Synthesis and evaluation of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl) nicotinamides as potential anticancer agents that inhibit tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2014.04.038

A series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)nicotinamides (4) was synthesized and tested for their anticancer activity against a panel of 60 human cancer cell lines. Some of the representative compounds such as 4a, 4b, 4f, 4g, 4i and 4t were selected for the five dose study and amongst them 4g and 4i displayed significant anticancer activity with GI₅₀ values ranging from 0.25 to 8.34 and 1.42 to 5.86 μM, respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. The most active compound in this series 4g also inhibited tubulin polymerization with IC₅₀ value 1.93 μM superior to that of E7010. Moreover, assay to investigate the effect on caspase-9, Hoechst staining and DNA fragmentation analysis suggested that these compounds induced cell death by apoptosis. Docking experiments showed that they interact and bind efficiently with tubulin protein. Overall, the results demonstrate that N-((1-benzyl-1H-1,2, 3-triazol-4-yl)methyl)nicotinamide scaffold possess anticancer property by inhibiting the tubulin polymerization.

Full text of DOI:10.1016/j.bmc.2014.04.038

Accepted Manuscript
Synthesis and Evaluation of N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)nicoti-
namides as Potential Anticancer Agents that Inhibit Tubulin Polymerization
Ahmed Kamal, N.V. SubbaReddy, V. LakshmaNayak, Narasimha Rao Bolla,
A.V. Subba Rao, B. Prasad
PII:
S0968-0896(14)00303-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.038
BMC 11533
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 February 2014
16 April 2014
18 April 2014
Please cite this article as: Kamal, A., SubbaReddy, N.V., LakshmaNayak, V., Bolla, N.R., Subba Rao, A.V., Prasad,
B., Synthesis and Evaluation of N-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)nicotinamides as Potential Anticancer
Agents that Inhibit Tubulin Polymerization, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.04.038
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Graphical Abstract
Synthesis and Evaluation of N-((1-Benzyl-1H-1,2,3-triazol-4-
yl)methyl)nicotinamides as Potential Anticancer Agents that Inhibit Tubulin
Polymerization
Ahmed Kamal,*a,b N. V. Subba Reddy,a V. Lakshma Nayak,a Narasimha Rao Bolla,b A.V. Subba
Raoa
A series of nicotinamides was synthesized and evaluated for their anticancer potential against a
panel of sixty human cancer cell lines by NCI. Some of the representative compounds like 4g
and 4i displayed good antiproliferative activity against MCF-7 cell line by inhibiting tubulin
polymerization and these compounds induced apoptosis.

Synthesis and Evaluation of N-((1-Benzyl-1H-1,2,3-triazol-4-
yl)methyl)nicotinamides as Potential Anticancer Agents that Inhibit
Tubulin Polymerization
Ahmed Kamal,*a,b N. V. SubbaReddy,a V. LakshmaNayak,a Narasimha Rao Bolla,bA.V.
Subba Rao,a B. Prasada
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 N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)nicotinamides (4) was
synthesized and tested for their anticancer activity against a panel of sixty human cancer cell
lines. Some of the representative compounds such as 4a, 4b, 4f, 4g, 4i and 4t were selected
for the five dose study and amongst them 4g and 4i displayed significant anticancer activity
with GI50 values ranging from 0.25 to 8.34 and 1.42 to 5.86 µM respectively. Cell cycle
analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7
cells. The most active compound in this series 4g also inhibited tubulin polymerization with
IC50 value 1.93 µM superior to that of E7010. Moreover, assay to investigate the effect on
Caspase-9, Hoechst staining and DNA fragmentation analysis suggested that these
compounds induced cell death by apoptosis. Docking experiments showed that they interact
and bind efficiently with tubulin protein. Overall, the results demonstrate that N-((1-benzyl-
1H-1,2,3-triazol-4-yl)methyl)nicotinamide scaffold possess anticancer property by inhibiting
the tubulin polymerization.
Keywords: E7010, cell cycle, cytotoxicity, triazoles, tubulin polymerization.
,aCorresponding authors. Tel.: +91-40-27193157; fax: +91-40-27193189 (A.K.); e-mail:
ahmedkamal@iict.res.in (A. Kamal).
1

1. Introduction
Small molecules which affect tubulin polymerization have drawn considerable
interest. Microtubules that consist of α- and β-tubulin heterodimers play a vital role in many
cellular processes, such as mitosis, formation and maintenance of cellular shape and cell
motility and intracellular transport.1 Their importance to cellular functions, especially mitosis
makes them prominent target for the development of anticancer agents.2 In general, antitumor
agents that inhibit the function of microtubules are known as antimitotic agents which exhibit
their anticancer properties by interfering with the dynamic process of microtubule assembly
and disassembly, inducing cell cycle arrest in the metaphase/anaphase transition and
subsequent apoptosis.33] Three distinct classes of antimitotic agents are identified by their
mechanism of action and their different binding sites on tubulin. The vinca alkaloids
(vincristine, vinblastine, vindesine, and vinorelbine) and colchicines are known as
microtubule-destabilizing agents or microtubule polymerization inhibitors but with a different
binding site and depolymerization mechanism. Vinca alkaloids are used in the treatment of
leukemias, lymphomas, small cell lung cancer and other cancers.4-6 The taxanes are
microtubule-stabilizing agents or polymerizing agents, which are used in the treatment of
lung, breast, ovarian, head and neck, and bladder carcinomas among others. However, the
problems of neurotoxicity, difficult syntheses, p-glycoprotein-mediated drug resistance and
isolation procedure5 with existing amtimitotic agents encouraged medicinal chemists to
develop novel antimitotic agents with novel modes of action.6,7
<Insert Figure 1>
E7010 (1), a sulphonamide that exhibits antitumor activity by inhibiting tubulin
polymerisation,8 causes cell cycle arrest and apoptosis in M phase.9,10 N-Phenyl nicotinamides
(2) are known as apoptosis inducers which blocks the cell cycle in G2/M phase and structure–
activity relationship (SAR) studies indicate that the 3-pyridyl group is crucial for their
activity.11 Whereas, 1,2,3-triazoles have displayed broad range of biological properties such
as antifungal, anticancer,12-15 anti-allergic, antibacterial, anti-HIV, anticonvulsant, anti-
inflammatory and antitubercular activities. Recently N-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)nicotinamides have been evaluated as antimicrotubule agents against selective
cancer cell lines.16 In continuation to our search for identifying potent and selective
anticancer agents, we incorporated triazole moiety to the 2-anilino pyridyl structural motif of
2

E7010 that resulted in triazolo linked nicotinamides (4a-t). These were evaluated for their
cytotoxic potential, effect on tubulin polymerization and cell-cycle effects.
2.
2.1. Chemistry.
1,4-Disubstituted triazoles can be prepared in high regioselectively by using Cu(I)-
Results and Discussions
catalyzed azide-alkyne cycloaddition (CuAAC) reaction.17,18 Following this strategy, the
synthesis of compounds 4a-t was carried out as shown in Scheme 1. Coupling of
propargylamine with 2-chloronicotinylchloride which was prepared from corresponding acid,
provided the precursor alkyne 6.This alkyne and substituted anilines 7a-k were refluxed in
ethylene glycol to afford the intermediates 2-anilino nicotinamides 8a-k. The azides 10a-d
that are required as another precursors were obtained from the reaction of benzyl bromides
with NaN3 in DMSO.19 The synthesis of 1,2,3-triazoles was carried out by exposing alkynes
to benzyl azides in the presence of catalytic Cu(I) and Na-Ascorbate in H2O/t-BuOH mixture.
<Insert Scheme 1>
3. Biology
3.1.Cytotoxicity:
20
MTT assay
was performed to evaluate the cytotoxic potential of the newly
synthesized compounds against five human cancer cell lines, A549 (lung), DU-145 (prostate),
MCF-7 (breast), ACHN (renal) and HT-29 (colon) in comparison to E7010 and the results are
summarized as IC50 values in Table 1. The results revealed that these compounds showed
promising cytotoxic activity with IC50 values ranging from 0.74-18.6 µM against different
cancer cell lines. The most active compounds 4g and 4i exhibited promising antiproliferative
activity with IC50 values ranging from 0.74-1.57 and 0.93-1.93 µM and significant activity in
MCF-7 cell line with IC50 values 0.74 and 0.93 µM respectively.
<Insert Table 1>
Further
all
the
newly
synthesizedN-((1-benzyl-1H-1,2,3-triazol-4-
yl)methyl)nicotinamides (4a-t) were evaluated for their cytotoxicity in preliminary screening
by the National Cancer Institute (NCI), against a panel of 60 human tumor cell lines derived
from nine different cancer types: leukemia, lung, colon, CNS, melanoma, ovarian, renal,
prostate and breast. These compounds can be considered as active, if they reduce the growth
3

