Anti-mycobacterial, cytotoxic activities of Knoevenagel and (E)-α,β-unsaturated esters and ketones from 2-chloronicotinaldehydes

Article abstract of DOI:10.1007/s00044-013-0622-4

Series of 2-chloronicotinaldehyde Knoevenagel derivatives 3a-r; (E)-α,β-unsaturated esters and ketones 5a-k were prepared and evaluated for their in vitro anti-mycobacterial activity against Mycobacterium tuberculosis H₃₇Rv (Mtb). Compounds 3e,

Full text of DOI:10.1007/s00044-013-0622-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0622-4
ORIGINAL RESEARCH
Anti-mycobacterial, cytotoxic activities of Knoevenagel
and (E)-a,b-unsaturated esters and ketones
from 2-chloronicotinaldehydes
•
Pathi Suman Rayala Nageswara Rao
•
•
•
Bhimapaka China Raju Dharmarajan Sriram
Pulla Venkat Koushik
Received: 9 November 2012 / Accepted: 6 May 2013
Ó Springer Science+Business Media New York 2013
Abstract Series of 2-chloronicotinaldehyde Knoevenagel
derivatives 3a–r; (E)-a,b-unsaturated esters and ketones
5a–k were prepared and evaluated for their in vitro anti-
mycobacterial activity against Mycobacterium tuberculosis
H37Rv (Mtb). Compounds 3e, 5b, 5d, 5e, 5g and 5i–k were
shown potent to significant activity. Compound 5j is the
most potent Mtb inhibitor (MIC: 4.89 lM) when compared
to standard drugs Ethambutol (MIC: 7.63 lM) and Cipro-
floxacin (MIC: 9.44 lM). Eight compounds displayed
potent/significant activity against M. tuberculosis were
chosen for the cytotoxicity against three cell lines (Raw
264.7, MCF7, and HeLa). Compound 5j displayed low
toxicity with high selective index (15–30) and is an
interesting new compound may serve for the development
of therapeutics targeted against anti-mycobacterial com-
pounds. This is the first report assigning in vitro anti-
mycobacterial inhibitory activity and structure–activity
relationship for this class of substituted 2-chloronicotin-
aldehyde derivatives and presents new family of
compounds.
Keywords Knoevenagel derivatives ꢀ
(E)-a,b-Unsaturated esters and ketones ꢀ
2-Chloronicotinaldehydes ꢀ Anti-mycobacterial activity ꢀ
Cytotoxicity
Introduction
Tuberculosis (TB) (Dover and Coxon, 2011) is an infectious
disease caused by Mycobacterium tuberculosis (or Koch
bacillus) is major health and challenging problem around
the world for more than five millennia. The most worrying
factor in the current TB problem is the prevalence of mul-
tidrug resistant (MDR) strains not only to the front line
drugs Isoniazid and Rifampicin but also second line drugs.
The numbers of deaths are occurring despite the availability
of effective chemotherapy and Bacillus Calmette-Guerin
(BCG) vaccine; therefore, there is an urgent need to isolate
an effective natural products or synthesis of new chemical
entities for anti-tuberculosis drugs. Similarly cancer the
uncontrolled, rapid and proliferation of abnormal cell is the
leading cause of human death, despite considerable pro-
gress of its biology and pharmacology (Jaracz et al., 2005).
Therefore safe, potent and selective search for new che-
motherapy agents are required for tuberculosis and cancer.
Our interest in design and synthesis of biologically active
heterocyclic compounds with potential biological activities
(Kumar et al., 2010, 2011; Raju et al., 2010; Gangadasu
et al., 2009a) and on anti-mycobacterial & anticancer
agents (Raju et al., 2011), in this study we report synthesis
of 2-chloronicotinaldehyde Knoevenagel derivatives and
(E)-a,b-unsaturated esters and ketones, their in vitro anti-
mycobacterial and cytotoxic activities as well as structure–
activity relationship in relation to various substitutions
present on 2-chloronicotinaldehyde for the first time.
P. Suman ꢀ R. N. Rao ꢀ B. C. Raju (&)
Natural Products Chemistry Division, CSIR-Indian Institute of
Chemical Technology, Hyderabad 500 007, India
e-mail: chinarajuiict@yahoo.co.in
D. Sriram ꢀ P. V. Koushik
Medicinal Chemistry Research Laboratory, Birla Institute of
Technology and Science, Pilani, Hyderabad Campus,
Hyderabad, India
e-mail: drdsriram@yahoo.com
123

Med Chem Res
Results and discussion
However, b-keto ester with chloro substituent at C(4) has
an impact on their reactivity and the reaction of 2-chloro-
nicotinaldehyde 1a with ethyl 4-chloro-3-oxobutanoate did
not afford the desired Knoevenagel derivative and pres-
ently the reaction is under investigation. Similarly, b-keto
ester with a trifluoro substituent at C(4) also has an
impact on their reactivity and the reaction of carbalde-
hydes with ethyl 4,4,4-trifluoro-3-oxobutanoate produced
(E)-a,b-unsaturated esters instead Knoevenagel derivative.
The reaction is proceeding through the Aldol-adduct
elimination under basic conditions produced geometri-
cally selective E product (Raju and Suman, 2010; Suman
et al., 2012). (E)-a,b-Unsaturated esters 5a–e were pre-
pared by the reaction of 2-chloronicotinaldehydes 1a–
e with ethyl 4,4,4-trifluoro-3-oxobutanoate 4a in presence
of piperidine in DCM at room temperature with good
yields (Scheme 2).
Chemistry
Substituted 2-chloronicotinaldehydes were synthesized by
Vilsmeier cyclization of enamide substrates according to
our earlier reported method (Gangadasu et al., 2006). Thus
prepared substituted 2-chloronicotinaldehydes are an
important synthons utilized for the preparation of various
biologically important heterocyclic compounds such as
Baylis–Hillman adducts (Ravinder et al., 2009; Narender
et al., 2005), quinolines (Narender et al., 2006), 1,8-
naphthyridines (Narender et al., 2009) and 2-chloro-5-
methylpyridine-3-olefin derivatives (Gangadasu et al.,
2009b) and Imidacloprid (Gangadasu et al., 2009a).
