Accepted Manuscript

Synthesis of 2-anilinopyridine dimers as microtubule targeting and apoptosisinducing agents

Ahmed Kamal, S.M. Ali Hussaini, V. Lakshma Nayak, M. Shaheer Malik, M.Lakshmi Sucharitha, Thokhir Basha Shaik, Md. Ashraf, Chandrakant Bagul

PII: S0968-0896(14)00775-5DOI: http://dx.doi.org/10.1016/j.bmc.2014.11.001Reference: BMC 11890

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 24 September 2014Revised Date: 1 November 2014Accepted Date: 1 November 2014

Please cite this article as: Kamal, A., Ali Hussaini, S.M., Lakshma Nayak, V., Shaheer Malik, M., LakshmiSucharitha, M., Shaik, T.B., Ashraf, Md., Bagul, C., Synthesis of 2-anilinopyridine dimers as microtubule targetingand apoptosis inducing agents, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.11.001

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Synthesis of 2-anilinopyridine dimers as microtubule targeting and

apoptosis inducing agents

Ahmed Kamal,*a,b S. M. Ali Hussaini,a V. Lakshma Nayak,a M. Shaheer Malik,a M. Lakshmi

Sucharitha,b Thokhir Basha Shaik,a Md. Ashraf,a Chandrakant Bagulb

aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,

Hyderabad 500007, India

bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and

Research (NIPER), Hyderabad 500 037, India

Abstract

A series of 2-anilinopyridine dimers have been synthesized and evaluated for their anticancer

potential. Most of the compounds have showed significant growth inhibition of the cell lines

tested and compound 4d was most effective amongst the series displaying a GI50 of 0.99 µM

specifically against the prostate cancer cell line (DU145). Studies to understand the

mechanism of action of 4d indicates that it disrupts microtubule dynamics by inhibiting

tubulin polymerization thereby arresting the cell cycle in G2/M phase. Competitive

colchicine binding assay suggests that 4d binds into colchicine binding site of the tubulin.

Further from some detailed biological studies like mitochondrial membrane potential,

caspase-3 assay, DNA fragmentation analysis and Annexin V-FITC assay it is evident that 4d

induces apoptosis. Molecular modeling studies provide an insight into the binding modes of

4d with colchicine binding site of tubulin and the data obtained correlates with the

antiproliferative activity.

Keywords: Microtubules, E7010, 2-anilinopyridine, dimers, tubulin polymerization, apoptosis

* Corresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189;

E-mail addresses: ahmedkamal@iict.res.in

Introduction

Microtubules are long, filamentous, tube-shaped protein polymers formed by head to

tail assembly of α and β tubulin heterodimers.1 These are essentially found in all eukaryotic

cells and are crucial for the development and maintenance of cell shape, intracellular

transport, cell signalling, cell division and mitosis. These are highly dynamic polymers and

their depolymerization to tubulin and polymerization back to form the microtubules provide

scope for the development of anticancer drugs that may intervene in such dynamics. In fact a

large number of structurally diverse antimitotic agents derived from natural sources or

synthetic libraries are known to interfere with microtubule dynamics.2 Agents that inhibit

tubulin, the main component of microtubules are known to interfere with their dynamics

thereby causing cell death by arresting cell cycle in G2/M phase.3 Another reason for

significant interest in developing agents interfering with microtubule dynamics is their role in

endothelial cell biology. Colchicine binding site on tubulin, named after the natural product

colchicine (1) isolated from Colchicum autumnale is one of the widely studied targets for the

development of anticancer agents.4 Molecules that bind into colchicine domain such as

combretastatin-A4 (CA-4, 2) not only inhibits tubulin polymerization but are also reported to

target tumor vasculature thereby serving as vascular disrupting agents5. Moreover, these

agents can also prevent the formation of new blood vessels (angiogenesis) cutting off the

supply of blood to the tumor cells and thereby function as inhibitors of angiogenesis.

In recent years, several molecules structurally distinct from colchicine have been

crystallized in the colchicine binding site. E7010 (3) is a sulfonamide that binds to colchicine

binding site on β tubulin subunit leading to cell cycle arrest in G2/M phase.6 It is an orally

bioavailable tubulin binding agent presently under phase II clinical trials.7 It was found to

possess a wide spectrum of antitumor activity and is also found to be effective against certain

multidrug and vincristine resistant cell lines. A close analysis of the structure of E7010

binding to colchicine binding site reveals that its pyridine and methoxy groups superimpose

with A and C rings of the colchicine respectively, while the sulphonamide bridge overlaps

with the B ring. Interestingly, it binds much deeply than colchicine in β tubulin pocket but

does not interact with α subunit of the tubulin.

On the other hand, dimeric structures of bioactive heterocyclic scaffolds are of

significant potential in medicinal chemistry.8 Many research groups have reported dimers of

various bioactive compounds such as pyrrolobenzodiazepine9a,b (SJG 136),9c monastrol,10

naphthalimides,11 flavanoids12 etc possessing better activity than the corresponding

monomers. In this approach the two active monomers are separated by different spacers that

include alkyl, piperizine, triazole and other groups. Most of these dimers were found to

possess improved affinity towards the corresponding receptors than the parent monomer. This

could be attributed to the existence of numerous proteins responsible for cell proliferation in

hetero or homodimeric states.8 Many such proteins have been identified to be crucial for

oncogenic signalling pathway and are targeted to reduce tumor cell proliferation. Since

microtubules are also composed of α and β tubulin heterodimers and continuing our search

for newer anticancer agents based on E7010 nucleus13-16 a new dimeric series of conjugates

comprising of two 2-anilinopyridinyl moieties connected with an amide bond have been

developed. We herein report the synthesis and biological evaluation of these 2-

anilinopyridinyl dimers that inhibit tubulin polymerization and induce cytotoxic effects.

<Figure 1>

Chemistry

The 2-anilinonicotinic acid derivatives required as precursors for the synthesis of

dimers (4a-p) were synthesized by following the synthetic route depicted in Scheme 1.

Commercially available 2-choloronicotinic acid (5) was converted to its ethyl ester (6) by

refluxing in ethanol with catalytic amount of conc. H2SO4. The ester (6) was then heated with

substituted anilines (7a-d) in ethylene glycol to afford the aniline coupled ethyl nicotinate

(8a-d) in very good yields (83-89%). In order to determine the effect of substitutents on the

cytotoxic activity, electron donating such as methoxy, trimethoxy, fluoro and strong electron

withdrawing substituents such as trifluoromethyl were studied on the aryl ring of the aniline

moiety. This coupled ester was hydrolysed under basic conditions by heating with 2N NaOH

to get 2-anilinonicotinic acid derivatives (9a-d).

<Scheme 1>

Another set of intermediates, 2-anilinopyridin-3-yl amines required for the synthesis

of dimers (4a-p) were synthesized according to Scheme 2. A mixture of 2-chloro-3-

nitropyridine (10) and substituted anilines (11a-d) was heated in ethylene glycol to obtain the

coupled product 2-anilino-3-nitropyridine derivatives (12a-d) in excellent yields (90-94%).

Differently substituted anilines with both electron donating as well as electron withdrawing

substituents such as methoxy, fluoro and chloro respectively were used to study their effect

on the cytotoxic activity. The nitro group of 12a-d was later reduced using stannous chloride

in refluxing methanol for 3 hours to obtain the 2-anilino-pyridin-3-amine intermediates (13a-

d) in 85-77% yields.

<Scheme 2>

Finally the synthesis of target compounds, 2-anilinopyridinyl dimers was

accomplished as outlined in Scheme 3. The acid derivatives (9a-d) obtained from Scheme 1

and the amines (13a-d) procured by following Scheme 2 were coupled using EDC and HOBt

in DMF to afford the target 2-anilinopyridinyl-2-anilinonicotinamides (4a-p) in good yields

(71-87%).

<Scheme 3>

Biology

Antiproliferative activity

All the synthesized 2-anilinopyridinyl dimers (4a-p) were tested for antiproliferative

activity against selected human cancer cell lines like A549 (lung), MCF-7 (breast) and DU-

145 (prostate) by using sulforhodamine B (SRB) method17 and the results are tabulated in

Table 1, wherein E7010 was used as the reference compound. Most of the compounds have

shown selectivity towards the DU-145 cell line and were found to be active below 10 µM

concentration. Several conjugates such as 4a, 4l and 4p have displayed good growth

inhibition towards all the cell lines tested with varying magnitude. Compound 4d was found

to be the most effective dimeric conjugate amongst the series exhibiting GI50 value of 0.99

µM against prostate cancer cell line (DU-145). However some of the dimeric conjugates were

found to be inactive even at 100 µM concentration.