of any of the cancer cell lines up to 60% or more in at least eight cancer cell lines screened.
From the preliminary screen, six compounds like 4a, 4b, 4f, 4g, 4i, and 4t were selected for
five dose screening to determine GI50 (the molar concentration required to cause 50% growth
inhibition). These results are expressed as GI50 values21 and listed in Table 2. These selected
compounds (4a, 4f, 4g, 4i, and 4t) exhibited GI50 values ranging from 2.1-20.4, 2.3-5.9, 0.25-
12.7, 2.40-10.5, and 2.1-9.03 µM respectively against majority of cell lines. Particularly, 4g
exhibited good cytotoxicity with GI50 values 0.25, 0.56, 0.62, 0.65, 0.72 and 0.75 µM against
MDA-MB-435, SF-295, PC-3, MCF7, SK-MEL-5 and K-562 cell lines respectively.
<Insert Table 2>
3.2. Cell cycle analysis
To better understand the cytotoxic effects of these nicotinamides, the cell cycle
analysis was performed on MCF-7 cell lines that were treated with 4g and 4i at 0.5 and 1µM
after 48 h by flow cytometry20. The results demonstrate that both 4g and 4i induced more
accumulation of cells in G2/M phase; 68.8% and 62.1% at 0.5 µM concentration and 69.9%
and 67.8% at 1 µM concentration respectively relative to control (Figure. 2, Table. 3).
However, in E7010, the percentage of cells (cell accumulation) at G2/M phase increased from
20.6% to 25.7% by increasing the concentration from 0.5 to 1µM. The effect of these
nicotinamides on cell cycle progression correlated well with their cytotoxic activity.
<Insert Figure 2>
<Insert Table 3>
3.3. Effect on tubulin polymerization
As these (4g and 4i) compounds showed G2/M cell cycle arrest which is a hallmark of
tubulin polymerization,22 we investigated the antiporliferative activities of these compounds
on their ability to inhibit tubulin polymerization. E7010 was used for comparision. In the
assembly assay,23,24 both 4g and 4i inhibited 65.2% and 67.9% respectively, whereas E7010
inhibited 58.0% tubulin polymerization compared to control (Figure. 3). Next these
compounds were evaluated for IC50 values for their ability to inhibit tubulin polymerization in
vitro and the results are summarized in Table 4. Compound 4g, which displayed maximum
tubulin inhibition assembly, was found to be more potent (IC50, 1.93 µM) than E7010 (2.88
4

µM), whereas 4i with IC50 value of 2.78 µM is equipotent to E7010. The effect of these
compounds on the inhibition tubulin assembly also correlated well with their significant
cytotoxicity.
<Insert Figure 3>
<Insert Table 4>
3.4. Effect on microtubules
The effects of 4g and 4i were further examined on the cellular microtubules in MCF-7
cancer cells by immunohistochemistry.25 It is clear from the cytotoxicity studies that these
compounds inhibited the growth of MCF-7 cell line. Therefore, MCF-7 cells were treated
with these nicotinamides along with the standard E7010 at 1 µM concentration for 48 h. As
shown in Figure 4, microtubules network displayed normal arrangement and organization in
untreated human breast cancer cells. However, cells treated with 4g and 4i showed disrupted
microtubule organization with spherical morphology thus demonstrating the inhibition of
tubulin polymerization.
<Insert Figure 4>
3.5. Hoechst staining for morphological analysis of apoptosis
Apoptosis is a continuous process in which the mitochondrial membrane potential is
depolarized and chromatin condensation takes place.25 Hoechst staining was performed to
investigate the apoptotic inducing effect of 4g and 4i in MCF-7 cell line at 1 µM
concentration for 24 h. These compounds showed significant effect on the nuclear
condensation in comparison to the untreated control cells as shown in Figure. 5. These results
demonstrated that these compounds are effective in inducing cellular apoptosis.
<Insert Figure 5>
3.6. Effect on caspase-9
The activation of caspases play an important role in the process of cell death.26 To
ascertain the cytotoxic effect of 4g and 4i were due to apoptotic cell death, we examined the
activitation of caspase 9 in MCF-7 cell line which lacks endogenous caspase-9.27 MCF-7 cells
were treated with these compounds along with standard E7010 at 1 and 2 µM concentrations.
5

The results demonstrate that there was 4 to 5 fold induction in caspase-9 activity by these
nicotinamides at 2 µM. The reference compound E7010 induced 3-fold induction in caspase
9 activity as compared to untreated cells as shown in Figure 6. Therefore activation of
caspase 9 by 4g and 4i suggest that they have ability to induce apoptosis in MCF-7 cells.
<Insert Figure 6>
3.7. DNA fragmentation assay
DNA fragmentation is 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..28 It is clear from the cytotoxic studies that these
compounds (4g and 4i) significantly inhibited the growth of MCF-7cell lines that were
treated with these compounds at 1 µM concentration for 48 h. DNA fragmentation revealed
that these compounds induced significant DNA fragmentation, however, no fragmentation
was observed in untreated cells as shown in Figure.7.
<Insert Figure 7>
4.0. Molecular modelling studies
To elucidate the mode of binding interactions with tubulin, we performed molecular
docking studies with most active molecules 4g and 4i. Interestingly, binding pose of
compound 4g shows that ether oxygen attached rings are in the hydrophobic pocket of the
colchicine binding domain in the β chain forming hydrophobic interaction with the Leu248,
Ala316, Leu255, Cys241, Val238 and Ala250. In addition to hydrophobic contacts, hydrogen
bonding is observed between the βCys241 and ether oxygen. Moreover, 2,4-
dimethoxyanilino group of 4g interact with the amino residues located deep in the colchicine
binding site where the A ring of colchicine (trimethoxybenzyl ring) interacts. The structure of
4g resembles to that of E7010, hence a comparison the docking pose of 4g with the binding
pose of E7010 was performed. Superimposed structure for cocrystallized pose of E7010
(PDB ID 3HKC) and docking pose of 4g shows that the 2-anilinopyridinyl group of 4g is in
opposite to that of the E7010. This may be due to the longer substitution on 2-anilinopyridyl
ring. In E7010, the 2-anilinopyridinyl group is in hydrophobic pocket of colchicine binding
site however, it is at α,β interface for 4g. Carbonyl oxygen forms a hydrogen bonding
6

interaction with the αAsn101 (Fig. 8). Some of the amino acids in hydrophobic contacts with
4g are αSer178, αTyr224, βGln247, βLeu248, βAla354, αGln11, and βLys254 at α, β
interface. Overall molecular docking studies provide information that these molecules fit well
at α,β interface of the tubulin and making favourable interactions. Figure 9 gives the
significant information about the interaction of the different amino acids with each part of the
molecule. These interactions could be useful in rationalizing the potency observed for
compound 4g in its inhibition of tubulin polymerization.
<Insert Figure 8>
<Insert Figure 9>
5. Conclusion
In summary, a series of N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)nicotinamides (4a-
t) was synthesized and evaluated for cytotoxic activity. Among them, six compounds were
selected for the 60 cell line anticancer screening programme by NCI, USA. We identified two
compounds (4g and 4i) as the most effective antiproliferative agents. They showed significant
activity in the breast cancer cell line MCF-7 and the FACS analysis showed that cell cycle
arrest at G2/M phase. These compounds 4g and 4i also inhibited tubulin polymerization with
IC50 1.93 and 2.78 µM. Detailed biological studies like Hoechst staining, caspase 9 activity
assay and DNA fragmentation analysis demonstrate that these compounds induced apoptotic
cell death. Overall, the current study demonstrated that 4g and 4i are promising tubulin
polymerization inhibitors and worthy of further evaluation as potential chemotherapeutic
agents.
6. Experimental Section
All chemicals and reagents were obtained from Aldrich (Sigma-Aldrich), St. Louis,
MO, USA), Lancaster (Alfa Aesar, 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 silica gel glass plate containing 60 GF-254, and
visualization was achieved by UV light or iodine indicator. Column chromatography was
1
performed with Merck 60–120 mesh silica gel. H and 13C NMR spectra were determined in
CDCl3 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,
7

