz, 1 H),7.90 (d, J = 15.1 Hz, 1 H), 7.83 (s, 1 H), 7.13 (s, 2 H), 7.01 (s, 2 H), 6.91–6.87 (m, 1 H), 6.76 (d, 1 H), 3.76 (s, 6 H), 3.74 (s, 6 H); 3.63 (s, 3 H),3.62 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 165.1, 160.3,155.4, 151.4, 149.6, 141.3, 139.6, 133.3, 132.1, 129.9, 128.1, 123.8,121.7, 120.5, 120.2, 110.1, 69.1, 58.2 ppm; MS (ESI): m/z 496 [M +

1]+ ; HRMS (ESI m/z) for C26H29N3O7, calcd: 495.2006, found:496.2136 [M + 1]+ ; IR (KBr, lmax): n= 3449.7, 3152.3, 3070.1, 2952.6,1679.8, 1601, 1580.8, 1517.4, 1498.9, 1466.6, 1349.5, 1224.5, 1181.4,972.5, 781.4, 758 cm�1; Anal. calcd for C26H29N3O7: C 63.02, H 5.90,N 8.48, found: C 63.12, H 5.98, N 8.78.

(E)-N-(3,4-Dimethoxyphenyl)-3-(2-((3,4,5-trimethoxyphenyl)ami-no)pyridin-3-yl)acrylamide (5 b): Compound 5 b was prepared ac-cording to the general method, using (E)-3-(2-((3,4,5-trimethoxy-phenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and 3,4-di-methoxyaniline (112 mg, 0.8 mmol) to obtain pure product 5 b asa yellow solid. Yield: 81 %; mp: 231–232 8C; 1H NMR (300 MHz,[D6]DMSO): d= 10.20 (s, 1 H), 8.25 (s, 1 H), 8.13 (d, J = 4.2 Hz, 1 H),7.87 (d, J = 15.3, 1 H), 7.86 (d, J = 8.4 Hz, 2 H), 7.27 (d, J = 8.3 Hz,1 H), 7.11 (s, 2 H), 6.91–6.86 (m, 3 H), 3.76 (s, 6 H), 3.72 (s, 6 H),3.65 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 163.2, 160.2,153.4, 150.2, 149.1, 144.3, 139.5, 129.3, 126.2, 122.9, 121.2, 119.8,116.7, 115.9, 110.2, 103.1, 983, 69.1, 65.2, 56.1 ppm; MS (ESI): m/z466 [M + 1]+ ; HRMS (ESI m/z) for C25H27N3O6, calcd: 465.1900,found: 466.2940 [M + 1]+ ; IR (KBr, lmax): n= 3419.6, 3162.2, 3070.1,2972.6, 1685.8, 1601, 1580.8, 1517.4, 1498.9, 1466.6, 1349.5, 1224.5,1181.4, 962.5, 775.4, 757 cm�1; Anal. calcd for C25H27N3O6 : C 64.50,H 5.85, N 9.03, found: C 64.56, H 5.95, N 9.73.

(E)-N-(4-Methoxyphenyl)-3-(2-((3,4,5-trimethoxyphenyl)amino)-pyridin-3-yl)acrylamide (5 c): Compound 5 c was prepared accord-ing to the general method, using (E)-3-(2-((3,4,5-trimethoxypheny-l)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and 4-methoxya-niline (98 mg, 0.8 mmol) to obtain pure product 5 c as a yellowsolid. Yield: 86 %; mp: 226–228 8C; 1H NMR (300 MHz, [D6]DMSO):d= 10.10 (s, 1 H), 8.73 (s, 1 H), 8.13 (d, J = 3.12 Hz, 1 H), 7.93 (d, J =15.4 Hz, 1 H), 7.84 (d, J = 6.2, 1.7 Hz, 1 H), 7.60–7.55 (m, 2 H), 7.12 (s,2 H), 7.11 (s, 2 H), 6.91–6.86 (m, 2 H), 3.86 (s, 6 H), 3.82 (s, 3 H),3.67 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 164.2, 161.3,153.4, 150.2, 148.1, 144.6, 139.7, 129.0, 126.0, 122.9, 120.2, 119.1,116.0, 115.5, 110.2, 103.1, 69.4, 55.2, 54.1 ppm; MS (ESI): m/z 436[M + 1]+ ; HRMS (ESI m/z) for C24H25N3O5, calcd: 435.1794, found:436.2454 [M + 1]+ ; IR (KBr, lmax): n= 3433.5, 3162.4, 3035.5, 2959.3,1681.4, 1599.3, 1587.4, 1556.5, 1515.3, 1498.3, 1440.5, 1348.5,




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1267.5, 1135.4, 1097.0, 782.1, 755.2 cm�1; Anal. calcd forC24H21N5O6S: C 66.19, H 5.79, N 9.65, found: C 66.59, H 5.99, N 9.55.

(E)-N-(4-Fluorophenyl)-3-(2-((3,4,5-trimethoxyphenyl)amino)pyri-din-3-yl)acrylamide (5 d): Compound 5 d was prepared accordingto the general method, using (E)-3-(2-((3,4,5-trimethoxyphenyl)ami-no)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and 4-fluoroaniline(89 mg, 0.8 mmol) to obtain pure product 5 d as a yellow solid.Yield: 85 %; mp: 220–222 8C; 1HNMR (300 MHz, [D6]DMSO): d=10.12 (s, 1 H), 8.62 (s, 1 H), 8.18 (s, 1 H), 8.12 (dd, J = 3.0, 1.7 Hz, 1 H),7.93 (d, J = 15.4 1 Hz, 1 H), 7.85 (dd, J = 6.0, 1.7 Hz, 1 H), 7.60–7.55(m, 1 H), 7.12 (s, 2 H), 7.02 (t, J = 8.8 Hz, 2 H), 6.88–6.84 (m, 1 H), 6.75(d, J = 15.2 Hz, 1 H), 3.79 (s, 6 H), 3.67 ppm (s, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 163.7, 159.3, 156.0, 152.9, 148.2, 136.1,135.4, 134.5, 133.6, 124.0, 121.8, 121.5, 116.7, 114.6, 97.2, 60.2,55.4 ppm; MS (ESI): m/z 424 [M + 1]+ ; HRMS (ESI m/z) forC23H22FN3O4, calcd: 423.1594, found: 424.1698 [M + 1]+ ; IR (KBr,lmax): n= 3376.2, 3167.8, 2923, 2853, 1672, 1602, 1574, 1507, 1474,1298, 1153, 812 cm�1; Anal. calcd for C23H22FN3O4 : C 65.24, H 5.24,N 9.92, found: C 65.64, H 5.84, N 9.91.

(E)-3-(2-((4-Fluorophenyl)amino)pyridin-3-yl)-N-(3,4,5-trimethox-yphenyl)acrylamide (5 e): Compound 5 e was prepared accordingto the general method, using (E)-3-(2-(4-fluorobenzyl)pyridin-3-yl)a-crylic acid (257 mg, 1 mmol) and 3,4,5-trimethoxyaniline (146 mg,0.8 mmol) to obtain pure product 5 ) as a yellow solid. Yield: 86 %;mp: 221–222 8C; 1H NMR (300 MHz, [D6]DMSO): d= 10.14 (s, 1 H),8.62 (s, 1 H), 8.18 (s, 1 H), 8.13 (dd, J = 3.1, 1.7 Hz, 1 H), 7.93 (d, J =15.4 1 Hz, 1 H), 7.84 (dd, J = 6.3, 1.7 Hz, 1 H), 7.62–7.57 (m, 1 H), 7.12(s, 2 H), 7.02 (t, J = 8.8 Hz, 2 H), 6.79–6.74 (m, 1 H), 6.75 (d, J =15.4 Hz, 1 H), 3.79 (s, 6 H), 3.67 ppm (s, 3 H); 13C NMR (75 MHz,[D6]DMSO): d= 163.8, 159.2, 156.1, 151.9, 148.2, 136.1, 135.2, 135.5,132.6, 124.0, 121.9, 121.5, 116.7, 114.6, 100.2, 60.4, 55.6 ppm; MS(ESI): m/z 424 [M + 1]+ ; HRMS (ESI m/z) for C23H22FN3O4, calcd:423.1594, found: 424.3688 [M + 1]+ ; IR (KBr, lmax): n= 3443.5,3163.4, 3035.5, 2960.3, 1688.4, 1597.3, 1587.4, 1556.5, 1515.3,1498.3, 1446.5, 1348.7, 1264.5, 1155.4, 1097.0, 792.1, 757.2 cm�1;Anal. calcd for C23H22FN3O4 : C 65.24, H 5.24, N 9.92, found: C 65.66,H 5.85, N 9.96.

