Synthesis of 2-anilinopyridine-arylpropenone conjugates as tubulin inhibitors and apoptotic inducers

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Graphical abstract

A library of (Z)-3-(arylamino)-1-(2-(arylamino) pyridin-3-yl)prop-2-en-1-one conjugates

conjugates were synthesised and investigated for their anticancer activity against various

human cancer cell lines. Some of the tested compounds like 9a, 9g and 9j exhibited

promising antiproliferative activity.

Tubulin Polymerization

Inhibitory Activity 9a: 1.34 µM

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Synthesis of 2-Anilinopyridine – Arylpropenone Conjugates as

Tubulin Inhibitors and Apoptotic Inducers

Ahmed Kamal,*^aVangala Santhosh Reddy,^aM. V. P. S. Vishnuvardhan,^aG. Bharath Kumar,^aAnver

Basha Shaik,^aSumit. S. Chourasiya^a, M. Kashi Reddy,^aIbrahim Bin Sayeed,^aPraveen Reddy Adiyala

,^aNishant Jain^b

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

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A series of new (Z)-3-(arylamino)-1-(2-(arylamino)pyridin-3-yl)prop-2-en-1-one conjugates 9a-p were

synthesized and evaluated for their cytotoxic activity against some human cancer cell lines. Some of the

treated compounds like 9a, 9g and 9j showed significant activity with IC₅₀values ranging from 0.51 to 1.29

μΜ. Flow cytometry results revealed that A549 cells by these compounds caused accumulation of cells in the

G2/M phase. Interestingly, compound 9a demonstrated a considerable inhibitory effect on tubulin

polymerization and showed significant inhibition of tubulin polymerization with IC₅₀value of 1.34 µM,

whereas, nocodazole showed antitubulin activity with IC₅₀value 2.64 µM. Further, Hoechst staining and

activation of caspase-3 suggested that these conjugates induced cell death by apoptosis. Fluorescence based

competitive colchicine binding assay and the docking studies suggests that these conjugates 9a and 9g binds

to the tubulin perfectly at the colchicine binding site.

resistant cell lines.¹⁵

Introduction

Microtubules are key components of eukaryotic cells in a

variety of elemental cellular processes such as formation, cell

division, maintenance of cell shape, regulation of motility, cell

signaling, secretion and cytoplasmic transport.^1-3Microtubules

have a fundamental role in the cellular processes that makes

them an important target for anticancer drugs; consequently,

the molecules that target tubulin also stops the mitosis of

cancer cells.^4-6A number of small molecules have been

described in the literature that inhibit polymerization of

tubulin with subsequent arrest of cells during mitosis and

thereby leading to apoptosis.⁷Microtubule polymerization

inhibitors are effective in the treatment of breast, lung,

ovarian and other cancers. Consequently, microtubules have

become an important target to discover the new anticancer

agents. However, peripheral neuropathy occurrence is a main

complication in the development of microtubule targeting

agents as anticancer drugs. To overcome this problem, the

discoveries of new molecules to defeat neuropathies are

required and some important ones are colchicine

(I),

combretastatin A-4 (II) nocodazole (III) and E-7010 (IV).^8-12

Combretastatin A-4

(CA-4) (II) is one of the well-known natural product, isolated

from the bark of the South African tree Combretum caﬀrum

that inhibits tubulin polymerization at the colchicine binding

site.^13,14CA-4 has shown significant cytotoxicity against a

variety of human cancer cell lines, including multidrug

Figure 1A. Chemical structures of microtubule targeting agents nocodazole (I),

combretastatin A-4 (II), E-7010 (III) and (Z)-3-(arylamino)-1-(2-(arylamino)pyridin-3-

yl)prop-2-en-1-one conjugates (9a-p).

A disodium phosphate derivative of CA-4 (CA-4P) has shown

promising cytotoxicity in human cancer and it is in clinical

trials.¹⁶Structure activity relationship (SAR) studies of CA-4

reveals that 3,4,5- trimethoxy phenyl ring and cis-double bond

are essential for activity.^17-19Similarly E7010 (III) was designed

and synthesized by Hiroshi Yoshino in 1992, it inhibits tubulin

polymerization due to its reversible binding at colchicine

binding site on tubulin²⁰and exhibits potent in vivo antitumor

^a.Medicinal Chemistry and Pharmacology Division, Indian Institute of Chemical

Technology, Hyderabad-500007 (India),Fax:(+91)40-2719-3189,E-mail:

ahmedkamal@iict.res.in

^b.Address here Centre for Chemical Biology, Indian Institute of Chemical

Technology, Hyderabad -500007( India)

Electronic Supplementary Information (ESI) available: ¹HNMR, ¹³CNMR and HRMS activity against tumor xenografts as well as various rodent

Spectrums of all final compounds.

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tumors²¹and is in phase II clinical studies.²²Therefore, a sulphuric acid in ethanol gave ethyl 2-chloron_Vi_ic_eo_wt_Ai_rn_tia_clt_ee_On(_li2_ne

)

variety of synthetic small molecules that contain different followed by nucleophilic substitutio^Dn^OI:w¹⁰i^.t¹h^039/s^Cu⁵b^Rs^At¹i⁹tu⁴⁶te^8Gd

heterocycle scaffolds like 2-anilinopyridine, thiazole, aromatic amines 3a-d provided ethyl 2-(arylamino) nicotinates

arylindoles, pyrazole, oxadiazoline, imidazoles and oxindoles^23-

have been reported as inhibitors of tubulin polymerization. THF

(

4a-d) in quantitative yield. This upon reduction with LAH in

26

afforded (2-(arylamino)pyridin-3-yl)methanol

Recently, we have reported a new family of 2-anilinonicotinyl intermediates (5a-d), followed by oxidation with IBX produces

arylsulfonyl

hydrazide

and

imidazopyridine-oxindole aldehydes 6a-d. These aldehydes 6a-d were treated with

conjugates that exhibit promising cytotoxic activity.^27,28ethynylmagnesium bromide in THF to obtain the intermediates

However, several clinically useful tubulin depolymerizing drugs 7a-d followed by once again oxidation with IBX in DMSO

have huge drawbacks. To overcome these problems, we have provided the corresponding precursors 8a-d. Consequently,

designed and synthesised a new class of (Z)-3-(arylamino)-1-(2- the target conjugates 9a-p were formed by reaction between

(arylamino)pyridin-3-yl)prop-2-en-1-one derivatives (9a-p) by corresponding precursors 8a-d and arylamines in ethanol.

keeping structural aspects of combretastatin A-4 (II) and E-

7010 (III). Further, evaluated them for their cytotoxicity against

various selected human cancer cell lines. These compounds

also showed significant inhibition of tubulin polymerization.

Biological studies

Antiproliferative activity

Compounds 9a-p have been evaluated for their cytotoxicity in

selected human cancer cell lines of MCF-7 (breast), A549

(lung), HCT116 (colon) and Hela (cervix) by using MTT assay

Results and Discussion

Chemistry

Synthesis of the (Z)-3-(arylamino)-1-(2-(arylamino) pyridin-3-

yl)prop-2-en-1-one conjugates (9a-p) is shown in Scheme 1.

The esterification of 2-chloronicotinic acid in presence of

Scheme 1: Reagents & Conditions: (i) Conc. H₂SO_4,Ethanol, rt, 75-85%; (ii) Ethylene glycol, 120 ^°C, 5-6 h, 70-80% ;(iii) LAH, THF, rt, 3-4 h, 70-75%; (iv) IBX, DMSO, 0 ^°C, 2 h,

65%; (v) Ethynylmagnesium bromide, THF, 0 ^°C,-rt, 3 h, 55-65%; (vi) IBX, DMSO, 0 ^°C, 2 h, 75%; (vii) arylamines, EtOH, 4 h, rt, 55-60%.

2 | RSC Advances 2015, 00, 1-3

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Table 1. Structures of compounds 9ap melting points and their yields.

Entry

9a

Ar

R¹

R²

R³

Melting point (^°C)

Yield% (mg)

5-indolyl

H

OCH₃

H

162164

68% (93)

9b

9c

9d

9e

9f

6-indolyl

H

OCH₃

F

H

227229

238240

260262

139141

156158

197199

242244

134136

196198

290292

291293

279281

278280

152154

165167

72% (99)

3,5-dimethoxyphenyl

4-methoxyphenyl

5-indolyl

H

80% (116)

79% (106)

81% (127)

69% (108)

70% (107)

79% (138)

76% (106)

65% (95)

H

OCH₃

H

OCH₃

H

6-indolyl

9g

9h

9i

4-methoxyphenyl

Trimethoxyphenyl

5-indolyl

9j

3,5-dimethoxyphenyl

4-methoxyphenyl

Trimethoxyphenyl

4-trifluoromethylphenyl

5-indolyl

H

F

H

9k

9l

H

F

H

81% (110)

80% (127)

78% (117)

75% (119)

79% (126)

71% (129)

H

F

H

9m

9n

9o

9p

H

F

H

6-indolyl

H

Trimethoxyphenyl

H

To examine the mechanism underlying the antiproliferative

method. Some of these (Z)-3-(aryl amino)-1-(2-(aryl activities of these (Z)-3-(aryl amino)-1-(2-(aryl amino) pyridin-

amino)pyridin-3-yl)prop-2-en-1-ones 9a, 9c, 9g and 9j

(

)

exhibited significant cytotoxic activity against various cancer

cell line (IC₅₀range 0.51-2.23 μM). From the structure activity

relationship (SAR) studies, some interesting trends have been

observed in these compounds. With respect to substitution 4-

methoxy on the B-ring derivatives, with 5-indolyl aryl group

containing compound (9a) showed promising cytotoxic activity

against most of the cancer cell lines compared to 9b, which

contain 6-indolyl aryl group and similar results were appeared

in compounds 9e, 9f, 9n and 9o and these results confirms

that 5-indolyl aryl group is essential for activity. Compounds 9c

and 9j which includes 3,5-dimethoxyphenyl group at aryl (Ar)

position showed potent anticancer activity with IC₅₀ranging

from 0.90 to 2.23 μM. 3,4,5-trimethoxy substituted on B-ring

with 4-methoxy phenyl group at aryl (Ar) position compound

9g showed potent anticancer activity against tested cancer cell

lines with IC₅₀ranging from 0.62-1.39 μM. Particularly,

Figure 2. Anti-mitotic effect of 9a, 9g and 9j by FACS analysis. Induction of cell

cycle G2/M arrest by compounds 9a, 9g and 9j. A549 cells were harvested after

treatment at 3 µM for 24 h. Untreated cells and DMSO treated cells served as

controls. The percentage of cells in each phase of cell cycle was quantified by Flow

Cytometry.

compounds 9a, 9g and 9j were shown promising anticancer

activity than positive control, nocodazole (III) against all tested

cell lines (Table 2). These encouraging cytotoxicity results of

compounds 9a, 9g and 9j promoted us to understand their

effect on cell cycle and tubulin polymerization.

3-yl)prop-2-en-1-ones (9a

alterations by treating A549 cells with some of the potential

compounds like 9a 9g and 9j at 3 µM concentration for 24 h

to know the phase distribution by flow cytometry. They caused

G2/M cell-cycle arrest with a large accumulation of cells

76.42%, 75.24% and 75.56% respectively in the G2/M phase

(Figure2).

-p), we analyzed the cell cycle

,

Effect on cell cycle arrest

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Table 2 IC₅₀values^a(in μM) for compounds 9a-p in selected

human cancer cell lines.