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.
6.1. Synthesis of 2-chloro-N-(prop-2-ynyl)nicotinamide (6)
To a solution of 2-chloronicotinic acid (5g, 31.84 mmol) in dry DCM under nitrogen,
oxalyl chloride (3.3 mL, 38.21 mmol) and catalytic amount of N,N-dimethylformamide were
added carefully with stirring. The reaction was stirred for 3 h. After completion of the
reaction, the reaction mixture was concentrated under vaccum to yield 2-chloronicotinyl
chloride as solid which was used for next step without purification. 2-Chloronicotinyl
chloride (4.6 g, 26.1 mmol) was dissolved in dry DCM, cooled to 0 °C, propargylamine
hydrochloride (26.1 mmol) and triethylamine (9.04 mL, 65.25 mmol) were added. The
reaction was warmed to room temperature. After stirring overnight, the reaction mixture was
diluted with water and extracted with DCM. The combined organic extracts were washed
with aq. NaHCO3 and dried with Na2SO4 and concentrated in vacuo.1H NMR (300 MHz,
CDCl3) δ 8.46 (dd, J = 3.0, 2.2 Hz, 1H), 8.10 (dd, J = 6.2, 3.0 Hz, 1H), 7.36 (d, J = 3.0 Hz,
1H), 6.94 (brs, 1H), 4.25 (m, 2H), 2.32 (t, J = 2.2 Hz, 1H).
6.2. 2-(Phenylamino)-N-(prop-2-ynyl)nicotinamide (8a): The compound 2-chloro-N-(prop-
2-ynyl)nicotinamide 6 (195 mg, 1 mmol) and aniline 7a (93 mg, 1 mmol) were taken in
ethylene glycol and heated at 120 °C for 6 h. After completion of the reaction as confirmed
by TLC, the reaction mixture was cooled, diluted with water and extracted with ethyl acetate
and concentrated in vacuo. The crude product was further purified by column
chromatography to afford compound 8a as a pale yellow solid (175 mg, 70%); 1H NMR (300
MHz, CDCl3) δ 10.30 (s, 1H), 8.30 (dd, J = 3.0, 1.5 Hz, 1H), 7.69-7.64 (m, 3H), 7.34-7.29
(m, 2H), 7.05-7.00 (m, 1H), 6.70-6.65 (m, 1H), 6.36 (brs, 1H), 4.23-4.21 (m, 2H), 2.31 (s,
1H); MS (ESI m/z): 252 [M+H]+ .
6.3. 2-(4-Fluorophenylamino)-N-(prop-2-ynyl)nicotinamide (8b):The title compound was
prepared according to the method described for compound 8a, employing 2-chloro-N-(prop-
2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 4-fluoroaniline (7b, 111 mg, 1 mmol) to obtain
1
the pure product 8b as solid (193 mg, 72%); H NMR (300 MHz, CDCl3) δ 10.50 (s, 1H),
8.37 (dd, J = 3.0, 2.2 Hz, 1H), 7.82-7.76 (m, 1H), 7.72 (dd, J = 6.0, 1.5 Hz, 1H), 7.28-7.19
(m, 2H), 6.77-6.67 (m, 2H), 6.34 (brs, 1H), 4.25-4.23 (m, 2H), 2.32 (s, 1H); MS (ESI m/z):
270 [M+H]+.
8

6.4. 2-(4-Chlorophenylamino)-N-(prop-2-ynyl)nicotinamide (8c):The title compound was
prepared according to the method described for compound 8a, employing 2-chloro-N-(prop-
2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 4-chloroaniline (7c, 127 mg, 1 mmol) to obtain
1
the pure product 8c as solid (200 mg, 70%); H NMR (300 MHz, CDCl3) δ 10.38 (s, 1H),
8.33 (dd, J = 3.0, 1.5 Hz, 1H), 7.71 (dd, J = 6.0, 1.5 Hz, 1H), 7.63 (m, 2H), 7.27-7.24 (m,
2H), 6.73-6.69 (m, 1H), 6.35 (brs, 1H), 4.24-4.21 (m, 2H), 2.31 (s, 1H); MS (ESI m/z): 286
[M+H]+.
6.5. 2-(4-Bromophenylamino)-N-(prop-2-ynyl)nicotinamide (8d):The title compound was
prepared according to the method described for compound 8a, employing 2-chloro-N-(prop-
2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 4-bromoaniline (7d, 172 mg, 1 mmol) to
1
obtain the pure product 8d as solid (236 mg, 70%); H NMR (300 MHz, CDCl3) δ 10.36 (s,
1H), 8.32 (dd, J = 3.0, 1.6 Hz, 1H), 7.69 (dd, J = 6.0, 1.6 Hz, 1H), 7.63-7.60 (m, 2H), 7.24-
7.20 (m, 2H), 6.72-6.69 (m, 1H), 6.34 (brs, 1H), 4.24-4.21 (m, 2H), 2.31 (s, 1H); MS (ESI
m/z): 330 [M+H]+.
6.6. 2-(4-Methoxyphenylamino)-N-(prop-2-ynyl)nicotinamide (8e): The title compound
was prepared according to the method described for compound 8a, employing 2-chloro-N-
(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 4-methoxyaniline (7e, 123 mg, 1 mmol)
to obtain the pure product 8e as solid (202 mg, 72%); 1H NMR (300 MHz, CDCl3) δ 10.20 (s,
1H), 8.27 (dd, J = 3.4, 1.3 Hz, 1H), 7.66 (dd, J = 6.2, 1.5 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H),
6.88 (d, J = 8.8 Hz, 2H), 6.65-6.60 (m, 1H), 6.36 (brs, 1H), 4.24-4.21 (m, 2H), 3.80 (s, 3H),
2.32 (s, 1H); MS (ESI m/z): 282 [M+H]+.
6.7. 2-(3-Fluorophenylamino)-N-(prop-2-ynyl)nicotinamide (8f): The title compound was
prepared according to the method described for compound 8a, employing 2-chloro-N-(prop-
2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 3-fluoroaniline (7f, 111 mg, 1 mmol) to obtain
1
the pure product 8f as solid (182 mg, 72%); H NMR (300 MHz, CDCl3) δ 10.49 (s, 1H),
8.37 (dd, J = 3.2, 1.5 Hz, 1H), 7.79 (d, J = 11.7 Hz, 1H), 7.72 (dd, J = 1.5, 6.2 Hz, 1H), 7.25-
7.19 (m, 3H), 6.78-6.67 (m, 2H), 6.32 (brs, 1H), 4.24-4.21 (m, 2H), 2.32 (s, 1H); MS (ESI
m/z): 270 [M+H]+.
6.8.
2-(2,4-Dimethoxyphenylamino)-N-(prop-2-ynyl)nicotinamide(8g):
The
title
compound was prepared according to the method described for compound 8a, employing 2-
chloro-N-(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 2,4-dimethoxyaniline (7g, 153
1
mg, 1 mmol) to obtain the pure product 8g as solid (217 mg, 70%); H NMR (300 MHz,
9

CDCl3) δ 10.21 (s, 1H), 8.30 (dd, J = 4.5, 1.5 Hz, 2H), 8.21 (d, J = 8.3 Hz, 1H), 7.69 (dd, J =
6.0, 1.5 Hz, 1H), 6.65-6.63 (m, 2H), 6.36 (brs, 1H), 4.26-4.24 (m, 2H), 3.91 (s, 3H), 3.81 (s,
3H), 2.36 (s, 1H); MS (ESI m/z): 312 [M+H]+.
6.9. 2-(2,5-Dimethoxyphenylamino)-N-(prop-2-ynyl)nicotinamide (8h): The title
compound was prepared according to the method described for compound 8a, employing 2-
chloro-N-(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 2,5-dimethoxyaniline (7h, 153
1
mg, 1 mmol) to obtain the pure product 8h as solid (211 mg, 68%); H NMR (300 MHz,
CDCl3) δ 10.60 (s, 1H), 8.36 (s, 2H), 7.71 (d, J = 7.3 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 6.69-
6.66 (m, 1H), 6.48 (dd, J = 8.6, 2.8 Hz, 1H), 6.36 (brs, 1H), 4.26-4.24 (m, 2H), 3.91 (s, 3H),
3.80 (s, 3H), 2.31 (s, 1H); MS (ESI m/z): 312 [M+H]+.
6.10. 2-(3,5-Dimethoxyphenylamino)-N-(prop-2-ynyl)nicotinamide (8i): The title
compound was prepared according to the method described for compound 8a, employing 2-
chloro-N-(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 3,5-dimethoxyaniline (7i, 153
1
mg, 1 mmol) to obtain the pure product 8i as solid (217 mg, 70%); H NMR (300 MHz,
CDCl3) δ 10.35 (s, 1H), 8.33 (dd, J = 3.4, 1.2 Hz, 1H), 7.72 (dd, J = 6.6, 1.2 Hz, 1H), 6.98 (s,
2H), 6.72-6.68 (m, 1H), 6.36 (brs, 1H), 6.14 (t, J = 2.0 Hz, 1H), 4.24-4.21 (m, 2H), 3.79 (s,
6H), 2.31 (s, 1H); MS (ESI m/z): 312 [M+H]+.
6.11. N-(prop-2-ynyl)-2-(3,4,5-trimethoxyphenylamino)nicotinamide (8j): The title
compound was prepared according to the method described for compound 8a, employing 2-
chloro-N-(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and 3,4,5-trimethoxyaniline (7j,
183 mg, 1 mmol) to obtain the pure product 8j as solid (235 mg, 65%); 1H NMR (300 MHz,
CDCl3) δ 10.32 (s, 1H), 8.34 (dd, J = 3.2, 1.5 Hz, 1H), 7.69 (dd, J = 6.2, 1.3 Hz, 1H), 6.95 (d,
J = 2.0 Hz, 2H), 6.72-6.68 (m, 1H), 6.36 (brs, 1H), 4.25-4.23 (m, 2H), 3.87 (s, 6H), 3.82 (s,
3H), 2.31 (s, 1H); MS (ESI m/z): 342 [M+H]+.
6.12. 2-(4-Chlorobenzylamino)-N-(prop-2-ynyl)nicotinamide (8k): The title compound
was prepared according to the method described for compound 8a, employing 2-chloro-N-
(prop-2-ynyl)nicotinamide (6, 195 mg, 1 mmol) and (4-chlorophenyl)methanamine (7k, 141
1
mg, 1 mmol) to obtain the pure product 8k as solid (212 mg, 71%); H NMR (300 MHz,
CDCl3) δ 10.21 (s, 1H), 8.48 (brs, 1H), 8.20 (dd, J = 2.9, 1.8 Hz, 1H), 7.60 (dd, J = 6.0, 1.83
Hz, 1H), 7.32-7.27 (m, 3H), 6.56-6.50 (m, 1H), 6.22 (brs, 1H), 4.66 (d, J = 5.8 Hz, 2H) 4.19-
4.16 (m, 2H), 2.28 (s, 1H); MS (ESI m/z): 300 [M+H]+.
10