(E)-N-(3,4-Dimethoxyphenyl)-3-(2-((4-fluorophenyl)amino)pyri-din-3-yl)acrylamide (5 f): Compound 5 f was prepared accordingto the general method, using (E)-3-(2-(4-fluorobenzyl)pyridin-3-yl)a-crylic acid (257 mg, 1 mmol) and 3,4-dimethoxyaniline (122 mg,0.8 mmol) to obtain pure product 5 f as a yellow solid. Yield: 80 %;mp: 218–220 8C; 1H NMR (300 MHz, [D6]DMSO): d= 10.24 (s, 1 H),8.63 (s, 1 H), 8.28 (s, 1 H), 8.13 (dd, J = 3.4, 1.7 Hz, 1 H), 7.94 (d, J =15.2, 1 Hz, 1 H), 7.84 (dd, J = 6.4, 1.7 Hz, 1 H), 7.62–7.57 (m, 2 H),7.22 (s, 2 H), 7.02 (t, J = 8.7 Hz, 2 H), 6.79–6.74 (m, 1 H), 6.75 (d, J =15.4 Hz, 1 H), 3.75 (s, 3 H), 3.68 ppm (s, 3 H); 13C NMR (75 MHz,[D6]DMSO): d= 163.2, 155.3, 154.4, 153.3, 148.2, 137.3, 135.4, 134.3,132.2, 124.9, 122.0, 121.8, 120.7, 117.9, 115.2, 114.8, 114.5, 113.8,59.8, 55.1 ppm; MS (ESI): m/z 393 [M]+ ; HRMS (ESI m/z) forC22H20FN3O3, calcd: 393.1489, found: 393.5489 [M]+ ; IR (KBr, lmax):n= 3323.5, 3143.4, 3035.5, 2958.3, 1683.4, 1577.8, 1537.4, 1557.5,1525.5, 1498.7, 1476.5, 1348.7, 1265.5, 1155.4, 1097.0, 792.2,757.9 cm�1; Anal. calcd for C22H20FN3O3 : C 67.17, H 5.12, N 10.68,found: C 67.19, H 5.67, N 10.65.

(E)-3-(2-((4-Fluorophenyl)amino)pyridin-3-yl)-N-(4-methoxyphe-nyl)acrylamide (5 g): Compound 5 g was prepared according tothe general method, using (E)-3-(2-(4-fluorobenzyl)pyridin-3-yl)a-crylic acid (257 mg, 1 mmol) and 4-trimethoxyaniline (98 mg,0.8 mmol) to obtain pure product 5 g as a yellow solid. Yield: 87 %;mp: 215–216 8C; 1H NMR (300 MHz, [D6]DMSO): d= 10.06 (s, 1 H),

8.60 (s, 1 H), 8.10 (d, J = 4.1 Hz, 1 H), 7.86–7.79 (m, 2 H), 7.62 (d, J =8.8 Hz, 2 H), 7.56 (dd, J = 5.0, 3.9 Hz, 1 H), 7.00 (s, 2 H), 6.87 (d, J =8.4 Hz, 3 H), 6.73 (d, J = 15.2 Hz, 1 H), 3.72 ppm (s, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 164.1, 155.8, 152.1, 149.3, 148.2, 137.8,134.4, 133.1, 132.7, 125.9, 122.4, 121.9, 121.1, 118.9, 115.6, 114.8,112.5, 110.8, 59.7 ppm; MS (ESI): m/z 364 [M + 1]+ ; HRMS (ESI m/z)for C21H18FN3O2, calcd: 363.1383, found: 364.2378 [M + 1]+ ; IR (KBr,lmax): n= 3343.7, 3153.4, 3015.5, 2956.3, 1679.4, 1583.3, 1587.3,1557.9, 1525.3, 1498.7, 1426.5, 1345.1, 1264.5, 1115.5, 1097.0, 791.1,758.2 cm�1; Anal. calcd for C21H18FN3O2 : C 69.41, H 4.99, N 11.56,found: C 69.48, H 5.99, N 11.66.

(E)-N-(4-Fluorophenyl)-3-(2-((4-fluorophenyl)amino)pyridin-3-yl)acrylamide (5 h): Compound 5 h was prepared according to thegeneral method, using (E)-3-(2-(4-fluorobenzyl)pyridin-3-yl)acrylicacid (257 mg, 1 mmol) and 4-fluoroaniline (89.0 mg, 0.8 mmol) toobtain pure product 5 h as a yellow solid. Yield: 84 %; mp: 214–216 8C; 1H NMR (300 MHz, [D6]DMSO): d= 10.32 (s, 1 H), 8.69 (s, 1 H),8.14 (dd, J = 3.02, 1.5 Hz, 1 H), 7.89–7.84 (m, 2 H), 7.74–7.69 (m, 2 H),7.55–7.50 (m, 2 H), 7.17 (t, J = 8.8 Hz, 2 H), 7.07 (t, J = 8.8 Hz, 2 H),6.90–6.85 (m, 1 H), 6.75 ppm (d, J = 15.4 Hz, 1 H); 13C NMR (75 MHz,[D6]DMSO): d= 163.1, 158.3, 153.6, 151.3, 149.6, 142.7, 139.7, 135.4,131.2, 129.5, 124.2, 123.8, 122.9, 120.9, 120.0, 119.1 ppm; MS (ESI):m/z 352 [M + 1]+ ; HRMS (ESI m/z) for C24H21N5O6S, calcd: 351.1183,found: 352.2481 [M + 1]+ ; IR (KBr): nmax = 3433.5, 3143.4, 3075.5,2930.7, 1687.6, 1587.9, 1584.4, 1526.5, 1511.5, 1498.9, 1446.9,1348.7, 1268.5, 1155.9, 1097.0, 792.1, 787.2 cm�1; Anal. calcd forC21H18FN3O2: C 68.37, H 4.30, N 11.96, found: C 68.87, H 4.49, N11.98.