<Table 1>

From the cytotoxicity data it has been noticed that conjugates resulting from the

reaction between acids and amines with electron donating substituents at 4-position have

appreciable anticancer potential. Similarly, stronger electron withdrawing groups on the acid

moiety bestows good anticancer activity. However, most of the compounds that possess a

methoxy substituent on the acid moiety were found to be inactive whereas compounds with

fluoro substituent have shown enhanced activity in some specific cell lines. Moreover,

compounds with fluoro and chloro substituents on the amine moiety possess appreciable

cytotoxic activity. Thus, it could be concluded that electron donating substituents such as

fluoro and chloro on the amine moiety is beneficial for the activity.

Cell cycle analysis

Many cytotoxic compounds exert their growth inhibitory effect either by arresting the

cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combined

effect of both cycle block and apoptosis.18 Furthermore, regulation of the cell cycle and

apoptosis are considered to be effective strategies in the development of cancer

therapeutics.19 SRB assay was performed to evaluate the cytotoxic potential of these dimers

against human prostate cancer cell line (DU-145) and compared with E7010. Results revealed

that 4d and E7010 showed significant cytotoxic activity against human prostate cancer cells

with IC50 values 1.31 and 1.99 µM respectively. Therefore, it was considered of interest to

understand whether this inhibition of cell growth was due to cell cycle arrest. In this study

DU-145 cells were treated with 4d at 1 and 2 µM concentrations for 48 h and E7010 was

used as reference compound in this study. The data obtained clearly indicates that 4d arrested

cell cycle at G2/M phase (Figure 2). Moreover, 4d showed 32.49 and 41.19 % of cell

accumulation in G2/M phase at 1 and 2 µM concentrations respectively, whereas E7010

showed 36.52 % cell accumulation in G2/M phase at 2 µM concentration.

<Figure 2>

<Table 2>

Effect of 4d on tubulin polymerization

In general G2/M cell cycle arrest is strongly associated with inhibition of tubulin

polymerization6 and since 4d caused cell cycle arrest at G2/M phase, it was considered of

interest to investigate its microtubule inhibitory function. Tubulin subunits are known to

heterodimerize and self-assemble to form microtubules in a time dependent manner. The

progression of tubulin polymerization20 was thus examined by monitoring the increase in

fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate for 1 h

at 37 oC. Compound 4d significantly inhibited tubulin polymerization by 64.28 %, whereas

E7010 inhibited it by 59.22 % (Figure 3).

<Figure 3>

This was followed by the evaluation of IC50 values for this compound and results

indicate that 4d showed better tubulin-assembly inhibition with an IC50 value of 2.16 µM and

was comparable to E7010 (IC50=2.40 µM). The effect of 4d on the inhibition of tubulin

assembly correlated well with its significant antiproliferative activity.

Competitive colchicine binding assay

This dimer (4d), showed good inhibitory effects on tubulin polymerization similar to

that of E7010 which is reported to bind into colchicine binding site of tubulin , therefore it

was considered of interest to investigate the binding of 4d on this site of the tubulin. Hence,

a fluorescence based assay was carried out21 taking E7010 as the positive control and taxol as

the negative control. Thus 4d, E7010 and taxol were separately coincubated with colchicine

at 37 ˚C for 60 minutes and their fluorescence were measured. As the tubulin-colchicine

complex gives fluorescence at 435 nm when excited at 350 nm the flurorescence was

measured at the same wavelength. As evident from the Figure 4, a significant decrease in the

fluorescence was observed in case of 4d and E7010. Whereas taxol exerts no effect on the

complex fluorescence as it binds at a different site on the tubulin. These observations indicate

that 4d and E7010 compete to bind to the tubulin at the colchicine binding site.

<Figure 4>

Measurement of mitochondrial membrane potential (∆Ψm)

The maintenance of mitochondrial membrane potential (∆Ψm) is significant for

mitochondrial integrity and bioenergetic function.22 Mitochondrial changes, including loss of

mitochondrial membrane potential (∆Ψm), are key events that take place during drug-induced

apoptosis. Mitochondrial injury by 4d was evaluated by detecting drops in mitochondrial

membrane potential (∆Ψm). In this study we have investigated the involvement of

mitochondria in the induction of apoptosis by 4d. After 48 h of drug treatment with 4d, it was

observed that reduced mitochondrial membrane potential (∆Ψm) of DU-145 cells, assessed

by JC-1 staining (Figure 5).

<Figure 5>

Effect on activation of caspase 3 activity

From previous reports, it is well established that molecules affecting microtubule

polymerization cause mitotic arrest and ultimately lead to apoptosis.23 Caspases, are a family

of cysteine-aspartic proteases that are crucial mediators of apoptosis. Among them, caspase-3

is the best understood in the mammalian caspases in terms of its specificity and role in

apoptosis. Furthermore, there are some reports24-26 that indicate that the cell cycle arrest at

G2/M phase takes place by the induction of cellular apoptosis. Hence, it was considered of

interest to understand the correlation of cytotoxicity with that to apoptosis by 4d. DU-145

cells were treated with 4d (1 and 2µM) and examined for the activation of caspase-3 activity.

Results indicate that there was nearly 2 to 3-fold induction in caspase-3 levels compared to

the control (Figure 6).

<Figure 6>

Annexin V-FITC for apoptosis

The apoptotic effect of 4d was further evaluated by Annexin V FITC/PI (AV/PI) dual

staining assay25 to examine the occurrence of phosphatidylserine externalization and also to

understand whether it is due to physiological apoptosis or nonspecific necrosis. In this study

DU-145 cells were treated with compound 4d for 48 h at 1 and 2 µM concentrations to

examine the apoptotic effect. It was observed that 4d showed significant apoptosis against

DU-145 cells as shown in Figure 7. Results indicated that 4d showed 18.3 % and 22.5 % of

apoptosis at 1 and 2 µM concentrations respectively, whereas 2.67 % of apoptosis was

observed in control (untreated cells). The standard E7010 showed 21.6 % of apoptosis at 2

µM concentration. This experiment suggests that 4d significantly induces apoptosis in DU-

145 cells.

<Figure 7>

<Table 3>

DNA fragmentation analysis

Apoptosis is a programmed cell death, characterized with chromatin condensation and

internucleosomal DNA fragmentation.27 DNA fragmentation is well known and a typical

biochemical hallmark of apoptotic cell death. During apoptosis DNA is cleaved into small

fragments by endonucleases and these fragments can be observed by gel electrophoresis as

ladders. To investigate the ability of 4d for the induction of intranucleosomal DNA

fragments, DU-145 cells were treated with this compound at 1 µM concentration for 48 h and

DNA was isolated from these cells. The DNA was run on 2% agarose gel electrophoresis

after staining with ethidium bromide under UV illumination. It is observed that 4d produced

significant DNA fragmentation (Figure 8), which is indicative of apoptosis.

<Figure 8>

Molecular modeling studies

In order to get an insight into the binding modes of 4d with tubulin, molecular

modelling studies have been carried out. The protein structure of tubulin was downloaded

from protein data bank (PDB code: 3E22)28a and the docking studies were performed using

the software Autodock 4.0.28b The docking studies were performed in the colchicine binding

domain of tubulin. The results suggest that 4d binds at the interface of α and β chains of

tubulin heterodimer which is depicted in Figure 9A. As evident from the figure several amino

acid residues such as Ser 178, Ala 180, Cys 241, Leu 248, Asn 258, Met 259, Val 315, Ala

317, Val 318, Lys 352, Thr 353 etc surround 4d in the binding pocket. A series of

hydrophobic interactions have been observed between most of these amino acid residues with

aryl and pyridine rings of 4d. Additionally the secondary NH of 2-anilinopyridine moiety

establishes a hydrogen bonding interaction with the carbonyl group of the amino acid residue

αThr 179 (distance 2.2 Å). This is contrary to the binding mode of E7010 which establishes a

hydrogen bonding interaction with β Val 238 (Figure 9B). This could be attributed to the

dissimilar orientations of these molecules in the binding pocket (Figure 9C) thereby showing

different effects on cytotoxicity. As shown in Figure 9D, one of the moiety of 2-

anilinopyridine of 4d is deeply buried in the β subunit of the tubulin while the other is

exposed outside towards the α chain, where it establishes several polar and hydrophobic

interactions including hydrogen bonding interactions with the surrounding amino acid

residues. All these observations illustrate that 4d exerts cytotoxic effect by interacting with

colchicine binding domain of the tubulin which is evident from the competitive colchicine

binding assay.