6.13. N-((1-(3-Phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(phenylamino)
nicotinamide (4a):To a solution of 2-(phenylamino)-N-(prop-2-ynyl)nicotinamide (8a, 150
mg, 0.59 mmol)and 1-(azidomethyl)-3-phenoxybenzene (10a, 147 mg, 0.65 mmol) in 2:1
mixture of water and tert-butyl alcohol, sodium ascorbate (12 mg, 0.06 mmol) and copper (II)
sulphate (7.5 mg, 0.03 mmol) were added sequentially. The reaction was stirred at room
temperature for overnight, TLC analysis indicated completion of reaction. The solvent was
concentrated under vacuum and extracted with EtOAc to give crude product. The crude was
purified by column chromatography to afford pure product 4a as brown solid (210 mg, 74%);
mp: 124-126 ºC; 1H NMR (300 MHz, CDCl3) δ 10.51 (s, 1H), 8.25 (dd, J = 2.2, 1.5 Hz, 1H),
7. 84 (dd, J = 2.2,1.5 Hz, 1H), 7.80 (brs, 1H), 7.66-7.60 (m, 3H), 7.33-7.25 (m, 4H), 7.08 (t, J
= 7.5 Hz, 1H), 7.00-6.90 (m, 7H), 6.61-6.59 (m, 1H ), 5.45 (s, 2H), 4.62 (d, J = 5.2 Hz, 2H);
13C NMR (75 MHz, CDCl3) δ 168.3, 158.1, 156.3, 155.3, 151.4, 140.0, 136.2, 136.1, 130.5,
129.9, 128.7, 123.9, 122.7, 122.5, 122.3, 120.5, 119.2, 118.6, 118.1, 113.0, 110.60, 53.9,
35.1; IR ( cm−1): 3237, 2951, 2360, 1641, 1596, 1488, 1304, 1251, 1054, 826; MS (ESI
m/z): 477 [M+H]+; HRMS (ESI m/z) calcd for C28H25N6O2: 477.2033 [M+H]+; found:
477.2002.
6.14.
2-(4-Fluorophenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol4-yl)methyl)
nicotinamide (4b): Starting from 2-(4-fluorophenylamino)-N-(prop-2-ynyl)nicotinamide (8b,
150mg, 0.55 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 138 mg, 0.61 mmol), 4b
was prepared according to the procedure described for 4a as white solid (220 mg, 80%); mp:
1
160-162 ºC; H NMR (300 MHz, CDCl3) δ 10.36 (s, 1H), 8.25 (dd, J = 3.0, 1.5 Hz, 1H ),
7.76 (dd, J = 3.0, 1.5 Hz, 2H), 7.61-7.59 (m, 3H), 7.35-7.28 (m, 3H), 7.22 (brs, 1H), 7.11 (t, J
= 7.5 Hz, 1H), 7.03-6.94 (m, 7H), 6.67-6.63 (m, 1H), 5.45 (s, 2H), 4.64 (d, J = 6.0 Hz, 1H);
13C NMR (75 MHz, CDCl3+DMSO) δ 166.5, 157.7, 155.9, 154.9, 154.5, 153.5, 149.1, 143.8,
136.0, 135.5, 135.0, 131.2, 128.6, 122.1, 121.5, 121.2, 119.9, 119.1, 117.0, 113.5, 111.6,
109.2, 51.6, 33.6; IR ( cm−1): 3282, 2918, 1642, 1612, 1589, 1458, 1225, 1053, 825; MS
(ESI m/z): 495 [M+H]+; HRMS (ESI m/z) calcd for C28H24O2N6F: 495.1939, [M+H]+; found:
495.1939.
6.15. 2-(4-Chlorophenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
nicotinamide (4c): Starting from 2-(4-chlorophenylamino)-N-(prop-2-ynyl)nicotinamide (8c,
150 mg, 0.52 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 130 mg, 0.57 mmol), 4c
was prepared according to the procedure described for 4a as yellow solid (204 mg, 76%); mp:
1
127-129 ºC; H NMR (300 MHz, CDCl3) δ 10.51 (s, 1H), 8.25 (dd, J = 2.2, 1.5 Hz, 1H), 7.
11

84 (dd, J = 2.2, 1.5 Hz, 2H), 7.61-7.58 (m, 3H), 7.35-7.23 (m, 5H), 7.08 (t, J = 7.5 Hz, 1H),
6.98-6.91 (m, 5H), 6.67-6.63 (m, 1H ), 5.45 (s, 2H), 4.62(d, J = 5.2 Hz, 2H); IR ( cm−1):
3283, 3146, 2360, 1641, 1613, 1589, 1488, 1309, 1256, 1052, 823; MS (ESI m/z): 511
[M+H]+
6.16. 2-(4-Bromophenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
nicotinamide (4d): Starting from 2-(4-bromophenylamino)-N-(prop-2-ynyl)nicotinamide
(8d, 150 mg, 0.45 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 112 mg, 0.5 mmol),
4d was prepared according to the procedure described for 4a as white solid (181 mg, 72%);
mp: 149-151 ºC; 1H NMR (300 MHz, CDCl3) δ 10.51 (s, 1H), 8.29 (dd, J = 2.2, 1.5 Hz, 1H),
7.78 (dd, J = 2.2,1.5 Hz, 1H), 7.60-7.55 (m, 3H), 7.40-7.28 (m, 6H), 7.14-7.08 (m, 1H), 6.99-
6.91 (m, 5H), 6.70-6.65 (m, 1H), 5.48 (s, 2H ), 4.66 (d, J = 5.2Hz, 2H); IR ( cm−1): 3295,
2951, 2360, 1645, 1614, 1583, 1486, 1310, 1258, 1046, 825; MS (ESI m/z): 555 [M+H]+;
HRMS (ESI m/z) calcd for C28H24O2N6Br: 555.1138, [M+H]+; found: 555.1124.
6.17. 2-(4-Methoxyphenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
nicotinamide (4f): Starting from 2-(4-methoxyphenylamino)-N-(prop-2-ynyl)nicotinamide
(8f, 150 mg, 0.53 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 132 mg, 0.58 mmol),
4f was prepared according to the procedure described for 4a as yellow solid (202 mg, 75%);
1
mp: 95-98 ºC; H NMR (300 MHz, CDCl3) δ 10.32 (s, 1H), 8.18 (dd, J = 3.0, 1.5 Hz, 1H),
7.80 (dd, J = 3.0, 1.5 Hz, 2H), 7.59(s, 1H), 7.49 (d, J = 8.3 Hz, 2H), 7.33-7.27 (m, 3H), 7.07
(t, J = 7.5 Hz, 1H), 6.96-6.90 (m, 5H), 6.85-6.79 (m, 2H), 6.54-6.50 (m, 1H), 5.45 (s, 2H),
4.60 (d, J = 5.2 Hz, 2H) 3.78(s, 3H); 13C NMR(75 MHz, CDCl3) δ 168.2, 158.0, 155.7, 155.3,
151.5, 144.8, 136.1, 135.9, 133.0, 132.7, 130.4, 129.8, 123.8, 122.8, 122.4, 120.7, 119.1,
118.5, 118.0, 113.9, 112.34, 109.9,55.4, 53.8, 35.0; IR ( cm−1): 3235, 3049, 2360, 1640,
1607, 1585, 1457, 1306, 1250, 1053, 827; MS (ESI m/z): 507 [M+H]+; HRMS (ESI m/z)
calcd for C29H27O3N6: 507.2145 [M+H]+; found: 507.2103.
6.18. 2-(3-Fluorophenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
nicotinamide (4e): Starting from 2-(3-fluorophenylamino)-N-(prop-2-ynyl)nicotinamide
(8e, 150 mg, 0.55 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 86 mg, 0.61 mmol),
4e was prepared according to the procedure described for 4a as white solid (220 mg, 80%);
mp: 130-132 ºC; 1H NMR (300 MHz, CDCl3) δ 10.62 (s, 1H), 8.31 (dd, J = 3.0, 1.7 Hz, 1H),
7. 83 (dd, J = 3.0, 1.7 Hz, 1H), 7.78-7.73 (m, 1H), 7.59-7.53 (m, 2H), 7.36-7.18 (m, 5H), 7.12
(t, J = 7.5 Hz, 1H), 6.99-6.91 (m, 5H), 6.71-6.66 (m, 2H ), 5.48 (s, 2H), 4.66 (d, J = 5.6Hz,
2H); IR ( cm−1): 3345, 3142, 2360, 1642, 1608, 1588, 1488, 1259, 1053, 827; MS (ESI
12