(E)-N-(6-Nitrobenzo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxyphen-yl)amino)pyridin-3-yl)acrylamide (6 a): Compound 6 a was pre-pared according to the general method, using (E)-3-(2-((3,4,5-trime-thoxyphenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and6-nitrobenzo[d]thiazol-2-amine (150 mg, 0.8 mmol) to obtain pureproduct 6 a as a yellow solid. Yield: 81 %; mp: 241–243 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.70 (s, 1 H), 8.75 (s, 1 H), 8.33 (d, J =4.1 Hz, 1 H), 8.13 (m, 2 H), 7.88 (d, J = 7.6 Hz, 1 H), 7.86 (d, J = 8.4 Hz,1 H), 7.47 (d, J = 8.3 Hz, 1 H), 7.01 (s, 2 H), 6.91–6.86 (m, 2 H), 3.76 (s,6 H), 3.62 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 166.2,160.3, 154.4, 150.3, 149.2, 142.3, 139.4, 135.3, 134.2, 129.9, 127.2,123.8, 122.7, 120.9, 120.2, 120.1, 118.3, 116.8, 79.1, 78.2, 78.1 ppm;MS (ESI): m/z 508 [M + 1]+ ; HRMS (ESI m/z) for C24H21N5O6S, calcd:507.1213, found: 508.1817 [M + 1]+ ; IR (KBr, lmax): n= 3413.5,3165.4, 3035.5, 2973.8, 1685.4, 1607.3, 1587.2, 1555.7, 1525.3,1498.1, 1444.7, 1368.7, 1264.5, 1175.4, 1097.0, 791.1, 756.2 cm�1;Anal. calcd for C24H21N5O6S: C 56.80, H 4.17, N 13.80, found: C56.94, H 4.25, N 13.90.

(E)-N-(6-Chlorobenzo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxyphe-nyl)amino)pyridin-3-yl)acrylamide (6 b): Compound 6 b was pre-pared according to the general method, using (E)-3-(2-((3,4,5-trime-thoxyphenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and6-chlorobenzo[d]thiazol-2-amine (146 mg, 0.8 mmol) to obtain pureproduct 6 b as a yellow solid. Yield: 83 %; mp: 240–242 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.60 (s, 1 H), 8.66 (s, 1 H), 8.23 (d, J =3.1 Hz, 1 H), 8.13 (m, 2 H), 7.88 (d, J = 7.0 Hz, 1 H), 7.76 (d, J = 8.4 Hz,1 H), 7.47 (d, J = 8.4 Hz, 1 H), 7.01 (s, 2 H), 6.91–6.86 (m, 2 H), 3.76 (s,6 H), 3.62 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 165.7,160.1, 152.4, 150.1, 147.2, 140.3, 137.2, 134.5, 134.2, 127.9, 125.2,122.8, 122.1, 120.9, 120.0, 119.1, 118.3, 116.4, 71.1, 68.2, 58.5 ppm;MS (ESI): m/z 499 [M + 1]+ ; HRMS (ESI m/z) for C24H21ClN4O4S, calcd:498.0943, found: 499.1953 [M + 1]+ ; IR (KBr, lmax): n= 3343.5,3125.4, 3015.5, 2972.8, 1686.4, 1627.3, 1567.3, 1575.7, 1525.7,1498.9, 1445.7, 1358.7, 1261.5, 1125.4, 1095.0, 874.2, 781.7,




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757.2 cm�1; Anal. calcd for C24H21ClN4O4S, C 58.00, H 4.26, N 11.27,found: C 58.44, H 4.15, N 11.49.

(E)-N-(6-Methoxybenzo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxy-phenyl)amino)pyridin-3-yl)acrylamide (6 c): Compound 6 c wasprepared according to the general method, using (E)-3-(2-((3,4,5-tri-methoxyphenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol)and 6-methoxybenzo[d]thiazol-2-amine (144 mg, 0.8 mmol) toobtain pure product 6 c as a yellow solid. Yield: 80 %; mp: 250–252 8C; 1H NMR (300 MHz, [D6]DMSO): d= 12.60 (s, 1 H), 8.64 (s, 1 H),8.22 (d, J = 4.5 Hz, 1 H), 8.07 (d, J = 15.4 Hz, 1 H), 7.88 (d, J = 7.55 Hz,1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.60 (d, J = 2.2 Hz, 1 H), 7.06–7.01 (m,3 H), 6.92–6.84 (m, 2 H), 3.81 (s, 3 H), 3.74 (s, 6 H), 3.62 ppm (s, 3 H);13C NMR (75 MHz, [D6]DMSO): d= 164.0, 158.7, 153.5, 150.3, 149.2,147.3, 141.0, 135.3, 138.2, 129.9, 135.4, 133.2, 127.7, 126.1, 121.2,120.9, 120.3, 116.8, 115.3, 77.1, 75.2, 74.8 ppm; MS (ESI): m/z 493[M + 1]+ ; HRMS (ESI m/z) for C25H24N4O5S, calcd: 492.5469, found:493.1534 [M + 1]+ ; IR (KBr, lmax): n= 3341.6, 3127.4, 3018.5, 2972.4,1696.4, 1607.3, 1567.7, 1555.7, 1507.7, 1493.9, 1441.7, 1352.7,1265.5, 1127.4, 1092.0, 871.2, 755.7, 737.8 cm�1; Anal. calcd forC25H24N4O5S: C 60.96, H 4.91, N 11.37, found: C 61.34, H 4.75, N12.59.

(E)-N-(6-Fluorobenzo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxyphe-nyl)amino)pyridin-3-yl)acrylamide (6 d): Compound 6 d was pre-pared according to the general method, using (E)-3-(2-((3,4,5-trime-thoxyphenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and6-fluorobenzo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pureproduct 6 d as a yellow solid. Yield: 92 %; mp: 234–236 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.60 (s, 1 H), 8.66 (s, 1 H), 8.23 (d, J =3.1 Hz, 1 H), 8.13 (m, 2 H), 7.88 (d, J = 7.0 Hz, 1 H), 7.76 (d, J = 8.4 Hz,1 H), 7.47 (d, J = 8.4 Hz, 1 H), 7.01 (s, 2 H), 6.91–6.86 (m, 2 H), 3.76 (s,6 H), 3.62 ppm (s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 164.6,160.3, 156.4, 150.9, 148.9, 140.4, 138.4, 136.4, 135.8, 130.9, 128.2,123.8, 121.9, 120.9, 120.2, 120.1, 118.3, 116.8, 73.1, 68.9, 65.1; MS(ESI): m/z 481 [M + 1]+ ; HRMS (ESI m/z) for C24H21FN4O4S, calcd:480.1268, found: 481.3468 [M + 1]+ ; IR (KBr, lmax): n= 3341.6,3127.4, 3018.5, 2972.4, 1696.4, 1607.3, 1567.7, 1555.7, 1507.7,1493.9, 1441.7, 1352.7, 1265.5, 1127.4, 1092.0, 871.2, 755.7,737.8 cm�1; Anal. calcd for C24H21FN4O4S, C 59.99, H 4.41, N 11.66,found: C 59.84, H 4.55, N 11.79.

(E)-N-(6-Ethoxybenzo[d]thiazol-2-yl)-3-(2-((3,4,5-trimethoxyphe-nyl)amino)pyridin-3-yl)acrylamide (6 e): Compound 6 e was pre-pared according to the general method, using (E)-3-(2-((3,4,5-trime-thoxyphenyl)amino)pyridin-3-yl)acrylic acid (330 mg, 1 mmol) and6-ethoxybenzo[d]thiazol-2-amine (155 mg, 0.8 mmol) to obtainpure product 6 e as a yellow solid. Yield: 92 %; mp: 270–272 8C;1H NMR (300 MHz, [D6]DMSO): d= 12.40 (s, 1 H), 8.65 (s, 1 H), 8.22(d, J = 4.5 Hz, 1 H), 8.08 (d, J = 15.2 Hz, 1 H), 7.87 (d, J = 7.5 Hz, 1 H),7.66 (d, J = 8.8 Hz, 1 H), 7.56 (d, J = 2.2 Hz, 1 H), 7.04 (m, 3 H), 6.91–6.84 (m, 2 H), 4.07 (q, J = 6.9, 6.7 Hz, 2 H), 1.3 ppm (t, J = 6.9 Hz,3 H); 13C NMR (75 MHz, [D6]DMSO): d= 165.3, 163.8, 154.8, 149.3,144.3, 139.5, 136.7, 135.4, 131.5, 129.2, 128.4, 124.3, 120.3, 119.5,118.6, 116.8, 115.7, 104.8, 65.8, 55.7, 15.9 ppm; MS (ESI): m/z 506[M]+ ; HRMS (ESI m/z) for C26H26N4O5S, calcd: 506.1624, found:506.2324 [M]+ ; IR (KBr, lmax): n= 3435.5, 3161.6, 3062.5, 2922.4,1681.4, 1592.4, 1586.5, 1493.9, 1431.7, 1352.7, 1215.5, 1127.4,1092.0, 872.8, 776.8, 732.8 cm�1; Anal. calcd for C24H21ClN4O4S: C61.65, H 5.17, N 11.06, found: C 61.44, H 5.45, N 11.40.