<Figure 9>

Conclusion

In conclusion, a series of 2-anilinopyridinyl dimers were synthesized and evaluated

for their cytotoxic activity against three human cancer cell lines i.e.; lung (A549), breast

(MCF-7) and prostate (DU-145). All the synthesized compounds showed good to moderate

activity and among the series, 4d showed significant cytotoxic activity against human

prostate cancer cell line (DU-145). This compound disrupted microtubule dynamics and

induced abnormal spindle structure and centrosome formation, which resulted in cell-cycle

arrest at the G2/M phase. Detailed biological studies like mitochondrial membrane potential,

caspase-3 assay, DNA fragmentation analysis and Annexin V-FITC assay suggested that this

compound induces apoptosis significantly. Molecular modeling studies indicate that the

antiproliferative activity of 4d is due to its binding into tubulin. The results demonstrate that

4d has the potential to be taken up for further development.

Experimental Section

All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich), St. Louis, MO,

USA), Lancaster (Alfa Aeser, Johnson Matthey Company, Ward Hill, MA, USA), or

Spectrochem Pvt. Ltd. (Mumbai, India) and were used without further purification. Reactions

were performer by TLC performed on silicagel glass plate containing 60 GF-254, and

visualization was achieved by UV light or iodine indicator. Column chromatography was

performed with Merck 60–120 mesh silica gel. 1H and 13C NMR spectra were determined in

CDCl3 and DMSO-d6 by using Varian and Avance instruments. Chemical shifts are expressed

in parts per million (δ in ppm) downfield from internal TMS and coupling constants are

expressed in Hz. 1H NMR spectroscopic data are reported in the following order: multiplicity

(s, singlet; brs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet),

coupling constants in Hz, number of protons. ESI mass spectra were recorded on a Micro

mass Quattro LC using ESI+ software with capillary voltage 3.98 kV and an ESI mode

positive ion trap detector. Melting points were determined with an Electro thermal melting

point apparatus, and are uncorrected.

Ethyl 2-(4-Fluorophenylamino)nicotinate (8a): The compound ethyl 2-chloronicotinate (6,

1850 mg, 10 mmol) was heated while stirring with 4-fluoro aniline (7a, 1111 mg, 10 mmol)

in ethylene glycol at 140 ˚C for 8h. After completion of the reaction, water was added and the

product was extracted using ethyl acetate. The compound was purified by column

chromatography (silica gel 60-120) to afford 8a as a yellow solid in good yield (89%). 1H

NMR (300 MHz, CDCl3): δ 10.19 (bs, 1H), 8.32 (dd, J=4.6, 2.0 Hz, 1H), 8.22 (dd, J=8.0, 2.0

Hz, 1H), 7.65 (m, 2H), 7.00 (m, 2H), 6.70 (dd, J=7.3, 4.6 Hz, 1H), 4.40 (q, J=7.3 Hz, 2H),

1.44 (t, J=7.3 Hz, 3H); ESI-MS: 261 [M+H]+.

Ethyl 2-(4-Methoxyphenylamino)nicotinate (8b): The compound 8b was prepared

according to the method described for compound 8a, employing ethyl 2-chloronicotinate (6,

1850 mg, 10 mmol) and 4-methoxy aniline (7b, 1230 mg, 10 mmol) to obtain the pure

product 8b as a yellow solid in good yield (89%). 1H NMR (300 MHz, CDCl3): δ 10.00 (s,

1H), 8.32 (dd, J=4.6, 2.3 Hz, 1H), 8.20 (dd, J=7.8, 2.3 Hz, 1H), 7.52 (dd, J=7.0, 2.3 Hz, 2H),

6.88 (dd, J=7.0, 2.3 Hz, 2H), 6.64 (dd, J=7.8, 4.6 Hz, 1H), 4.38 (q, J=7.0 Hz, 2H), 3.82 (s,

3H), 1.43 (t, J=7.0 Hz, 3H); ESI-MS: 273 [M+H]+.

Ethyl 2-(3,4,5-Trimethoxyphenylamino)nicotinate (8c): The compound 8c was prepared

according to the method described for compound 8a, employing ethyl 2-chloronicotinate (6,

1850 mg, 10 mmol) and 3,4,5-trimethoxy aniline (7c, 1230 mg, 10 mmol) to obtain the pure

product 8c as a yellow solid in good yield (83%). 1H NMR (300 MHz, CDCl3): δ 10.09 (s,

1H), 8.28 (dd, J=4.5, 2.3 Hz, 1H), 8.18 (dd, J=7.8, 2.8 Hz, 1H), 6.88 (s, 2H), 6.64 (dd, J=7.8,

4.6 Hz, 1H), 4.40 (q, J=7.2 Hz, 2H), 3.89 (s, 6H), 3.82 (s, 3H), 1.44 (t, J=7.2 Hz, 3H); ESI-

MS: 333 [M+H]+.

Ethyl 2-(3,5-Bis(trifluoromethyl)phenylamino)nicotinate (8d): The compound 8d was

prepared according to the method described for compound 8a, employing ethyl 2-

chloronicotinate (6, 1850 mg, 10 mmol) and 3,5-bis(trifluoromethyl)aniline (7d, 2290 mg, 10

mmol) to obtain the pure product 8d as a yellow solid in good yield (87%). 1H NMR (300

MHz, CDCl3): δ 10.68 (s, 1H), 8.45 (dd, J=2.8, 2.0 Hz, 1H), 8.34-8.27 (m, 3H), 7.49 (s, 1H),

6.87 (dd, J=4.9, 3.0 Hz, 2H), 4.42 (q, J=7.2 Hz, 2H), 3.82 (s, 3H), 1.44 (t, J=7.0 Hz, 3H);

ESI-MS: 379 [M+H]+.

General procedure for the synthesis of substituted 2-anilinonicotinic acids (9a-d): Ethyl-

2-anilino nicotinyl esters (8a-d, 1 mmol) were refluxed in 2N NaOH for 2h. After completion

of the reaction, the mixture was cooled and neutralized with 2N HCl. A white solid appears

which was filtered and washed with water to give pure acid products (9a-d).

N-(4-Fluorophenyl)-3-nitropyridin-2-amine (12a): 2-choloro-3-nitropyridine (10, 1585

mg, 10 mmol) was heated while stirring with 4-fluoro aniline (11a, 1111 mg, 10 mmol) in

ethylene glycol at 140 ˚C for 8h. After completion of the reaction, water was added and the

product was extracted using ethyl acetate. The compound was purified by column

chromatography (silica gel 60-120) to afford 12a as a red solid in excellent yield (92%). 1H

NMR (300 MHz, CDCl3): δ 10.16 (s, 1H), 8.53 (dd, J=6.7, 1.8 Hz, 1H), 8.44 (dd, J=2.6, 1.7

Hz, 1H), 7.52 (dt, J=8.1, 1.3 Hz, 2H), 6.96 (dt, J=8.1, 1.3 Hz, 2H), 6.82 (dd, J=4.5, 3.7 Hz,

1H); ESI-MS: 234 [M+H]+.

N-(4-Methoxyphenyl)-3-nitropyridin-2-amine (12b): The compound 12b was synthesized

following the procedure described for compound 12a employing 2-choloro-3-nitropyridine

(10, 1585 mg, 10 mmol) and 4-methoxy aniline (11b, 1231 mg, 10 mmol) to afford the pure

product 12b as a red solid in excellent yield (94%). 1H NMR (300 MHz, CDCl3): δ 9.96 (s,

1H), 8.50 (dd, J=6.6, 1.8 Hz, 1H), 8.44 (dd, J=2.6, 1.8 Hz, 1H), 7.48 (d, J=9.0 Hz, 2H), 6.94

(d, J=9.0 Hz, 2H), 6.77 (dd, J=4.5, 3.8 Hz, 1H), 3.83 (s, 3H); ESI-MS: 246 [M+H]+.