m/z): 495 [M+H]+; HRMS (ESI m/z) calcd for C28H24O2N6F: 495.1939 [M+H]+; found:
495.1912.
6.19.
2-(2,4-Dimethoxyphenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)nicotinamide (4g): Starting from 2-(2,4-dimethoxyphenylamino)-N-(prop-2-
ynyl)nicotinamide (8g, 150 mg, 0.48 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a,
119 mg, 0.53 mmol), 4g was prepared according to the procedure described for 4a as yellow
1
solid (201 mg, 78%); mp: 127-129 ºC; H NMR (300 MHz, CDCl3) δ 10.30 (s, 1H), 8.25-
8.21 (m, 2H), 7.73 (d, J = 7.5 Hz 1H), 7.56 (s, 1H), 7.35-7.28 (m, 4H), 7.11 (t, J = 7.5 Hz,
1H), 6.99-6.90 (m, 5H), 6.59-6.47 (m, 3H), 5.45 (s, 2H), 4.67 (d, J = 5.2 Hz, 2H), 3.87 (s,
13
3H), 3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ 168.0, 158.3, 157.9, 155.3, 151.3, 150.8,
144.8, 136.1, 135.7, 133.9, 130.4, 129.8, 127.1, 123.7, 123.0, 122.3, 121.4, 119.1, 118.5,
117.9, 112.1, 110.8, 103.4, 98.8, 55.8, 55.4, 53.8, 35.0; IR ( cm−1): 3366, 2927, 2359, 1632,
1602, 1560, 1489, 1254, 1055, 827; MS (ESI m/z): 537 [M+H] +; HRMS (ESI m/z) calcd for
C30H29O4N6: 537.2229 [M+H]+; found: 537.2244.
6.20.
2-(2,5-Dimethoxyphenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)nicotinamide (4h): Starting from 2-(2,5-dimethoxyphenylamino)-N-(prop-2-
ynyl)nicotinamide (8h, 150 mg, 0.48 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a,
119 mg, 0.53 mmol), 4h was prepared according to the procedure described for 4a as yellow
solid (201 mg, 78%); mp: 154-156 ºC; 1H NMR (300 MHz, CDCl3) δ 10.71 (s, 1H), 8.34 (d,
J = 2.2Hz, 2H), 8.32 (dd, J = 3.0, 1.5 Hz, 1H) ,7.76 (dd, J = 2.2, 1.5 Hz, 1H), 7.54 (s, 1H),
7.32 (d, J = 7.5 Hz, 2H), 7.28 (d, J = 2.2 Hz, 1H), 7.18-7.06 (m, 2H), 6.97-6.84 (m, 4H), 6.76
(d, J = 8.3 Hz 1H), 6.66-6.62 (m, 1H), 6.42-6.38 (m, 1H), 5.45 (s, 2H), 4.66 (d, J = 5.2 Hz,
2H), 3.90 (s, 3H), 3.79(s, 3H); 13C NMR (75 MHz, CDCl3) δ 168.4, 158.4, 156.7, 155.1,
154.2, 151.5, 145.2, 143.9, 136.7, 136.2, 131.3, 130.8, 130.2, 124.2, 122.8, 121.2, 119.6,
118.9, 118.4, 113.4, 112.1, 111.4, 107.1, 105.5, 57.2, 56.0, 54.2, 35.5; IR ( cm−1): 3335,
2942, 2360, 1635, 1602, 1578, 1488, 1291, 1054, 827; MS (ESI m/z): 537 [M+H] +; HRMS
(ESI m/z) calcd for C30H29O4N6: 537.2244 [M+H]+; found: 537.2229.
6.21.
2-(3,5-Dimethoxyphenylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-
yl)methyl)nicotinamide (4i):. Starting from 2-(3,5-dimethoxyphenylamino)-N-(prop-2-
ynyl)nicotinamide (8i, 150 mg, 0.48 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a,
119 mg, 0.53 mmol), 4i was prepared according to the procedure described for 4a as white
solid (201 mg, 78%); mp: 123-125 ºC; 1H NMR (300 MHz, CDCl3) δ 10.55 (s, 1H), 8.27 (dd,
13

J = 2.2, 1.5 Hz, 1H), 7.82 (dd, J = 2.2, 1.5 Hz, 1H), 7.72 (t, J = 6.0 Hz, 1H), 7.58 (s, 1H),
7.33-7.28 (m, 3H), 7.08 (t, J = 7.5 Hz, 1H), 6.97-6.90 (m, 7H), 6.62-6.58 (m, 1H), 6.08 (t, J =
2.2 Hz 1H), 5.46 (s, 2H ), 4.61 (d, J = 5.2 Hz, 2H), 3.80 (s, 6H); IR ( cm−1): 3245, 3038,
2360, 1624, 1602, 1526, 1457, 1319, 1259, 1058, 835; MS (ESI m/z): 537 [M+H] +; HRMS
(ESI m/z) calcd for C30H29O4N6: 537.2244 [M+H]+; found: 537.2229.
6.22.
N-((1-(3-Phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(3,4,5-
trimethoxyphenylamino)nicotinamide(4j): Starting from N-(prop-2-ynyl)-2-(3,4,5-
trimethoxyphenylamino)nicotinamide (8j, 150 mg, 0.41 mmol) and 1-(azidomethyl)-3-
phenoxybenzene (10a, 102 mg, 0.45 mmol), 4j was prepared according to the procedure
1
described for 4a as brown solid (186 mg, 75%); mp: 142-144 ºC; H NMR (300 MHz,
CDCl3) δ 10.42 (s, 1H), 8.27 (d, J = 7.5, Hz, 1H ), 7.85 (d, J = 7.5 Hz, 1H), 7.75 (brs, 1H),
7.60 (s, 1H), 7.39 -7.27 (m, 3H), 7.11 (t, J = 7.5 Hz, 1H), 6.99-6.83 (m, 7H), 6.65 (m, 1H),
5.47 (s, 2H), 4.66 (d, J = 5.2 Hz, 2H), 3.85 (s, 6H), 3.81 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 168.1, 158.3, 156.3, 153.3, 153.0, 151.4, 144.7, 136.1, 133.4, 132.7, 130.4, 129.8, 123.8,
122.8, 122.3, 120.7, 119.1, 118.5, 118.0, 112.8, 110.4, 98.3, 60.8, 56.0, 53.8, 35.0; MS (ESI
m/z): 567 [M+H]+; HRMS (ESI m/z) calcd for C31H31N6O5: 567.2350 [M+H]+; found:
567.2336.
6.23.
N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(4-fluorophenylamino)
nicotinamide (4k): Starting from 2-(4-fluorophenylamino)-N-(prop-2-ynyl)nicotinamide (8b,
150 mg, 0.55 mmol) and 1-(azidomethyl)-4-fluorobenzene (10b, 91 mg, 0.61 mmol), 4k was
prepared according to the procedure described for 4a as yellow solid (187 mg, 80%); mp:
1
197-199 ºC; H NMR (300 MHz, CDCl3) δ 10.61 (s, 1H), 8.63 (brs, 1H), 8.24 (dd, J = 4.4,
1.4 Hz, 1H), 7. 98 (dd, J =, 8.0, 1.8 Hz, 1H), 7.64 (s, 1H), 7.63-7.58 (m, 2H), 7.41 (s, 1H),
7.23 (s, 1H), 6.98 (t, J = 8.4 Hz, 2H), 6.88 (t, J = 8.4 Hz, 2H), 6.69-6.65 (m, 1H ), 5.44 (s,
2H), 4.63 (d, J = 5.5 Hz, 2H); IR ( cm−1): 3245, 2936, 2359, 1646, 1609, 1586, 1458, 1301,
1259, 1057, 827; MS (ESI m/z): 421 [M+H]+; HRMS (ESI m/z) calcd for C22H19ON6F2:
421.1558[M+H] +; found: 421.1556.
6.24.
2-(4-Fluorophenylamino)-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-
(4l): Starting from 2-(4-fluorophenylamino)-N-(prop-2-
yl)methyl)nicotinamide
ynyl)nicotinamide (8b, 150 mg, 0.55 mmol) and 1-(azidomethyl)-4-methoxybenzene (10c, 99
mg, 0.61 mmol), 4l was prepared according to the procedure described for 4a as brown solid
(192 mg, 80%); mp: 201-204 ºC; 1H NMR (300 MHz, CDCl3) δ 10.57 (s, 1H), 8.45 (brs, 1H),
8.25 (dd, J = 4.7, 1.4 Hz, 1H), 7. 94 (dd, J = 7.7, 1.4 Hz, 1H), 7.62-7.57 (m, 3H), 7.41 (s,
14