However preparation of 2-chloronicotinaldehyde Knoeve-
nagel derivatives and (E)-a,b-unsaturated esters/ketones
and their biological activities were not reported so far in
the literature. Knoevenagel condensation is most important
basic reaction in synthetic organic chemistry for the prep-
aration of substituted alkenes and it has numerous appli-
cations in the synthesis of fine chemicals & target
molecules by the condensation of carbonyl compounds
with compounds containing a methylene activated by one
or two electron-withdrawing substituents (Trost, 1991).
Scheme 1 describes the synthesis of Knoevenagel deriva-
tives 3a–r by the reaction of 2-chloronicotinaldehydes 1a–
d with ethyl 3-oxobutanoate, ethyl 3-oxo-3-phenylpropan-
oate, 2-methoxyethyl 3-oxobutanoate, diethyl malonate,
ethyl 2-cyanoacetate and methyl 2-cyanoacetate 2a–f in
presence of piperidine in DCM at room temperature.
The method is new, general, practical and established
method was successfully applied for the synthesis of
(E)-a,b-unsaturated ketones 5f–k by the reaction of 2-chlo-
ronicotinaldehydes 1a–c with 1,1,1-trifluoropentane-2,4-
dione 4b and 4,4,4-trifluoro-1-phenylbutane-1,3-dione 4c.
Thus synthesized compounds 3a–r (new) and 5a–k (Raju
et al., 2010) are well characterized by spectral data.
In-vitro anti-mycobacterial activity
The synthesized compounds 3a–r and 5a–k were screened
for their in vitro anti-mycobacterial activity against
M. tuberculosis H37Rv (MTB) by agar dilution method rec-
ommended by National Committee for Clinical Laboratory
Scheme 1 Synthesis of
Knoevenagel derivatives 3a–r
O
R1
R2
CHO
Cl
R1
R2
O
R4
Piperidine
DCM, r.t
R5
R4
+
R5
N
N
Cl
1a-d
2a-f
3a-r
1a, R1 = Me, R2 = H
1b, R1 = Ph, R2 = H
2a, R4 = OEt, R5 = COMe
2b, R4 = OEt, , R5 = COPh
1c, R1 = H, R2 = CO2Me 2c, R4 = OCH2CH2OMe, R5 = COMe
1d, R1 = Propyl, R2 = H
2d, R4 = OEt, R5 = CO2Et
2e, R4 = OEt,R5 = CN
2f, R4 = OMe, R5 = CN
Scheme 2 Synthesis of E-(a,b)
unsaturated esters and ketones
5a–k
O
R1
R2
CHO
Cl
R1
R2
R3 CF3
R3
Piperidine
DCM, r.t
+
O
O
N
N
Cl
4a-c
1a-e
5a-k
1a, R1 = Me, R2 = H
1b, R1 = Ph, R2 = H
4a, R3 = OEt
4b, R3 = Me
1c, R1 = H, R2 = CO2Me 4c, R3 = Ph
1d, R1 = Propyl, R2 = H
1e, R1 = Ethyl, R2 = H
123

Med Chem Res
Table 1 Synthesis and anti-mycobacterial activity of Knoevenagel derivatives 3a–r
Entry
Compounds
R1
R2
R4
R5
Yielda (%)
CLogPb
MIC (lg/mL)
MIC (lM)
1
3a
Me
Ph
H
H
OEt
COMe
COMe
COMe
COPh
COPh
COPh
COMe
COMe
CO2Et
CO2Et
CO2Et
CO2Et
CN
72
76
74
75
80
81
72
78
80
83
85
84
82
85
87
78
87
87
2.26
3.65
1.43
3.68
5.02
2.79
1.76
3.10
3.01
4.04
2.13
4.07
2.11
3.50
1.20
1.58
2.97
0.75
25.0
25.0
93.63
75.98
[80.38
37.99
15.98
67.02
80.38
33.51
84.17
34.81
73.31
38.46
[100
80.12
[85.03
[105
41.94
[89.28
0.66
2
3b
H
OEt
3
3c
CO2Me
OEt
[25.0
12.5
4
3d
Me
Ph
H
H
OEt
5
3e
H
OEt
6.25
6
3f
CO2Me
OEt
25.0
7
3g
Me
Ph
Me
Ph
H
H
OC2H4OMe
OC2H4OMe
OEt
25.0
8
3h
H
12.5
9
3i
H
25.0
10
11
12
13
14
15
16
17
18
3j
H
OEt
12.5
3k
CO2Me
OEt
25.0
3l
Propyl
Me
Ph
H
H
OEt
12.5
3m
H
OEt
[25.0
25.0
3n
H
OEt
CN
3o
CO2Me
H
OEt
CN
[25.0
[25.0
12.5
3p
Me
Ph
H
OMe
OMe
OMe
CN
3q
H
CN
3r
CO2Me
CN
[25.0
0.05
Isoniazid
Ethambutol
Ciprofloxacin
1.56
7.63
3.13
9.44
a
Isolated & unoptimized yields
b
CLogP calculated using Chemdraw Ultra 11.0 software by Cambridge Soft
Standards for the determination of MIC and the values of
the synthesized compounds along with standard drugs
Isoniazid, Ethambutol and Ciprofloxacin for comparison
are presented in Tables 1 and 2. Based on MIC values we
could observe structure–activity relationship by the influ-
ence of substitutions present on 2-chloronicotinaldehyde
derivatives. All the 29 compounds 3a–r and 5a–k were
screened in present study and MIC’s ranging from 1.56 to
25.0 lg/mL.
