Synthesis of amidine and bis amidine derivatives and their evaluation for anti-inflammatory and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2012.10.046

2-Cyanopyridine (1a), 4-cyanopyridine (1b), 2-cyanopyrazine (1c) on condensation with mono amines (2a-c) and diamines (4a-c) in the presence of sodium methoxide as catalyst gave amidine derivatives (3a-i) and bis amidine derivatives (5a-i) in good yields.

Full text of DOI:10.1016/j.ejmech.2012.10.046

European Journal of Medicinal Chemistry 59 (2013) 7e14
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of amidine and bis amidine derivatives and their evaluation
for anti-inflammatory and anticancer activity
,
Surbhi Arya a, Nikhil Kumar b, Partha Roy b, S.M. Sondhi a
*
a Department of Chemistry, Indian Institute of Technology-Roorkee, Roorkee, 247667 UK, India
b Department of Biotechnology, Indian Institute of Technology-Roorkee, Roorkee, 247667 UK, India
a r t i c l e i n f o
a b s t r a c t
Article history:
2-Cyanopyridine (1a), 4-cyanopyridine (1b), 2-cyanopyrazine (1c) on condensation with mono amines
(2aec) and diamines (4aec) in the presence of sodium methoxide as catalyst gave amidine derivatives
(3aei) and bis amidine derivatives (5aei) in good yields. All these compounds were fully characterized
by spectroscopic means and elemental analysis. On screening for anti-inflammatory activity and for
in vitro anticancer activity compounds 5c and 5d exhibited good anti-inflammatory activity whereas
compounds 5d breast (T47D), 5h, 5i lung (NCI H-522), 5i colon (HCT-15), 3c, 3h, 5i ovary (PA-1) and 3c,
5b, 5h liver (HepG2) exhibited good anticancer activity.
Received 25 May 2012
Received in revised form
21 September 2012
Accepted 26 October 2012
Available online 3 November 2012
Keywords:
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
Amidines
Bis amidines
Anti-inflammatory
Anticancer
1. Introduction
Tempted by wide variety of biological activities exhibited by ami-
dine and bis amidine derivatives and in continuation [28e31] of our
Inflammatory diseases such as asthma, allergy, arthritis,
multiple sclerosis etc. are quite common form which human beings
suffer worldwide. For the treatment of inflammatory diseases
various anti-inflammatory drugs i.e. indomethacin, diclofenac,
aspirin, ibuprofen, nimisulide, celecoxib and roficoxib etc. are
available in the market [1]. These drugs cannot be used continu-
ously for long time as they can cause serious side effects such as
ulceration, gastrointestinal bleeding and heart strock [2,3]. Another
disease which is responsible for many deaths every year is cancer.
At present various research groups worldwide are involved in
search of safer anti-inflammatory and anticancer agents [4e7].
Amidine and bis amidine derivatives possessing anti-inflammatory
[8e10], antimicrobial [11], antiparasitic [12], antibacterial [13],
antiprotozoal [14], antimalarial [15], anticancer [16e22], anti HIV
[23], antidegenerative [24], and urokinase inhibitor [25] activities
are well documented in literature. Amidine derivatives also act as
drug carrier [26] and as starting materials for various heterocyclic
molecules [27].
efforts in search of potent molecules exhibiting anti-inflammatory
and anticancer activities we have synthesized a number of amidine
and bis amidine derivatives and screened them for anti-inflammatory
and anticancer activities which we wish to report in this paper.
2. Results and discussion
2.1. Chemistry
Amidine and bis amidine derivatives can be synthesized by
condensation of nitrile with mono and diamines, but in most of the
cases activation of nitrile is needed [32e38]. 2-Cyanopyridine (1a)
4-cyanopyridine (1b) and 2-cyanopyrazine (1c) were first allowed
to react with sodium methoxide by stirring at room temperature for
2 h using absolute methanol as solvent of reaction to give insitu
intermediate [39] 1 (Scheme 1). Intermediate 1 (Scheme 1)
undergoes substitution reaction with various mono and diamines to
give amidine and bis amidine derivatives in good yields. Conden-
sation of 2-cyanopyridine (1a), 4-cyanopyridine (1b) and 2-
cyanopyrazine (1c) with 4-(2-aminoethyl) morpholine (2a), 3-
(aminomethyl) pyridine (2b) and 1-(2-aminoethyl) piperazine (2c)
in equimolar ratio and in the presence of sodium methoxide by
refluxing for 8 h using absolute methanol as solvent of reaction gave
* Corresponding author. Tel.: þ91 1332 285811; fax: þ91 1332 273650.