1H), 7.23 (s, 1H), 6.98 (t, J = 8.4 Hz, 2H), 6.88 (t J = 8.4 Hz, 2H), 6.69-6.65 (m, 1H ), 5.44
13
(s, 2H), 4.63 (d, J = 5.5 Hz, 2H), 3.78 (s, 3H); C NMR (75 MHz, CDCl3) δ 167.1, 158.6,
154.13, 149.7, 135.9, 135.5, 128.6, 126.0, 121.4, 120.6, 120.5, 113.8, 113.2, 112.0, 114.1,
109.7, 54.2, 52.3, 34.2 ; IR ( cm−1): 3235, 2978, 2359, 1635, 1584, 1456, 1305, 1248,
1056, 826; MS (ESI m/z): 433 [M+H]+; HRMS (ESI m/z) calcd for C23H22FN6O2:
433.1782[M+H] +; found: 433.17633.
6.25.
N-((1-(3,5-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(4-
fluorophenylamino)nicotinamide (4m): Starting from 2-(4-fluorophenylamino)-N-(prop-2-
ynyl)nicotinamide (8b, 150 mg, 0.55 mmol) and 1-(azidomethyl)-3,5-dimethoxybenzene
(10d, 116 mg, 0.61 mmol), 4m was prepared according to the procedure described for 4a as
solid (182 mg, 71%); mp: 209-211 ºC;1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1H), 8.19 (dd,
J = 3.0, 1.5 Hz, 1H), 7.81 (dd, J = 3.0, 1.5 Hz, 2H), 7.52-7.47 (m, 3H), 7.20 (d, J = 9.0 Hz,
2H), 6.83 (t, J = 7.5 Hz, 3H), 6.55-6.51 (m, 1H), 5.41 (s, 2H), 4.58 (d, J = 4.5Hz, 2H), 3.78
(s, 3H) , 3.77 (s, 3H);13C NMR (75 MHz, CDCl3) δ 166.2, 160.0, 157.4, 154.2, 153.2, 148.9,
143.5, 135.3, 134.8, 130.1, 128.5, 121.3, 119.6, 113.6, 111.4, 108.9, 54.2, 50.8, 33.3; IR (
cm−1): 3289, 2930, 2359, 1618, 1590, 1476, 1321, 1299, 1055, 825; MS (ESI m/z): 463
[M+H] +; HRMS (ESI m/z) calcd for C24H24O3N6F: 463.1888 [M+H] +; found: 463.1856.
6.26. N-((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(4-methoxyphenylamino)
nicotinamide (4n): Starting from 2-(4-methoxyphenylamino)-N-(prop-2-ynyl)nicotinamide
(8e, 150 mg, 0.53 mmol) and 1-(azidomethyl)-4-fluorobenzene (10b, 88 mg, 0.58 mmol), 4n
was prepared according to the procedure described for 4a as yellow solid (173 mg, 75%); mp:
169-171 ºC; 1H NMR (500 MHz, CDCl3) δ 10.26 (s, 1H), 8.23 (dd, J = 4.0, 1.5Hz, 1H), 7.74
(dd, J = 8.0,1.5 Hz, 1H), 7.57 (brs, 1H), 7.50 (d, J = 9.0 Hz, 2H), 7.29-7.24 (m, 3H), 7.06 (t, J
= 9.0 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 6.59-6.50 (m, 1H), 5.47 (s, 2H), 4.64 (d, J = 5.2 Hz,
13
2H), 3.78 (s, 3H); C NMR (75 MHz, CDCl3) δ 168.3, 164.4, 161.1, 155.7, 155.3, 151.5,
135.9, 132.9, 130.0, 129.93, 122.7, 116.2, 115.9, 114.0, 112.3, 109.9, 55.4, 53.4, 35.0; IR (
cm−1): 3282, 3147, 2360, 1646, 1602, 1585, 1458, 1303, 1246, 1057, 826; MS (ESI m/z):433
[M+H]+; HRMS (ESI m/z) calcd forC23H22O2N6F:433.1763[M+H]+found:433.1763.
6.27. N-((1-(4-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(4-methoxyphenylamino)
nicotinamide (4o): Starting from 2-(4-methoxyphenylamino)-N-(prop-2-ynyl)nicotinamide
(8e, 150 mg, 0.53 mmol) and 1-(azidomethyl)-4-methoxybenzene (10c, 95 mg, 0.58 mmol),
4o was prepared according to the procedure described for 4a as white solid (189 mg, 80%);1H
15

NMR (300 MHz, CDCl3) δ 10.31 (s, 1H), 8.19 (dd, J = 4.9, 1.7 Hz, 1H), 7.81 (dd, J = 7.7, 1.7
Hz, 2H), 7.52-7.48 (m, 3H), 7.25-7.19 (m, 2H), 6.85-6.81 (m, 4H), 6.55-6.51 (m, 1H), 5.42
(s, 2H), 4.55 (d, J = 5.4 Hz, 2H), 3.78 (s, 3H), 3.77 (s, 3H); IR ( cm−1): 3254, 3049, 2359,
1637, 1608, 1506, 1457, 1246, 1035, 827;
6.28.
N-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(4-
methoxyphenylamino)nicotinamide (4p): Starting from 2-(4-methoxyphenylamino)-N-
(prop-2-ynyl)nicotinamide (8e, 150 mg, 0.53 mmol) and 1-(azidomethyl)-3,5-
dimethoxybenzene (10d, 102 mg, 0.58 mmol), 4p was prepared according to the procedure
described for 4a as white solid (197 mg, 78%); mp: 147-149 ºC; 1H NMR (300 MHz, CDCl3)
δ 10.30 (s, 1H), 8.24 (dd, J = 4.9, 1.7 Hz, 2H), 7.70 (m, 1H), 7.53 (s, 1H), 7.32-7.26 (m,1H),
7.10-7.05 (m,1H), 6.97-6.87 (m, 3H), 6.58-6.55 (m, 1H), 6.47-6.44(m, 2H), 5.42 (s, 2H), 4.66
13
(d, J = 5.4 Hz, 2H), 3.79 (s, 3H), 3.75(s, 6H); C NMR (75 MHz, CDCl3) δ 166.4, 159.3,
153.7, 153.1, 149.1, 143.7, 136.1, 135.3, 131.8, 121.4, 120.1, 112.2, 110.8, 108.6, 104.4,
98.2, 53.7, 53.6, 51.7, 33.5; IR ( cm−1): 3264, 2948, 1621, 1599, 1584, 1459, 1310, 1295,
1049, 824; MS (ESI m/z): 475 [M+H]+; HRMS (ESI m/z) calcd for C25H27N6O4:
475.2088[M+H]+; found: 475.2057.
6.29.
2-(2,4-Dimethoxyphenylamino)-N-((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-
2-(2,4-dimethoxyphenylamino)-N-(prop-2-
yl)methyl)nicotinamide(4q): Starting from
ynyl)nicotinamide (8g, 150 mg, 0.48 mmol) and 1-(azidomethyl)-4-methoxybenzene (10c, 86
mg, 0.53 mmol), 4q was prepared according to the procedure described for 4a as white
solid (180 mg, 79%); mp: 178-180 ºC; 1H NMR (500 MHz, CDCl3) δ 10.30 (s, 1H), 8.23 (m,
2H), 7.74 (dd, J = 6.0,2.0 Hz, 1H), 7.51 (s, 1H), 7.34 (brs, 1H), 7.20 (d, J = 9.0 Hz, 2H), 6.86
(d, J = 9.0 Hz, 2H), 6.56-6.48 (m, 3H), 5.45 (s, 2H), 4.64 (d, J = 6.0 Hz, 2H), 3.87 (s, 3H),
3.79 (s, 3H), 3.79 (s, 3H); MS (ESI m/z): 475 [M+H]+; HRMS (ESI m/z) calcd for
C25H27O4N6: 475.2088 [M+H] +; found: 475.2057
6.30.
2-(2,4-Dimethoxyphenylamino)-N-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-
yl)methyl)nicotinamide (4r): Starting from 2-(2,4-dimethoxyphenylamino)-N-(prop-2-
ynyl)nicotinamide (8g, 150 mg, 0.48 mmol) and 1-(azidomethyl)-4-fluorobenzene (10b, 79
mg, 0.53 mmol), 4r was prepared according to the procedure described for 4a as brown
solid (167 mg, 70%); mp: 146-148 ºC; 1H NMR (300 MHz, CDCl3) δ 10.33 (s, 1H), 8.25 (dd,
J = 7.5, 1.5 Hz, 2H), 7.72 (dd, J = 7.5,1.5 Hz, 1H), 7.53 (s, 1H), 7.28-7.23 (m, 3H), 7.18-7.13
(m, 1H), 7.03 (t, J = 8.3 Hz, 2H), 6.58-6.54 (m, 1H), 6.46 (s, 1H), 5.45 (s, 2H), 4.64 (d, J =
16