3-Oxobutanoate condensation products 3a–c displayed
moderate activity, however ethyl 3-oxo-3-phenylpropano-
ate condensation Knoevenagel derivative 3e displayed
potent activity due to the presence of phenyl substitution at
fifth position on pyridyl ring where as methyl substitution
3d displayed significant activity. Similarly 2-methoxyethyl
3-oxobutanoate condensation Knoevenagel products 3g and
3h; 3h displayed significant activity due to presence of
phenyl at fifth position, however in 3g (5-methyl) drasti-
cally decreased and displayed moderate activity. Diethyl
malonate condensation products such as 3i–l, among these
3j (5-phenyl) and 3l (5-propyl) displayed significant
activity. Methyl and ethyl 2-cyanoacetates condensation
products 3m–r, among these, compound 3q displayed
significant activity due to presence of phenyl substitution
at fifth position on pyridyl ring and remaining compounds
exhibited moderate activity. Overall phenyl substitution on
pyridyl and or ethyl 3-oxo-3-phenylpropanoate condensa-
tion derivatives displayed potent/significant activity when
compared to other substitutions (Table 1).
Amongst 5a–e, 5b, 5e and 5d with phenyl, ethyl and
propyl moieties at fifth position displayed significant
in vitro anti-mycobacterial activity when compared to 5a
and 5c with methyl and methylcarboxy moieties respec-
tively (Table 2). Phenyl, ethyl, propyl substitutions has an
impact over methyl and methylcarboxy on pyridyl moiety
in improving the activity.
Nicotinaldehyde derivatives of (E)-a,b-unsaturated
ketones 5f–k (Table 2; Fig. 1) were interesting compounds
in present study and displayed the potent activities. Com-
pound 5g (5-phenyl) displayed potent activity (MIC:
12.17 lM) due to the presence of phenyl substitution on
pyridyl ring, however 5b (5-methyl), and 5h (6-methyl
carboxy) displayed moderate activity. Compound 5j (5-
phenyl, MIC: 4.89 lM) is the most potent in present series of
compounds when compared to standard drugs Ethambutol
(MIC: 7.63 lM) and Ciprofloxacin (MIC: 9.44 lM). How-
ever 5i (5-methyl) and 5k (6-methylcarboxy) displayed
significant activity. Phenyl substitution at fifth position and
a,b-unsaturated benzoyl substitution at third position on
pyridyl displayed most potent and may serve as an
123

Med Chem Res
Table 2 Synthesis and anti-mycobacterial activity of E-(a,b) unsaturated esters and ketones 5a–k
Entry
Compounds
R1
R2
R3
Yielda (%)
CLogPb
MIC (lg/mL)
MIC (lM)
1
5a
Me
Ph
H
OEt
OEt
OEt
OEt
OEt
Me
Me
Me
Ph
87
90
90
89
90
91
91
93
92
92
92
2.79
4.18
1.96
3.82
3.32
1.94
3.33
1.11
3.43
4.81
2.55
25.0
6.25
12.5
6.25
6.25
12.5
3.13
12.5
6.25
1.56
6.25
0.05
1.56
3.13
111.1
21.77
46.46
24.70
26.15
64.10
12.17
52.30
24.31
4.89
2
5b
H
3
5c
H
CO2Me
4
5d
Propyl
Ethyl
Me
Ph
H
5
5e
H
6
5f
H
7
5g
H
8
5h
H
CO2Me
H
9
5i
Me
Ph
10
11
5j
H
Ph
5k
H
CO2Me
Ph
20.76
0.66
Isoniazid
Ethambutol
Ciprofloxacin
7.63
9.44
a
Isolated & unoptimized yields
b
CLogP calculated using Chemdraw Ultra 11.0 software by Cambridge Soft
Table 3 Cytotoxicity (IC50) of Knoevenagel & E-(a,b)-unsaturated
esters and ketones against Raw 246.7, MCF7 and HeLa cell lines in
lM
O
R1
R2
R3
Substitutions
play a major role
S. no. Compound
Raw 264.7
MCF7
HeLa
61 1.3
N
Cl
Crucial
for activity
1
2
3
4
5
6
7
8
3e
5b
5d
5e
5g
5i
68 5.7
21 1.3
106 6.0
11 5.5
R1 = Ph, R2 = H;
R3 = Me, Ph
28 3.9
31 6.5
8
7
1.8
1.1
9
9
1.2
4.3
Fig. 1 Illustration of model derivatives as potent anti-mycobacterial
activity
7
2.1
127 1.1
16 4.5
138 2.2
14 3.2
82 8.6
52 1.9
104 3.7
38 5.9
interesting new compound for the development of thera-
peutics targeted against anti-mycobacterial compounds.
5j
102 2.03
35 1.6
152 7.0
104 10.2
5k
Gemcitabine 0.025 0.009 0.15 0.007 0.395 0.034
Cytotoxic evaluation
Knoevenagel derivative 3e and (E)-a,b-unsaturated esters
and ketones 5b, 5d–e, 5g and 5i–k had displayed potent/
significant anti-mycobacterial activity were chosen for the
cytotoxicity studies assessed by MTT (3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide) protein assay
against Raw 264.7 (Mouse monocyte macrophages), MCF7
(human breast cancer) and HeLa (human epithelial cervical
cancer) cell lines. The inhibitory activities (IC50) of the
compounds in lM are presented in Table 3. The ratio
between in vitro cytotoxicity (IC50 in lM) and tuberculosis
activities (MIC in lM) determine the selectivity index (SI).
SI values of compounds greater than 10 in all three cell
lines were considered to be nontoxic. Comparing the tox-
icity exhibited by the potent/significant antitubercular
2-chloronicotinaldehyde derivatives 3e, 5b, 5d–e, 5g and
5i–k on three cell lines, two compounds 5g and 5j shown
low toxicity i.e. higher SI values ([10) and the remaining
Selectivity index (SI)
35
30
25
20
15
10
5
Raw 264.7
MCF7
HELA
0
3e
5b
5d
5e
5g
5i
5j
5k
Nicotinaldehyde derivatives
Fig. 2 Selectivity index (SI) for 2-chloronicotinaldehyde derivatives
3e, 5b, 5d–e, 5g, and 5i–k against Raw 264.7, MCF7 and HeLa cell
lines. SI is the ratio of cytotoxicity (IC50 in lM) to in vitro activity
against Mycobacterium tuberculosis H37Rv expressed by MIC in lM
123

Med Chem Res
compounds shown lower SI value (\10) in all examined
cell lines (Fig. 2). The results indicated that the compounds
5g and 5j were shown high inhibitory activity against Mtb
also exhibited low toxicity (high selectivity index, 10–30)
against examined cell lines.