IC₅₀values (μM)^a

Comp

A549^b

Hela^c

MCF-7^d

HCT116^e

9a

9b

9c

9d

9e

9f

0.51± 0.11

1.47±0.51

1.52±0.13

4.36±0.21

1.94±0.06

3.17±0.61

1.12±0.12

4.61±0.31

2.81±0.28

0.91±0.05

5.57±0.71

5.73±0.39

3.37±0.25

2.67±0.17

4.79±0.18

5.61±0.09

0.65±0.08

1.33±0.31

1.39±0.05

4.58±0.09

2.27±0.37

3.32±0.29

1.29±0.18

4.10±0.36

2.69±0.21

0.90±0.27

2.56±0.16

4.86±0.39

5.16±0.17

2.38±0.17

4.01±0.08

6.75±0.16

0.71±0.23

1.37±0.06

2.23±0.28

2.85±0.11

2.42±0.09

2.81±0.37

0.62±0.21

3.38±0.08

2.33±0.13

1.21±0.19

2.78±0.28

2.21±0.65

2.09±0.31

2.17±0.29

3.18±0.07

4.47±0.05

0.99±0.20

3.31±0.29

2.05±0.11

4.14±0.22

2.49±0.32

5.06±0.40

1.39±0.43

5.35±0.39

3.01±0.26

1.16±0.19

6.01±0.38

3.78±0.08

3.86±0.16

3.02±0.21

3.81±0.38

7.53±0.71

9g

9h

9i

Figure 3. Effect of 9a, 9g and 9j on microtubules and nuclear condensation. A549 cells

were treated with 9a, 9g and 9j concentrations of 3 μM for 24h, followed by fixation,

permeabilization and indirect immune fluorescent analysis with an anti-α-tubulin-FITC.

Nuclei were stained with DAPI. The merged images of cells stained for tubulin and DAPI

are represented.

9j

9k

9l

Effect on inhibition of tubulin polymerization

9m

9n

9o

9p

To investigate whether the antiproliferative activity of these

synthesized conjugates (9a, 9g and 9j) has any relationship to

the interaction with tubulin, we evaluated their effect on the

inhibition of tubulin polymerization. Among these compounds,

9a exhibited potent inhibition of microtubule assembly with

IC₅₀value 1.34 µM. However, compounds 9g and 9j also

appreciably inhibited tubulin polymerization with IC₅₀values

5.68 and 5.34 µM respectively (Table 3). Thus, these results

suggest that 9a is a potent inhibitor of tubulin polymerization

than nocodazole, a known tubulin polymerization inhibitor and

similar to combretastatin A-4.²³

Noc

f

1.50±0.0.6

0.92±0.37

1.80±0.19

-

(III)

^a50% growth inhibition and the values are mean of three

determinations.^blung cancer cell line, ^ccervical cancer cell line,

^dbreast cancer cell line, ^ecolon cancer cell line, ^fnot tested.

Table 3. Effect of compounds 9a, 9g and 9j on Tubulin

Polymerization.

Effect on microtubule network

Compound

Inhibition of tubulin polymerization,^aIC₅₀(µM)

Compounds that alter cell-cycle factors with preferential G2/M

phase are known to exhibit effects on tubulin assembly.

Furthermore, inhibition of tubulin polymerization is strongly

correlated with G2/M cell-cycle arrest.²⁹Therefore as these

9a

9g

1.34 ± 0.3

5.68 ± 1.0

5.34 ± 1.0

1.30 ± 0.12

2.64 ± 0.4

9j

compounds 9a, 9g and 9j exhibited G2/M cell-cycle arrest, it

was considered of interest to understand the microtubule

inhibitory function. Thus, A549 cells were treated with 9a, 9g

and 9j at 3 µM for 24 h and the results indicate that there is

dispersed microtubule organization relative to the control

cells, as observed by immunofluorescence and microscopy

studies using anti- -tubulin antibody (Figure 3).

CA-4

Noc (III)

[a] Compounds (final concentration: 3 µM and final volume: 10µl)

were pre-incubated with tubulin at a final conc. of 10µm

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Analysis of free versus polymerized tubulin by

Western blotting

The microtubules continuously undergo polymerization and

depolymerization which are mediated by α and -tubulin, and

changing this dynamic balance is considered as a potential

target for cancer drug development (e.g; paclitaxel or

colchicines).^30,31In order to extend the in vitro effects of these

conjugates on tubulin polymerization to the cellular effects

i.e., ratio of free vs polymerized tubulin. Western blot analysis

of α-tubulin in A549 cells was performed by treating the cells

with nocodazole, paclitaxel, 9a, 9g and 9j at 2 µM for 24 h.

Immunoblot results show that the cells exposed to these

compounds 9a contain more tubulin in the soluble fraction of

the cells, when compared to insoluble fraction. Hence,

increased tubulin in soluble fraction of cells treated by these

conjugates also supports the inhibition of tubulin

polymerization (Figure 4).

Figure 5. Hoechst staining of 9a, 9g and 9j in A549 cells at 2 μM concentration.

3. The results indicate that there is nearly 3 to 4-fold induction

in caspase-3 activity in cells treated with 2 µM concentration

by these compounds when compared to control, thus

suggesting the activation of caspase-3 by 9a, 9g and 9j indicate

that they have the ability to induce apoptosis in A549 cells

(Figure 6).

Figure 4. Distribution of tubulin in polymerized vs soluble fractions as analyzed by

Immunobloting in 9a and 9j treated A549 cells. A549 cells were treated with 2 μM of

9a, 9j, nocodazole (III) and Paclitaxel for 24 h. The fractions containing soluble and

polymerised tubulin were collected and separated by SDS-PAGE. Tubulin was detected

by Immunoblot analysis.

Hoechst staining

Apoptosis is one of the major pathways that leads to the

process of cell death. Chromatin condensation and fragmented

nuclei are known as the classic characteristics of apoptosis.³²It

was considered of interest to investigate the apoptotic

Figure 6. Effect of compounds 9a, 9g and 9j on caspase-3 activity: A549 cells were

treatedfor 48 h with 2 µM concentrations of compounds 9a, 9g and 9j. Values indicated

are the mean_SD of two different experimentsperformed in triplicates.

inducing effect of the potent compounds 9a

Hoechst staining (H 33258) method in A549 cancer cell line.

Therefore cells were treated with 9a 9g and 9j at 2 µM

, 9g and 9j by

,

concentrations for 48 h. Manual field quantification of

apoptotic cells based on cytoplasmic condensation, presence

of apoptotic bodies, nuclear fragmentation and relative

fluorescence of the test compounds (9a, 9g and 9j) revealed

that there was significant increase in the percentage of

apoptotic cells as shown in Figure 5.

Competitive tubulin-binding assay

To confirm the above observations, we investigated the effect

of 9a, 9g and 9j on tubulin polymerization using an in vitro

tubulin polymerization assay. Compounds 9a, 9g and 9j

inhibited polymerization of tubulin similar to that of

nocodazole (I). Therefore, we further assessed the ability of

compounds 9a, 9g and 9j to compete with the colchicine for

binding to tubulin via competitive binding assays. Nocodazole

(I) was used as a positive control and paclitaxel as a negative

Caspase-3 activation

It is well known that the cell cycle arrest at G2/M phase is

shown to induce cellular apoptosis, hence it was considered of

control. Because the intrinsic fluorescence of colchicine

increases upon binding to tubulin,³⁴it was used as an index for

9a, 9g and 9j competition with colchicine in tubulin binding. As

shown in Figure 7, paclitaxel did not affect the binding to

tubulin. However, the fluorescence of colchicine-tubulin

interest to examine whether the cytotoxicity of 9a, 9g and 9j is

by virtue of apoptotic cell death.³³Cysteine aspartase group

namely, caspases play a crucial role in the induction of

apoptosis and amongst them caspase-3 happens to be one of

the effector caspase. This prompted us to treat A549 cells with

complex was reduced in the presence of compounds 9a, 9g, 9j

and nocodazole (III) inhibit the binding of colchicine to tubulin,

there by suggesting that the compounds 9a, 9g and 9j binds at

the colchicine binding site.

compounds 9a, 9g and 9j to examine the activation of caspase-

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respectively. A) Represent the binding mode of 9a and B) Represent the binding

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mode of 9g.

hydrophobic contacts within the binding pocket of the β-chain.

In case of 9a, the methoxy benzene is directed towards

Cysβ241, the amino acid with which methoxy groups (Ring A)

of colchicine form hydrogen bond interactions. The carbonyl

group showed hydrogen bonding interaction with Valα181 (2.0

Å) and the –NH group of indole nucleus showed hydrogen

bonding interaction with Gluβ247 (1.8 Å). Further, the protein

ligand complex was stabilized by hydrophobic interactions like

Leuβ242, Alaβ250, Asnβ258, Valβ315, Alaβ316, Ileβ318 and

Ileβ378. The trimethoxy benzene ring of 9g showed key

hydrogen bond interactions with the side chain -SH of Cysβ241

(hydrogen bond distance 2.2 Å and 2.3 Å). In addition to this -

NH group of 9g showed hydrogen bonding interaction with

Lysβ254 (2.3 Å). Other residues like Leuβ242, Asnβ258,

Valβ315, Alaβ316, Ileβ318 and Ileβ378 were found to be

involved in hydrophobic interactions. So, the docking studies

revealed that the conjugates 9a and 9g binds to the colchicine

binding site of tubulin by forming favourable interactions.

Figure 7. Fluorescence based colchicine competitive binding assay of conjugates

9a, 9g and 9j were carried out at various concentrations containing 3 µM of

tubulin and colchicine for 60 min at 37 ⁰C. Nocodazole (I) was used as a positive

control where as taxol was used as negative control which binds at taxane site.

Docking Studies

In order to understand the binding interactions, we have

performed the molecular docking study of active compounds

obtained from competitive tubulin-binding assay by using

Glide 5.8 module in maestro 9.3. These compounds 9a, 9g Conclusions

have been docked against the tubulin protien (PDB code:

3UT5)³⁵. The molecular docking study suggest that these

compounds bind in the colchicine binding pocket with

hydrogen bonds with the α-chain, β-chain and extensive

In conclusions, we have designed and synthesized new

analogues of E-7010 and tested for their in vitro anticancer

activities against variety of different human cancer cell lines.

All the compounds showed moderate to good anticancer

potency against most of the tested cancer cell lines, however

some of the compounds 9a

anticancer potency IC₅₀values in the range of 0.51 to 2.23 µM.

This investigation reveals that compounds 9a 9g and 9j have

shown promising anticancer activity in comparison with

nocodazole ( ). FACS results reveals that compounds 9a 9g and

, 9c, 9g and 9j exhibited significant

,

I

,

9j caused cell cycle arrest and accumulated cells in the G2/M

phase. These compounds inhibited tubulin assembly as shown

by the tubilin polymerization assay. Docking studies also

supported the fact that these compounds binds to colchicines

binding site of the tubulin. Interestingly, it was observed that

IC50 of compound 9a was less than nocodazole (I), a well

known tubulin polymerization inhibitor. Hoechst 33258

staining and DNA fragmentation assay revealed that these

compounds induce cell death by apoptosis. Further, activation

of caspase-3 also suggested that these compounds produce

apoptotic cell death. Based on the results from the biological

studies it is evident that compounds 9a, 9g and 9j have the

potential to be taken up for further detailed studies either

alone or in combination with existing therapies as potential

tubulin polymerization inhibitors.

Experimental Section

Chemistry

Figure 8.: Binding model of ligands at the interface of α,β-tubulin heterodimer.