E-mail address: sondifcy@iitr.ernet.in (S.M. Sondhi).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.046

8
S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
X
Y
X
Y
Abs MeOH/MeONa
RT, 2hr stirring
OMe
NNa
CN
Z
Z
1a-c
I
a, X= Y=CH Z=N
b, X=Z=CH Y=N
c, Y=CH X=Z=N
Refluxing, 8 hrs
Refluxing, 12 hrs
H2N R' NH2
R NH2
4a-c
2a-c
a, R=
a, R'=
(CH2)4
O
N
N
CH2 CH2
N
H2
C
(CH2)3
N
N (CH2)3
b, R=
c, R=
b, R'=
c, R'=
HN
CH2 CH2
NH
NH
X
NH
X
Y
X
Y
H
C
Y
C
N R' NH C
Z
Z
Z
NH R
3a-i
5a-i
X
Y
Z
R
X
Y
Z
R'
O
O
N
N
CH2 CH2
CH2 CH2
(CH2)4
(CH2)4
a
b
c
d
e
f
CH
CH
N
CH
N
N
a
CH
CH
N
CH
N
N
CH
N
b
c
d
e
f
CH
N
O
N
CH2 CH2
(CH2)4
CH
CH
N
CH
CH
N
N
H2
C
(CH2)3
N
N
N
N
(CH2)3
(CH2)3
CH
CH
N
N
CH
CH
N
N
N
N
H2
C
(CH2)3
(CH2)3
CH
N
CH
N
H2
C
N
N
(CH2)3
CH
CH
N
CH
CH
N
HN
N
CH2 CH2
CH2 CH2
CH2 CH2
g
h
i
CH
CH
N
N
g
h
i
CH
CH
N
N
HN
HN
N
N
CH
N
CH
N
CH
CH
Scheme 1. Synthesis of amidine 3aei and bis amidine 5aei derivatives.
amidine derivatives (3aei) (Scheme 1) in good yields. All the
compounds were purified by crystallization and structures assigned
to (3aei) are fully supported by spectral data i.e. IR, 1H NMR, 13C
NMR and GCeMS and elemental analysis reported in experimental
section of this paper.
Synthesis of bis amidine derivatives was carried out by
condensation of (1ae1c) with 1,4-diaminobutane (4a), 1,4-bis(3-
aminopropyl) piperazine (4b) and benzidine (4c) in 2:1 molar
ratio, in the presence of sodium methoxide, by refluxing for 12 h
using absolute methanol as solvent of reaction. This condensation
reaction gave bis amidine derivatives (5aei) in good yields. All the
compounds were purified by crystallization. Spectral (IR, 1H NMR,
13C NMR and GCeMS) data and elemental analysis of 5aei (Scheme
1) reported in experimental section of this paper is in agreement
with structures assigned to them.
compounds were administered orally (p.o) and assayed at a dose of
at 50 mg/kg body weight. Standard drug used for comparison was
ibuprofen. Results of pharmacological evaluation are summarized
in Table 1. A look at Table 1 indicates that bis amidine derivatives 5c
and 5d possess good anti-inflammatory activity i.e. 37% and 38% at
50 mg/kg p.o. as compared to standard drug ibuprofen which
exhibited 39% activity at 50 mg/kg p.o.
Amidine (3aei) and bis amidine derivatives (5aei) were
screened in vitro for anticancer activity [41] against five human
cancer cell lines i.e. breast (T47D), lung (NCI H-522), colon (HCT-15),
ovary (PA-1) and liver (HepG2) at a concentration of 1 ꢀ 10ꢁ5
M
concentration and results are summarized in Table 1. A look at
Table 1 indicates that compounds 5d breast (T47D), 5h, 5i lung (NCI
H-522), 5i colon (HCT-15), 3c, 3h, 5i ovary (PA-1) and 3c, 5b, 5h
liver (HepG2) exhibited good anticancer activities against various
cancer cell lines mentioned above. Compounds 3c, 3h, 5b, 5d, 5h
and 5i which exhibited good anticancer activity against various cell
lines were further studied and their IC50 values were determined.