1.13 (t, 3H, J = 7.17 Hz, CH3), 2.26 (s, 3H, CH3), 2.40 (s,
3H, CH3), 4.18 (q, 2H, J = 7.17 Hz, OCH2), 7.52 (s, 1H,
heteroaromatic), 7.66 (s, 1H, olefin), 8.16 (s, 1H, hetero-
aromatic). 13C NMR (75.5 MHz, CDCl3) d: 13.86, 26.81,
29.61, 61.72, 128.03, 132.28, 136.30, 137.77, 138.30,
148.04, 150.78, 166.20, 194.21. IR (KBr, cm-1): 2,922 (s),
2,852 (m), 1,727 (s), 1,674 (s), 1,418 (m), 1,377 (s), 1,213
(s), 1,044 (m). ESI-MS: m/z 268 (M??1).
Conclusion
The present invention describes the synthesis of Knoevenagel
derivatives 3a–r and a,b-unsaturated esters and ketones 5a–
k from 2-chloronicotinaldehydes and evaluated for their
in vitro anti-mycobacterial activity against M. tuberculosis
H37Rv (MTB) for the first time. Compound 5j is the most
potent than standard drugs Ethambutol and Ciprofloxacin in
present series of compounds and may serve as an interesting
new compound for the development of therapeutics targeted
against anti-mycobacterial compounds. The synthesis and
biological activities of 2-chloronicotinaldehyde derived
chalcones and flavanones were currently under investigation.
Ethyl 2-((2-chloro-5-phenylpyridin-3-yl)methylene)-3-oxo-
1
butanoate (3b) Liquid. H NMR (300 MHz, CDCl3) d:
1.39 (t, 3H, J = 7.17 Hz, CH3), 2.34 (s, 3H, CH3), 4.34 (q,
2H, J = 7.17 Hz, OCH2), 7.37-7.54 (m, 5H, aromatic),
7.82 (s, 1H, olefin), 7.92 (d, 1H, J = 2.07 Hz, heteroaro-
matic), 8.58 (d, 1H, J = 2.47 Hz, heteroaromatic). 13C
NMR (75.5 MHz, CDCl3) d: 14.20, 31.15, 61.81, 126.87,
127.00, 128.26, 128.80, 129.38, 135.61, 135.72, 137.30,
138.27, 148.36, 149.39, 163.38, 200.34. IR (KBr, cm-1):
2,925 (s), 2,860 (m), 1,727 (s), 1,680 (w), 1,217 (s), 1,048
(s). ESI-MS: m/z 330 (M??1).
Methyl-6-chloro-5-(2-(ethoxycarbonyl)-3-oxobut-1-enyl)-
1
picolinate (3c) Liquid. H NMR (300 MHz, CDCl3) d:
Experimental
1.38 (t, 3H, J = 7.17 Hz, CH3), 2.27 (s, 3H, CH3), 4.00 (s,
3H, OCH3), 4.35 (q, 2H, J = 6.98 Hz, OCH2), 7.78 (s, 1H,
olefin), 7.86 (d, 1H, J = 7.93 Hz, heteroaromatic), 8.06 (d,
1H, J = 7.93 Hz, heteroaromatic). 13C NMR (75.5 MHz,
CDCl3) d: 14.23, 29.75, 51.34, 61.81, 123.47, 123.59,
132.59, 134.17, 135.15, 138.64, 139.81, 163.70, 165.55,
192.93. IR (KBr, cm-1): 2,930 (s), 2,860 (m), 1,727 (s),
1,675 (m), 1,087 (s), 1,045 (m). ESI-MS: m/z 312 (M??1).
Chemistry
1H NMR and 13C NMR spectra were recorded on a Varian
Gemini 200 MHz and Avance 300 MHz spectrometer in
CDCl3 using TMS as internal standard. IR spectra were
recorded on a Nicollet 740 FTIR spectrometer. Mass spectra
were obtained on Agilent LCMS instrument. Melting points
were determined in open glass capillary tubes on a Metler FP
51 melting point apparatus and are uncorrected. All reac-
tions were monitored by thin layer chromatography (TLC)
on pre-coated silica gel 60 F254 (mesh); spots were visual-
ized under UV light. Merck silica gel (60–120; 100–200
mesh) was used for column chromatography. All the reac-
tions were carried out using reagent-grade solvents, and the
reagents were purchased from Sigma-Aldrich.
Ethyl 2-benzoyl-3-(2-chloro-5-methylpyridine-3-yl) acry-
late (3d) Liquid. 1H NMR (300 MHz, CDCl3) d: 1.12 (t,
3H, J = 7.17 Hz, CH3), 2.13 (s, 3H, CH3), 4.28 (q, 2H,
J = 7.17 Hz, OCH2), 7.38–7.62 (m, 5H, aromatic), 7.86 (d,
1H, J = 7.36 Hz, olefin), 8.06 (d, 1H, J = 6.65 Hz, het-
eroaromatic), 8.12 (s, 1H, heteroaromatic). 13C NMR
(75.5 MHz, CDCl3) d: 13.91, 29.72, 61.92, 127.72, 128.74,
128.86, 128.94, 133.72, 134.15, 137.21, 139.21, 150.50,
181.50, 193.86. IR (KBr, cm-1): 2,922 (s), 2,854 (m),
1,732 (s), 1,680 (s), 1,240 (s), 1,083 (m). ESI-MS: m/z 330
(M??1).
General procedure for the synthesis of Knoevenagel
products (3a–r)
Piperidine (1.0 mmol) was added to a stirred solution of
2-chloro-5-methylnicotinaldehyde (1a, 1.0 mmol), ethyl
3-oxobutanoate (2a 1.0 mmol) in dichloromethane (4 mL)
at room temperature, and the contents were stirred for 6 h.