Ligands are shown in stick models (magenta colour). Hydrogen bonding

interactions are shown as black dashes, and residues involved are represented

also in stick models, while residues involved in hydrophobic interactions

are represented as lines. Cyan and salmon color represents α- and β-tubulin,

All chemicals were purchased from Spectrochem Pvt. Ltd

(Mumbai, India), Aldrich (Sigma–Aldrich, St. Louis, MO, USA),

Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill,

MA, USA) and were used without further purification.

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Reactions were monitored by TLC, performed on silica gel glass 3H, J=7.1 Hz, CH₃), 3.81 (s, 3H, -OCH₃), 4.35-4.41_V(_ieq_w, _A2_rtH_ic,_leJ_O=_n7_lin.1_e

plates containing 60 GF-254, and visualization on TLC was Hz, CH ), 6.64-6.67 (dd, 1H, J=7.8 Hz, J=4.7 Hz,

^{DOI: 10.10}A³⁹r^/H^C)⁵,^R6^A.¹9⁹0⁴⁶(⁸d^G,

2

attained by UV light or iodine indicator. Column 2H, J=9.0 Hz, ArH), 7.53 (d, 2H, J=9.0 Hz, ArH), 8.22 (dd, 1H,

chromatography was performed with Merck 60–120 mesh J=7.8 Hz, J=2.1 Hz, ArH), 8.32 (dd, 1H, J=2.0 Hz, J=4.7 Hz, ArH),

silica gel. ¹H NMR and ¹³C NMR spectra were recorded on 9.96 (s, 1H, NH) ppm; MS (ESI) m/z 273 [M+H].

Bruker UXNMR/XWIN-NMR (300 MHz) instruments. Chemical

shifts are reported in parts per million ( in ppm) relative to Ethyl 2-(4-fluorophenylamino)nicotinate (4c)

the peak for TMS (tetramethylsilane) as an internal standard, Ethyl 2-(4-fluorophenylamino)nicotinate 4c was obtained by

coupling constants are reported in hertz (Hz). ESI spectra were using above described method by dissolving ethyl 2-

recorded on Micro mass, Quattro LC using ESI⁺software with chloronicotinate (3 g, 16.2 mmol) and 4-fluoroaniline (1.80 g,

capillary voltage 3.98 kV and ESI mode positive ion trap 16.2 mmol) in ethylene glycol (20 mL) as yellow coloured solid

1

detector. High-resolution mass spectra (HRMS) were recorded (3.08 g, 73%): H NMR (300 MHz, CDCl₃);



1.42 (t, 3H, J=7.2

), 6.69-6.72 (dd, 1H,

points were determined with an Electro thermal melting point J=5.6 Hz, J=1.9 Hz, ArH), 7.00-7.06 (m, 2H, ArH), 7.62 (dd, 2h,

on a QSTAR XL Hybrid MS–MS mass spectrometer. Melting Hz, CH₃), 4.36-4.41 (q, 2H, J=7.2 Hz, CH

2

apparatus and are uncorrected.

J=4.3 Hz, J=4.7 Hz, ArH), 8.24 (dd, 1H, J=5.6 Hz, J=2.1 Hz, ArH),

8.34 (dd, 1H, J=2.7 Hz, J=1.9 Hz, ArH), 10.12 (s, 1H, NH) ppm;

MS (ESI) m/z 261 [M+H].

Preparation of ethyl 2-chloronicotinate (2)

A solution of 2-Chloro nicotinic acid (10 g, 64 mmol) and

ethanol (100 mL) was stirred at 0°C for 15 minutes. To this Ethyl 2-(phenylamino)nicotinate (4d)

solution concentrated sulfuric acid (3 mL) was added drop wise Ethyl 2-(phenylamino)nicotinate 4d was obtained by using

and refluxed for 4 h. After completion of reaction (checked by above described method by dissolving ethyl 2-chloronicotinate

TLC), ethanol was removed by vacuum followed by (2.5 g, 13.5 mmol) and aniline (1.26 g, 13.5 mmol) in

neutralization with sodium bicarbonate solution and ethyl etheleneglycol (20 mL) as yellow coloured solid(2.49 g, yield

1

acetate was added. The organic layer was extracted, washed 76%): H NMR (300 MHz, CDCl₃);



1.41 (t, 3H, J=7.2 Hz, CH₃),

), 6.68-6.73 (dd, 1H, J=5.8 Hz,

evaporated in vacuum to obtain pure ethyl 2-chloronicotinate J=2.8 Hz, ArH), 7.05 (t, 1H, J=7.4 Hz, ArH), 7.34 (t, 2H, J=7.5 Hz,

) and it was used directly to next step without further ArH), 7.70 (d, 2H, J=7.7 Hz, ArH), 8.24 (dd, 1H, J=5.8 Hz, J=2.1

with water, dried over anhydrous sodium sulphate and 4.34-4.43 (q, 2H, J=7.2 Hz, CH

2

(2

purification.

Hz, ArH), 8.37 (dd, 1H, J=2.8 Hz, J=1.9 Hz, ArH), 10.20 (s, 1H,

NH) ppm; MS (ESI) m/z 243 [M+H].

Preparation of ethyl 2-(aryl amino) nicotinate (4a-d)

A solution of ethyl 2-chloronicotinate and substituted anilines General method for synthesis of (2-(aryl amino) pyridin-3-yl)

were dissolved in ethylene glycol and heated at 120°C for 5-6 methanol (5a-d)

h. After completion of reaction, the reaction mixture was To the ethyl 2-(aryl amino) nicotinate 4a-d, obtained in the

cooled to room temperature followed by water and ethyl above step was added LiAH₄(0.5 mol) in dry THF at 0 °C and

acetate was added. The organic layer was separated, dried stirred for 2 h at room temperature. Added saturated NH₄Cl

over anhydrous Na₂SO₄, evaporated in vacuum and it was solution drop wise to quench the unreacted LiAlH₄and

purified

by

column

chromatography

(20%

ethyl removed the THF under vacuum then extracted with ethyl

acetate. The organic layer was dried on anhydrous NaSO₄and

evaporated ethyl acetate to obtain brown solid products of (2-

(aryl amino) pyridin-3-yl) methanol 5a-d (yield 73-81%). The

acetate/hexane) to get pure compounds 4a-d.

Ethyl 2-(3,4,5-trimethoxyphenylamino)nicotinate (4a)

Ethyl 2-(3,4,5-trimethoxyphenylamino)nicotinate 4a was alcohols produced in this step were pure, and no further

obtained by using above described method by dissolving ethyl purification was required. These compounds were taken as

2-chloronicotinate (3 g, 16.2 mmol) and 3,4,5- such for the next step.

trimethoxyaniline (2.97 g, 16.2 mmol) in ethylene glycol (20

1

mL) as yellow coloured solid (4.2 g, yield 78%): H NMR (300 (2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)methanol (5a)

MHz, CDCl₃);

3.91 (s, 6H, -OCH₃), 4.21-4.29 (q, 2H, J=7.1 Hz, CH

(dd, 1H, J=2.8 Hz, J=5.7 Hz, Ar ), 7.01 (s, 2H, ArH), 8.20 (dd, ethyl 2-(3,4,5-trimethoxyphenylamino)nicotinate 4a (4.1 g, 12

1H, J=5.7 Hz, J=2.2 Hz, ArH), 8.31 (dd, 1H, J=2.8 Hz, J=1.9 Hz, mmol) to LiAlH₄(234 mg, 6 mmol) in THF (30 mL) as brown



1.39 (t, 3H, J=7.1 Hz, CH₃), 3.83 (s, 3H, -OCH₃), (2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)methanol

5a

2

), 6.62-6.66 was obtained according to above described method by adding

H

ArH), 10.01 (s, 1H, NH) ppm; MS (ESI) m/z 333 [M+H].

coloured solid, (2.65 g, yield 74%): ¹H NMR (300 MHz, CDCl₃);



3.83 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 4.62 (s, 2H, -CH₂OH),

6.61-6.65 (dd, 1H, J=3.1Hz, J=6.6 Hz, ArH), 6.92 (s, 2H, ArH),

Ethyl 2-(4-methoxyphenylamino) nicotinate (4b)

Ethyl 2-(4-methoxyphenylamino)nicotinate 4b was obtained 7.21-7.26 (m, 1H, ArH), 7.80 (brs, 1H, NH), 8.16 (dd, 1H, J=3.1

by using above described method by dissolving ethyl 2- Hz, J=1.9 Hz, ArH); ppm; MS (ESI) m/z 291 [M+H].

chloronicotinate (3 g, 16.2 mmol) and 4-methoxyaniline (1.99

g, 16.2 mmol) in etheleneglycol (20 mL) as yellow coloured (2-(4-Methoxyphenylamino)pyridin-3-yl)methanol (5b)

1

solid (3.57 g, yield 81%): H NMR (300 MHz, CDCl₃);

 1.41 (t,

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ARTICLE

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(2-(4-Methoxyphenylamino)pyridin-3-yl)methanol 5b was 2-(4-Methoxyphenylamino) nicotinaldehyde 6b was prepared

View Article Online

DOI: 10.1039/C5RA19468G

obtained according to above described method by adding according to above explained method by the addition of (2-(4-

ethyl 2-(4-methoxyphenylamino)nicotinate 4b (3.4 g, 12.5 methoxyphenylamino)pyridin-3-yl)methanol 5b (1.9 g. 8.3

mmol) to LiAlH₄(238 mg, 6 mmol) in THF (30 mL) as brown mmol) to IBX (3.47 g 12.4 mmol) in DMSO solution. Yellow

1

coloured solid, (2.01 g, yield 70%); H NMR (300 MHz, DMSO- colored solid; (1.36 g, yield 72%): H NMR (300 MHz, CDCl₃);



d₆); 3.78 (s, 3H, OCH₃), 4.60 (s, 2H, -CH₂OH), 6.58-6.66 (dd, 3.82 (s, 3H, OCH₃), 6.75-6.81 (dd, 1H, J=5.3 Hz, J=2.3 Hz, ArH),



1H, J=2.1 Hz, J=5.1 Hz, ArH), 6.87 (d, 2H, J=9.0 Hz, ArH), 7.24- 6.92 (d, 2H, J=9.0 Hz, ArH), 7.58 (d, 2H, J=9.0 Hz, ArH), 7.82-

7.29 (dd, 1H, J=1.7 Hz, J=5.1 Hz, ArH), 7.40 (d, 2H, J=9.0 Hz, 7.87 (dd, 1H, J=6.0 Hz, J=1.5 Hz, ArH), 8.36-8.39 (dd, 1H, J=3.0

ArH), 7.46 (brs, 1H, NH), 8.09 (dd, 1H, J=3.2 Hz, J=1.9 Hz, ArH); Hz, J=1.5 Hz, ArH), 9.87 (s, 1H, CHO), 10.27 (brs, 1H, NH); ppm;

ppm; MS (ESI) m/z 231 [M+H].

MS (ESI) m/z 229 [M+H].