(E)-N-(6-Nitrobenzo[d]thiazol-2-yl)-3-(2-(phenylamino)pyridin-3-yl)acrylamide (6 f): Compound 6 f was prepared according to thegeneral method, using (E)-3-(2-(phenylamino)pyridin-3-yl)acrylicacid (240 mg, 1 mmol) and 6-nitrobenzo[d]thiazol-2-amine

(150 mg, 0.8 mmol) to obtain pure product 6 f as a yellow solid.Yield: 90 %; mp: 220–222 8C; 1H NMR (300 MHz, [D6]DMSO): d=12.42 (s, 1 H), 8.65 (s, 1 H), 8.16–8.13 (m, 1 H), 8.10 (d, J = 6.0 Hz,1 H), 7.93 (d, J = 2.0 Hz, 1 H), 7.83 (dd, J = 6.2, 1.3 Hz, 1 H), 7.68 (d,J = 8.6 Hz, 1 H), 7.56 (d, J = 7.9 Hz, 2 H), 7.37 (dd, J = 6.4, 2.0 Hz, 1 H),7.18 (t, J = 7.9, 7.7 Hz, 2 H), 6.92–6.80 ppm (m, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 174.5, 166.7, 160.1, 159.3, 148.0, 143.9,140.9, 135.0, 131.3, 129.5, 128.2, 124.3, 121.3, 119.4, 117.8, 116.4,113.6, 113.3 ppm; MS (ESI): m/z 418 [M + 1]+ ; HRMS (ESI m/z) forC21H15N5O3S, calcd: 417.0896, found: 417.1996 [M + 1]+ ; IR (KBr,lmax): n= 3342.1, 3173.4, 2923.8, 1686.9, 1607.1, 1582.4, 1507.5,1498.4, 1298.5, 1153.7, 976.3, 812.4, 789.2 cm�1; Anal. calcd forC21H15N5O3S: C 60.42, H 3.62, N 16.78, found: C 60.58, H 3.61, N16.74.

(E)-N-(6-Chlorobenzo[d]thiazol-2-yl)-3-(2-(phenylamino)pyridin-3-yl)acrylamide (6 g): Compound 6 g was prepared according to thegeneral method, using (E)-3-(2-(phenylamino)pyridin-3-yl)acrylicacid (240 mg, 1 mmol) and 6-clorobenzo[d]thiazol-2-amine(144 mg, 0.8 mmol) to obtain pure product 6 g as a yellow solid.Yield: 90 %; mp: 228–230 8C; 1H NMR (300 MHz, [D6]DMSO): d=12.40 (s, 1 H), 8.79 (s, 1 H), 8.21 (d, J = 6.0 Hz, 1 H), 8.12 (d, J =15.4 Hz, 1 H), 7.94–7.88 (m, 2 H), 7.78–7.75 (m, 1 H), 7.57 (d, J =7.7 Hz, 2 H), 7.29–7.24 (m, 3 H), 6.96–6.86 ppm (m, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 164.5, 160.7, 159.1, 151.3, 148.0, 143.9,140.9, 136.0, 133.3, 129.9, 129.5, 125.6, 124.8, 122.4, 118.3, 117.8,116.2, 113.8 ppm; MS (ESI): m/z 408 [M + 1]+ ; HRMS (ESI m/z) forC21H15ClN4OS, calcd: 407.0689, found: 408.5689 [M + 1]+ ; IR (KBr,lmax): n= 3452.1, 3182.0, 2922.4, 1687.5, 1606, 1562, 1507, 1494,1398, 1153, 985.3, 812, 783.5 cm�1; Anal. calcd for C21H15ClN4OS: C61.99, H 3.72, N 13.77, found: C 61.54, H 3.65, N 13.89.

(E)-N-(6-Methoxybenzo[d]thiazol-2-yl)-3-(2-(phenylamino)pyri-din-3-yl)acrylamide (6 h): Compound 6 h was prepared accordingto the general method, using (E)-3-(2-(phenylamino)pyridin-3-yl)a-crylic acid (240 mg, 1 mmol) and 6-methoxybenzo[d]thiazol-2-amine (144 mg, 0.8 mmol) to obtain pure product 6 h as a yellowsolid. Yield: 85 %; mp: 234–236 8C; 1H NMR (300 MHz, [D6]DMSO):d= 12.49 (s, 1 H), 8.78 (s, 1 H), 8.20 (d, J = 3.3 Hz, 1 H), 8.09 (d, J =

15.4 Hz, 1 H), 7.90 (d, J = 7.3 Hz, 1 H), 7.67 (d, J = 8.8 Hz, 1 H), 7.60–7.55 (m,3 H), 7.27 (t, J = 7.9, 7.5 Hz, 2 H), 7.05 (dd, J = 6.4, 2.4 Hz,1 H), 6.96–6.86 (m, 3 H), 3.81 ppm (s, 3 H); 13C NMR (75 MHz,[D6]DMSO): d= 163.8, 156.1, 156.0, 153.5, 149.3, 142.7, 141.2, 137.9,135.6, 132.9, 128.2, 121.3, 121.1, 120.8, 116.7, 115.6, 104.6,55.6 ppm; MS (ESI): m/z 403 [M + 1]+ ; HRMS (ESI m/z) forC22H18N4O2S, calcd: 402.1150, found: 403.1754 [M + 1]+ ; IR (KBr,lmax): n= 3321.4, 3089.3, 2923.5, 2853.3, 1692.1, 1612, 1562, 1507,1494, 1394.3, 1298, 1153, 894.2, 812 cm�1; Anal. calcd forC22H18N4O2S: C 65.65, H 4.51, N 13.92, found: C 65.58, H 4.65, N14.10.

(E)-N-(6-Fluorobenzo[d]thiazol-2-yl)-3-(2-(phenylamino)pyridin-3-yl)acrylamide (6 i): Compound 6 i was prepared according to thegeneral method, using (E)-3-(2-(phenylamino)pyridin-3-yl)acrylicacid (240 mg, 1 mmol) and 6-methoxybenzo[d]thiazol-2-amine(144 mg, 0.8 mmol) to obtain pure product 6 i as a yellow solid.Yield: 86 %; mp: 224–226 8C; 1H NMR (300 MHz, [D6]DMSO): d=12.42 (s, 1 H), 8.65 (s, 1 H), 8.16–8.13 (m, 1 H), 8.10 (d, J = 6.0 Hz,1 H), 7.93 (d, J = 2.0 Hz, 1 H), 7.83 (dd, J = 6.2, 1.3 Hz, 1 H), 7.68 (d,J = 8.6 Hz, 1 H), 7.56 (d, J = 7.9 Hz, 2 H), 7.37 (dd, J = 6.4, 2.0 Hz, 1 H),7.18 (t, J = 7.9, 7.7 Hz, 2 H), 6.92–6.80 ppm (m, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 166.5, 161.5, 159.4, 152.3, 148.1, 143.8,140.4, 136.3, 133.4, 129.8, 129.1, 125.2, 124.9, 122.9, 118.3, 117.8,116.2, 112.8, 102.3 ppm; MS (ESI): m/z 391 [M + 1]+ ; HRMS (ESI m/z)for C21H15FN4OS, calcd: 390.0951, found: 391.0981 [M + 1]+ ; IR (KBr,




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lmax): n= 3462.6, 3184.0, 2922.4, 1687.5, 1606, 1562, 1507, 1498,1398, 1158, 985.3, 822.6, 787.5 cm�1; Anal. calcd for C21H15FN4OS: C64.60, H 3.87, N 14.35, found: C 64.57, H 4.65, N 14.74.