After completion of reaction (TLC), the reaction mixture
was purified by column chromatography using silica gel
(60:120) afford.
Ethyl 2-benzoyl-3-(2-chloro-5-phenylpyridin-3-yl)acrylate
(3e) Liquid. 1H NMR (300 MHz, CDCl3) d: 1.26 (t, 3H,
J = 6.98 Hz, CH3), 4.30 (q, 2H, J = 6.98 Hz, OCH2),
7.24–7.32 (m, 2H, aromatic), 7.36–7.60 (m, 6H, aromatic),
7.80 (d, 1H, J = 6.98 Hz, heteroaromatic), 7.92 (d, 2H,
J = 7.17 Hz, aromatic), 8.20 (s, 1H, olefin), 8.46 (d, 1H,
J = 2.45 Hz, heteroaromatic). 13C NMR (75.5 MHz,
CDCl3) d: 13.97, 61.99, 126.77, 128.23, 128.65, 128.93,
128.97, 129.15, 134.23, 135.38, 135.54, 135.77, 135.98,
Ethyl 2-((2-chloro-5-methylpyridin-3-yl)methylene)-3-oxo-
1
butanoate (3a) Liquid. H NMR (300 MHz, CDCl3) d:
123

Med Chem Res
Diethyl 2-((2-chloro-5-phenylpyridin-3-yl)methylene)mal-
onate (3j) Liquid. 1H NMR (300 MHz, CDCl3) d: 1.10 (t,
3H, J = 7.17 Hz, CH3), 1.31 (t, 3H, J = 7.17 Hz, CH3),
4.15 (q, 2H, J = 7.17 Hz, OCH2), 4.28 (q, 2H,
J = 7.17 Hz, OCH2), 7.31–7.48 (m, 5H, aromatic), 7.85 (s,
1H, olefin), 7.92 (d, 1H, J = 2.45 Hz, heteroaromatic),
8.52 (d, 1H, J = 2.45 Hz, heteroaromatic). 13C NMR
(75.5 MHz, CDCl3) d: 14.27, 14.03, 61.81, 61.60, 126.87,
127.10, 128.31, 128.78, 129.40, 135.65, 135.75, 137.40,
138.30, 148.48, 149.40, 163.56, 165.11. IR (KBr, cm-1):
2,983 (m), 2,925 (m), 1,727 (s), 1,635 (w), 1,070 (m),
1,048 (s). ESI-MS: m/z 360 (M??1).
137.04, 137.09, 148.22, 149.37, 163.96, 193.91. IR (KBr,
cm-1): 2,927 (s), 2,847 (m), 1,733 (s), 1,685 (s), 1,085 (s).
ESI-MS: m/z 392 (M??1).
Methyl 5-(2-benzoyl-3-ethoxy-3-oxoprop-1-enyl)-6-chlo-
1
ropicolinate (3f) Liquid. H NMR (300 MHz, CDCl3) d:
1.23 (t, 3H, J = 7.17 Hz, CH3), 3.95 (s, 3H, OCH3), 4.28
(q, 2H, J = 7.17 Hz, OCH2), 7.41 (m, 2H, aromatic), 7.56
(d, 1H, J = 7.36 Hz heteroaromatic), 7.75–7.90 (m, 4H,
aromatic), 8.14 (s, 1H, heteroaromatic). 13C NMR
(75.5 MHz, CDCl3). d: 13.94, 53.19, 62.18, 123.62,
128.94, 129.34, 132.17, 134.45, 135.43, 136.07, 137.14,
139.61, 148.03, 150.71, 163.65, 163.83, 193.46. IR (KBr,
cm-1): 2,928 (s), 2,850 (m), 1,732 (s), 1,680 (m), 1,085 (s),
1,025 (m). ESI-MS: m/z 374 (M??1).
Diethyl 2-((2-chloro-6-(methoxycarbonyl)pyridin-3-yl)met-
Liquid. 1H NMR (300 MHz,
hylene)malonate (3k)
CDCl3) d: 1.22 (t, 3H, J = 7.17 Hz, CH3), 1.38 (t, 3H,
J = 7.17 Hz, CH3), 4.02 (s, 3H, OCH3), 4.22 (q, 2H,
J = 6.98 Hz, OCH2), 4.36 (q, 2H, J = 6.98 Hz, OCH2),
7.87 (s, 1H, olefin), 7.92 (d, 1H, J = 7.93 Hz, heteroaro-
matic), 8.04 (d, 1H, J = 7.93 Hz, heteroaromatic). 13C
NMR (75.5 MHz, CDCl3) d: 13.83, 14.03, 53.25, 62.04,
62.23, 123.56, 131.90, 132.38, 136.25, 139.20, 148.29,
150.77, 162.87, 163.96, 164.67. IR (KBr, cm-1): 2,982
(m), 2,923 (m), 1,728 (s), 1,634 (w), 1,247 (s), 1,069 (s).
ESI-MS: m/z 298 (M??1).
2-Methoxyethyl 2-((2-chloro-5-methylpyridin-3-yl)methy-
1
lene)-3-oxobutanoate (3g) Liquid. H NMR (300 MHz,
CDCl3) d. 2.33 (s, 3H, CH3), 2.48 (s, 3H, CH3), 3.30 (s, 3H,
OCH3), 3.56 (t, 2H, J = 4.72 Hz, OCH2), 4.36 (t, 2H,
J = 4.52 Hz, OCH2), 7.64 (s, 1H, olefin), 7.75 (s, 1H,
heteroaromatic), 8.23 (s, 1H, heteroaromatic). 13C NMR
(75.5 MHz, CDCl3) d: 26.74, 29.64, 58.73, 64.51, 69.81,
132.62, 136.35, 136.35, 136.69, 138.44, 139.29, 150.72,
166.22, 194.22. IR (KBr, cm-1): 2,922 (s), 2,854 (m),
1,725 (s), 1,676 (m), 1,215 (s), 1,085 (s), 1,045 (m). ESI-
MS: m/z 298 (M??1).