2-(4-Fluorophenylamino) pyridin-3-yl)methanol (5c)

2-(4-Fluorophenylamino) nicotinaldehyde (6c)

2-(4-Fluorophenylamino)pyridin-3-yl)methanol

5c

was 2-(4-Fluorophenylamino) nicotinaldehyde 6c was prepared

obtained according to above described method by adding according to above explained method by the addition of (2-(4-

ethyl 2-(4-fluorophenylamino)nicotinate 4c (3.0 g, 11.5 mmol) fluorophenylamino)pyridin-3-yl)methanol 5c (1.9 g. 8.7 mmol)

to LiAlH4 (219 mg, 6 mmol) in THF (30 mL) as brown coloured to IBX (3.66 g 13 mmol) in DMSO solution. Yellow colored

1

solid, (1.99 g, yield 79%); ¹H NMR (300 MHz, DMSO-d6);



4.59 solid; (1.50 g. yield 81%): H NMR (300 MHz, CDCl₃);



6.64-

(s, 2H, -CH₂OH), 6.62-6.69 (dd, 1H, J=4.5 Hz, J=2.3 Hz, ArH), 6.70 (dd, 1H, J=6.1 Hz, J=1.8 Hz, ArH), 6.91 (d, 2H, J=7.6 Hz,

6.94-7.04 (m, 2H, ArH), 7.24-7.29 (dd, 1H, J=5.3 Hz, J=1.5 Hz, ArH), 7.21-7.29 (m, 2H, ArH), 7.67 (dd, 1H, J=6.1 Hz, J=2.0 Hz,

ArH), 7.39-7.48 (dd, 2H, J=9.0 Hz, J=4.5 Hz, ArH), 7.61 (brs, 1H, ArH), 8.39 (dd, 1H, J=3.1 Hz, J=1.8 Hz, ArH), 9.81 (s, 1H, CHO),

NH), 8.10 (dd, 1H, J=3.0 Hz, J=2.3 Hz, ArH); ppm; MS (ESI) m/z 10.35 (brs, 1H, NH); ppm; MS (ESI) m/z 217 [M+H].

219 [M+H].

2-(Phenyl amino) nicotinaldehyde (6d)

(2-(Phenyl amino) pyridin-3-yl)methanol (5d)

2-(Phenyl amino) nicotinaldehyde 6d was prepared according

(2-(Phenyl amino)pyridin-3-yl)methanol 5d was obtained to above explained method by the addition of (2-(phenyl

according to above described method by adding ethyl 2- amino)pyridin-3-yl)methanol 5d (1.4 g. 7 mmol) to IBX (2.93 g

(phenyl amino)nicotinate 4d (2.4 g, 10 mmol) to LiAlH4 10.5 mmol) in DMSO solution. Yellow coloured solid; (1.00 g,

(188mg, 5 mmol) in THF (30 mL) as brown coloured solid, yield 72%): ¹H NMR (300 MHz, CDCl₃);

 6.82-6.86 (dd, 1H,

1

(1.45g, yield 73%); H NMR (300 MHz, CDCl₃);

 4.66 (s, 2H, - J=4.9 Hz, J=2.7 Hz, ArH), 7.10 (t, 1H, J=7.5 Hz, ArH), 7.36 (t, 2H,

CH₂OH), 6.67-6.71 (dd, 1H, J=5.0 Hz, J=2.1 Hz, ArH), 6.99 (t, 1H, J=7.5 Hz, ArH), 7.74 (d, 2H, J=7.6 Hz, ArH), 7.85-7.88 (dd, 1h,

J=7.4 Hz, ArH), 7.28-7.34 (m, 3H, ArH), 7.54 (d, 2H, J=8.1 Hz, J=5.5 Hz, J=2.0 Hz, ArH), 8.40 -8.43 (dd, 1H, J=2.9 Hz, J=2.0 Hz,

ArH), 7.68 (brs, 1H, NH), 8.15-8.18 (dd, 1H, J=1.7 Hz, J=5.0 Hz, ArH), 9.88 (s, 1H, CHO), 10.44 (brs, 1H, NH); ppm; MS (ESI) m/z

ArH); ppm; MS (ESI) m/z 201 [M+H].

199 [M+H].

General method for synthesis of 2-(Aryl amino)nicotinaldehyde

(6a-d)

General procedure for the Synthesis of 1-(2-(arylphenylamino)

pyridin-3-yl)prop-2-yn-1-ol (7a-d)

To the (2-(aryl amino) pyridin-3-yl)methanol 5a-d produced in A solution of aldehydes 6a-d in dry tetrahydrofuran (THF) was

the above step was added IBX (1.2 mol) in DMSO and stirred added to stirred solution of ethynylmagnesium bromide in

for 1 h at room temperature. Added ice cold water to the tetrahydrofuran (0.5 M) at 0 ^oC for 2 h and then stirred at

reaction mixture and extracted with ethyl acetate. The organic room temperature for another 5-6 h. After completion of

layer was dried on anhydrous NaSO₄and evaporated the ethyl reaction saturated aqueous ammonium chloride solution 5-10

acetate

to

obtain

pure

corresponding

2-(Aryl mL was added, and the THF was removed in vacuum followed

by ethyl acetate was added. The organic layer was extracted

and washed with brine solution, dried over anhydrous Na₂SO₄

and evaporated in vacuum to obtain pure compounds and

amino)nicotinaldehyde 6a-d in good yields (72-81%).

2-(3,4,5-Trimethoxyphenylamino)nicotinaldehyde (6a)

2-(3,4,5-Trimethoxyphenylamino)nicotinaldehyde 6a was these were used for next step without purification.

prepared according to above explained method by the

addition

of

(2-(3,4,5-trimethoxyphenylamino)pyridin-3- 1-(2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol

yl)methanol 5a (2.4 g. 8.3 mmol) to IBX (3.47 g 12.4 mmol) in (7a)

1

DMSO solution. Yellow coloured solid; (1.88 g, yield 79%); H 1-(2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol

NMR (300 MHz, CDCl₃);  3.84 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 7a was obtained using above described method by adding 2-

6.83-6.87 (dd, 1H, J=4.9 Hz, J=2.7 Hz, ArH), 7.04 (s, 2H, ArH), (3,4,5-trimethoxyphenylamino)nicotinaldehyde 6a (1.75 g, 6

7.86-7.89 (dd, 1H, J=5.6 Hz, J=2.0 Hz, ArH), 8.41-8.44 (dd, 1H, mmol) to the ethynylmagnesium bromide (18.2 ml, 9 mmol) in

1

J=2.9 Hz, J=2.0 Hz, ArH), 9.89 (s, 1H, CHO), 10.38 (brs, 1H, NH); dry tetrahydrofuran solution as brown solid (1.32 g, 69%): H

ppm; MS (ESI) m/z 289 [M+H].

NMR (300 MHz, CDCl₃);  2.79 (d, 1H, J=2.1 Hz, ≡H), 3.81 (s,

3H, OCH₃), 3.84 (s, 6H, OCH₃), 5.47 (d, 1H, J=2.0 Hz, H-C-OH),

6.73-6.77 (dd, 1H, J=4.9 Hz, J=2.4 Hz, ArH), 6.78 (s, 2H, ArH),

2-(4-Methoxyphenylamino) nicotinaldehyde (6b)

8 | RSC Advances 2015, 00, 1-3

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7.57 (brs, 1H, NH), 7.71-7.75 (dd, 1H, J=5.8 Hz, J=1.5 Hz, ArH), 1-(2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)prop-2-yn-1-one

View Article Online

DOI: 10.1039/C5RA19468G

8.17-8.20 (dd, 1H, J=4.9 Hz, J=1.6 Hz, ArH) ppm; MS (ESI) m/z (8a)

315 [M+H].

1-(2-(3,4,5-Trimethoxyphenylamino)pyridin-3-yl)prop-2-yn-1-

one 8a was obtained using above described method by adding

1-(2-(3,4,5-trimethoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol

7b 7a (1.20 g, 3.8 mmol) to the 2- iodoxy benzoic acid (1.60 g, 5.7

1-(2-(4-Methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol (7b)

1-(2-(4-Methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol

was obtained using above described method by adding 2-(4- mmol) in dimethyl sulfoxide solution as yellow solid (970 mg,

methoxyphenylamino)nicotinaldehyde 6b (1.25 g, 5.5 mmol) yield 81%): ¹H NMR (300 MHz, CDCl₃);



3.55 (s, 1H, ≡ 퐇), 3.84

to the ethynylmagnesium bromide (16.4 mL, 8 mmol) in dry (s, 3H, –OCH3), 3.89 (s, 6H, –OCH3), 6.78-6.84 (dd, 1H, J=5.3

tetrahydrofuran solution as brown solid (920 mg, yield 66%): Hz, J=3.0 Hz, ArH), 6.99 (s, 2H, ArH), 8.42-8.46 (dd, 1H J=2.2 Hz,

¹H NMR (300MHz, CDCl₃);

 2.70 (d, 1H, J=2.2 Hz, ≡H), 3.87 (s, J=2.3 Hz, ArH), 8.47-8.51 (dd, 1H, J=6.0 Hz, J=2.3 Hz, ArH),

3H, OCH3), 6.03-6.11 (m, 1H, H-C-OH), 6.88-6.97 (dd, 1H, J=6.8 10.74 (brs, 1H, NH) ppm; MS (ESI) m/z 313 [M+H].

Hz, J=6.0 Hz, ArH), 6.99-7.10 (m, 2H, ArH), 7.27-7.40 (m, 1H,

ArH), 7.55-7.68 (m, 3H, ArH, NH), 8.71-8.82 (dd, 1H, J=6.8 Hz, 1-(2-(4-Methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-one (8b)

J=6.6 Hz, ArH); ppm; MS (ESI) m/z 255 [M+H].

1-(2-(4-Methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-one 8b

was obtained using above described method by adding 1-(2-(4-

methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-ol 7b (800 mg,

1-(2-(4-Fluorophenylamino)pyridin-3-yl)prop-2-yn-1-ol (7c)

1-(2-(4-Fluorophenylamino)pyridin-3-yl)prop-2-yn-1-ol 7c was 3.2 mmol) to the 2- iodoxy benzoic acid (1.32 g, 4.7 mmol) in

obtained using above described method by adding 2-(4- dimethyl sulfoxide solution as yellow solid (600 mg, yield

fluorophenylamino)nicotinaldehyde 6c (1.40 g, 6.5 mmol) to 76%): ¹H NMR (300 MHz, CDCl₃);

 3.51 (s, 1H, ≡ 퐇), 3.81 (s,

the ethynylmagnesium bromide (19.4 ml, 9.3 mmol) in dry 3H, –OCH3), 6.71-6.78 (dd, 1H, J=4.5 hz, J=3.2 Hz, ArH), 6.92 (d,

tetrahydrofuran solution as brown solid (960 mg, yield 61%): 2H, J=8.9 Hz, ArH), 7.55 (d, 2H, J=8.9 Hz, ArH), 8.36-8.41 (dd,

¹H NMR (300 MHz, CDCl₃);

 2.77 (d, 1H, J=2.3 Hz, ≡H), 5.43 (d, 1H, J=4.5 Hz, J=1.9 Hz, ArH), 8.42-8.48 (dd, 1H, J=5.8 Hz, J=1.9

1H, J=1.5 Hz, H-C-OH), 6.71-6.78 (dd, 1H, J=5.3 Hz, J=2.3 Hz, Hz, ArH), 10.63 (brs, 1h, NH) ppm; MS (ESI) m/z 253 [M+H].