These IC50 values are summarized in Table 2. IC50 value for all above
mentioned compounds for normal cell COS-1 is also reported in
Table 2.
2.2. Biological results
Purified and fully characterized amidine derivatives (3aei) and
bis amidine derivatives (5aei) were screened for anti-inflammatory
activity [40] using carrageenan induced paw oedema model. All the

S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
9
Table 1
Anti-inflammatory and in vitro anticancer activity of amidine and bis amidine 3aei and 5aei derivatives.
Comp No.
Anti-inflammatory activity
(%) at 50 mg/kg p.o
aAnticancer activity (% growth inhibition) at a concentration of 1 ꢀ 10ꢁ5
M
Breast T47D
Lung NCl H-522
Colon HCT-15
Ovary PA-1
Liver HepG2
3a
3b
3c
3d
3e
3f
3g
3h
3i
5a
5b
5c
5d
5e
5f
5g
00
28
15
10
00
18
11
00
20
14
15
37
38
31
27
26
15
31
39
e
21
12
13
23
22
23
14
06
22
29
27
19
39
02
28
02
12
14
e
31
11
35
09
07
11
08
17
09
38
20
05
39
31
15
32
36
43
e
12
17
06
23
22
03
24
13
28
05
05
09
31
09
12
16
03
45
e
43
43
51
36
21
23
40
46
36
27
10
15
31
13
26
37
07
43
e
40
09
52
42
20
26
27
10
23
26
50
17
34
06
22
23
48
37
e
5h
5i
Ibuprofen
b5-FU
cCYC-PHO
dCYC-HEXI
15
09
11
13
11
09
19
04
16
22
12
34
32
18
18
e
e
Bold values represent compounds showing good anti-inflammatory and good anticancer activity.
a
Compounds tested in triplicate, data expressed as mean value of three independent experiments.
5-FU 5-Fluorouracil.
CYC-PHO Cyclophosphamide.
CYC-HEXI Cycloheximide.
b
c
d
2.3. Structure activity relationship
5c and 5d exhibited good anti-inflammatory and 3c, 3h, 5b, 5d, 5h,
5i exhibited good anticancer activity.
Two series of compounds i.e. amidines (3aei) and bis amidines
(5aei) are screened for anti-inflammatory and anticancer activities.
Two compounds of bis amidine series i.e. 5c and 5d exhibited good
anti-inflammatory activity whereas compounds 3c, 3h, 5b, 5d, 5h
and 5i exhibited good anticancer activity. A look at the structures of
compounds showing good anti-inflammatory and anticancer
activity, we can say that pyridine and pyrazine derivatives do
possess sites for interaction with the targets but compounds 5c, 5d;
3c, 3h, 5b, 5d, 5h and 5i have structures which can effectively
interact with the targets both from electronic and stereochemical
point of view and hence possess good anti-inflammatory and
anticancer activity.
4. Experimental protocols
4.1. General
Melting points (mp) were determined on a JSGW apparatus and
are uncorrected. IR spectra were recorded using a Perkin Elmer
1600 FT spectrometer. 1H and 13C NMR spectra were recorded on
a Bruker WH-500 spectrometer at a ca 5e15% (w/v) solution in
DMSO-d6, and D2O. GCeMS was recorded on Perkin Elmer Clarus
500 gas chromatograph where built in MS detector was used.
Elemental analysis was carried out on a Vario EL III elementor. Thin
layer chromatography (TLC) was performed on silica gel G for TLC
(Merck) and spots were visualized by iodine vapor or by irradiation
with ultraviolet light (short wave length, 254 nm). During inter-
pretation of 13C NMR a few abbreviations has been used which are
morp (morpholine), py (pyridine), pyra (pyrazine), piper (pipera-
zine) and Ph (phenyl).
3. Conclusion
2-Cyanopyridine, 4-cyanopyridine and 2-cyanopyrazine interact
with mono and diamines in the presence of sodium methoxide to
give amidine (3aei) and bis amidine (5aei) derivatives. Compounds
Table 2
IC50 valuesa,b of in vitro antitumor activity of active compounds.
Comp No.