N-(4-Chlorophenyl)-3-nitropyridin-2-amine (12c): The compound 12c was synthesized

following the procedure described for compound 12a employing 2-choloro-3-nitropyridine

(10, 1585 mg, 10 mmol) and 4-chloro aniline (11c, 1275 mg, 10 mmol) to afford the pure

product 12c as a red solid in excellent yield (90%). 1H NMR (300 MHz, CDCl3): δ 10.14 (s,

1H), 8.49 (dd, J=6.3, 1.6 Hz, 1H), 8.45 (dd, J=2.8, 1.7 Hz, 1H), 7.52 (d, J=8.1 Hz, 2H), 7.02

(d, J=8.1 Hz, 2H), 6.85 (dd, J=4.6, 3.8 Hz, 1H); ESI-MS: 250 [M+H]+.

3-Nitro-N-phenylpyridin-2-amine (12d): The compound 12d was synthesized following the

procedure described for compound 12a employing 2-choloro-3-nitropyridine (10, 1585 mg,

10 mmol) and aniline (11d, 931 mg, 10 mmol) to afford the pure product 12d as a red solid in

excellent yield (90%). 1H NMR (300 MHz, CDCl3): δ 10.12 (s, 1H), 8.53 (dd, J=6.8, 1.5 Hz,

1H), 8.49 (dd, J=2.8, 1.5 Hz, 1H), 7.65 (d, J=7.7 Hz, 2H), 7.40 (t, J=7.5 Hz, 2H), 7.19 (t,

J=7.4 Hz, 1H), 6.83 (dd, J=4.5, 3.7 Hz, 1H); ESI-MS: 216 [M+H]+.

N2-(4-Fluorophenyl)pyridine-2,3-diamine (13a): N-(4-fluorophenyl)-3-nitropyridin-2-

amine (12a, 2330 mg, 10 mmol)) were refluxed with SnCl2.2H2O (6750 mg, 30 mmol) in

methanol for 2h. After completion of the reaction methanol was evaporated under reduced

pressure. The reaction mixture was neutralized using saturated NaHCO3 solution and ethyl

acetate was added. After filtering the junk over celite, the reaction mixture was extracted

using ethyl acetate. The organic layer was dried over anhydrous Na2SO4 and concentrated to

obtain the products 13a in good yield (88%). The amine products were used directly without

further purification. 1H NMR (300 MHz, CDCl3): δ 7.81 (dd, J=3.6, 1.3 Hz, 1H), 7.29-7.22

(m, 2H), 7.04-6.95 (m, 3H), 6.75 (dd, J=5.1, 2.5, 1H), 6.18 (bs, 1H); ESI-MS: 204 [M+H]+.

N2-(4-Methoxyphenyl)pyridine-2,3-diamine (13b): The compound 13b was synthesized

following the protocol used for the synthesis of 13a employing N-(4-methoxyphenyl)-3-

nitropyridin-2-amine (12b, 2450 mg, 10mmol) and SnCl2.2H2O (6750 mg, 30 mmol) to

afford 13b in good yield (89%). 1H NMR (300 MHz, CDCl3): δ 7.79 (d, J=3.0 Hz, 1H), 7.21

(d, J=8.8 Hz, 2H), 6.98 (dd, J=6.4, 1.1 Hz, 1H), 6.86 (d, J=8.8 Hz, 2H), 6.71 (dd, J=4.8, 2.6

Hz, 1H), 6.08 (bs, 1H), 3.78 (s, 3H); ESI-MS: 216 [M+H]+.

N2-(4-Chlorophenyl)pyridine-2,3-diamine (13c): The compound 13c was synthesized

following the protocol used for the synthesis of 13a employing N-(4-chlorophenyl)-3-

nitropyridin-2-amine (12c, 2310 mg, 10mmol) and SnCl2.2H2O (6750 mg, 30 mmol) to

afford 13c in good yield (85%). 1H NMR (300 MHz, CDCl3): δ 7.80 (dd, J=3.6, 1.4 Hz, 1H),

7.29-7.22 (m, 2H), 7.06-6.94 (m, 3H), 6.72 (dd, J=5.2, 2.5, 1H), 6.21 (bs, 1H); ESI-MS: 220

[M+H]+.

N2-Phenylpyridine-2,3-diamine (13d): The compound 13d was synthesized following the

protocol used for the synthesis of 13a employing 3-nitro-N-phenylpyridin-2-amine (12d,

2152 mg, 10mmol) and SnCl2.2H2O (6750 mg, 30 mmol) to afford 13e in good yield (87%). 1H NMR (300 MHz, CDCl3): δ 7.83 (dd, J=3.4, 1.5 Hz, 1H), 7.23 (s, 4H), 7.02 (dd, J= 6.0,

1.5 Hz, 1H), 6.78 (dd, J=4.9, 2.6 Hz, 1H), 6.26 (bs, 1H); ESI-MS: 186 [M+H]+.

2-(4-Fluorophenylamino)-N-(2-(4-fluorophenylamino)pyridin-3-yl)nicotinamide (4a): 2-

(4-fluorophenylamino)nicotinic acid (9a, 232 mg, 1 mmol) was taken in a RB flask equipped

with a magnetic stirring bar containing dry DMF under inert conditions. To this flask HOBt

(183 mg, 1.2 mmol) followed by EDC (229 mg, 1.2 mmol) were added while stirring. After 5

minutes N2-(4-fluorophenyl)pyridine-2,3-diamine (13a, 203mg, 1 mmol) was added and the

reaction mixture was left to stir for 12h. After completion of the reaction, ice water was

added to the flask and the compound was extracted using CHCl3. The organic layer was dried

over anhydrous Na2SO4, concentrated and purified using column chromatography (silica gel,

60-120) to afford the pure compound (4a). Yield: 85%; M.P: 171-173 °C; 1H NMR (500

MHz, CDCl3): δ 10.21 (s, 1H), 8.33 (d, J=3.6 Hz, 1H), 8.17 (d, J=3.6 Hz, 1H), 7.87 (d, J=7.5

Hz, 1H), 7.73 (s, 1H), 7.63-7.50 (m, 3H), 7.23-7.17 (m, 2H), 7.05-6.93 (m, 5H), 6.66 (q,

J=4.7, 2.8 Hz, 1H), 6.59 (s, 1H); ESI-MS: 418 [M+H]+, HRMS Calcd for C23H18ON5F2

[M+H]+ 418.1474 Found: 418.1468.

N-(2-(4-Fluorophenylamino)pyridin-3-yl)-2-(4-methoxyphenylamino)nicotinamide (4b):

The titled compound 4b was synthesized following the procedure described for compound 4a

using 2-(4-methoxyphenylamino)nicotinic acid (9b, 244 mg, 1 mmol) and N2-(4-

fluorophenyl)pyridine-2,3-diamine (13a, 203 mg, 1 mmol) to afford the pure compound 4b.

Yield: 84%; M.P: 131-133 °C; 1H NMR (300 MHz, CDCl3): δ 10.06 (s, 1H), 8.32 (dd, J=3.0,

1.7 Hz, 1H), 8.19 (dd, J=3.4, 1.5 Hz, 1H), 7.89 (dd, J=6.4, 1.5 Hz, 1H), 7.66 (s, 1H), 7.51 (d,

J=9.1 Hz, 2H), 7.25-7.19 (m, 2H), 7.04-6.94 (m, 3H), 6.89 (d, J=8.9 Hz, 2H), 6.66-6.60 (m,

1H), 6.54 (s, 1H), 3.80 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 167.2, 156.0, 155.8, 152.5,

149.4, 145.6, 136.7, 135.7, 132.9, 132.4, 123.2, 121.4, 116.7, 115.8, 115.5, 114.1, 112.4,

109.2, 55.4; ESI-MS: 430 [M+H]+, HRMS Calcd for C24H21O2N5F [M+H]+ 430.1674 Found:

430.1664.

N-(2-(4-Fluorophenylamino)pyridin-3-yl)-2-(3,4,5-

trimethoxyphenylamino)nicotinamide (4c): The titled compound 4c was synthesized

following the procedure described for compound 4a using 2-(3,4,5-

trimethoxyphenylamino)nicotinic acid (9c, 304 mg, 1mmol) and N2-(4-

fluorophenyl)pyridine-2,3-diamine (13a, 203 mg, 1 mmol) to afford the pure compound 4c.