(E)-N-(6-Ethoxybenzo[d]thiazol-2-yl)-3-(2-(phenylamino)pyridin-3-yl)acrylamide (6 j): Compound 6 j was prepared according to thegeneral method, using (E)-3-(2-(phenylamino)pyridin-3-yl)acrylicacid (240 mg, 1 mmol) and 6-methoxybenzo[d]thiazol-2-amine(144 mg, 0.8 mmol) to obtain pure product 6 j as a yellow solid.Yield: 85 %; mp: 242–244 8C; 1H NMR (300 MHz, [D6]DMSO): d=12.50 (s, 1 H), 8.78 (s, 1 H), 8.21 (d, J = 3.7 Hz, 1 H), 8.09 (d, J =

15.4 Hz, 1 H), 7.90 (d, J = 7.3 Hz, 1 H), 7.65 (d, J = 8.8 Hz, 1 H), 7.57–7.55 (m, 3 H), 7.26 (t, J = 8.1, 7.5 Hz, 2 H), 7.03 (dd, J = Hz, 1 H), 6.96–6.85 (m, 3 H), 4.07 (q, J = 6.9, 6.7 Hz, 2 H), 1.35 ppm (t, J = 6.9 Hz,3 H); 13C NMR (75 MHz, [D6]DMSO): d= 163.9, 156.1, 155.4, 153.6,149.4, 142.7, 141.3, 137.9, 135.7, 133.0, 128.2, 121.1, 120.9, 120.1,116.8, 115.7, 115.3, 105.3, 63.6, 14.7 ppm; MS (ESI): m/z 416 [M]+ ;HRMS (ESI m/z) for C23H20N4O2S, calcd: 416.1307, found: 416.2357[M]+ ; IR (KBr, lmax): n= 3424.1, 3013.3, 2923.3, 1682.0, 1602.5,1572.0, 1507.0, 1499.4, 1298.3, 1053, 986.8, 812 cm�1; Anal. calcdfor C23H20N4O2S: C 66.33, H 4.84, N 13.45, found: C 66.58, H 4.98, N13.87.

(E)-3-(2-((4-Chlorophenyl)amino)pyridin-3-yl)-N-(6-nitrobenzo[d]-thiazol-2-yl)acrylamide (6 k): Compound 6 k was prepared accord-ing to the general method, using (E)-3-(2-((4-chlorophenyl)amino)-pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-nitrobenzo[d]thia-zol-2-amine (156 mg, 0.8 mmol) to obtain pure product 6 k asa yellow solid. Yield: 76 %; mp: 245–247 8C; 1H NMR (300 MHz,[D6]DMSO): d= 12.40 (s, 1 H), 8.93 (s, 1 H), 8.23 (dd, J = 3.2, 1.5 Hz,1 H), 8.16 (d, J = 1.8 Hz, 1 H), 8.15 (d, J = 15.3 Hz, 1 H), 7.88 (dd, J =6.2, 1.5 Hz, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 7.63 (d, J = 8.8 Hz, 2 H), 7.48(dd, J = 6.4, 2.0 Hz, 1 H), 7.32 (d, J = 8.8 Hz, 2 H), 6.98–6.92 (m, 1 H),6.87 ppm (d, J = 15.5 Hz, 1 H); 13C NMR (75 MHz, [D6]DMSO): d=164.5, 162.7, 160.1, 159.3, 148.0, 144.3, 143.9, 139.0, 135.0, 131.3,129.6, 127.7, 124.8, 122.4, 121.5, 119.5, 117.3, 116.2, 112.3 ppm; MS(ESI): m/z 452 [M + 1]+ ; HRMS (ESI m/z) for C21H14ClN5O3S, calcd:451.0506, found: 452.6507 [M + 1]+ ; IR (KBr, lmax): n= 3396.2,3102.4, 2923.0, 1688.9, 1624.5, 1585.3, 1507.3, 1498.4, 1298.5,1153.0, 1028.2, 874.4, 835.0 cm�1; Anal. calcd for C21H14ClN5O3S: C55.82, H 3.12, N 15.50, found: C 55.22, H 3.26, N 15.19.

(E)-N-(6-Chlorobenzo[d]thiazol-2-yl)-3-(2-((4-chlorophenyl)ami-no)pyridin-3-yl)acrylamide (6 l): Compound 6 l was prepared ac-cording to the general method, using (E)-3-(2-((4-chlorophenyl)ami-no)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-chlorobenzo[d]-thiazol-2-amine (146 mg, 0.8 mmol) to obtain pure product 6 l asa yellow solid. Yield: 86 %; mp: 248–250 8C; 1H NMR (300 MHz,[D6]DMSO): d= 12.60 (s, 1 H), 8.92 (s, 1 H), 8.23 (dd, J = 3.2, 1.5 Hz,1 H), 8.15 (d, J = 1.8 Hz, 1 H), 8.11 (d, J = 15.4 Hz, 1 H), 7.92 (dd, J =6.32, 1.5 Hz, 1 H), 7.77 (d, J = 8.4 Hz, 1 H), 7.63 (d, J = 8.8 Hz, 2 H),7.48 (dd, J = 6.4, 2.0 Hz, 1 H), 7.32 (d, J = 8.8 Hz, 2 H), 6.98–6.93 (m,1 H), 6.87 ppm (d, J = 15.4 Hz, 1 H); 13C NMR (75 MHz, [D6]DMSO):d= 165.6, 160.7, 158.1, 154.3, 148.7, 144.7, 143.1, 139.4, 135.5,131.3, 129.6, 127.7, 124.5, 121.4, 120.5, 119.6, 116.3, 115.2 ppm; MS(ESI): m/z 441 [M + 1]+ ; HRMS (ESI m/z) for C21H14Cl2N4OS, calcd:440.0265, found: 441.1235 [M + 1]+ ; IR (KBr, lmax): n= 3456.2,3089.0, 2923, 1672, 1602, 1572, 1507, 1469.4, 1291.8, 1143.3, 934.2,812 cm�1; Anal. calcd for C21H14Cl2N4OS: C 57.15, H 3.20, N 12.69,found: C 57.24, H 3.15, N 12.49.

(E)-3-(2-((4-Chlorophenyl)amino)pyridin-3-yl)-N-(6-methoxyben-zo[d]thiazol-2-yl)acrylamide (6 m): Compound 6 m was preparedaccording to the general method, using (E)-3-(2-((4-chlorophenyl)a-mino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-chloroben-

zo[d]thiazol-2-amine (146 mg, 0.8 mmol) to obtain pure product6 m as a yellow solid. Yield: 86 %; mp: 255–256 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.60 (s, 1 H), 8.96 (s, 1 H), 8.23 (dd, J =3.2, 1.3 Hz, 1 H), 8.08 (d, J = 15.4 Hz, 1 H), 7.92 (dd, J = 1.3, 6.2 Hz,1 H), 7.67–7.59 (m, 4 H), 7.32 (d, J = 8.8 Hz, 2 H), 7.06 (dd, J = 1.3,6.2 Hz, 1 H), 6.97–6.93 (m, 1 H), 6.86 (d, J = 15.4 Hz, 1 H), 3.81 ppm(s, 3 H); 13C NMR (75 MHz, [D6]DMSO): d= 166.7, 160.1, 159.4, 148.0,143.9, 139.0, 138.4, 135.0, 131.3, 129.3, 128.5, 127.4, 126.4, 124.3,122.4, 122.1, 116.3, 115.4, 113.6, 109.4, 55.8 ppm; MS (ESI): m/z 437[M]+ ; HRMS (ESI m/z) for C22H17ClN4O2S, calcd: 437.0794, found:437.1794 [M]+ ; IR (KBr, lmax): n= 3382.3, 3057.1, 2923, 1685.5,1602.5, 1572.6, 1507.4, 1494.3, 1268.3, 1158, 894.3, 812 cm�1; Anal.calcd for C22H17ClN4O2S: C 60.48, H 3.92, N 12.82, found: C 61.14, H3.15, N 12.09.