IC50 (mm)
Breast T47D
Lung NCI H-522
Colon HCT-15
Ovary PA-1
Liver HepG2
Normal Cell COS-1
3c
3h
5b
5d
5h
5i
5-FU
CYC-PHO
CYC-HEXI
55.38 ꢂ 1.81
184.13 ꢂ 14.8
42.95 ꢂ 4.8
22.52 1.59
34.09 ꢂ 0.72
62.41 ꢂ 5.4
51.8 ꢂ 2.34
70.1 ꢂ 2.32
65.13 ꢂ 7.31
18.25 ꢂ 1.16
35.30 ꢂ 4.56
36.62 ꢂ 4.75
21.98 ꢂ 1.34
13.43 2.89
14.05 2.57
56.76 ꢂ 3.4
67.9 ꢂ 3.09
60.1 ꢂ 5.34
78.30 ꢂ 12.94
35.37 ꢂ 4.61
87.8 ꢂ 5.32
13.55 1.11
19.28 1.87
49.18 ꢂ 3.09
39.70 ꢂ 3.40
42.45 ꢂ 3.61
17.08 0.70
39.5 ꢂ 4.32
64.12 ꢂ 5.43
40.6 ꢂ 2.09
9.40 2.45
191.16 ꢂ 9.87
11.176 2.65
20.77 ꢂ 4.46
11.92 1.17
20.33 ꢂ 2.22
29.87 ꢂ 1.82
55.3 ꢂ 3.59
173 ꢂ 5.98
147.1 ꢂ 11.30
246.2 ꢂ 9.38
203 ꢂ 17.102
50 ꢂ 10.76
25.09 ꢂ 5.51
123.76 ꢂ 9.45
11.92 0.60
45.01 ꢂ 1.45
74.32 ꢂ 4.98
54.13 ꢂ 4.65
100.3 ꢂ 6.43
110 ꢂ 8.98
125.43 ꢂ 9.24
128.31 ꢂ 7.89
57.12 ꢂ 4.65
a
50% growth inhibition as determined by MTT assay (24 h drug exposure).
Compounds tested in triplicate, data expressed as mean value ꢂ SD of three independent experiments.
b

10
S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
4.2. General procedure for the synthesis of amidine derivatives [3]
4.2.4. N-(Pyridin-3-ylmethyl)-pyridine-2-carboxamidine (3d)
Yield 76%. semisolid. IR (KBr) nmax: 3444 & 3379 (NH), 1664 (e
4.2.1. Synthesis of N-(2-morpholin-4-yl-ethyl)-pyridin-2-
CaNe), 1526 & 1469 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm):
carboxamidine (3a)
4.483 (s, 2H, CH2), 7.247e7.273 (m, 1H, Ar), 7.434e7.475 (m, 1H, Ar),
7.656e7.679 (m, 1H, Ar), 7.820e7.838 (m, 2H, Ar), 8.265e8.277 (q,
1H, Ar), 8.336e8.352 (t, 1H, Ar), 8.433e8.462 (m, 1H, Ar). 13C NMR
Sodium metal (23 mg) was dissolved in absolute methanol
(20 ml) and was labeled as sodium methoxide solution in methanol.
2-Cyanopyridine (0.210 ml, 2 mmol) was dissolved in absolute
methanol (10 ml) and to it was added sodium methoxide solution
(0.5 ml) prepared above, the reaction contents were stirred at room
temperature for 2 h. 4-(2-Aminoethyl) morpholine (0.26 ml,
2 mmol) was added to the reaction mixture. The reaction contents
were heated under reflux for 8 h. Solvent was removed under
reduced pressure and to the residue left behind was added diethyl
ether, solid so obtained was filtered and washed with diethyl ether
to give crude product. This crude product was purified by crystal-
lization from ethyl acetate/methanol to give pure N-(2-morpholin-
4-yl-ethyl)-pyridine-2-carboxamidine (3a). Yield 370 mg (79%) mp
125e126 ꢃC. IR (KBr) nmax: 3428 (NH),1646 (eCaNe), cmꢁ1. 1H NMR
(125 MHz, DMSO-d6) d: 43.16 (CH2), 123.21 (py), 127.83 (py), 129.10
(py), 132.70 (py), 134.74 (py), 137.90 (py), 139.31 (py), 147.48 (py),
148.72 (py), 151.22 (py) and 164.62 (amidine). GCeMS m/z 212 (Mþ,
NH2
C N CH2
H2
C
+
C
+
30%), 196 (
, 100%), 134 (
, 7%),
N
N
N
N
+
92 (
CH2, 6%), Anal. Calcd. for C12H12N4 C 67.92; H 5.66; N
N
26.41%. Found C 67.97; H 5.70; N 26.45%.