Diethyl 2-((2-chloro-5-propylpyridin-3-yl)methylene)mal-
1
onate (3l) Liquid. H NMR (300 MHz, CDCl3) d: 0.94
(t, 3H, J = 7.3 Hz, CH3), 1.20 (t, 3H, J = 6.98 Hz, CH3),
1.35 (t, J = 7.07 Hz, 3H, CH3), 1.61 (sextect, 2H, CH2),
2.57 (t, J = 7.3 Hz, 2H, CH2), 4.24 (q, 2H, J = 6.98 Hz,
OCH2), 4.34 (q, 2H, J = 7.07 Hz, OCH2), 7.60 (d, 1H,
J = 2.26 Hz, heteroaromatic), 7.90 (s, 1H, olefin), 8.20 (d,
1H, J = 2.07 Hz, heteroaromatic). 13C NMR (75.5 MHz,
CDCl3) d: 13.46, 13.84, 14.07, 23.98, 29.66, 34.01, 61.79,
62.00, 128.05, 130.25, 136.91, 137.38, 137.72, 150.44,
163.23, 165.29. IR (KBr, cm-1): 2,983 (m), 2,925 (m),
1,729 (s), 1,635 (w), 1,250 (s), 1,090 (s), 1,069 (s). ESI-
MS: m/z 326 (M??1).
2-Methoxyethyl 2-((2-chloro-5-phenylpyridin-3-yl)methy-
1
lene)-3-oxobutanoate (3h) Liquid. H NMR (300 MHz,
CDCl3) d: 2.48 (s, 3H, CH3), 3.13 (s, 3H, OCH3), 3.16
(t, 2H, J = 4.72 Hz, OCH2), 4.29 (t, 2H, J = 4.72 Hz,
OCH2), 7.36–7.55 (m, 5H, aromatic), 7.77 (s, 1H, olefin),
7.97 (d, 1H, J = 2.46 Hz, heteroaromatic), 8.60 (d, 1H,
J = 2.46 Hz, heteroaromatic). 13C NMR (75.5 MHz,
CDCl3) d: 29.77, 58.58, 64.47, 69.79, 127.01, 128.74,
129.31, 129.36, 135.79, 136.14, 136.29, 137.38, 137.75,
148.33, 148.43, 165.96, 192.94. IR (KBr, cm-1): 2,927 (s),
2,857 (m), 1,725 (s), 1,678 (m), 1,220 (s), 1,085 (s), 1,050
(m). ESI-MS: m/z 360 (M??1).
Ethyl 3-(2-chloro-5-methylpyridin-3-yl)-2-cyanoacrylate
1
(3m) Yellow solid; M.P. 75–77 °C. H NMR (300 MHz,
Diethyl 2-((2-chloro-5-methylpyridin-3-yl)methylene)mal-
1
CDCl3) d: 1.42 (t, 3H, J = 7.17 Hz, CH3), 2.42 (s, 3H,
CH3), 4.42 (q, 2H, J = 7.17 Hz, OCH2), 8.34 (d, 2H,
J = 7.05 Hz, heteroaromatic), 8.58 (s, 1H, olefin). 13C NMR
(75.5 MHz, CDCl3) d: 14.20, 29.61, 63.19, 107.78, 114.17,
125.87, 128.84, 133.18, 138.40, 149.47, 153.06, 161.17. IR
(KBr, cm-1): 2,923 (s), 2,854 (w), 2,227 (w), 1,725 (s), 1,640
(m), 1,266 (s), 1,084 (m). ESI-MS: m/z 251 (M??1).
onate (3i) Liquid. H NMR (300 MHz, CDCl3) d: 1.22
(t, 3H, J = 7.17 Hz, CH3), 1.36 (t, 3H, J = 7.17 Hz, CH3),
2.32 (s, 3H, CH3), 4.26 (q, 2H, J = 7.17 Hz, OCH2), 4.36
(q, 2H, J = 7.17 Hz, OCH2), 7.58 (d, 1H, J = 1.32 Hz,
heteroaromatic), 7.90 (s, 1H, olefin), 8.22 (d, 1H,
J = 1.32 Hz, heteroaromatic). 13C NMR (75.5 MHz,
CDCl3) d: 13.82, 14.02, 29.39, 61.66, 61.80, 127.76,
130.08, 132.45, 137.13, 138.36, 148.10, 150.60, 163.22,
165.21. IR (KBr, cm-1): 2,982 (m), 2,923 (m), 1,728 (s),
1,634 (w), 1,247 (s), 1,069 (s). ESI-MS: m/z 298 (M??1).
Ethyl 3-(2-chloro-5-phenylpyridin-3-yl)-2-cyanoacrylate
(3n) Yellow solid. M.P. 106–108 °C. 1H NMR (300 MHz,
123

Med Chem Res
General procedure for the synthesis of (E)-a,b-unsaturated
esters and ketones (5a–k)
CDCl3) d: 1.45 (t, 3H, J = 6.98 Hz, CH3), 4.44 (q, 2H,
J = 7.17 Hz, OCH2), 7.40–7.66 (m, 5H, aromatic), 8.62 (s,
1H, olefin), 8.72 (d, 2H, J = 1.32 Hz, heteroaromatic). 13
C
Piperidine (1.0 mmol) was added slowly to a stirred solu-
tion of 2-chloro-5-methylnicotinaldehyde (1a, 1.0 mmol),
ethyl 4,4,4-trifluoro-3-oxobutanoate (4a, 1.0 mmol) in
DCM (4 mL) at room temperature and the contents were
stirred for 6 h. After completion of reaction (TLC), the
reaction mixture was purified by column chromatography
using silica gel (60:120) afforded ethyl (E)-3-(2-chloro-
5-methyl-3-pyridyl)-2-propenoate 5a as colorless solid in
NMR (75.5 MHz, CDCl3) d: 14.25, 63.08, 108.53, 114.26,
126.44, 127.17, 129.12, 129.53, 135.30, 136.17, 136.35,
149.33, 150.46, 150.87, 160.88. IR (KBr, cm-1): 2,925 (s),
2,852 (m), 2,229 (m), 1,727 (s), 1,684 (m), 1,270 (s), 1,090
(s). ESI-MS: m/z 313 (M??1).