(E)-3-(2-((4-Chlorophenyl)amino)pyridin-3-yl)-N-(6-fluoroben-zo[d]thiazol-2-yl)acrylamide (6 n): Compound 6 n was preparedaccording to the general method, using (E)-3-(2-((4-chlorophenyl)a-mino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-fluoroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product6 n as a yellow solid. Yield: 86 %; mp: 240–242 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.40 (s, 1 H), 8.93 (s, 1 H), 8.23 (dd, J =3.2, 1.5 Hz, 1 H), 8.16 (d, J = 1.8 Hz, 1 H), 8.15 (d, J = 15.3 Hz, 1 H),7.88 (dd, J = 6.2, 1.5 Hz, 1 H), 7.78 (d, J = 8.4 Hz, 1 H), 7.63 (d, J =8.8 Hz, 2 H), 7.48 (dd, J = 6.4, 2.0 Hz, 1 H), 7.32 (d, J = 8.8 Hz, 2 H),6.98–6.92 (m, 1 H), 6.87 ppm (d, J = 15.5 Hz, 1 H); 13C NMR (75 MHz,[D6]DMSO): d= 164.4, 162.9, 160.5, 159.8, 148.1, 142.3, 140.9, 139.1,135.5, 131.4, 129.6, 127.3, 123.8, 122.4, 121.6, 119.7, 117.8, 116.1,112.8 ppm; MS (ESI): m/z 425 [M + 1]+ ; HRMS (ESI m/z) for-C21H14ClFN4OS, calcd: 424.0561, found: 425.2530 [M + 1]+ ; IR (KBr,lmax): n= 34562.7, 3104.2, 2923, 1692.7, 1602.3, 1572.8, 1507.2,1494.2, 1295.3, 1153, 947.2, 812 783.2 cm�1; Anal. calcd forC21H14ClFN4OS: C 59.36, H 3.32, N 13.19, found: C 59.24, H 3.16, N13.39.

(E)-3-(2-((4-Chlorophenyl)amino)pyridin-3-yl)-N-(6-ethoxyben-zo[d]thiazol-2-yl)acrylamide (6 o): Compound 6 o was preparedaccording to the general method, using (E)-3-(2-((4-chlorophenyl)a-mino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-fluoroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product6 o as a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.60 (s, 1 H), 8.65 (s, 1 H), 8.22 (d, J =4.5 Hz, 1 H), 8.08 (d, J = 15.2 Hz, 1 H), 7.85 (d, J = 6.6 Hz, 1 H), 7.66(d, J = 8.8 Hz, 1 H), 7.56 (d, J = 2.2 Hz, 1 H), 7.05–7.01 (m, 3 H), 6.91–6.84 (m, 2 H), 4.07 (q, J = 6.9, 6.7 Hz, 2 H), 1.34 ppm (t, J = 6.9 Hz,3 H); 13C NMR (75 MHz, [D6]DMSO): d= 165.6, 160.9, 159.5, 149.0,145.9, 139.9, 138.4, 135.6, 131.7, 129.3, 128.5, 127.8, 126.4, 124.3,122.6, 122.6, 116.8, 114.4, 113.6, 109.4, 55.8, 16.3 ppm; MS (ESI): m/z453 [M + 1]+ ; HRMS (ESI m/z) for C23H19ClN4O2S, calcd: 452.0917,found: 453.0917 [M + 1]+ ; IR (KBr, lmax): n= 3324.6, 3109.6, 2923,1672, 1686.2, 1609.4, 1572, 1507, 1464, 1298, 1153, 812 cm�1; Anal.calcd for C23H19ClN4O2S: C 61.26, H 4.25, N 12.42;, found: C 61.34, H4.75, N 12.59.

(E)-3-(2-((4-Methoxyphenyl)amino)pyridin-3-yl)-N-(6-nitroben-zo[d]thiazol-2-yl)acrylamide (6 p): Compound 6 p was preparedaccording to the general method, using (E)-3-(2-((4-methoxypheny-l)amino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-nitroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product6 p as a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.65 (s, 1 H), 8.65 (s, 1 H), 8.24 (d, J =3.6 Hz, 2 H), 8.11 (d, J = 15.3 Hz, 1 H), 7.95 (dd, J = 6.5, 2.2 Hz,1 H),7.84 (d, J = 7.0 Hz,1 H),7.78–7.75 (m, 1 H), 7.46 (d, J = 8.6 Hz, 2 H),7.32–7.28 (m, 2 H), 6.88–6.82 (m, 3 H), 3.73 ppm (s, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 166.1, 161.2, 159.3, 140.3, 138.9, 137.2,




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136.2, 128.9, 126.5, 126.0, 125.2, 121.2, 120.1, 117.8, 116.4, 115.8,115.0, 114.9, 104.3, 55.9 ppm; MS (ESI): m/z 448 [M + 1]+ ; HRMS (ESIm/z) for C22H17N5O4S, calcd: 447.1001, found: 448.1239 [M + 1]+ ; IR(KBr, lmax): n= 3451.4, 3089.1, 2932.1, 1682.0, 1602.4, 1592.3,1507.2, 1464.1, 1218.6, 1053.4, 812 cm�1; Anal. calcd forC22H17FN4O2S: C 59.05, H 3.83, N 15.65, found: C 59.98, H 3.64, N15.45.

(E)-N-(6-Chlorobenzo[d]thiazol-2-yl)-3-(2-((4-methoxyphenyl)ami-no)pyridin-3-yl)acrylamide (6 q): Compound 6 q was prepared ac-cording to the general method, using (E)-3-(2-((4-methoxyphenyl)a-mino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-chloroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product6 q as a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.57 (s, 1 H), 8.62 (s, 1 H), 8.13 (d, J =3.4 Hz, 2 H), 8.10 (d, J = 15.3 Hz, 1 H), 7.91 (dd, J = 6.5, 2.1 Hz,1 H),7.83 (d, J = 7.0 Hz,1 H),7.78–7.74 (m, 1 H), 7.46 (d, J = 8.8 Hz, 2 H),7.32–7.18 (m, 1 H), 6.88–6.82 (m, 4 H), 3.72 ppm (s, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 164.4, 159.1, 154.6, 154.2, 149.8, 147.6,138.7, 135.8, 134.1, 133.4, 127.3, 126.8, 122.8, 121.8, 121.5, 120.3,115.8, 114.9, 113.6, 55.2 ppm; MS (ESI): m/z 448 [M + 1]+ ; HRMS (ESIm/z) for C22H17N5O4S, calcd: 447.1001, found: 448.1605 [M + 1]+ ; IR(KBr, lmax): n= 3378.2, 3056.8, 2998.5„ 1689.3, 1601.4, 1572.8,1507.6, 1464.6, 1248.3, 1153, 1089.4, 907.5, 812 cm�1; Anal. calcdfor C22H17N5O4S: C 59.05, H 3.83, N 15.65, found: C 59.57, H 3.77, N15.47.