Yield: 81%; M.P: 204-206 °C; 1H NMR (500 MHz, CDCl3): δ 10.23 (s, 1H), 8.36 (dd, J=3.2,

1.5 Hz, 1H), 8.19 (dd, J=3.5, 1.1 Hz, 1H), 7.87 (dd, J=6.4, 1.2 Hz, 1H), 7.77 (s, 1H), 7.60

(dd, J=6.2, 1.5 Hz, 1H), 7.24-7.22 (m, 2H), 7.01-6.99 (m, 2H), 6.97-6.95 (m, 2H), 6.67 (q,

J=4.7, 2.9 Hz, 1H), 6.59 (s, 1H), 3.84 (s, 6H), 3.81 (s, 3H); ESI-MS: 490 [M+H]+, HRMS

Calcd for C26H25O4N5F [M+H]+ 490.1885 Found: 490.1875.

2-(3,5-Bis(trifluoromethyl)phenylamino)-N-(2-(4-fluorophenylamino)pyridin-3-

yl)nicotinamide (4d): The titled compound 4d was synthesized following the procedure

described for compound 4a using 2-(3,5-bis(trifluoromethyl)phenylamino)nicotinic acid (9d,

350 mg, 1mmol) and N2-(4-fluorophenyl)pyridine-2,3-diamine (13a, 203 mg, 1 mmol) to

afford the pure compound 4d. Yield: 87%; M.P: 164-166 °C; 1H NMR (500 MHz, CDCl3): δ

10.83 (s, 1H), 10.59 (s, 1H), 10.28 (s, 1H), 8.68 (d, J=7.3 Hz, 1H), 8.42 (d, J=3.7 Hz, 1H),

8.32 (d, J=7.5 Hz, 1H), 8.23 (s, 2H), 7.82 (s, 1H), 7.75 (d, J=5.6 Hz, 1H), 7.46 (s, 1H), 7.32-

7.29 (m, 2H), 7.05 (t, J=6.7 Hz, 2H), 6.96 (t, J=8.4 Hz, 2H), 6.86 (q, J=4.7, 3.0 Hz, 1H); 13C

NMR (75 MHz, CDCl3+DMSO-d6): δ 169.3, 166.8, 153.1, 150.1, 147.8, 140.6, 138.4, 134.6,

133.5, 130.8, 130.4, 128.3, 125.1, 123.9, 121.9, 117.7, 115.5, 113.7, 112.8; ESI-MS: 536

[M+H]+, HRMS Calcd for C25H17ON5F7 [M+H]+ 536.1316 Found: 536.1308.

2-(4-Fluorophenylamino)-N-(2-(4-methoxyphenylamino)pyridin-3-yl)nicotinamide (4e):

The titled compound 4e was synthesized following the procedure described for compound 11

using 2-(4-fluorophenylamino)nicotinic acid (9a, 232 mg, 1 mmol) and N2-(4-

methoxyphenyl)pyridine-2,3-diamine (13b, 215 mg, 1 mmol) to afford the pure compound

4e. Yield: 84%; M.P: 150-152 °C; 1H NMR (500 MHz, CDCl3): δ 10.22 (s, 1H), 8.28 (d,

J=3.4 Hz, 1H), 8.11 (d, J=3.4 Hz, 1H), 7.88 (d, J=7.6 Hz, 1H), 7.84 (s, 1H), 7.57 (q, J=4.7,

3.9 Hz, 2H), 7.35 (d, J=6.9 Hz, 1H), 7.12 (d, J=8.7 Hz, 2H), 7.00 (t, J=8.7 Hz, 2H), 6.89 (q,

J=4.8, 2.5 Hz, 1H), 6.83 (d, J=8.7 Hz, 2H), 6.58 (q, J=4.7, 2.7 Hz, 1H), 6.51 (bs, 1H), 3.73 (s,

3H); 13C NMR (75 MHz, CDCl3): δ 167.2, 160.1, 156.9, 155.7, 155.4, 151.9, 150.0, 145.3,

35.9, 133.6, 133.0, 122.1, 122.4, 122.3, 115.4, 115.1, 114.4, 112.8, 109.8, 55.4; ESI-MS: 430

[M+H]+, HRMS Calcd for C24H21O2N5F [M+H]+ 430.1674 Found: 430.1664.

2-(4-Methoxyphenylamino)-N-(2-(4-methoxyphenylamino)pyridin-3-yl)nicotinamide

(4f): The titled compound 4f was synthesized following the procedure described for

compound 4a using 2-(4-methoxyphenylamino)nicotinic acid (9b, 244 mg, 1 mmol) and N2-

(4-methoxyphenyl)pyridine-2,3-diamine (13b, 215 mg, 1 mmol) to afford the pure compound

4f. Yield: 81%; 1H NMR (300 MHz, CDCl3+DMSO-d6): δ 10.08 (s, 1H), 8.28 (dd, J=3.0, 1.5

Hz, 1H), 8.16 (d, J=4.5 Hz, 1H), 8.00 (dd, J=6.0, 1.5 Hz, 1H), 7.61 (s, 1H), 7.51 (d, J=9.1

Hz, 2H), 7.31 (dd, J=6.8, 1.5 Hz, 1H), 7.15 (d, J=9.1 Hz, 2H), 6.98-6.62 (m, 1H), 6.88 (t,

J=8.3 Hz, 4H), 6.60-6.54 (m, 1H), 6.37 (s, 1H), 3.80 (s, 3H), 3.76 (s, 3H); 13C NMR (75

MHz, CDCl3): δ 167.2, 155.5, 152.0, 145.3, 135.9, 132.9, 123.0, 122.1, 121.2, 115.8, 114.3,

114.0, 112.2, 109.5, 55.4; ESI-MS: 442 [M+H]+, HRMS Calcd for C25H24O3N5 [M+H]+

442.1874 Found: 442.1862.

N-(2-(4-Methoxyphenylamino)pyridin-3-yl)-2-(3,4,5-trimethoxyphenylamino)

nicotinamide (4g): The titled compound 4g was synthesized following the procedure

described for compound 4a using 2-(3,4,5-trimethoxyphenylamino)nicotinic acid (9c, 304

mg, 1mmol) and N2-(4-methoxyphenyl)pyridine-2,3-diamine (13b, 215 mg, 1 mmol) to

afford the pure compound 4g. Yield: 78%; M.P: 149-151 °C; 1H NMR (500 MHz, CDCl3): δ

10.26 (s, 1H), 8.33 (d, J=3.9 Hz, 1H), 8.14 (d, J=3.6 Hz, 1H), 7.93 (d, J=7.6 Hz, 1H), 7.82 (s,

1H), 7.42 (d, J=7.2 Hz, 1H), 7.17 (d, J=8.5 Hz, 2H), 6.97 (s, 2H), 6.92 (q, J=4.8, 2.4 Hz, 1H),

6.85 (d, J=8.5 Hz, 2H), 6.61 (q, J=4.8, 2.3 Hz, 1H), 6.51 (s, 1H), 3.84 (s, 6H), 3.81 (s, 3H),

3.75 (s, 3H); 13C NMR (125 MHz, CDCl3): δ 167.1, 155.4, 153.1, 152.1, 145.6, 135.8, 133.6,

132.9, 125.4, 122.2, 116.2, 114.5, 112.8, 109.9, 98.3, 60.9, 56.0, 55.5; ESI-MS: 502 [M+H]+,

HRMS Calcd for C27H28O5N5 [M+H]+ 502.2085 Found: 502.2074.

2-(3,5-Bis(trifluoromethyl)phenylamino)-N-(2-(4-methoxyphenylamino)pyridin-3-

yl)nicotinamide (4h): The titled compound 4h was synthesized following the procedure

described for compound 4a using 2-(3,5-bis(trifluoromethyl)phenylamino)nicotinic acid (9d,

350 mg, 1mmol) and N2-(4-methoxyphenyl)pyridine-2,3-diamine (13b, 215 mg, 1 mmol) to

afford the pure compound 4h. Yield: 82%; 1H NMR (500 MHz, CDCl3+DMSO-d6): δ 11.13

(s, 1H), 10.95 (s, 1H), 10.49 (s, 1H), 8.72 (d, J=7.5 Hz, 1H), 8.35-8.29 (m, 1H), 8.19 (s, 2H),

8.13-8.03 (m, 1H), 7.67 (s, 1H), 7.59 (d, J=6.2 Hz, 1H), 7.35-7.15 (m, 3H), 6.97-6.84 (m,

3H), 3.74 (s, 3H); ESI-MS: 548 [M+H]+, HRMS Calcd for C26H20O2N5F6 [M+H]+ 548.1516

Found: 548.1510.