ArH), 6.95-7.04 (m, 2H, ArH), 7.38-7.46 (dd, 2H, J=4.5 Hz, J=9.0

Hz, ArH), 7.54 (brs, 1H, NH), 7.67-7.73 (dd, 1H, J=6.0 Hz, J=1.5 1-(2-(4-Fluorophenylamino)pyridin-3-yl)prop-2-yn-1-one (8c)

Hz, ArH), 8.13-8.18 (dd, 1H, J=3.0 Hz, J=1.5 Hz, ArH); ppm; MS 1-(2-(4-Fluorophenylamino)pyridin-3-yl)prop-2-yn-1-one

8c

(ESI) m/z 243 [M+H].

was obtained using above described method by adding 1-(2-(4-

fluorophenylamino)pyridin-3-yl)prop-2-yn-1-ol 7c (900mg, 3.7

mmol) to the 2- iodoxy benzoic acid (1.56g, 5.6 mmol) in

1-(2-(Phenylamino)pyridin-3-yl)prop-2-yn-1-ol (7d)

1-(2-(Phenylamino)pyridin-3-yl)prop-2-yn-1-ol 7d was obtained dimethyl sulfoxide solution as yellow solid (640mg, yield 72%):

using above described method by adding 2- 1H NMR (300 MHz, CDCl3); 3.54 (s, 1H, ≡ 퐇), 6.76-6.84 (dd,

(phenylamino)nicotinaldehyde 6d (900 mg, 4.5 mmol) to the 1H, J=4.5 Hz, J=3.8 Hz, ArH), 7.00-7.11 (m, 2H, ArH), 7.59-7.68

ethynylmagnesium bromide (13.6 mL, mmol) in dry (dd, 2H, J=3.7 Hz, J=9.0 Hz, ArH), 8.38-8.42 (dd, 1H, J=3.0 Hz,

tetrahydrofuran solution as brown solid (710 mg, yield 70%): J=1.5 Hz, ArH), 8.45-8.50 (dd, 1H, J=6.0 Hz, J=1.5 Hz, ArH),

¹H NMR (300 MHz, CDCl₃);

2.79 (d, 1H, J=2.3 Hz, H), 5.48 (d, 10.72 (brs, 1H, NH) ppm; MS (ESI) m/z 241 [M+H].



7



≡

1H, J=2.1 Hz, H-C-OH), 6.73-6.79 (dd, 1H, J=5.1 Hz, J=2.5 Hz,

ArH), 7.00 (t, 1H, J=7.4 Hz, ArH), 7.31 (t, 2H, J=7.6 Hz, ArH), 1-(2-(Phenylamino)pyridin-3-yl)prop-2-yn-1-one (8d)

7.51 (d, 2H, J=7.6 Hz, ArH), 7.61 (brs, 1H, NH), 7.72-7.78 (dd, 1-(2-(Phenylamino)pyridin-3-yl)prop-2-yn-1-one

8d

was

1H, J=5.8 Hz, J=1.7 Hz, ArH), 8.18-8.28 (dd, 1H, J=3.2 Hz, J=1.7 obtained using above described method by adding 1-(2-

Hz, ArH); ppm; MS (ESI) m/z 225 [M+H].

(phenylamino)pyridin-3-yl)prop-2-yn-1-ol 7d (600mg, 2.7

mmol) to the 2- iodoxy benzoic acid (1.12g, 4 mmol) in

dimethyl sulfoxide solution as yellow solid (420 mg, yield

General method for synthesis of 1-(2-(arylamino)pyridin-3-yl)prop-

2-yn-1-one (8a-d)

70%): ¹H NMR (300 MHz, CDCl₃);

 3.50 (s, 1H, ≡ 퐇), 6.71-6.76

A solution of 2-iodoxy benzoic acid (quantity) and dimethyl (m, 1H, ArH), 7.05 (t, 1H, J=7.3 Hz, ArH), 7.39 (t, 2H, J=7.2 Hz,

sulfoxide (DMSO) (quantity) was stirred for 10 min at room ArH), 7.45 (d, 2H, J=7.2 Hz, ArH), 8.40-8.46 (dd, 1H, J=5.6 Hz,

temperature until homogeneous solution. A solution of J=1.5 Hz, ArH), 8.48-8.56 (dd, 1H, J=4.5 Hz, J=1.5 Hz, ArH) ppm;

alcohols 7a-d in dimethyl sulfoxide was added slowly, and it MS (ESI) m/z 223 [M+H].

was stirred for 4-5 h. After completion of reaction, ice water

was added to reaction mixture and the mixture was stirred for

another 10 min. To this mixture ethyl acetate was added and

General method for synthesis of (Z)-3-(Aryl amino)-1-(2-(aryl

amino) pyridin-3-yl)prop-2-en-1-one (9a-p)

filtered through celite. The organic layer was separated and The ethynyl ketone (8a-d) and aryl amine were dissolved in

washed with water subsequently saturated Na₂CO₃solution absolute ethanol and the mixture was stirred for 4 -5 h at

and brine, after that dried over anhydrous Na₂SO₄and room temperature. After the completion of the reaction

evaporated in vacuum to attain the pure compound and was (confirm by TLC), water was added to the reaction mixture and

used directly for next step.

the crude product filtered. It was recrystallized from methanol

to afford pure compound 9a-p

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(d, 2H, J=9.0 Hz, ArH), 8.00-8.04 (dd, 1H, J=1.1 Hz, J=6.4 Hz,

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(Z)-3-(1H-indol-5-ylamino)-1-(2-(4-methoxyphenylamino)pyridin-3- ArH), 8.26-8.29 (dd, 1H, J=1.8 Hz, J=3.0^DH^Oz^I:,¹⁰A^.r¹⁰H³)⁹,^/1^C1^5R.0^A7¹⁹⁴(b⁶⁸rs^G,

yl)prop-2-en-1-one (9a)

(Z)-3-(1H-Indol-5-ylamino)-1-(2-(4-methoxyphenylamino)

1H, NH), 11.76 (d, 1H, J=12.5 Hz, NH-CH=) ppm; ¹³C NMR (300

MHz, CDCl₃): 192.0, 161.9, 156.0, 155.5, 152.1, 144.1, 141.9,



pyridin-3-yl)prop-2-en-1-one 9a was obtained according to 137.9, 133.1, 128.5, 123.1, 115.0, 114.7, 114.1, 112.4, 95.7,

above described method by using starting materials 1-(2-(4- 95.1, 94.0, 55.45 ppm; IR (KBr) (



_max/cm^-1): 3213, 2922, 1621,

methoxyphenylamino)pyridin-3-yl)prop-2-yn-1-one 8b (90 mg, 1549, 1510, 1460, 1291, 1235, 1107, 1014, 827; MS (ESI): 406

0.4 mmol) and 5-amino indole (47 mg, 0.4 mmol) as yellow [M+H]+; HRMS (ESI): calcd. for C₂₃H₂₄N₃O₄(M+H)⁺406.1761;

solid. ¹H NMR (300 MHz, CDCl₃);

 3.81 (s, 3H, OCH₃), 5.94 (d, found: 406.1752.

1H, J=7.9 Hz, COCH=), 6.54 (s, 1H, ArH), 6.62-6.68 (dd, 1H,

J=3.0 Hz, J= 4.7 Hz, ArH), 6.91 (d, 2H, J=8.8 Hz, ArH), 7.03 (dd, (Z)-3-(4-Methoxyphenylamino)-1-(2-(4-

1H, J=6.4 Hz, J=2.2 Hz, ArH), 7.23-7.27 (m, 1H, ArH), 7.35-7.42 methoxyphenylamino)pyridin-3-yl)prop-2-en-1-one (9d)

(m, 2H, ArH), 7.50-7.58 (dd, 1H, J=7.9 Hz, J=12.6 Hz, =CH-NH), (Z)-3-(4-Methoxyphenylamino)-1-(2-(4-

7.61 (d, 2H, J=8.8 Hz, ArH), 7.98-8.04 (dd, 1H, J=1.7 Hz, J=6.2 methoxyphenylamino)pyridin-3-yl)prop-2-en-1-one 9d was

Hz, ArH), 8.22 (brs, 1H, NH), 8.25-8.29 (dd, 1H, J=1.7 Hz, J=3.0 obtained according to above described method by using

Hz, ArH), 11.14 (brs, 1H, NH), 12.06 (d, 1H, J=12.6 Hz, NH-CH=) starting materials

ppm; ¹³C NMR(300 MHz,CDCl₃):

190.2, 154.8, 154.3, 150.5, yl)prop-2-yn-1-one 8b (90 mg, 0.4 mmol) and 4-

136.8, 132.9, 132.0, 127.8, 125.6, 121.7, 114.7, 113.2, 111.7, methoxyaniline (44 mg, 0.4 mmol) as yellow solid. H NMR

106.9, 100.7, 91.82, 54.7; IR (KBr) ( 3.82 (s, 6H, OCH₃), 5.93 (d, 1H, J=7.9 Hz,

_max/cm^-1): 3346, 2923, (300 MHz, CDCl₃);

1-(2-(4-methoxyphenylamino)pyridin-3-



1





2851, 1630, 1576, 1491, 1460, 1282, 1236, 1125, 1005, 769; COCH=), 6.63-6.67 (dd, 1H, J=4.1 Hz, J=7.5 Hz, ArH), 6.88-6.94

MS (ESI): 385 [M+H]+; HRMS (ESI): calcd for C₂₃H₂₁N₄O₂(M+H)⁺(m, 4H, ArH), 7.08 (d, 2H, J=9.0 Hz, ArH), 7.37-7.44 (m, 1H,

385.1659; found: 385.1658.

=CH-NH), 7.60 (d, 2H,J=8.7 Hz, ArH), 8.00 (d, 1H, J=7.5 Hz,

ArH), 8.27 (d, 1H, J=6.4 Hz, ArH), 11.09 (brs, 1H, NH), 11.89 (d,

(Z)-3-(1H-indol-6-ylamino)-1-(2-(4-methoxyphenylamino)pyridin-3- 1H, J=12.1 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz, CDCl₃):



yl)prop-2-en-1-one (9b)

(Z)-3-(1H-Indol-6-ylamino)-1-(2-(4-

190.7, 154.7, 150.9, 146.1, 137.2, 133.2, 132.5, 132.5, 128.2,

125.8, 122.3, 115.1, 113.6, 112.2, 112.0, 107.4, 92.2, 55.1; IR

_max/cm^-1): 3238, 3002, 2844, 1636, 1498, 1341, 1157,

methoxyphenylamino)pyridin-3-yl)prop-2-en-1-one 9b was (KBr) (

obtained according to above described method by using 1140, 1068, 1014, 925, 871; MS (ESI): 376 [M+H]+; HRMS (ESI)

starting materials 1-(2-(4-methoxyphenylamino)pyridin-3- calcd for C₂₂H₂₂N₃O₃[M+H]+ 376.1655, found: 376.1852.

yl)prop-2-yn-1-one 8b (90 mg, 0.4 mmol) and 6-amino indole

(47 mg, 0.4 mmol) as yellow solid. ¹H NMR (300 MHz, CDCl₃);





(Z)-3-(1H-Indol-5-ylamino)-1-(2-(3,4,5-

3.81 (s, 3H, OCH₃), 5.96 (d, 1h, J=7.4 Hz, COCH=), 6.53 (s, 1H, trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one (9e)

ArH), 6.63-6.67 (m, 1H, ArH), 6.91 (d, 2H, J=8.9 Hz, ArH), 6.96 (Z)-3-(1H-Indol-5-ylamino)-1-(2-(3,4,5-

(d, 1H, J=6.7 Hz, ArH), 7.13 (brs, 1H, ArH), 7.18 (t, 1H, J=6.7 Hz, trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one 9e was

ArH), 7.51-7.56 (dd, 1H, J=7.4 Hz, J=12.1 Hz, =CH-NH), 7.59- obtained according to above described method by using

7.63 (m, 3H, ArH), 7.99-8.02 (dd, 1H, J=1.2 Hz, J=6.7 Hz, ArH), starting materials 1-(2-(3,4,5-trimethoxyphenylamino)pyridin-

