Synthesis of 2-anilinopyridine dimers as microtubule targeting and apoptosis inducing agents
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Transcript of Synthesis of 2-anilinopyridine dimers as microtubule targeting and apoptosis inducing agents
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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
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting proof before it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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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: [email protected]
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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
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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
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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>
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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>
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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>
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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
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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
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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.
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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,
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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
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(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]+.
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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-
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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
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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
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(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,
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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
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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
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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,
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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
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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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28. a) Cormier, A.; Marchand, M.; Ravelli, R. B. G.; Knossow, M.; Gigant, B. EMBO
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Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.;
Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth,
G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö.;
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30. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.
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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).
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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
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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.
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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).
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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.
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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
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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
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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.
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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.