Ethyl 6-chloro-5-(2-cyano-3-ethoxy-3-oxoprop-1-enyl)pico-
linate (3o) Yellow solid. M.P. 140–142 °C. 1H NMR
(300 MHz, CDCl3) d: 1.44 (t, 3H, J = 6.98 Hz, CH3), 4.04
(s, 3H, OCH3), 4.44 (q, 2H, J = 7.17 Hz, OCH2), 8.20 (d,
1H, J = 7.93 Hz, heteroaromatic), 8.56 (s, 1H, olefin),
8.64 (d, 1H, J = 7.93 Hz, heteroaromatic). 13C NMR
(75.5 MHz, CDCl3) d: 14.09, 53.13, 63.18, 109.91, 113.54,
123.91, 129.74, 138.96, 147.96, 149.89, 151.94, 160.40,
163.21. IR (KBr, cm-1): 2,926 (s), 2,851 (m), 2,230 (m),
1,725 (s), 1,683 (m), 1,090 (s), 1,041 (m). ESI-MS: m/z 295
(M??1).
1
87 % yield. M.P. 69–71 °C. H NMR (300 MHz, CDCl3)
d: 1.30 (t, 3H, J = 7.0 Hz, CH3), 2.32 (s, 3H, CH3), 4.24
(q, 2H, J = 7.0 Hz, OCH2), 6.38 (d, 1H, J = 15.6 Hz,
HC = CH), 7.64 (d, 1H, J = 2.2 Hz, heteroaromatic), 7.86
(d, 1H, J = 15.6 Hz, HC = CH), 7.14 (d, 1H, J = 1.7 Hz,
heteroaromatic). 13C NMR (75.5 MHz, CDCl3) d: 14.17,
29.60, 60.80, 122.55, 128.61, 132.65, 136.39, 138.84,
148.42, 150.72, 165.80. IR (KBr, cm-1): 2,925 (m), 2,855
(w), 1,715 (s), 1,603 (m), 1,414 (m), 1,315 (m), 1,284 (m),
1,181 (s), 1,027 (m), 985 (m), 868 (w), 775 (m), 699 (m).
ESI-MS: m/z 226 (M??1). ESI–HR–MS: m/z calcd for
C11H13ClNO2 [M?H]? 226.0,556, found 226.0637.
Methyl 3-(2-chloro-5-methylpyridin-3-yl)-2-cyanoacrylate
1
(3p) Yellow solid. M.P. 80–82 °C; H NMR (300 MHz,
CDCl3) d: 2.44 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 8.36
(s, 2H, heteroaromatic), 8.58 (s, 1H, olefin). 13C NMR
(75.5 MHz, CDCl3) d: 29.45, 53.81, 107.36, 114.36,
125.98, 127.74, 132.98, 138.44, 149.96, 152.91, 161.42.
IR (KBr, cm-1): 2,922 (s), 2,852 (w), 2,225 (w), 1,727
(s), 1,637 (m), 1,264 (s), 1,087 (m). ESI-MS: m/z 237
(M??1).
Mycobacterial growth assay
Ten fold serial dilutions of each test compound/drug were
prepared and incorporated into Middlebrook 7H11 agar
medium with OADC Growth Supplement. Inoculum of
M. tuberculosis H37Rv was prepared from fresh Middle-
brook 7H11 agar slants with OADC Growth Supplement
adjusted to 1 mg/mL (wet weight) in Tween 80 (0.05 %)
saline diluted to 10-2 to give a concentration of *107 cfu/
mL. A 5 lL amount of bacterial suspension was spotted into
7H11 agar tubes containing tenfold serial dilutions of drugs
per mL. The tubes were incubated at 37 °C, and final read-
ings were recorded after 28 days. The minimum inhibitory
concentration (MIC) is defined as the minimum concentra-
tion of compound required to give complete inhibition of
bacterial growth. This method is similar to that recom-
mended by the National Committee for Clinical Laboratory
Standards for the determination of MIC in triplicate.
Methyl 3-(2-chloro-5-phenylpyridin-3-yl)-2-cyanoacrylate
(3q)
Yellow solid. M.P. 110–112 °C. 1H NMR
(300 MHz, CDCl3) d: 3.98 (s, 3H, OCH3), 7.40-7.64 (m,
5H, aromatic), 8.62 (s, 1H, olefin), 8.72 (d, 1H,
J = 2.45 Hz, heteroaromatic), 8.74 (d, 1H, J = 2.07 Hz,
heteroaromatic). 13C NMR (75.5 MHz, CDCl3) d: 53.63,
108.06, 114.19, 126.36, 127.19, 129.16, 129.57, 135.32,
136.15, 136.37, 149.51, 150.55, 151.00, 161.37. IR
(KBr, cm-1): 2,925 (s), 2,849 (m), 2,226 (m), 1,726 (s),
1,645 (m), 1,265 (s), 1,090 (m). ESI-MS: m/z 321
(M??1).
Methyl 6-chloro-5-(2-cyano-3-methoxy-3-oxoprop-1-enyl)-
picolinate (3r) Yellow solid. M.P. 144–146 °C. 1H NMR
(300 MHz, CDCl3) d: 4.00 (s, 3H, OCH3), 4.04 (s, 3H,
OCH3), 8.20 (d, 1H, J = 7.93 Hz, heteroaromatic), 8.56 (s,
Cell culture maintenance and antiproliferative assays
Cell lines used in this study were procured from the
American Type Culture Collection (ATCC, USA). HeLa
(human epithelial cervical cancer), Raw 264.7 (Mouse
monocyte macrophages) were grown in Dulbecco’s modi-
fied Eagles’s medium (containing 10 % FBS in a humidi-
fied atmosphere of 5 % CO2 at 37 °C). MCF-7 (human
breast cancer cell line) were grown in Eagle’s minimal
essential medium (MEM) containing nonessential amino
1H, olefin), 8.64 (d, 1H, J = 7.93 Hz, heteroaromatic). 13
C
NMR (75.5 MHz, CDCl3) d: 53.13, 53.81, 109.91, 113.54,
123.91, 129.74, 138.96, 147.96, 149.89, 151.94, 160.40,
163.21. IR (KBr, cm-1): 2,923 (s), 2,850 (m), 2,226 (m),
1,728 (s), 1,679 (m), 1,087 (s), 1,039 (m). ESI-MS: m/z
281(M??1).