13
5.2 Hz, 2H), 3.90 (s, 3H), 3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ 168.1, 164.3, 161.0,
155.2, 151.3, 150.8, 144.9, 135.8, 129.8, 122.9, 122.3, 121.4, 116.1, 115.8, 112.1, 110.7,
103.4, 98.7, 55.8, 55.4, 53.3, 35.0; IR ( cm−1): 3336, 2939, 2360, 1634, 1599, 1577, 1459,
1236, 1048, 827; MS (ESI m/z): 463 [M+H]+; HRMS (ESI m/z) calcd forC24H24O3N6F:
463.1888 [M+H]+; found: 463.1856.
6.31.
N-((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)-2-(2,4-
dimethoxyphenylamino)nicotinamide (4s): Starting from 2-(2,4-dimethoxyphenylamino)-
N-(prop-2-ynyl)nicotinamide (8g, 150 mg, 0.48 mmol) and 1-(azidomethyl)-3,5-
dimethoxybenzene (10d, 102 mg, 0.53 mmol), 4s was prepared according to the procedure
1
described for 4a as yellow solid (175 mg, 72%); mp: 147-149 ºC; H NMR (500 MHz,
CDCl3) δ 10.36 (s, 1H), 8.25 (dd, J = 5.0,3.0 Hz, 2H), 7.73 (dd, J = 7.0 Hz, 1H), 7.53 (s, 1H),
7.15 (brs, 1H), 6.58-6.54 (m, 1H), 6.47-6.43 (m, 2H), 6.36 (s, 3H), 5.41 (s, 2H), 4.66 (d, J =
5.2 Hz, 2H), 3.91 (s, 3H), 3.80 (s, 3H),3.74 (s, 6H); IR ( cm−1): 3358, 2924, 2360, 1631,
1599, 1585, 142552, 1306, 1258, 1053, 827; MS (ESI m/z): 505 [M+H]+; HRMS (ESI m/z)
calcd for C26H29O5N6: 505.2193 [M+H]+ found: 505.2169.
6.32. 2-(4-Chlorobenzylamino)-N-((1-(3-phenoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)
nicotinamide (4t): Starting from 2-(4-chlorobenzylamino)-N-(prop-2-ynyl)nicotinamide
(8k, 150 mg, 0.50 mmol) and 1-(azidomethyl)-3-phenoxybenzene (10a, 124 mg, 0.55 mmol),
4t was prepared according to the procedure described for 4a as white solid (184 mg, 60%);
mp: 209-211 ºC; 1H NMR (300 MHz, CDCl3) δ 8.45 (t, J = 5.2 Hz, 1H), 8.14-8.11 (m, 1H),
7.63 (d, J = 7.5 Hz, 1H), 7.50 (brs, 1H), 7.45 (s, 1H), 7.27-7.13 (m, 7H), 7.02 (t, J = 7.5 Hz,
1H), 6.90-6.80 (m, 5H), 6.36-6.32 (m, 1H), 5.33 (s, 2H), 4.60 (d, J = 5.2 Hz, 2H), 4.49 (d, J =
13
6.0 Hz, 2H); C NMR (75 MHz, CDCl3) δ 168.4, 158.0, 157.8, 156.3, 151.9, 145.1,139.7,
136.2, 135.8, 130.5, 129.9, 128.5, 127.5, 126.9, 123.8, 122.6, 122.4, 119.2, 118.6, 118.1,
1108, 109.4, 53.8, 44.8, 34.9; IR ( cm−1): 3288, 3054, 2359, 1629, 1579, 1458, 1307, 1252,
1055, 822; MS (ESI m/z): 525 [M+H]+; HRMS (ESI m/z) calcd for C29H26O2N6Cl: 525.1800
[M+H]+; found: 525.1778.
7. Biology
7.1. Evaluation of in vitro anti-cancer activity
The anticancer activity of the compounds was determined using MTT (3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay.20 1×106 cells/well
were seeded in 100 µL DMEM, supplemented with 10% FBS in each well of 96-well micro
17

culture plates and incubated for 24 h at 37 °C in a CO2 incubator. Compounds, diluted to the
desired concentrations in culture medium, were added to the wells with respective vehicle
control. After 48 h of incubation, MTT (10 µL, 5 mg/mL) was added to each well and the
plates were further incubated for 4 h. The supernatant from each well was carefully removed,
formazon crystals were dissolved in DMSO (100 µL) and absorbance at 540 nm wavelength
was recorded.
7.2. In vitro growth inhibition
The screening of anticancer activity was evaluated by the NCI, USA, according to
standard procedures (http://dtp.nci.nih.gov/ branches/btb/ivclsp.html).21
7.3. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the
cells through the cell-cycle phases. MCF-7 cells were incubated for 48 h with compounds 4g
and 4i at concentrations of 0.5 and 1µM. and also with E7010 in the same concentrations.
Untreated and treated cells were harvested, washed with phosphate-buffered saline (PBS),
fixed in ice-cold 70% ethanol, and stained with propidium iodide(Sigma–Aldrich). Cell-cycle
analysis was performed by flow cytometry (Becton Dickinson FACS Caliber instrument).21
7.4. 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 10µL contained PEM buffer, GTP (1µM) in the presence or absence of test
compounds 4g and 4i (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 Vario scan multimode plate reader (Thermo
scientific Inc.). E7010 was used as positive control in each assay. The IC50 value is 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
µM), in the presence or absence of tested compounds at 0.5 and 1 µM concentrations.
Polymerization was monitored by increase in the fluorescence as mentioned above at 37°C.
7.5. Immunohistochemistry
MCF-7 cells were seeded on glass cover slips, incubated for 48 h inthe presence or
absence of test compounds 4g, 4i and E7010 (1 µM). Following the termination of
incubation, cells were fixed with 3% paraformaldehyde, 0.02% glutaraldehyde in PBS and
permeabilized by dipping the cells in 100% methanol (20 °C). The cover slips were left in
18

100% MeOH overnight at 4 °C. Subsequently, the cover slips were blocked with a 1% bovine
serum albumin (BSA) solution for 60 min and then incubated with anti-α-tubulin antibody
(1:1000) at room temperature for 2 h. The slides were washed three times for 5 min each with
PBST. Next, the cover slips were incubated with FITC-conjugated anti-mouse secondary
antibody (Sigma–Aldrich) for 1 h and then washed three times with PBST solution. Finally,
the cells were observed under a fluorescence microscope (Leica, Germany), and the pictures
were analyzed for the integrity of the microtubule network.
7.6. Morphological analysis for apoptosis with Hoechst staining
MCF-7 Cells were seeded at a density of 10,000 cells over 18-mm cover slips and
incubated for 24 h. The medium was replaced, and cells were treated with compounds, 4g and
4i at 0.5 and 1 µM concentrations for 48 h. Cells treated with vehicle (0.001% DMSO) were
included as controls for all experiments. After overnight treatment, Hoechst 33258 (Sigma
Aldrich) was added to medium at a concentration of 0.5 mg/mL containing 4% Para
formaldehyde. After incubation for 30 min, cells from each dish were captured from
randomly selected fields under fluorescent microscope (Leica, Germany) to qualitatively
determine the proportion of viable and apoptotic cells based on their relative fluorescence and
nuclear fragmentation.
7.7. Caspase 9 activity
To determine the caspase-9 activity of 4g, 4i and E7010 for detection of apoptosis in
breast cancer cell line (MCF-7), the commercially available apoptosis detection kit (Sigma-
Caspase 9 Assay kit, Florometric) was used. MCF-7 cells were treated with compounds
4gand 4i at 1µM concentration for 48 h. Here the substrate used is Ac-LEHD-AFC to the cell
lysate and incubation was carried out at 37 °C for 1 h. Readings were taken at excitation
wavelength 400 nm and emission wavelength 505 nm.
7.8. Determination of the Apoptotic DNA Ladder.
MCF-7 Cells were seeded (1×106) in six-well plates. After incubation for 24 h, cells
were treated with compound 4g, 4i and E7010 at 1 µM 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
dodecylsulfate (SDS, 10%, 10 mL) and 50 mg/mL RNase A (10 mL) were then added, and
the mixture was incubated for 2 h at 568C. After incubation, proteinase K (25 mg/mL) was
added and further incubated at 37 °C for 2 h. Ammonium acetate (10M, 65 µL) and ice-cold
ethanol (500 µL) were then added, and mixed well. These samples were incubated at -80 °C
19