8.18 (brs, 1H, NH), 8.26-8.28 (dd, 1H, J=1.5 Hz, J=3.1 Hz, ArH), 3-yl)prop-2-yn-1-one 8a (110 mg, 0.35 mmol) and 5-amino

11.13 (brs, 1H, NH), 12.06 (d, 1H, J=12.1 Hz, NH-CH=) ppm; ¹³C indole (47 mg, 0.35 mmol) as yellow solid. ¹H NMR (300 MHz,

NMR(300 MHz,CDCl₃):

145.0, 144.5, 141.8, 138.0, 136.1, 122.7, 115.4, 115.1, 113.0, J=7.9 Hz, COCH=), 6.54 (s, 1H, ArH), 6.67-6.74 (dd, 1H, J=2.6

95.8, 95.2, 93.9, 55.5 ppm; IR (KBr) (

_max/cm^-1): 3074, 2929, Hz, J=4.9 Hz, ArH), 7.01-7.11 (m, 4H, ArH), 7.36-7.45 (m, 2H,

 192.0, 161.9, 155.6, 151.9, 150.5, CDCl₃);  3.84 (s, 3H, OCH₃), 3.91 (s, 6H, OCH₃), 5.95 (d, 1H,



2835, 1633, 1609, 1589, 1514, 1288, 1236, 1140, 1068, 1012, ArH), 7.51-7.61 (dd, 1H, J=8.1 Hz, J=12.3 Hz, =CH-NH), 8.04 (d,

824; MS (ESI): 385 [M+H]+; HRMS (ESI): calcd. for C₂₃H₂₁N₄O₂1H, J=6.9 Hz, ArH), 8.23 (brs, 1H, NH), 8.31 (d, 1H, J=5.8 Hz,

(M+H)⁺385.1659; found: 385.1657.

ArH), 11.27 (brs, 1H, NH), 12.07 (d, 1H, J=13.7 Hz, NH-CH=)

ppm; ¹³C NMR (300 MHz, CDCl₃):

192.1, 155.6, 153.1, 152.1,

144.3, 138.7, 138.0, 136.0, 122.7, 117.3, 115.1, 112.9, 98.7,

94.4, 60.9, 56.0 ppm; IR (KBr) (

_max/cm^-1): 3109, 2920, 2837,



(Z)-3-(3,5-Dimethoxyphenylamino)-1-(2-(4-

methoxyphenylamino)pyridin-3-yl)prop-2-en-1-one (9c)

(Z)-3-(3,5-Dimethoxyphenylamino)-1-(2-(4-



1631, 1581, 1512, 1481, 1292, 1210, 1028, 833, 765; MS (ESI):

methoxyphenylamino) pyridin-3-yl)prop-2-en-1-one 9c was 445 [M+H]+; HRMS (ESI): calcd for C₂₅H₂₅N₄O₄(M+H)⁺

obtained according to above described method by using 445.1870; found: 445.1863.

starting materials

1-(2-(4-methoxyphenylamino)pyridin-3-

yl)prop-2-yn-1-one 8b (90 mg, 0.4 mmol) and 3,5-

1

dimethoxyaniline (55 mg, 0.4 mmol) as yellow solid. H NMR

(300 MHz, CDCl₃);

 3.81 (s, 3H, OCH₃), 3.83 (s, 6H, OCH₃), 5.98 (Z)-3-(1H-Indol-6-ylamino)-1-(2-(3,4,5-

(d, 1H, J=7.9 Hz, COCH=), 6.21 (s, 1H, ArH), 6.27 (s, 2H, ArH), trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one (9f)

6.64-6.68 (dd, 1H, J=3.0 Hz, J=6.4 Hz, ArH), 6.91 (d, 2H, J=9.0 (Z)-3-(1H-Indol-6-ylamino)-1-(2-(3,4,5-

Hz, ArH), 7.41-7.46 (dd, 1H, J=7.9 Hz, J=12.5 Hz, =CH-NH), 7.60 trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one 9f was

10 | RSC Advances 2015, 00, 1-3

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obtained according to above described method by using

starting materials 1-(2-(3,4,5-trimethoxyphenylamino) (Z)-3-(1H-Indol-5-ylamino)-1-(2-(4-fluorophenylamino)pyridin-3-

View Article Online

DOI: 10.1039/C5RA19468G

pyridine-3-yl)prop-2-yn-1-one 8a (110 mg, 0.35 mmol) and 6- yl)prop-2-en-1-one (9i)

amino indole (47 mg, 0.35 mmol) as yellow solid. ¹H NMR (300 (Z)-3-(1H-Indol-5-ylamino)-1-(2-(4-fluorophenylamino)

MHz, CDCl₃);  3.83 (s, 3H, OCH₃), 3.89 (s, 6H, OCH₃), 5.92 (d, 1H, pyridin-3-yl)prop-2-en-1-one 9i was obtained according to

J=7.9 Hz, COCH=), 6.52 (brs, 1H, ArH), 6.66-6.73 (m, 1H, ArH), 6.91- above described method by using starting materials 1-(2-(4-

6.98 (dd, 1H, J=6.9 Hz, J=1.5 Hz, ArH), 7.02-7.10 (m, 3H, ArH), 7.17 fluorophenylamino)pyridin-3-yl)prop-2-yn-1-one 8c (90mg,

(brs, 1h, ArH), 7.46-7.56 (dd, 1H, J=7.9 Hz, J=12.5 Hz, =CH-NH ), 7.59 0.37 mmol) and 6-amino indole (50mg, 0.37 mmol) as yellow

1

(d, 1h, J=8.5 Hz, ArH), 7.97-8.03 (dd, 1H, J=1.3 Hz, J=6.4 Hz, ArH), solid: H NMR (300 MHz, CDCl₃);  5.97 (d, 1H, J=7.9 Hz, COCH=),

8.28-8.33 (dd, 1H, J=3.1 Hz, J=1.5 Hz, ArH), 8.49 (brs, 1H, NH), 11.28 6.54 (s, 1H, ArH), 6.69-6.73 (dd, 1H, J=4.8 Hz, J= 1.8 Hz, ArH), 6.96-

(brs, 1H, NH), 12.06 (d, 1H, J=12.6 Hz, NH-CH=) ppm; ¹³C NMR (300 7.00 (dd, 1H, J=1.8 Hz, J=6.7 Hz, ArH), 7.01-7.06 (m, 2H, ArH), 7.15 -

MHz, CDCl₃):  191.2, 155.5, 153.1, 151.4, 145.8, 137.7, 136.4, 7.21 (m, 2H, ArH), 7.54 -7.59 (dd, 1H, J=7.9 Hz, J=12.4 Hz, =CH-NH),

135.2, 125.1, 124.7, 115.8, 112.9, 110.7, 102.5, 98.9, 98.5, 93.1, 7.62 (d, 1H, J=8.6 Hz, ArH), 7.68-7.71 (m, 2H, ArH), 8.01-8.05 (dd,

60.9, 56.0 ppm; IR (KBr) (_max/cm^-1): 3233, 2922, 1633, 1581, 1496, 1H, J=1.8 Hz, J=6.1 Hz, ArH), 8.16 (brs, 1H, NH), 8.28-8.31 (dd, 1H,

1414, 1296, 1244, 1144, 1092, 1018, 845; MS (ESI): 445 [M+H]+; J=1.8 Hz, J=4.8 Hz, ArH), 11.27 (brs, 1H, NH), 12.08 (d, 1H, J=12.7 Hz,

HRMS (ESI): calcd for C₂₅H₂₅N₄O₄(M+H)⁺445.1870; found: NH-CH=) ppm; ¹³C NMR(300 MHz,CDCl₃+ DMSO-d₆):  188.9, 157.6,

445.1866.

153.5, 153.3, 149.4, 144.9, 136.3, 134.9, 132.7, 123.8, 123.5, 120.1,

119.6, 113.8, 113.5, 111.8, 108.3, 99.8, 97.5, 91.2 PPM; IR (KBr)

(_max/cm^-1): 3064, 2967, 2838, 1630, 1581, 1481, 1415, 1284, 1247,

1168, 1023, 809; MS (ESI): 373 [M+H]+; HRMS (ESI) calcd for

C₂₂H₁₈FN₄O [M+H]+ 373.1459, found: 373.1456.

(Z)-3-(4-Methoxyphenylamino)-1-(2-(3,4,5-

trimethoxyphenylamino)pyridin-3-yl) prop-2-en-1-one (9g)

(

Z)-3-(4-Methoxyphenylamino)-1-(2-(3,4,5-

trimethoxyphenylamino)pyridin-3-yl) prop-2-en-1-one 9g was

obtained according to above described method by using (Z)-3-(3,5-Dimethoxyphenylamino)-1-(2-(4-

starting materials 1-(2-(3,4,5-trimethoxyphenylamino)pyridin fluorophenylamino)pyridin-3-yl)prop-2-en-1-one (9j)

-3-yl)prop-2-yn-1-one 8a (110 mg, 0.35 mmol) and 4- (Z)-3-(3,5-Dimethoxyphenylamino)-1-(2-(4-

1

methoxyaniline (43 mg, 0.35 mmol) as yellow solid. H NMR fluorophenylamino)pyridin-3-yl)prop-2-en-1-one

9j

was

(300 MHz, CDCl₃);  3.82 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.90 (s, obtained according to above described method by using

6H, OCH₃), 5.94 (d, 1H, J=7.9 Hz, COCH=), 6.67-6.75 (dd, 1H, J=2.6 starting materials 1-(2-(4-fluorophenylamino)pyridin-3-

Hz, J=4.9 Hz, ArH), 6.92 (d, 2H, J=8.4 Hz, ArH), 7.03-7.13 (m, 4H, yl)prop-2-yn-1-one 8c (90mg, 0.37 mmol) and 3, 5-

1

ArH), 7.37-7.47 (dd, 1H, J=7.9 Hz, J=12.1 Hz, =CH-NH), 8.02 (d, 1H, dimethoxyaniline (57mg, 0.37mmol) as yellow solid: H NMR

J=7.7 Hz, ArH), 8.31 (d, 1H, J=7.6 Hz, ArH), 11.22 (brs, 1H, NH), (300MHz, CDCl₃);  3.82 (s, 6H, OCH₃), 5.98 (d, 1H, J=8.1 Hz,

11.89 (d, 1H, J=12.6 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz, CDCl₃):  COCH=), 6.19-6.33 (m, 3H, ArH), 6.67-6.76 (dd, 1H, J=3.0 Hz, J=4.7

192.3, 153.3, 152.3, 145.2, 144.5, 139.0, 138.3, 136.2, 122.9, 117.5, Hz, ArH), 6.98-7.10 (m, 2H, ArH), 7.40-7.51 (dd, 1H, J=7.9 Hz, J=12.1

115.4, 113.1, 98.9, 61.1, 56.2 ppm; IR (KBr) (_max/cm^-1): 3337, 2925, Hz, =CH-NH), 7.64-7.73 (m, 2H, ArH), 8.04 (d, 1H, J=7.8 Hz, ArH),

1614, 1588, 1549, 1504, 1479, 1414, 1279, 1241, 1126, 1014, 897; 8.30 (dd, 1H, J=1.7 Hz, J=4.5 Hz, ArH), 11.21 (brs, 1H, NH), 11.77 (d,

MS (ESI): 436 [M+H]+; HRMS (ESI): calcd for C₂₄H₂₆N₃O₅(M+H)⁺1H, J=12.1 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz, CDCl₃):  192.0,

436.1867; found: 445.1852.