123

Med Chem Res
chloronicotinaldehydes by the Vilsmeier reaction. Tetrahedron
62:8398–8403
Gangadasu B, Raju BC, Rao VJ (2009a) Synthesis of imidacloprid
analogues from novel chloronicotinaldehydes. J Heterocycl
Chem 46:1213–1217
Gangadasu B, Reddy MJR, Ravinder M, Kumar SB, Raju BC, Kumar
KP, Murthy USN, Rao VJ (2009b) Synthesis, photochemical E
(trans)/Z (cis) isomerization and antimicrobial activity of
2-chloro-5-methylpyridine-3-olefin derivatives. Eur J Med Chem
44:4661–4667
Jaracz S, Chen J, Kuznetsova LV, Ojima I (2005) Recent advances in
tumor-targeting anticancer drug conjugates. Bioorg Med Chem
13:5043–5054
Kumar JA, Tiwari AK, Ali AZ, Madhusudana K, Reddy BS,
Ramakrishna S, Raju BC (2010) New antihyperglycemic,
a-glucosidase inhibitory and cytotoxic derivatives of benzimi-
dazoles. J Enzyme Inhib Med Chem 25:80–86
Kumar JA, Tiwari AK, Ali AZ, Rao RR, Raju BC (2011) Synthesis
and a-glucosidase inhibitory, DPPH scavenging activity of
substituted 2-Oxo-2H-chromen-7-yl-dihydrogen phosphate
derivatives. J Heterocycl Chem 48:1251–1257
Narender P, Srinivas U, Gangadasu B, Biswas S, Rao VJ (2005) Anti-
malarial activity of Baylis–Hillman adducts from substituted
2-chloronicotinaldehydes. Bioorg Med Chem Lett 15:5378–5381
Narender P, Gangadasu B, Ravinder M, Srinivas U, Swamy GYSK,
Ravikumar K, Rao VJ (2006) Baylis–Hillman adducts between
pyridine carboxaldehyde derivatives and cyclic enones. Tetra-
hedron 62:954–959
Narender P, Ravinder M, Sadhu PS, Raju BC, Ramesh C, Rao VJ
(2009) Synthesis of substituted 1,8-naphthyridine-3-carboxylates
from Baylis–Hillman adducts of substituted 2-chloronicotinal-
dehydes. Helvetica Chim Acta 92:959–966
acids, 1 mM sodium pyruvate and 10 % FBS. Cells were
trypsinized when confluent from T75 flasks and seeded in
96-well microtiter plates in 200-lL aliquots at plating
densities of 5,000 cells/well. MTT (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay was then
performed to determine the cell viability. The synthesized
compounds were tested for cytotoxicity in three different
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 dif-
ferent drug dilutions were added to the appropriate
microtiter wells resulting in the required final drug con-
centrations. For each compound, four concentrations (10,
25, 50 and 100 lM) were evaluated and each was done in
triplicate wells. Plates were later incubated for another
48 h, later 10 lL of 10 mg/mL concentration of MTT was
added and incubated for 3 h at 37 °C. At the end of incu-
bation period formazon crystals were formed, the plates
were then centrifuged and the media from microtiter plates
were removed. Later plates were air dried. The bound
crystals were subsequently dissolved by adding 100 lL
DMSO. The absorbance was then read on ELISA plate
reader at a wavelength of 560 nm. Relative to the control
wells the percent growth was calculated for each well.
Sensitivity of the cells to the drug treatments was expressed
in terms of IC50, it is the concentration of a compound at
which 50 % growth reduction in growth is seen when
compared to the control. The percentage of cells killed was
obtained from the formula.
Raju BC, Suman P (2010) New and facile approach for the synthesis
of (E)-a, b-unsaturated esters and ketones. Chem A Eur
16:11840–11842
Raju BC, Tiwari AK, Kumar JA, Ali AZ, Agawane SB, Saidachary G,
Madhusudana K (2010) a-Glucosidase inhibitory antihypergly-
cemic activity of substituted chromenone derivatives. Bioorg
Med Chem 18:358–365
100 ꢁ mean ODsample
% percentage cells killed ¼
ꢂ 100
mean OD day 0
Raju BC, Rao RN, Suman P, Yogeeswari P, Sriram D, Shaik TB,
Kalivendi SV (2011) Synthesis, structure–activity relationship of
novel substituted 4H-chromen-1,2,3,4-tetrahydropyrimidine-5-
carboxylates as potential anti-mycobacterial and anticancer
agents. Bioorg Med Chem Lett 21:2855–2859
Acknowledgments The authors thank Dr. J. S. Yadav, Director, &
Dr. S. Chandrasekhar, Head, Natural Products Chemistry Division,
CSIR-IICT for their constant encouragement and support of this
work. PS and RNR thank CSIR, New Delhi for financial assistance.
Ravinder M, Sadhu PS, Rao VJ (2009) Simple, facile and one-pot
conversion of the Baylis–Hillman acetates into 3,5,6-trisubsti-
tuted-2-pyridones. Tetrahedron Lett 50:4229–4232
Suman P, Rao RN, Raju BC (2012) Microwave-assisted convenient
synthesis of a, b-unsaturated esters and ketones via Aldol-adduct
elimination. Helvetica Chim Acta. doi:10.1002/hlca.201200526
Trost BM (1991) Comprehensive organic synthesis, vol 2. Pergamon
press, Oxford, pp 133–340
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123