N-(2-(4-Chlorophenylamino)pyridin-3-yl)-2-(4-fluorophenylamino)nicotinamide (4i):

The titled compound 4i was synthesized following the procedure described for compound 4a

using 2-(4-fluorophenylamino)nicotinic acid (9a, 232 mg, 1 mmol) and N2-(4-

chlorophenyl)pyridine-2,3-diamine (13c, 219 mg, 1 mmol) to afford the pure compound 4i.

Yield: 86%; 1H NMR (500 MHz, CDCl3): δ 10.22 (s, 1H), 8.33 (dd, J=3.2, 1.5 Hz, 1H), 8.18

(s, 1H), 8.04 (s, 1H), 7.85 (dd, J=6.5, 1.2 Hz, 1H), 7.65 (dd, J=6.3, 1.5 Hz, 1H), 7.59-7.56

(m, 2H), 7.23 (s, 4H), 7.01 (t, J=8.7 Hz, 2H), 6.96 (q, J=4.8, 2.8 Hz, 1H), 6.83 (s, 1H), 6.68

(q, J=4.7, 2.9 Hz, 1H); 13C NMR (75 MHz, CDCl3): δ 167.1, 154.2, 150.2, 149.2, 143.9,

138.8, 136.8, 133.9, 135.2, 127.2, 124.5, 120.7, 119.7, 118.9, 114.2, 113.8, 112.0, 109.7;

ESI-MS: 434 [M+H]+, HRMS Calcd for C23H18ON5ClF [M+H]+ 434.1178 Found: 434.1174.

N-(2-(4-Chlorophenylamino)pyridin-3-yl)-2-(4-methoxyphenylamino)nicotinamide (4j):

The titled compound 4j was synthesized following the procedure described for compound 4a

using 2-(4-methoxyphenylamino)nicotinic acid (9b, 244 mg, 1 mmol) and N2-(4-

chlorophenyl)pyridine-2,3-diamine (13c, 219 mg, 1 mmol) to afford the pure compound 4j.

Yield: 81%; M.P: 132-134 °C; 1H NMR (500 MHz, CDCl3): δ 10.01 (s, 1H), 8.28 (d, J=3.0

Hz, 1H), 8.17 (d, J=3.4 Hz, 1H), 7.84 (d, J=7.5 Hz, 1H), 7.73 (s, 1H), 7.53 (d, J=7.3 Hz,

1H), 7.48 (d, J=8.5 Hz, 2H), 7.24-7.19 (m, 4H), 6.95 (t, J=5.0 Hz, 1H), 6.85 (d, J=8.4 Hz,

2H), 6.77 (s, 1H), 6.60 (t, J=4.8 Hz, 1H), 3.77 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 167.2,

155.8, 152.3, 145.4, 139.4, 135.8, 132.8, 132.1, 128.9, 127.0, 123.4, 121.4, 120.5, 116.9,

114.0, 112.4, 55.4; ESI-MS: 446 [M+H]+, HRMS Calcd for C24H21O2N5Cl [M+H]+ 446.1378

Found: 446.1367.

N-(2-(4-Chlorophenylamino)pyridin-3-yl)-2-(3,4,5-

trimethoxyphenylamino)nicotinamide (4k): The titled compound 4k was synthesized

following the procedure described for compound 4a using 2-(3,4,5-

trimethoxyphenylamino)nicotinic acid (9c, 304 mg, 1mmol) and N2-(4-

chlorophenyl)pyridine-2,3-diamine (13c, 219 mg, 1 mmol) to afford the pure compound 4k.

Yield: 82%; M.P: 198-200 °C; 1H NMR (300 MHz, CDCl3): δ 10.22 (s, 1H), 8.35 (dd, J=3.0,

1.5 Hz, 1H), 8.19 (dd, J=3.0, 1.5 Hz, 1H), 7.91 (s, 1H), 7.83 (dd, J=6.0, 1.5 Hz, 1H), 7.63

(dd, J=6.0, 1.5 Hz, 1H), 7.23 (s, 4H), 6.95 (s, 3H), 6.79 (s, 1H), 6.69-6.63 (m, 1H), 3.82 (s,

6H), 3.80 (s, 3H); 13C NMR (75 MHz, CDCl3): δ 167.5, 155.3, 152.8, 152.1, 149.3, 145.6,

139.2, 136.1, 135.8, 135.6, 133.6, 130.0, 128.8, 126.9, 120.5, 116.6, 112.8, 109.6, 98.1, 60.9,

55.8; ESI-MS: 506 [M+H]+, HRMS Calcd for C26H25O4N5Cl [M+H]+ 506.1589 Found:

506.1579.

2-(3,5-Bis(trifluoromethyl)phenylamino)-N-(2-(4-chlorophenylamino)pyridin-3-

yl)nicotinamide (4l): The titled compound 4l was synthesized following the procedure

described for compound 4a using 2-(3,5-bis(trifluoromethyl)phenylamino)nicotinic acid (9d,

350 mg, 1mmol) and N2-(4-chlorophenyl)pyridine-2,3-diamine (13c, 219 mg, 1 mmol) to

afford the pure compound 40l. Yield: 72%; 1H NMR (300 MHz, CDCl3): δ 10.81 (s, 1H),

8.47 (d, J=4.7 Hz, 1H), 8.27-8.21 (m, 3H), 7.96 (d, J=7.7 Hz, 1H), 7.86 (s, 1H), 7.73 (d,

J=8.7 Hz, 1H), 7.64-7.53 (m, 1H), 7.49 (s, 1H), 7.25 (t, J=7.5 Hz, 2H), 7.19 (d, J=8.9 Hz,

1H), 7.06-7.00 (m, 1H), 6.84 (q, J=4.9, 2.8 Hz, 1H), 6.61 (s, 1H); ESI-MS: 552 [M+H]+,

HRMS Calcd for C25H16N5OF6Cl [M+H]+ 552.0948 Found: 552.1017.

2-(4-Fluorophenylamino)-N-(2-(phenylamino)pyridin-3-yl)nicotinamide (4m): The titled

compound 4m was synthesized following the procedure described for compound 4a using 2-

(4-fluorophenylamino)nicotinic acid (9a, 232 mg, 1 mmol) and N2-phenylpyridine-2,3-

diamine (13d, 185 mg, 1 mmol) to afford the pure compound 4m. Yield: 86%; M.P: 186-188

°C; 1H NMR (300 MHz, CDCl3): δ 10.21 (s, 1H), 8.33 (dd, J=3.2, 1.5 Hz, 1H), 8.18 (s, 1H),

8.04 (s, 1H), 7.85 (dd, J=6.5, 1.2 Hz, 1H), 7.65 (dd, J=6.3, 1.5 Hz, 1H), 7.59-7.56 (m, 2H),

7.23 (s, 4H), 7.01 (t, J=8.7 Hz, 2H), 6.96 (q, J=4.8, 2.8 Hz, 1H), 6.83 (s, 2H), 6.68 (q, J=4.7,

2.9 Hz, 1H); 13C NMR (75 MHz, CDCl3): δ 166.9, 160.2, 155.5, 152.1, 148.3, 145.3, 135.6,

132.3, 129.2, 122.5, 119.0, 117.4, 115.4, 115.1, 112.9, 109.7; ESI-MS: 400 [M+H]+, HRMS

Calcd for C23H19ON5F [M+H]+ 400.1568 Found: 400.1566.

2-(4-Methoxyphenylamino)-N-(2-(phenylamino)pyridin-3-yl)nicotinamide (4n): The

titled compound 4n was synthesized following the procedure described for compound 4a

using 2-(4-methoxyphenylamino)nicotinic acid (9b, 244 mg, 1 mmol) and N2-

phenylpyridine-2,3-diamine (13d, 185 mg, 1 mmol) to afford the pure compound 4n. Yield:

79%; M.P: 160-162 °C; 1H NMR (300 MHz, CDCl3): δ 10.06 (s, 1H), 8.27 (dd, J=3.0, 1.7

Hz, 1H), 8.20 (dd, J=3.4, 1.3 Hz, 1H), 8.11 (dd, J=6.6, 1.3 Hz, 1H), 7.67 (s, 1H), 7.51 (d,

J=8.9 Hz, 2H), 7.33-7.27 (m, 2H), 7.17 (d, J=7.7 Hz, 2H) 7.05-6.99 (m, 2H), 6.88 (d, J=9.1

Hz, 2H), 6.53 (q, J=4.9, 2.8 Hz, 1H), 6.50 (s, 1H), 3.80 (s, 3H); ESI-MS: 412 [M+H]+,

HRMS Calcd for C24H22O2N5 [M+H]+ 412.1768 Found: 412.1763.