161.9, 155.6, 151.9, 144.5, 141.8, 137.9, 136.1, 129.5 122.6, 115.3,

115.0, 113.0, 95.8, 95.1, 93.9, 55.5 ppm; IR (KBr) (_max/cm^-1): 3427,

2928, 2833, 2356, 1628, 1576, 1506, 1484, 1414, 1236, 1124, 1012,

822;MS (ESI): 394 [M+H]+; HRMS (ESI) calcd for C₂₂H₂₁FN₃O₃[M+H]+

394.1561, found: 394.1559.

(Z)-3-(3,4,5-Trimethoxyphenylamino)-1-(2-(3,4,5-

trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one (9h)

(Z)-3-(3,4,5-Trimethoxyphenylamino)-1-(2-(3,4,5-

trimethoxyphenylamino)pyridin-3-yl)prop-2-en-1-one 9h was

obtained according to above described method by using (Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(4-

starting materials 1-(2-(3,4,5-trimethoxyphenylamino)pyridin methoxyphenylamino)prop-2-en-1-one (9k)

-3-yl)prop-2-yn-1-one 8a (110 mg, 0.35 mmol) and 3, 4, 5- (Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(4-

trimethoxyaniline (64 mg, 0.35 mmol) as yellow solid. ¹H NMR methoxyphenylamino)prop-2-en-1-one 9k was obtained

(300 MHz, CDCl₃);  3.84 (s, 6H, OCH₃), 3.91 (s, 12H, OCH₃), 5.98 (d, according to above described method by using starting

1H, J=7.8 Hz, COCH=), 6.35 (s, 2H, ArH), 6.71-6.74 (dd, 1H, J=3.0 Hz, materials 1-(2-(4-fluorophenylamino)pyridin-3-yl)prop-2-yn-1-

J=4.9 Hz, ArH), 7.05 (s, 2H, ArH), 7.41-7.46 (dd, 1H, J=7.8 Hz, J=12.4 one 8c (90mg, 0.37 mmol) and 4-methoxyaniline (46 mg, 0.37

Hz, =CH-NH), 8.02-8.05 (dd, 1H, J=1.1 Hz, J=6.8 Hz, ArH), 8.32-8.34 mmol) as yellow solid: ¹H NMR (300MHz, CDCl₃);  3.82 (s, 3H,

(dd, 1H, J=1.5 Hz, J=3.3 Hz, ArH), 11.16 (brs, 1H, NH), 11.83 (d, 1H, OCH₃), 5.94 (d, 1H, J=7.9 Hz, COCH=), 6.69-6.72 (dd, 1H, J=3.0 Hz,

J=12.1 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz, CDCl₃):  191.8, J=4.8 Hz, ArH), 6.92 (d, 2H, J=9.0 Hz, ArH), 7.00-7.06 (m, 2H, ArH),

155.6, 154.2, 153.2, 151.8, 144.9, 137.9, 136.2, 115.5, 113.9, 112.9, 7.08 (d, 2H, J=9.0 Hz, ArH), 7.39-7.45 (dd, 1H, J=7.9 Hz, J=12.4 Hz,

99.0, 94.4, 93.7, 61.0, 56.2 ppm; IR (KBr) (_max/cm^-1): 3419, 3072 =CH-NH), 7.65-7.71 (m, 2H, ArH), 8.02 (d, 1H, J=4.8 Hz, ArH), 8.29

2941, 1642, 1504, 1448, 1417, 1384, 1236, 1124, 1009, 837; MS (d, 1H, J=6.3 Hz, ArH), 11.23 (brs, 1H, NH), 11.92 (d, 1H, J=12.7 Hz,

(ESI): 496 [M+H]+; HRMS (ESI) calcd for C₂₆H₃₀N₃O₇[M+H]+ NH-CH=) ppm; IR (KBr) (_max/cm^-1): 3241, 2922, 2851, 1634, 1573,

496.2078, found: 496.2083.

1498, 1439, 1324, 1240, 1074, 1017, 976, 889, 817; MS (ESI): 364

J. Name., 2013, 00, 1-3 | 11

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ARTICLE

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[M+H]+; HRMS (ESI) calcd for C₂₁H₁₉FN₃O₂[M+H]+ 364.1455, found: J=3.0 Hz, ArH), 11.34 (brs, 1H, NH), 12.08 (d,1H, J=13.5 Hz, NH

-

View Article Online

364.1452.

CH=) ppm; IR (KBr) (



_max/cm^-1): 3180, 29^D2^O3,^I:1¹⁰6^.2¹⁰6³,⁹1^/C5⁵7^R4^A,¹⁹1⁴4⁶9⁸1^G,

1287, 1217, 1132, 1014, 887, 863; MS (ESI): 355 [M+H]+;

HRMS (ESI) calcd for C₂₂H₁₉N₄O [M+H]+ 355.1553, found:

355.1551.

(Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(3,4,5-

trimethoxyphenylamino)prop-2-en-1-one (9l)

(Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(3,4,5-

trimethoxyphenylamino)prop-2-en-1-one 9l was obtained (Z)-3-(1H-Indol-6-ylamino)-1-(2-(phenylamino)pyridin-3-yl)prop-2-

according to above described method by using starting en-1-one (9o)

materials 1-(2-(4-fluorophenylamino)pyridin-3-yl)prop-2-yn-1- (Z)-3-(1H-Indol-6-ylamino)-1-(2-(phenylamino)pyridin-3-

one 8c (90 mg, 0.37 mmol) and 3, 4, 5-trimethoxyaniline yl)prop-2-en-1-one 9o was obtained according to above

(69mg, 0.37 mmol) as yellow solid: ¹H NMR (300MHz, CDCl₃);



described method by using starting materials

1-(2-

3.83 (s, 3H, OCH₃), 3.91 (s, 6H, OCH₃), 5.98 (d, 1H, J=8.1 Hz, (phenylamino)pyridin-3-yl)prop-2-yn-1-one 8d (100mg, 0.45

COCH=), 6.34 (s, 2H, ArH), 6.68-6.76 (dd, 1H, J=3.0 Hz, J=4.7 mmol) and 6-amino indole (59mg, 0.45 mmol) as yellow solid:

Hz, ArH), 7.01-7.07 (m, 2H, ArH), 7.39-7.47 (dd, 1H, J=8.1 Hz, ¹H NMR (300MHz, CDCl₃);  5.97 (d, 1H, J=8.1 Hz, COCH=), 6.54 (brs,

J=12.4 Hz, =CH-NH), 7.65-7.71 (m, 2H, ArH), 8.01-8.06 (dd, 1H, 1H, ArH), 6.70-6.74 (m, 1H, ArH), 6.97-7.00 (dd, 1H, J=1.8 Hz, J=6.5

J=1.5 Hz, J=6.2 Hz, ArH), 8.28-8.32 (dd, 1H, J=1.8 Hz, J=2.8 Hz, Hz, ArH), 7.03 (t, 1H, J=7.5 Hz, ArH), 7.12 (brs, 1H, ArH), 7.15-7.23

ArH), 11.21 (brs, 1H, NH), 11.85 (d, 1H, J=12.6 Hz, NH-CH=) (m, 2H, ArH), 7.34 (t, 2H, J=7.5 Hz, ArH), 7.53-7.58 (dd, 1H, J=7.9 Hz,

ppm; ¹³C NMR (300 MHz,CDCl₃):

144.9, 137.9, 136.2, 122.6, 115.4, 115.1, 113.1, 94.4, 93.6, Hz, ArH), 8.02-8.05 (dd, 1h, J=1.5 Hz, J=6.5 Hz, ArH), 8.17 (brs, 1H,

 191.8, 155.6, 154.3, 151.8, J=12.4 Hz, =CH-NH), 7.62 (d, 1H, J=8.3 Hz, ArH), 7.77 (d, 2H, J=7.8

61.1, 56.2 ppm; IR (KBr) (

_max/cm^-1): 3417, 3001, 2923, 2851, NH), 8.30-8.33 (dd, 1H, J=1.8 Hz, J=3.3 Hz, ArH), 11.32 (brs, 1H, NH),

1635, 1578, 1488, 1415, 1285, 1233, 1125, 1011, 815; MS (ESI): 12.09 (d, 1H, J=12.4 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz,CDCl₃): 

424 [M+H]+; HRMS (ESI) calcd for C₂₃H₂₃FN₃O₄[M+H]+ 189.2, 149.6, 144.9, 138.7, 136.6, 132.8, 127.1,123.7, 120.3, 119.7,

424.1667, found: 424.1657.

118.4, 112.0, 108.4, 99.9, 91.3 ppm; IR (KBr) (_max/cm^-1): 3194,

2922, 1619, 1575, 1511, 1468, 1279, 1218, 1135, 1018, 866, 814;

MS (ESI): 355 [M+H]+, HRMS (ESI) calcd for C₂₂H₁₉N₄O [M+H]+

355.1553, found: 355.1554.

(Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(4-

trifluoromethyl)phenylamino)prop-2-en-1-one (9m)

(Z)-1-(2-(4-Fluorophenylamino)pyridin-3-yl)-3-(4-

(trifluoromethyl)phenylamino) prop-2-en-1-one 9m was (Z)-1-(2-(Phenylamino)pyridin-3-yl)-3-(3,4,5-

obtained according to above described method by using trimethoxyphenylamino)prop-2-en-1-one (9p)

starting materials

1-(2-(4-fluorophenylamino)pyridin-3- (Z)-1-(2-(Phenylamino)pyridin-3-yl)-3-(3,4,5-

yl)prop-2-yn-1-one 8c (90mg, 0.37 mmol) and 4- trimethoxyphenylamino)prop-2-en-1-one 9p was obtained

1

trifluoromethylaniline (60mg, 0.37 mmol) as yellow solid: H according to above described method by using starting

NMR (300MHz, CDCl₃);

1H, J=6.7 Hz, ArH), 7.06 (d, 2H, J=6.9 Hz, ArH), 7.14-7.35 (m, (100mg, 0.45 mmol) and 3, 4, 5-trimethoxyaniline (82mg, 0.45

2H, ArH), 7.42-7.52 (dd, 1H, J=7.3 Hz, J=12.3 Hz, =CH-NH), mmol) as yellow solid: ¹H NMR (300 MHz, CDCl₃);

3.40 (S, 9H,

 6.09 (d, 1H, J=7.3 Hz, COCH=), 6.74 (t, materials 1-(2-(phenylamino)pyridin-3-yl)prop-2-yn-1-one 8d



7.57-7.81 (m, 4H, ArH), 8.06 (d, 1H, J=6.7 Hz, ArH), 8.34 (d, 1H, OCH₃), 5.93 (d, 1H, J=7.9 Hz, COCH=), 6.51 (s, 2H, ArH), 6.70-

J=6.4 Hz, ArH), 11.21 (brs, 1H, NH), 11.91 (d, 1H, J=12.3 Hz, 6.76 (dd, 1H, J=2.8 Hz, J=4.9 Hz, ArH), 7.04 (t, 2H, J=8.1 Hz,

NH-CH=) ppm; ¹³C NMR (300 MHz,CDCl₃):



192.4, 160.2, ArH), 7.30-7.45 (m, 2H, ArH, =CH-NH ), 7.72 (d, 2H, J=8.1 Hz,

157.0, 155.7, 152.4, 143.2, 142.9, 138.2, 136.0, 127.1, 122.8, ArH), 8.02-8.06 (dd, 1H, J=1.3 Hz, J=6.4 Hz, ArH), 8.24-829 (dd,

122.7, 115.8, 115.2, 114.9, 113.0, 95.3 ppm; IR (KBr) (

_max/cm^-1H, J=1.3 Hz, J=3.2 Hz, ArH), 11.28 (brs, 1H, NH), 12.09 (d, 1H,

¹): 3419, 3089, 2928, 1635, 1573, 1514, 1483, 1280, 1237, J=13.7 Hz, NH-CH=) ppm; ¹³C NMR (300 MHz, CDCl₃):

191.2,

1198, 1125, 1017, 849; MS (ESI): 402 [M+H]+; HRMS (ESI) calcd 155.4, 153.1, 151.3, 145.8, 137.8, 136.3, 135.2. 125.1, 124.7,





for C₂₁H₁₅F₄N₃O [M+H]+ 402.1224, found: 402.1218.