4.2.5. N-(Pyridin-3-ylmethyl)-isonicotinamidine (3e)
Solvent of crystallization ethyl acetate/methanol. Yield 72%. mp
(500 MHz; DMSO-d6)
d
(ppm): 2.46 (bs, 4H, 2 ꢀ CH2), 2.574e2.602
278 ꢃC. IR (KBr) nmax: 3425 (NH), 1586 (eCaNe), 1547 & 1485
(t, 2H, J ¼ 7 Hz, eCH2e), 3.294e3.322 (t, 2H, J ¼ 7 Hz, eCH2e),
3.576e3.594 (t, 4H, J ¼ 4.5 Hz, eCH2e), 6.724 (bs, 2H, NH þ NH,
exch), 7.454e7.478 (q, 1H, J ¼ 5.5 Hz, 6.5 Hz, Ar), 7.853e7.884 (t, 1H,
J ¼ 7.5 Hz, Ar), 8.118e8.133 (d,1H, J ¼ 7.5 Hz, Ar), 8.563e8.572 (d,1H,
(Ar) cmꢁ1 1H NMR (500 MHz; D2O)
. d (ppm): 3.855 (s, 2H, CH2),
7.391e7.416 (q,1H, J ¼ 5 Hz, 7.5 Hz, Ar), 7.675e7.687 (q,1H, J ¼ 1.5 Hz,
4.5 Hz, Ar), 7.795e7.811 (d, 1H, J ¼ 8 Hz, Ar), 8.392e8.402 (q, 2H,
J ¼ 1.5 Hz, 3 Hz, py), 8.444e8.447 (d, 1H, J ¼ 1.5 Hz, py), 8.550e8.562
J ¼ 4.5 Hz, Ar). 13C NMR (125 MHz, DMSO-d6)
d: 36.21 (morp), 53.45
(q, 2H, J ¼ 1.5 Hz, 4.5 Hz, py). 13C NMR (125 MHz, DMSO-d6)
d: 43.14
(morp), 58.32 (morp), 66.30 (morp), 121.59 (py), 123.85 (py), 137.93
(CH2), 119.41 (py), 123.23 (py), 125.31 (py), 134.74 (py), 139.22 (py),
(py), 148.64 (py), 151.23 (py) and 163.87 (amidine). GCeMS m/z 235
147.46 (py),148.74 (py)150.70 (py)and 164.32(amidine). GCeMSm/z
.
+
.
+
(MHþ, 0.17%), 148 (
, 6%), 113 (
,
,
H2
+
O
N C CH2
H
O
N C CH2
212 (Mþ, 0.61%). 213 (MHþ, 18.15%), 107 (
, 100%), 105
NH
C
H
N
+
+
+
.
+
C NH
65%),105 (
,15%),100 (
,100%), 78 (
O
N CH2
+
+
N
N
CH
(
N
, 5.15%), 92 (
2,11.32%), 79 (
, 85.62%), 78
C NH
N
12%). Anal. Calcd for C12H18N4O C 61.53; H 7.69; N 23.93%. Found C
61.50; H 7.70; N 23.90%.
Similarly were synthesized other amidine derivatives 3bei.
Physical constants and spectral data of 3bei are summarized below.
N
+
(
, 70.35%) Anal. Calcd. for C12H12N4 C 67.92; H 5.66; N 26.41%.
N
4.2.2. N-(2-Morpholin-4-yl-ethyl)-isonicotinamidine (3b)
Found C 67.98; H 5.68; N 26.40%.