(E)-N-(6-Methoxybenzo[d]thiazol-2-yl)-3-(2-((4-methoxyphenyl)-amino)pyridin-3-yl)acrylamide (6 r): Compound 6 r was preparedaccording to the general method, using (E)-3-(2-((4-methoxypheny-l)amino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-chloroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product 6 ras a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR (300 MHz,[D6]DMSO): d= 12.52 (s, 1 H), 8.61 (s, 1 H), 8.13 (dd, J = 3.0, 1.5 Hz,1 H), 8.09 (d, J = 15.4 Hz, 1 H), 7.84 (dd, J = 6.0, 1.5 Hz, 1 H), 7.67 (d,J = 8.8 Hz, 1 H), 7.60 (d, J = 2.4 Hz, 1 H), 7.47 (d, J = 9.0 Hz, 2 H), 7.05(dd, J = 6.2, 2.6 Hz, 1 H), 6.88–6.81 (m, 4 H), 3.81 (s, 3 H), 3.73 ppm(s, 1 H); 13C NMR (75 MHz, [D6]DMSO): d= 163.8, 156.0, 154.0, 149.5,142.7, 138.0, 135.5, 134.1, 132.9, 122.5, 121.1, 120.5, 115.8, 114.9,114.7, 113.4, 104.6, 55.6, 55.1 ppm; MS (ESI): m/z 433 [M + 1]+ ;HRMS (ESI m/z) for C23H20N4O3S, calcd: 432.1256, found: 433.5421[M + 1]+ ; IR (KBr, lmax): n= 3321.5, 3079.3, 2923.0, 1672.3, 1612.5,1592.5, 1507.9, 1494.0, 1238.7, 1153.2, 1043.2, 978.5, 815.3 cm�1;Anal. calcd for C23H20N4O3S: C 63.87, H 4.66, N 12.95, found: C63.08, H 4.64, N 12.91.

(E)-N-(6-Fluorobenzo[d]thiazol-2-yl)-3-(2-((4-methoxyphenyl)ami-no)pyridin-3-yl)acrylamide (6 s): Compound 6 s was prepared ac-cording to the general method described, using (E)-3-(2-((4-me-thoxyphenyl)amino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and6-chlorobenzo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pureproduct 6 s as a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR(300 MHz, [D6]DMSO): d= 12.61 (s, 1 H), 8.63 (s, 1 H), 8.14 (d, J =3.6 Hz, 2 H), 8.10 (d, J = 15.4 Hz, 1 H), 7.93 (dd, J = 6.4, 2.1 Hz,1 H),7.84 (d, J = 7.0 Hz,1 H),7.78–7.75 (m, 1 H), 7.46 (d, J = 8.6 Hz, 2 H),7.32–7.28 (m, 1 H), 6.88–6.82 (m, 4 H), 3.73 ppm (s, 3 H); 13C NMR(75 MHz, [D6]DMSO): d= 164.4, 159.4, 155.3, 154.2, 150.1, 148.3,139.1, 135.9, 134.5, 133.9, 128.3, 127.8, 124.2, 121.9, 121.6, 120.3,116.3, 115.7, 114.3, 55.5 ppm; MS (ESI): m/z 421 [M + 1]+ ; HRMS (ESIm/z) for C22H17FN4O2S, calcd: 420.1056, found: 421.1451 [M + 1]+ ;IR (KBr, lmax): n= 3376.3, 3027.2, 2924.2, 1689.0, 1632.6, 1573.7,1517.3, 1484.3, 1289.3, 1123.8, 989.9, 876.4, 812 cm�1; Anal. calcdfor C22H17FN4O2S: C 62.84, H 4.08, N 13.33, found: C 62.08, H 4.74,N 13.41.

(E)-N-(6-Ethoxybenzo[d]thiazol-2-yl)-3-(2-((4-methoxyphenyl)ami-no)pyridin-3-yl)acrylamide (6 t): Compound 6 t was prepared ac-cording to the general method, using (E)-3-(2-((4-methoxyphenyl)a-mino)pyridin-3-yl)acrylic acid (274 mg, 1 mmol) and 6-chloroben-zo[d]thiazol-2-amine (134 mg, 0.8 mmol) to obtain pure product 6 tas a yellow solid. Yield: 86 %; mp: 260–262 8C; 1H NMR (300 MHz,[D6]DMSO): d= 12.45 (s, 1 H), 8.62 (s, 1 H), 8.14 (d, J = 3.5 Hz, 1 H),8.09 (d, J = 15.2 Hz, 1 H), 7.84 (d, J = 7.5 Hz, 1 H), 7.65 (d, J = 8.6 Hz,1H0, 7.56 (d, J = 2.2 Hz, 1 H), 7.47 (d, J = 8.8 Hz, 2 H), 7.04 (dd, J =6.4, 2.4 Hz, 1 H), 6.88–6.80 (m, 4 H), 4.05 (q, J = 6.9 Hz, 2 H), 3.72 (s,1 H), 1.34 ppm (t, J = 6.9, 3 H); 13C NMR (75 MHz, [D6]DMSO): d=163.8, 156.0, 155.3, 154.4, 154.0, 149.4, 142.6, 138.0, 135.4, 134.1,132.9, 122.5, 121.0, 120.5, 115.8, 115.2, 114.7, 113.4, 105.2, 63.5,55.0, 14.6 ppm; MS (ESI): m/z 447 [M + 1]+ ; HRMS (ESI m/z) forC24H22N4O3S, calcd: 446.1413, found: 447.8621 [M + 1]+ ; IR (KBr,lmax): n= 3457.1, 3191.0, 2921, 1682, 1602, 1582, 1507, 1494, 1298,1153, 812, 759.3, 743.6 cm�1; Anal. calcd for C24H22N4O3S: C 64.56, H4.97, N 12.55, found: C 64.58, H 4.65, N 12.81.

Biology

In vitro antitumor screening : The synthesized compounds (5 a–hand 6 a–t) were evaluated for their in vitro cytotoxicity in fourhuman cancer cell lines (A549, DU-145, HeLa, and HepG2). Drug ex-posure was continuous for 48 h, and a sulforhodamine B (SRB) pro-tein assay was used to estimate cell viability or growth. The celllines were grown in Dulbecco’s modified Eagle’s medium contain-ing 10 % fetal bovine serum and 2 mm l-glutamine and wereseeded into 96-well plates in 100 mL at plating densities dependingon the doubling time of individual cell lines. The plates were incu-bated at 37 8C, 5 % CO2, 95 % air, and 100 % relative humidity for24 h prior to the addition of experimental drugs. Aliquots (3 mL) ofthe drug dilutions were added to cells resulting in the requiredfinal drug concentrations. For each compound four concentrations(0.1, 1, 5, and 10 mm) were evaluated, and each were done in tripli-cate wells. Plates were incubated for a further 48 h, and the assaywas terminated by the addition of 50 mL cold trichloroacetic acid(TCA; final concentration, 10 % TCA) and incubated for 60 min at4 8C. The plates were washed five times with water and air-dried.SRB solution (50 mL) at 0.4 % (w/v) in 1 % acetic acid was added toeach of the wells, and plates were incubated for 20 min at roomtemperature. The residual dye was removed by washing five timeswith 1 % acetic acid. The plates were air-dried. Bound stain wassubsequently eluted with 10 mm Trizma base, and the absorbancewas read on an ELISA plate reader at a wavelength of 560 nm. Per-cent growth was calculated on a plate-by-plate basis for test wellsrelative to control wells. The above determinations were repeatedthree times. Percentage growth was expressed as the ratio of theaverage absorbance of the test well to the average absorbance ofthe control wells, � 100. Growth inhibition of 50 % (GI50), which isthe drug concentration resulting in a 50 % decrease in the net pro-tein increase (as measured by SRB staining) in control cells duringthe drug incubation, was calculated from [(t0�t)/(C�t0)] � 100 = 50,for which t0 is the optical density (OD) at time zero, C is the OD ofcontrol, and t is the OD of test growth in the presence of drug.