121.7, 115.8, 113.0, 110.7, 102.6, 98.5, 93.1, 60.9, 56.0 ppm; IR

(KBr) (



_max/cm^-1): 3429, 2937, 1633, 1577, 1506, 1485, 1415,

(Z)-3-(1H-Indol-5-ylamino)-1-(2-(phenylamino)pyridin-3-yl)prop-2-

en-1-one (9n)

1231, 1123, 1013, 918, 768; MS (ESI): 406 [M+H]+. HRMS (ESI)

calcd for C₂₃H₂₄N₃O₄[M+H]+ 406.1761, found: 406.1756.

(Z)-3-(1H-Indol-5-ylamino)-1-(2-(phenylamino)pyridin-3-

yl)prop-2-en-1-one 9n was obtained according to above

described method by using starting materials

1-(2-

(phenylamino)pyridin-3-yl)prop-2-yn-1-one 8d (100mg, 0.45

mmol) and 5-amino indole (59 mg, 0.45 mmol) as yellow solid:

¹H NMR (300 MHz, CDCl₃);

 5.94 (d, 1H, J=7.8 Hz, COCH=),

Biology

6.54 (brs, 1H, ArH), 6.69-6.73 (m, 1H, ArH), 6.99-7.06 (m, 3H,

ArH), 7.34 (t, 2H, J=8.2 Hz, ArH), 7.39 (d, 1H, J=8.6 Hz, ArH),

7.42 (brs, 1H, ArH), 7.52-7.58 (dd, 1H, J=7.8 Hz, J=12.7 Hz, =CH-

NH ), 7.77 (d, 2H, J=8.2 Hz, ArH), 8.02-8.05 (dd, 1H, J=1.5 Hz,

J=6.4 Hz, ArH), 8.19 (brs, 1H, NH), 8.30-8.33 (dd, 1H, J=1.8 Hz,

Cell Cultures, Maintenance and Antiproliferative Evaluation

All cell lines used in this study were purchased from the

American Type Culture Collection (ATCC, United States). A-549,

12 | RSC Advances 2015, 00, 1-3

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HeLa, MCF-7 and HCT-116 were grown in Dulbecco's modified PEM buffer (80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl , pH 6.9)

View Article Online

2

DOI: 10.1039/C5RA19468G

Eagle's medium (containing 10% FBS in

a

humidified in 384 well plates contained 2 mg/mL bovine brain tubulin, 10

atmosphere of 5% CO₂at 37 °C). A-549 cells were cultured in µM fluorescent reporter, 1 mM GTP in the presence or

o

Eagle's minimal essential medium (MEM) containing non- absence of test compounds at 37 C. Tubulin polymerization

essential amino acids, 1 mM sodium pyruvate, 10 mg/mL was followed by monitoring the fluorescence enhancement

bovine insulin, and 10% FBS. Cells were trypsinized when sub- due to the incorporation of a fluorescence reporter into

confluent from T25 flasks/60 mm dishes and seeded in 96-wel microtubules as polymerization proceeds. Fluorescence

plates. The synthesized test compounds were evaluated for emission at 420 nm (excitation wavelength is 360 nm) was

their in vitro antiproliferative in four different human cancer measured for 1 h at 1-min intervals in a multimode plate

cell lines. A protocol of 48 h continuous drug exposure was reader (Tecan M200). To determine the IC₅₀values of the

used, and a MTT cell proliferation assay was used to estimate compounds against tubulin polymerization, the compounds

cell viability or growth. The cell lines were grown in their were pre-incubated with tubulin at varying concentrations

respective media containing 10% fetal bovine serum and were (0.1, 1, 2, 5 and 10 µM). Assays were performed under similar

seeded into 96-well microtiter plates in 200 μL aliquots at conditions as employed for polymerization assays as described

plating densities depending on the doubling time of individual above.

cell lines. The microtiter plates were incubated at 37 °C, 5%

CO₂, 95% air, and 100% relative humidity for 24 h prior to

addition of experimental drugs. Aliquots of 2 μL of the test

Immunohistochemistry of Tubulin and Analysis of Nuclear

Morphology

compounds were added to the wells already containing 198 μL A549 cells were seeded on glass cover slip, incubated for 24 h

of cells, resulting in the required final drug concentrations. For in the presence or absence of test compounds 9a, 9g and 9j at

each compound, four concentrations (0.1, 1, 2, 10 and 50 μM) concentrations of 3 μM. Cells grown on cover slips were fixed

were evaluated, and each was done in triplicate wells. Plates in 3.5% formaldehyde in phosphate-buffered saline (PBS) pH

were incubated further for 48 h, and the assay was terminated 7.4 for 10 minutes at room temperature. Cells were

by the addition of 10 μL of 5% MTT and incubated for 60 min permeablized for 6 minutes in PBS containing 0.5% Triton X-

at 37 °C. Later, the plates were air-dried. Bound stain was 100 (Sigma) and 0.05% Tween-20 (Sigma). The permeablized

subsequently eluted with 100 μL of DMSO, and the absorbance cells were blocked with 2% BSA (Sigma) in PBS for 1h. Later,

was read on multimode plate reader (Tecan M200) at a the cells were incubated with primary antibody for tubulin

wavelength of 560 nm. Percent growth was calculated on a from (sigma) at (1:200) diluted in blocking solution for 4h at

plate by plate basis for test wells relative to control wells. The room temperature. Subsequently the antibodies were

above determinations were repeated thrice. The growth removed and the cells were washed thrice with PBS. Cells were

inhibitory effects of the compounds were analyzed by then incubated with FITC labeled anti-mouse secondary

generating dose response curves as a plot of the percentage antibody (1:500) for 1h at room temperature. Cells were

surviving cells versus compound concentration. The sensitivity washed thrice with PBS and mounted in medium containing

of the cancer cells to the test compound was expressed in DAPI. Images were captured using the Olympus confocal

terms of IC₅₀, a value defined as the concentration of microscope and analyzed with Provision software.

compound that produced 50% reduction as compared to the

Morphological analysis for apoptosis with Hoechst staining

control absorbance. IC₅₀values are indicated as means ± SD of

three independent experiments.

A549 cells were seeded at a density of 10,000 cells over 18-

mm cover slips and incubated for 24 h. Then, the medium was

replaced, and cells were treated with compounds 9a, 9g and 9j

Cell Cycle Analysis

A-549 cells in 60 mm dishes were incubated for 24 h in the at 2 µM for 24 h. Cells treated with vehicle (0.001% DMSO)

presence or absence of test compounds 9a 9g and 9j at 3 µM were included as controls for all experiments. An overnight

,

concentrations. Cells were harvested with trypsin-EDTA, fixed treatment, Hoechst 33342 (Sigma–Aldrich) were added to

with ice-cold 70% ethanol at 4 °C for 30 min, ethanol was medium at a concentration of 0.5 mg/mL containing 4% para

removed by centrifugation and cells were stained with 1 mL of formaldehyde. After incubation for 30 min at 37 °C, cells from

DNA staining solution [0.2 mg of propidium Iodide (PI), and 2 each dish were captured from randomly selected fields under

mg RNase A] for 30 min as described earlier . The DNA fluorescent microscope (Leica, Germany) to qualitatively

contents of 20,000 events were measured by flow cytometer determine the proportion of viable and apoptotic cells based

(BD FACS Canto II). Histograms were analyzed using FCS on their relative fluorescence and nuclear fragmentation

.

express 4 plus.

Caspase-3 activity assay

Tubulin Polymerization Assay

AFC conjugated Ac-DEVD substrate was used to determine the

An in vitro assay for monitoring the time-dependent caspase-3 activity of the potent compounds 9a, 9g and 9j at 2

polymerization of tubulin to microtubules was performed µM. A549 cells were seeded in six well plates with the

employing a fluorescence-based tubulin polymerization assay confluence of per (2.5 × 10^-6) well and are treated with the

kit (BK011, Cytoskeleton, Inc.) according to the manufacturer’s compounds at 2 µM concentration. The cells were washed

protocol. The reaction mixture in a final volume of 10 µl in with PBS after incubation for 48 h and then scraped in to the

J. Name., 2013, 00, 1-3 | 13

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ARTICLE

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PBS and centrifuged at 2000 rpm for 10 min at 4 °C. Pellet was validation of docking protocol 9a and 9g were docked into the

View Article Online

DOI: 10.1039/C5RA19468G

resuspended in 80 ml of lysis buffer. Then the pellet was active site of tubulin. A maximum of 10 docking poses per

passed through insulin syringe followed by incubation of ligand were generated in each case and analyzed further for

suspension on ice for 20–30 min centrifuged the lysate at the binding mode and intermolecular interactions.

13,200 rpm for 20 min at 4 °C and transferred the supernatant

to fresh tubes. In a 96 well black polystyrene plate, 50 ml cell

lysate, 50 ml of 2X assay buffer and 2 ml of caspase-3substrate

were taken. The reaction was allowed to take place for 1 h.

The fluorescence generated by the release of the fluorogenic

group AFC on cleavage by caspase-3 was measured by

excitation at 400 nm and emission at 505 nm for every 5 min

over 1 h. Protein was estimated by Bradford’s method and

normalized consequently.

Acknowledgements

V.S.R., A.B.S, G.B.K, M.K.R, I.B.S, S.V and P.R.A acknowledge

CSIR-UGC, New Delhi and 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). The authors thank Prof. P. V.

Bharatam, NIPER Mohali for discussion on molecular docking.

Competitive tubulin binding assay

Notes and references

The various concentrations (0 μM, 5 μM, 10μM and 15 μM ) of

conjugates 9a, 9g and 9j were coincubated with 3 μM

colchicines in 30 mM Tris buffer containing 3 μM tubulin at 37

1

Electronic Supplementary Information (ESI) includes H NMR,

¹³C NMR and HRMS spectra of final compounds (9a

-9p).

^oC for 60 min. The standard nocodazole (

I) was employed as a

positive control whereas peclitaxel was used as the negative

control. After incubation, the fluorescence of tubulin-

colchicine complex was measured by using Tecan multimode

reader at excitation wavelength of 380 nm and emission

wavelength of 435 nm; whereas 30 mM Tris buffer was used as

a blank. The raw fluorescence values were normalized first by

subtracting the fluorescence of the buffer and then setting the

fluorescence of 3 μM tubulin with 3 μM colchicine to 100%.

Values represented were ± SD of at least three independent

experiments.

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Docking

(6)

(7)

(8)

For the purpose of molecular docking studies, recently

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(PDB ID: 3UT5, resolution: 2.73 Å)³⁵was selected. This PDB ID

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Synthesis of 2-anilinopyridine-arylpropenone conjugates as tubulin inhibitors and apoptotic inducers

DOI: 10.1039/c5ra19468g

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