Solvent of crystallization ethyl acetate/methanol. Yield 80%. mp
122e125 ꢃC. IR (KBr) nmax: 3402 (NH), 1657 (eCaNe), 1551 & 1456
4.2.6. N-(Pyridin-3-ylmethyl)-pyrazine-2-carboxamidine (3f)
Solvent of crystallization ethyl acetate/methanol. Yield 81%. mp
135e136 ꢃC. IR (KBr) nmax: 3437 & 3334 (NH), 1655 (eCaNe), 1599,
(Ar) cmꢁ1 1H NMR (500 MHz; D2O)
. d (ppm): 2.359e2.388 (t, 4H,
J ¼ 7 Hz, 2 ꢀ CH2), 2.669e2.698 (t, 2H, J ¼ 7 Hz, CH2), 3.224e3.231
1574 & 1476 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm): 4.474 (s,
(d, 2H, J ¼ 3.5 Hz, eCH2e), 3.657 (bs, 4H, 2 ꢀ CH2), 7.652e7.664 (t,
2H, py), 8.617e8.628 (t, 2H, py). 13C NMR (125 MHz, DMSO-d6)
d:
2H, CH2), 7.348e7.374 (q, 1H, J ¼ 5 Hz, 10 Hz, Ar), 7.789e7.804 (d,
1H, J ¼ 7.5 Hz, Ar), 8.348e8.358 (t, 1H, Ar), 8.456e8.459 (d, 1H,
J ¼ 1.5 Hz, Ar), 8.605e8.636 (m, 2H, Ar), 9.031e9.033 (d, 1H,
43.66 (morp), 53.13 (morp), 58.77 (morp), 66.14 (morp), 121.18 (py),
143.42 (py),149.65 (py) and 164.67 (amidine). GCeMS m/z 234 (Mþ,
J ¼ 1.0 Hz, Ar). 13C NMR (125 MHz, DMSO-d6)
d: 43.21 (CH2), 123.34
+
0.65%), 100 (
, 100%), 78 (
, 12.59%). Anal. Calcd.
+
N
O
N CH2
(py), 135.22 (py), 136.82 (py), 142.54 (py), 143.04 (py), 145.36 (pyra),
147.03 (pyra), 147.49 (pyra), 148.95 (pyra) and 164.32 (amidine).
for C12H18N4O C 61.53; H 7.69; N 23.93%. Found C 61.55; H 7.67; N
23.95%.
NH
C
N
+
GCeMS m/z 213 (Mþ, 8%), 135 (
, 7%), 106
NH CH2
N
4.2.3. N-(2-Morpholin-4-yl-ethyl)-pyrazine-2-carboxamidine (3c)
Solvent of crystallization ethyl acetate/methanol. Yield 83%. mp
135e137 ꢃC. IR (KBr) nmax: 3432 & 3331 (NH), 1646 (eCaNe), 1595
.
+
N
N
+
+
N
(
, 11%), 105 (
NH
, 5%), 79 (
, 5%), 78
C
& 1469 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm): 2.559 (s, 4H,
C
N
N
N
N
2 ꢀ CH2), 2.683 (s, 2H, CH2), 3.401 (s, 2H, CH2), 3.689 (s, 4H,
2 ꢀ CH2), 8.601e8.630 (d, 2H, J ¼ 14.5 Hz, Ar), 8.998 (s, 1H, Ar). 13
C
+
(
, 25%). Anal. Calcd. for C11H11N5 C 61.97; H 5.16; N 32.86%.
NMR (125 MHz, DMSO-d6) d: 36.82 (morp), 54.05 (morp), 59.57
N
(morp), 66.65 (morp), 143.09 (pyra), 143.27 (pyra), 145.16 (pyra),
Found C 61.70; H 5.18; N 32.89%.
147.31 (pyra) and 164.22 (amidine). GCeMS m/z 236 (M þ 1, 0.5%),
.
+
4.2.7. N-(2-Piperazin-1-yl-ethyl)-pyridine-2-carboxamidine (3g)
Yield79%.Semisolid.IR(KBr)nmax:3391(NH),1647(eCaNe),1586
+
113 (
, 20%), 100 (
, 100%) Anal. Calcd.
O
N CH2
O
N C CH2
& 1464 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm): 2.461e2.475 (d,
H
for C11H17N5O C 56.17; H 7.23; N 29.78%. Found C 57.20; H 7.25; N
29.95%.