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.
7.9. Molecular modelling 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).30 Docking
studies were performed using AutoDock 4.2 software.31 The Analysis of intermolecular
interactions has been performed using Pymol, v. 0.99.32
Aknowledgement
N.V.S.R and A.V.S acknowledge CSIR-UGC, New Delhi, for the award of Senior Research
Fellowship. We also acknowledge CSIR for financial support under the 12th Five Year plan
project “Affordable Cancer Therapeutics (ACT)” (CSC0301).
References and notes
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12. Kamal, A.; Prabhakar, S.; Ramaiah, M.J. ; Reddy, P.V.; Reddy, C.R.; Mallareddy, A.;
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18. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
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21. http://dtp.nci.nih.gov/branches/btb/ivclsp.html.
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J. Pathol.2004, 165, 1401.
23. Huber, K.; Patel, P.;Zhang, L.;Evans, H.; Westwell, A. D.; Fischer, P. M.; Chan, S.;
Martin, S. Mol. Cancer Ther. 2008, 7, 143.
24. Kamal, A.;Srikanth, Y. V. V.;Shaik, T. B.;Khan, M. N. A.;Ashraf, M.; Reddy,M. K.;
Kumar, K. A.;Kalivendi, S. V. Med. Chem. Commun.2011, 2, 819.
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26. Costantini, P.;Bruey, J. M.;Castedo, M.;Metivier, D.;M. Loeffler, D.;Susin, S.
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J.;Zheng, G.;Sonnenberg, J. L.;Hada, M.;Ehara, M.;Toyota, K.;Fukuda, R.;Hasegawa,
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31. http://autodock.scripps.edu/
32. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.
22

O
H
OC2H5
N S
O
OOCH3
O
O
N
NH
N
H
NO2
N
2
E7010 (1
)
OH
N
N
N
H
N
N
O
N
C
B
N
N
N
D
NH
NH
RR2
OPh
O
A
R1
3
4a-t
Figure 1: Chemical structures of
E
7010 (1), N-phenyl nicotinamides (2), and compounds 3-4..
Figure 2: Flow cytometric analysis in thee MCF-7 cancer cell line aftertreatment with compounds 4g,4iand
E7010 at 0.5 and 1µM for 48 h.

140
120
100
80
Control
E7010
4g
4i
60
40
20
0
1
4
7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58
-20
Time (min)
80
70
60
50
40
30
20
10
0
Control
4g
4i
E7010
Figure 3: Effect of compounds 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 °C. All the compounds were
included at a final concentration of 3 µM. Values indicated are the mean ± SD of two different experiments
performed in triplicates.
24

Figure 4: IHC analysis of compounds on the microtubule network: Panel, I: Phase-contrast microsc
oopic view;
Panel II: fluorescence microscopic view: A: MCF-7 control cells, B: E7010 (1µM), C:4g (1µM), D:4i (1µM).
Figure 5: Hoechst staining in MCF-7 Breast cancer cell line. A: MCF-7 control cells, B: E7010 (1µ
(1µM), D: 4g (1µM).
MM), C: 4i

120000
100000
80000
60000
40000
20000
0
Figure 6: Effect of compounds 4g and 4i on caspase-9 activity: MCF-7 cells were treated with
compounds at 1 and 2 µM concentration for 48 h. Values indicated are the mean ± SD of two different
experiments performed in triplicates.
1
2
3
4
5
Figure 7: DNA fragmentation assay. Lane-1: Control (untreated), Lane-2: Marker (100 bp), Lane-3: E7010
(1µM), Lane-4: 4g (1 µM), Lane-5: 4i (1 µM).
Figure 8: Binding pose for 4g in colchicine binding site of tubulin and superimposition of 4g and 4i in
colchicine binding site of tubulin.

Figure 9: 2D interaction diagram of colchicine, E7010 and 4g.
Tables
Table 1: Cytotoxicity of compounds 4a-t and E7010 against a panel of cancercelllines.
CompoundIC50a (µM)
A549b
1.54
1.69
3.98
1.90
6.60
1.90
1.57
6.45
1.93
8.70
7.41
9.31
1.25
7.58
18.62
8.51
14.45
4.57
1.31
1.79
1.31
DU-145c
1.13
1.65
1.81
1.89
7.07
1.75
1.54
3.46
1.61
7.24
5.49
7.94
1.34
4.69
10.47
7.24
9.55
4.57
1.02
1.57
1.81
ACHNd
7.37
3.09
6.30
1.25
6.91
1.14
0.86
5.37
0.98
7.41
11.2
11.61
8.31
8.70
13.98
7.07
10.96
5.88
3.98
3.0
MCF-7e
1.65
1.65
2.51
0.97
7.24
1.78
0.74
3.71
0.93
7.53
6.60
8.31
1.0
5.49
11.74
8.21
9.41
7.58
1.17
2.51
1.25
HT-29f
3.09
6.76
4.36
1.39
7.41
1.27
0.89
9.54
1.34
11.74
11.01
9.37
1.41
8.70
11.22
9.77
10.23
7.76
3.38
0.96
1.62
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
E7010
2.13
a50% Inhibitory concentration and the values are average of three individual
experiments.blung cancer, cprostatecancer, drenal cell carcinoma, ebreast cancer,
f
colon cancer.
27

Table 2: Anticancer activity of N-((1-benzyl-1H-1, 2, 3-triazol-4-yl) methyl) nicotinamides against
panel of 60 human cancer cell lines.
Cancer panel/Cell lines
GI50 (µM)a
b4a
c4b
d4f
e4g
f4i
g4t
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
3.34
3.74
3.69
3.74
3.61
2.56
NA
NA
NA
NA
NA
NA
3.80
2.66
3.48
2.91
4.84
NT
1.20
2.21
0.75
3.65
3.03
NT
4.19
3.97
5.05
4.85
5.25
NT
3.05
3.39
3.24
3.28
3.12
3.31
Non-small-cell- lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon
4.81
5.18
5.58
NT
3.24
3.73
NT
6.11
25.3
60.2
10.7
38.6
29.9
5.29
5.25
10.8
3.44
4.16
5.07
5.67
3.92
3.45
5.24
3.52
2.13
4.15
5.09
12.7
NT
2.24
2.24
NT
4.02
6.33
6.31
4.67
9.82
6.22
6.15
3.98
3.31
3.21
3.65
5.69
4.23
3.66
NT
NT
2.85
2.18
2.85
2.11
3.05
1.82
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
2.79
3.70
3.40
3.81
3.65
3.63
3.97
6.22
NA
5.02
NT
5.60
5.61
6. 58
2.86
2.53
4.17
3.93
3.66
3.43
3.86
2.14
3.33
3.08
2.89
3.26
1.65
2.12
4.12
9.60
4.34
3.89
3.88
3.84
3.88
2.18
4.17
3.18
3.39
2.96
3.62
4.04
SW-620
CNS
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
3.22
3.69
3.10
3.27
1.94
3.61
2.1
4.59
2.94
2.79
3.55
3.09
3.91
3.88
0.56
2.65
3.33
3.41
3.60
5.43
4.06
4.39
9.64
3.98
4.58
4.82
2.80
2.24
3.91
2.02
2.66
5.66
15.1
20.7
3.81
7.12
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian
4.54
1.61
2.86
2.65
6.05
4.93
3.89
1.42
3.02
61.3
15.1
13.7
2.79
34.2
7.98
4.48
6.87
6.51
3.60
3.80
3.15
1.71
3.66
3.88
2.90
4.82
3.13
3.01
4.07
2.13
0.25
7.67
2.60
0.72
11.9
1.43
5.10
5.81
4.80
2.23
6.69
4.14
3.11
4.86
3.86
3.90
5.69
2.49
2.33
4.44
2.95
2.53
3.83
2.08
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
5.86
2.28
5.41
5.72
4.39
2.47
3.79
30.7
17.7
23.0
NA
7.44
14.0
5.94
5.89
2.98
5.19
4.23
3.72
2.12
3.11
5.96
1.54
5.11
8.34
4.10
2.12
2.59
NA
3.69
5.42
10.0
5.96
3.21
4.33
9.03
3.48
3.37
5.62
3.63
2.86
3.32
28