N-(2-(Phenylamino)pyridin-3-yl)-2-(3,4,5-trimethoxyphenylamino)nicotinamide (4o):

The titled compound 4o was synthesized following the procedure described for compound 4a

using 2-(3,4,5-trimethoxyphenylamino)nicotinic acid (9c, 304 mg, 1mmol) and N2-

phenylpyridine-2,3-diamine (13d, 185 mg, 1 mmol) to afford the pure compound 40o. Yield:

75%; M.P: 196-198 °C; 1H NMR (300 MHz, CDCl3): δ 10.23 (s, 1H), 8.33 (d, J=3.2 Hz, 1H),

8.21 (d, J=4.2 Hz, 1H), 8.07 (d, J=7.5 Hz, 1H), 7.76 (s, 1H), 7.34-7.25 (m, 3H), 7.18 (d,

J=7.7 Hz, 1H), 7.05-6.96 (m, 4H), 6.60 (q, J=4.9, 2.6 Hz, 1H), 6.54 (s, 1H), 3.85 (s, 6H),

3.81 (s, 3H); ESI-MS: 472 [M+H]+, HRMS Calcd for C26H26O4N5 [M+H]+ 472.1979 Found:

472.1967.

2-(3,5-Bis(trifluoromethyl)phenylamino)-N-(2-(phenylamino)pyridin-3-yl)nicotinamide

(4p): The titled compound 4p was synthesized following the procedure described for

compound 4a using 2-(3,5-bis(trifluoromethyl)phenylamino)nicotinic acid (9d, 350 mg,

1mmol) and N2-phenylpyridine-2,3-diamine (13d, 185 mg, 1 mmol) to afford the pure

compound 4p. Yield: 71%; M.P: 233-235 °C; 1H NMR (300 MHz, CDCl3): δ 11.09 (s, 1H),

10.78 (s, 1H), 8.63 (d, J=6.6 Hz, 1H), 8.48 (d, J=4.7 Hz, 1H), 8.31 (s, 2H), 8.08 (d, J=7.2

Hz, 1H), 7.83 (s, 2H), 7.50-7.42 (m, 5H), 7.28 (s, 1H), 7.06-6.98 (m, 2H); 13C NMR (75

MHz, CDCl3+DMSO-d6): δ 166.9, 153.1, 150.2, 147.6, 139.3, 138.2, 134.8, 134.1, 130.5,

128.8, 125.7, 124.0, 123.1, 122.5, 120.4, 117.8, 113.8, 112.8, 110.1; ESI-MS: 518 [M+Na]+,

HRMS Calcd for C23H19ON5F6Na[M+Na]+ 518.1386 Found: 518.1397.

Biology

In vitro antiproliferative activity

The antiproliferative activity of the compounds was determined using Sulphorhodamine B

(SRB) assay.17 Cells grown in DMEM, supplemented with 10% FBS were seeded in each

well of 96-well microculture plates and incubated for 24 h at 37 °C in a CO2 incubator.

Compounds, diluted to the desired concentrations (0.1, 1, 5 and 10 µM) in DMSO, were

added to the wells with respective control. After 48 h cells were fixed with 10% trichloro

acetic acid (TCA) solution and were further incubated for 60 min at 4 °C. The plates were

washed with tap water and airdried. Later Sulforhodamine B (SRB) solution (50 µL) at 0.4%

(w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 min

at room temperature. The residual dye was removed by washing with 1% acetic acid and the

plates were airdried. Bound stain was subsequently eluted with 10 mM trizma base, and the

absorbance was recorded on multimode reader (TECAN) at a wavelength of 540 nm.

Cell cycle analysis

Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells

through the cell cycle phases. DU-145, human prostate cancer cells were incubated with

compound 4d at 1 and 2 µM concentrations for 48 h. E7010 (2 µM) was used as reference

compound. Untreated and treated cells were harvested, washed with PBS, fixed in ice-cold

70% ethanol and stained with propidium iodide (Sigma Aldrich). Cell cycle was performed

by flow cytometry (Becton Dickinson FACS Caliber) as earlier described.

Tubulin polymerization assay

A fluorescence based in vitro tubulin polymerization assay was performed according to the

manufacturer’s protocol (BK011, Cytoskeleton, Inc.). Briefly, the reaction mixture in a total

volume of 10 µl contained PEM buffer, GTP (1 µM) in the presence or absence of test

compound 4d (final concentration of 3 µM). Tubulin polymerization was followed by a time

dependent increase in fluorescence due to the incorporation of a fluorescence reporter into

microtubules as polymerization proceeds. Fluorescence emission at 420 nm (excitation

wavelength is 360 nm) was measured by using a Varioscan multimode plate reader (Thermo

scientific Inc.). E7010 was used as positive control in each assay. The IC50 value was defined

as the drug concentration required inhibiting 50% of tubulin assembly compared to control.

The reaction mixture for these experiments include: tubulin (3 mg/mL) in PEM buffer, GTP

(1 mM), in the presence or absence of test compounds at 2.5, 5, 10, and 15 µM

concentrations. Polymerization was monitored by increase in the Fluorescence as mentioned

above at 37 °C.

Colchicine competitive binding assay

The test compound (4d) of various concentrations 5 µM, 10 µM, 15 µM, 20 µM and 25 µM

were co- incubated with 4 µM colchicine in 30 mM Tris buffer containing 3 µM tubulin for

60 min at 37 oC. Nocodazole was used as a positive control where as taxol was used as

negative control which doesn’t bind at colchicine site. After incubation the fluorescence of

tubulin-colchicine complex was determined by using Tecan multimode reader at excitation

wavelength at 350 nm and emission wavelength at 435 nm. 30 mM Tris buffer was used as

blank which was subtracted from all the samples. Fluorescence values are normalized to

DMSO control.

Mitochondrial membrane potential

DU-145 (1×106 cells/well) cells were cultured in six-well plates after treatment with

compound 4d at 1 and 2 µM concentrations for 48 h. After 48 h of treatment, cells were

collected by trypsinization and washed with PBS followed by resuspending in JC-1 (10

mM/L) and incubated at 37 °C for 15 min. Cells were rinsed three times with medium and

suspended in pre warmed medium. The cells were then subjected to flow cytometric analysis

on a FACstar Plus Flow Cytometer (Becton Dickinson) in the FL1, FL2 channel to detect

mitochondrial potential.

Caspase 3 activity

Caspase-3 assay was conducted for detection of apoptosis in prostate cancer cell line (DU-

145). The commercially available apoptosis detection kit (Sigma-Caspase 3 Assay kit,

Colorimetric) was used. DU-145 cells were treated with compound 4d at 1 and 2 µM

concentrations for 48 h. After 48 h of treatment, cells were collected by centrifugation,

washed once with PBS, and cell pellets were collected. Suspended the cell pellet in lysis

buffer and incubated for 15 min. After incubation, cells were centrifuge at 20,000 rpm for 15

min and collected the supernatant. Supernatants were used for measuring caspase 3 activity

using an ELISA-based assay, according to the manufacturer’s instructions.

Annexin V–FITC assay

DU-145 cells (1×106) were seeded in six-well plates and allowed to grow overnight. The

medium was then replaced with complete medium containing 1 and 2 µM concentrations of

compound 4d for 48 h along with vehicle alone (0.001% DMSO) as control. After 48 h of

drug treatment, cells from the supernatant and adherent monolayer cells were harvested by

trypsinization, washed with PBS at 3000 rpm. Then the cells (1×106) were stained with

Annexin V-FITC and propidium iodide using the Annexin-V-PI apoptosis detection kit

(Sigma Aldrich-India). Flow cytometry was performed using a FACScan (Becton Dickinson)

equipped with a single 488 nm argon laser. Annexin V-FITC was analyzed using excitation

and emission settings of 488 nm and 535 nm (FL-1 channel); PI, 488 nm and 610 nm (FL-2

channel). Debris and clumps were gated out using forward and orthogonal light scatter.