1H, J ¼ 7 Hz, one H of CH2), 2.535 (bs, 4H, 2 ꢀ CH2), 2.706e2.732 (t,1H,
J ¼ 6.5 Hz, one H of CH2), 2.812 (bs, 4H, 2 ꢀ CH2), 3.526e3.570 (q, 2H,

S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
11
J ¼ 7 Hz,15 Hz, CH2), 7.556e7.623 (m,1H, Ar), 7.883e7.996 (m, 2H, Ar),
8.532e8.605 (m, 1H, Ar). 13C NMR (125 MHz, DMSO-d6)
: 38.35
pyridine-2-carboxamidine (5a). Yield 480 mg (81%). IR (KBr) nmax
:
d
3438 (NH),1637 (eCaNe),1570,1488 (Ar) cmꢁ1. 1H NMR (500 MHz;
(piper), 45.59 (piper), 54.33 (piper), 61.79 (piper), 127.57 (py), 128.88
(py), 132.71 (py), 137.68 (py) 151.03 (py) and 164.91 (amidine). No
DMSO þ D2O)
d
(ppm): 1.648e1.676 (d, 4H, 2 ꢀ CH2), 3.166e3.246
(m, 4H, 2 ꢀ CH2), 7.484e7.509 (m, 2H, Ar), 7.867e7.901 (m, 2H,
Ar), 8.005e8.040 (q, 2H, Ar), 8.565e8.574 (d, 2H, Ar). 13C NMR
(125 MHz, DMSO-d6) d: 28.28 (CH2), 29.96 (CH2), 41.12 (CH2), 46.34
.
+
NH
molecular ion peak. m/z 149 (
, 20%), 112
C NH CH2CH3
(CH2), 127.69 (py), 128.90 (py), 132.51 (py), 137.78 (py) 151.04 (py)
N
and 163.07 (amidine). GCeMS m/z 296 (Mþ, 3.60%), 192
.
.
.
+
+
NH
NH
+
(
, 3%), 99 (
, 100%), 84 (
NH
,
+
+
H2C N
H2C C N
NH
+
(
, 33.07%),176 (
,
C NH (CH2)3 CH2
N
NH
C NH (CH2)4 NH2
H
N
N
.
+
NH
15%), 78 (
, 25%) Anal. Calcd. for C12H19N5 C 61.80; H 8.15; N
20.66%), 175
(
,
15.69%), 133
N
C NH (CH2)2 C CH2
H
30.04%. Found C 61.82; H 8.13; N 30.08%.
N
+
.
+
NH
C N CH2
N
4.2.8. N-(2-Piperazin-1-yl-ethyl)-isonicotinamidine (3h)
(
(
, 10.83%), 132 (
, 36.19%), 105
C N CH2
Yield75%. Semisolid. IR (KBr) nmax:3397(NH),1681(eCaNe),1550
N
N
N
& 1470 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm): 2.395e2.629 (m,
+
6H, 3 ꢀ CH2), 2.741e2.765 (t, 4H, 2 ꢀ CH2), 3.471e3.485 (t, 2H, CH2),
NH
C
, 100%), 78 (
, 70.29%). Anal. Calcd. for C16H20N6
7.592e7.641 (m, 2H, py), 8.554e8.580 (m, 2H, py).13C NMR (125 MHz,
+
N
DMSO-d6)
d: 39.13 (piper), 45.62 (piper), 54.43 (piper), 61.87 (piper),
C 64.86; H 6.75; N 28.37%. Found C 64.88; H 6.78; N 28.39%.
Similarly were synthesized other bis amidine derivatives
5bei. Physical constants and spectral data of 5bei are summa-
rized below.
125.48(py),144.29(py),150.63(py)and163.98(amidine). GCeMS m/z
NH
+
234 (MHþ, 0.2%), 149
(
,
5.15%), 106
C NH CH2CH2
N
.
.
+
+
4.3.2. N-(4-Isonicotinimidoylamino-butyl)-isonicotinamidine (5b)
Yield 78%. mp 240e242 ꢃC. IR (KBr)nmax:3400 (NH),1673 (eCaNe),
(
, 8.47%), 99 (
, 100%), 84 (
,
+
H2C N
NH
N
C NH
N
NH
H
1600 &1545 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
d (ppm): 1.489e1.535
NH
+
(d, 4H, 2 ꢀ CH2), 2.714e2.741 (t, 2H, CH2), 3.264e3.289 (t, 2H, CH2),
5.2%), 78 (
, 12.96%), 70 (
, 22.60%). Anal.
+
N
7.672e7.739 (m, 4H, py), 8.673e8.721 (dd, 4H, py). 13C NMR
HC NH CH2CH2
Calcd. for C12H19N5 C 61.80; H 8.15; N 30.04%. Found C 61.82; H 8.13; N
30.06%.