Tubulin polymerization assay : An in vitro assay for monitoringthe time-dependent polymerization of tubulin to microtubules wasperformed using a fluorescence-based tubulin polymerizationassay kit (BK011P, Cytoskeleton Inc.) according to the manufactur-er’s protocol. The reaction mixture, in a final volume of 10 mL inPEM buffer (80 mm PIPES, 0.5 mm EGTA, 2 mm MgCl2, pH 6.9), con-tained 2 mg mL�1 porcine brain tubulin, fluorescent reporter, and1 mm GTP in the presence or absence of test compounds (6 d and




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6 p ; 3 mm) at 37 8C. Tubulin polymerization was followed by moni-toring the fluorescence enhancement due to the incorporation ofa fluorescence reporter into microtubules as polymerization pro-ceeds. Fluorescence emission at 420 nm (excitation wavelength360 nm) was measured for 1 h at 1-min intervals in a multimodeplate reader (Tecan multimode reader). E7010 was used as a posi-tive control under similar experimental conditions. The IC50 value isdefined as the concentration required to inhibit 50 % of the poly-merization of tubulin assembly relative to control.

Cell-cycle analysis : Flow cytometric analysis (FACS) was performedto evaluate the distribution of the cells through the cell-cyclephases. A549 lung cancer cells were incubated with compounds6 d and 6 p at a concentration of 2 mm for 48 h along with E7010at the same concentration. Untreated and treated cells were har-vested, washed with phosphate-buffered saline (PBS), fixed in ice-cold 70 % ethanol and stained with propidium iodide (Sigma–Al-drich). Cell-cycle distribution was gauged by flow cytometry(Becton Dickinson FACS Caliber) as described earlier.[34]

Analysis of soluble versus polymerized tubulin in cells : Cellswere plated in 24-well dishes, grown to 60–80 % confluency, andtreated with either no drug, or 2 mm 6 d or 6 p ; nocodozole andpaclitaxel (each at 1 mm) were used as positive and negative con-trols, respectively. Cells were incubated with drugs for 48 h, thenthe media was removed, cells were rinsed in 1 � PBS at 22 8C, har-vested at the same temperature in lysis buffer containing 0.1 m

PIPES, 1 mm EGTA, 1 mm MgSO4, 30 % glycerol, 5 % DMSO, 5 mm

GTP, 0.125 % NP-40, and protease inhibitors, including aprotinin[200 U mL�1] , pH 6.9 and then centrifuged at 13 200 g at 22 8C for30 min in a Sorval Legendmicro 21R model temperature-controlledcentrifuge (Thermo scientific), to separate polymerized (P) fromsoluble (S) tubulin. Pellets of polymerized P tubulin were resus-pended in a volume of lysis buffer equal to the soluble S fraction,and resolved by 10 % SDS-PAGE as described earlier.[34] After trans-fer to NC membrane immunoblotting was performed with mouseanti-a-tubulin antibody [DMIA, Sigma, St. Louis, MO, USA], followedby a horseradish peroxidase (HRP)-conjugated secondary antibody(Sigma). The blot was imaged using a Phosphorimager (Fujifilm,Japan). Quantitative analysis of the S and P fractions was done bydensitometry using Gene-box (Syngene).

Caspase assay : A549 cells were plated in six-well plates, grown to60–80 % confluency, and treated with either no drug or test com-pounds 6 d and 6 p each at 2 mm. E7010 was used as positive con-trol. After 24 and 48 h, cells were collected by scraping, washedwith PBS, and centrifuged to collect pellet. The cells were lysed in200 mL 1 � lysis buffer by purging at least through an insulin sy-ringe followed by incubation on ice for 10–20 min. The lysate wascentrifuged at 13 200 rpm (15 600 g) for 20 min at 4 8C and trans-ferred the supernatant to fresh tubes. In a 96-well black polystyr-ene plate, 50 mL 2 � assay buffer, 50 mL cell lysate, and 2 mL cas-pase-3 substrates were taken. The reaction was allowed to proceedfor 1 h. The fluorescence generated by the release of the fluoro-genic group AFC upon cleavage by caspase-3 was measured by ex-citation at 400 nm and emission at 505 nm for every 5 min over1 h. Protein was estimated by the Bradford method and normal-ized accordingly using Tecan multimode reader.

Hoechst staining : A549 cells were seeded at a density of 10 000cells over 18 mm cover slips and incubated for 24 h. The mediumwas then replaced, and cells were treated with compounds 6 d and6 p at 2 mm for 48 h. Cells treated with vehicle (0.001 % DMSO)were included as controls for all experiments. After 48 h of treat-ment, Hoechst 33258 (Sigma–Aldrich) were added to the medium

at a concentration of 0.5 mg mL�1 containing 4 % paraformalde-hyde. After incubation for 30 min at 37 8C, cells from each dishwere captured from randomly selected fields under a fluorescencemicroscope (Leica, Germany) to qualitatively determine the apop-totic cells based on their relative fluorescence, chromatin conden-sation, and nuclear fragmentation.

Immunohistochemistry and microscopy : A549 cells were seededon glass cover slips, incubated for 48 h in the presence or absenceof test compounds 6 d and 6 p (2 mm). Following the terminationof incubation, cells were fixed with 3 % paraformaldehyde, 0.02 %glutaraldehyde in PBS, and permeabilized by dipping the cells in100 % methanol (�20 8C). Later, cover slips were blocked with 1 %BSA in PBS for 1 h followed by incubation with a primary anti-tu-bulin (mouse monoclonal) antibody and FITC-conjugated secon-dary mouse anti-IgG antibody. Photographs were taken using theconfocal microscope, equipped with FITC settings, and the pictureswere analyzed for the integrity of microtubule network. In parallelexperiments, E7010 (1 mm) was used as positive control for analyz-ing microtubule integrity.

Molecular modeling : All the geometries were optimized in Gaussi-an 09 using the PM3 semi-empirical method.[35] The protein struc-ture was downloaded from the RCSB Protein Data Bank (PDB ID:3E22).[32] Docking studies were performed with AutoDock 4.2 soft-ware.[33] The analysis of intermolecular interactions was performedwith PyMOL v. 0.99.[36]

Acknowledgements

M.A. acknowledges CSIR-UGC New Delhi for the award ofa senior research fellowship. We also acknowledge CSIR for finan-cial support under the 12th Five-Year plan project “AffordableCancer Therapeutics (ACT)” (CSC0301).

Keywords: 2-anilinopyridine-3-acrylamides · apoptosis ·growth inhibition · molecular docking · tubulin polymerization

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FULL PAPERS

A. Kamal,* M. Ashraf, M. N. A. Khan,V. D. Nimbarte, S. Faazil,N. V. Subba Reddy, S. Taj

&& –&&

Synthesis and Cytotoxic Activity of 2-Anilinopyridine-3-Acrylamides asTubulin Polymerization Inhibitors

Preventing assembly: A series of 2-ani-linopyridine-3-acrylamides was synthe-sized and evaluated for their anticancerpotential against four human cancer celllines. Representative compounds suchas 6 d and 6 p displayed good anti-pro-liferative activity in the micromolarrange against the A549 cell line byinhibiting tubulin polymerization.



retracted: synthesis and cytotoxic activity of 2-anilinopyridine-3-acrylamides as tubulin...

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