DNA fragmentation analysis

DU-145 cells were seeded (1×106) in six-well plates. After incubation of 24 h cells were

treated with compound 4d and E7010 at 1µM concentration. After 48 h of treatment, cells

were collected and centrifuged at 2500 rpm for 5 min at 4 °C. Pellet was collected and

washed with Phosphate buffered saline (PBS). Lysis buffer was added, the pellet was

collected centrifuged at 3000 rpm for 5 min at 4 °C and the supernant was collected. Sodium

dodecyl sulfate (SDS, 10%, 10 mL) and 50 mg/mL RNase A (10 mL) were then added, and

the mixture was incubated for 2 h at 56 °C. After incubation, proteinase K (25 mg/mL) was

added and the mixture was further incubated at 37 oC for 2 h. Ammonium acetate (10m, 65

µL) and ice-cold ethanol (500 µL) were then added, and the reaction was mixed well. These

samples were incubated at -80 °C for 1 h. After incubation, the samples were centrifuged at

12000 rpm for 20 min at 4 °C. After centrifugation, the pellet was washed with 80% ethanol

and air dried for 10 min at room temperature. The pellet was dissolved in 50 mL TE buffer,

and DNA laddering was determined by using 2% agarose gel electrophoresis in TE buffer.

Molecular modeling procedure:

All the geometries are optimized in Gaussian 09 using PM3 semi-empirical method.29 Protein

structure was downloaded from Protein Data Bank (PDB ID: 3E22). Docking studies were

performed using AutoDock 4.2 software. The analysis of intermolecular interactions has been

performed using Pymol, v. 0.99.30

Aknowledgement

S.M.A.H and MA thank CSIR and UGC, New Delhi for the award of research fellowship and

for the financial support under the 12th Five Year plan project “Affordable Cancer

Therapeutics (ACT)” (CSC0301).

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30. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.

Figures

H3CO

H3CO

OCH3

OCH3

OH

2

3

MeO

MeO

OMe

OMe

O

NH

1

O

N

HN

NH

S

O O

OH

OCH3

N NH

NH

O

N

HN

R1

R2

R3

R4

4a-p

Figure 1: Colchicine binding site inhibitors

Figure 2: Cell cycle analysis of 4d on DU-145 cells. A: Control cells (DU-145), B: E7010 (2 µM), C: 4d (1 µM) and D: 4d (2 µM).

Figure 3: Effect of 4d on the tubulin polymerization: tubulin polymerization was monitored by the increase in fluorescence at 360 nm (excitation) and 420 nm (emission) for 1 h at 37 oC. E7010 was used as a positive control. Values indicated are the mean ± SD of two different experiments performed in triplicates.

Figure 4: Fluorescence based colchicine competitive binding assay of 4d were carried out at various concentrations containing 3 µM of tubulin and colchicine for 60 min at 37 ˚C. E7010 was used as a positive control where as taxol was used as negative control which binds at taxane site. Fluorescence values are normalized to DMSO (control).

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20 25 30

F/F

0

Concentration (µM)

4d

E7010

Taxol

Figure 5: Compound 4d triggers mitochondrial injury. Drops in membrane potential (∆Ψm) was assessed by JC-1 staining of DU-145 cells treated with test compound and samples were then subjected to flow cytometry analysis on a FACScan (Becton Dickinson) in the FL1, FL2 channel to detect mitochondrial potential. Shown are representative dot plots (left panels) and quantification of membrane potential (right panel). A: Untreated control cells (DU-145), B: E7010 (2 µM), C: 4d (1 µM) and D: 4d (2 µM).

Figure 6: Effect of compound 4d on caspase-3 activity: DU-145 cells were treated with compound 4d at 1 and 2 µM concentrations for 48 h. Values indicated are the mean ± SD of two different experiments performed in triplicates.

Figure 7: Annexin V-FITC staining. A: Untreated control cells (DU-145), B: E7010 (2 µM), C: 4d (1 µM) and D: 4d (2 µM).

Figure 8: DNA laddering assay: Lane-1: 4d (1 µM), Lane-2: E7010 (1 µM), Lane-3: Marker (100 bp) and Lane-4: Untreated control DNA (DU-145).

Figure 9: A) Binding pose of 4d (green) with tubulin (yellow). Amino acid residues that are in close proximity or that make interactions with the compound are indicated in blue color. B) Ligplot of interactions of E7010. C) 4d (green) and E7010 (cyan) superimposed on each other. D) Binding pose of 4d in β subunit of tubulin.

Tables

Table: 1: Cytotoxic activity (GI50) data of compounds 4a-p expressed in µM.

GI50 values in (µM)

Entry Comp R1 R2 R3 R4 A549a

MCF 7b

DU145c

1 4a H F H F 15.2 ± 0.27 16.0 ± 0.31 8.4 ± 0.12

2 4b H OCH3 H F 18.5 ± 0.33 17.3 ± 0.22 >100

3 4c OCH3 OCH3 OCH3 F >100 >100 7.0 ± 0.14

4 4d CF3 H CF3 F 39.2 ± 0.26 >100 0.99 ± 0.09

5 4e H F H OCH3 4.1 ± 0.12 >100 6.6 ± 0.32

6 4f H OCH3 H OCH3 >100 >100 21.2 ± 0.14

7 4g OCH3 OCH3 OCH3 OCH3 >100 >100 >100

8 4h CF3 H CF3 OCH3 >100 >100 9.8 ± 0.19

9 4i H F H Cl >100 >100 4.1 ± 0.08

10 4j H OCH3 H Cl >100 28.8 ± 0.16 16.6 ± 0.36

11 4k OCH3 OCH3 OCH3 Cl >100 >100 10.5 ± 0.21

12 4l CF3 H CF3 Cl 3.9 ± 0.14 10.5 ± 0.12 3.1 ± 0.16

13 4m H F H H >100 >100 7.7 ± 0.31

14 4n H OCH3 H H >100 >100 8.1 ± 0.10

15 4o OCH3 OCH3 OCH3 H 6.0 ± 0.22 >100 >100

16 4p CF3 H CF3 H 3.2 ± 0.11 5.4 ± 0.18 4.8 ± 0.22

17 E7010 - - - - 1.31± 0.12 1.25± 0.08 1.81± 0.13 a- Lung cancer cell line, b- breast cancer cell line, c- prostate cancer cell line

Table 2: Distribution of DU-145 cells in various phases of cell cycle

Sample Sub G1 % G0/G1 % S % G2/M %

A: Control (DU-145) 3.52 84.75 4.18 6.81

B: E7010 (2 µM) 4.11 54.38 2.94 36.52

C: 4d (1 µM) 4.64 57.93 1.63 32.49

D: 4d (2 µM) 2.41 53.38 1.77 41.19

Table 3: Distribution of apoptotic cells in Annexin-V FITC experiment

Sample UL % UR % LL% LR %

A: Control 0.22 1.96 97.12 0.71

B: E7010 (2 µM) 1.01 17.72 77.34 3.93

C: 4d (1 µM) 1.27 14.03 80.43 4.28

D: 4d (2 µM) 1.84 16.53 75.67 5.97

Schemes

Scheme 1: Synthesis of 2-anilinonicotinic acid derivatives.

Reagents and conditions: a) cat conc. H2SO4, ethanol, reflux, 2h b) substituted anilines (7a-d) ethylene glycol, 140 ˚C, 8h c) 2N NaOH, reflux, 2h.

Scheme 2: Synthesis of 2-anilinopyridin-3-yl amines.

Reagents and conditions: a) ethylene glycol, 140 ˚C, 8h b) SnCl2.2H2O, methanol, 80 ˚C, 3h.

Scheme 3: Synthesis of final 2-anilinopyridine dimers.

Reagents and conditions: a) EDC, HOBt, DMF, rt, 12h.

Graphical Abstract

Synthesis of 2-anilinopyridine dimers as microtubule targeting and apoptosis

inducing agents

Ahmed Kamal,* S. M. Ali Hussaini, V. Lakshma Nayak, M. Shaheer Malik, M. Lakshmi Sucharitha,

Thokhir Basha Shaik, Md. Ashraf, Chandrakant Bagul.

A series of 2-anilinopyridine dimers were synthesized and evaluated for their anticancer potential

against selected human cancer cell lines. One of the compounds 4d displayed good antiproliferative

activity against DU-145 cell line by inhibiting tubulin polymerization and induced apoptosis.