(125 MHz, DMSO-d6)
46.12 (CH2), 125.65 (py), 149.93 (py) and 150.72 (py), 163.34 (amidine).
d: 27.92 (CH2), 30.08 (CH2), 45.63 (CH2),
NH
GCeMS m/z 296 (Mþ, 2.27%), 191 (
, 14.24%),
+
4.2.9. N-(2-Piperazin-1-yl-ethyl)-pyrazine-2-carboxamidine (3i)
Yield 80%. Semisolid. IR (KBr) nmax:3431(NH),1633(eCaNe),1606,
N
C NH (CH2)4 NH
.
+
1548 (Ar) cmꢁ1.1H NMR (500 MHz; D2O)
d (ppm): 2.474e2.864 (m, 9H,
NH
149
(
,
29.05%),
148
N
C NH CH2CH3
4 ꢀ CH2 þ one H of CH2), 3.331e3.474 (m, 3H,1 ꢀ CH2 þ one H of CH2),
8.633e8.703 (m, 2H, Ar), 9.013e9.029 (d, 1H, J ¼ 8 Hz, Ar). 13C NMR
NH
C NH
NH
+
+
(
,11.95%),134 (
,15.09%),105
(125 MHz, DMSO-d6) d: 35.95 (piper), 45.18 (piper), 53.64 (piper),
N
N
C NH CH2
CH
2CH2
57.16 (piper), 143.52 (pyra), 144.52 (pyra), 147.30 (pyra), 162.67
(pyra) and 165.14 (amidine). GCeMS m/z 235(
. , 5%), 150
.
.
M+1 +
.
+
+
+
(
, 34.35%), 104 (
, 43.28%), 77 (
,
.
+
N
N
C NH
NH
N
CN
H
N
+
(
, 12%), 107
(
, 6%), 99
NH
C
N
C NH CH2CH3
N
N
98.56%). Anal. Calcd. for C16H20N6 C 64.86; H 6.75; N 28.37%. Found C
64.88; H 6.79; N 28.40%.
.
+
N
+
(
, 100%), 84 (
, 11%), 79 (
, 8%). Anal.
+
HN
N CH2
N
NH
N
4.3.3. N-(2-Pyrazineimidoylamino-butyl)-pyrazine-2-
carboxamidine (5c)
Calcd. for C11H18N6 C 56.41; H 7.69; N 35.89%. Found C 56.42; H 7.70; N
35.90%.
Yield 83%, mp 157e160 ꢃC, IR (KBr) nmax: 3423 & 3336 (NH),1648
(eCaNe), 1598 &1474 (Ar) cmꢁ1 1H NMR (500 MHz; DMSO-d6)
.
4.3. General procedure for synthesis of bis amidine derivatives [5]
d
(ppm): 1.738 (s, 4H, 2 ꢀ CH2), 3.305 (s, 4H, 2 ꢀ CH2), 8.567e8.575 (t,
2H, pyrazine), 8.613e8.618 (d, 2H, J ¼ 2.5 Hz, pyrazine), 8.959e8.961
(d, 2H, J ¼ 1 Hz, pyrazine). 13C NMR (125 MHz, DMSO-d6)
d: 26.73
4.3.1. Synthesis of N-(2-pyridineimidoylamino-butyl)-pyridine-2-
carboxamidine (5a)
(CH2), 44.52 (CH2), 142.17 (pyra), 143.19 (pyra), 145.47 (pyra), 146.27
(pyra) and 163.12 (amidine). GCeMS m/z 298 (Mþ, 3.70%), 176
2-Cyanopyridine (0.420 ml; 4 mmol) was dissolved in absolute
methanol (20 ml) and to it was added sodium methoxide solution in
methanol (1.0 ml) (previously prepared). The reaction contents were
stirred at room temperature for 2 h 1,4-diaminobutane (0.18 ml,
2 mmol) was added to the reaction mixture. Reaction contents were
heated under reflux for 12 h. Solvent was removed under reduced
pressure. Crude product so obtained was washed with diethyl ether
and then with ethyl acetate to give thick mass. Solvent traces from
this thick mass was removed by applying high vacuum for 15 min to
give semisolid product i.e. N-(2-pyridineimidoylamino-butyl)-
NH
C NH (CH )
N
N
+
(
(
,
9.12%),
163
2 3 CH2
N
N
.
NH
C NHCH2
NH
N
H
+
+
,
5.11%), 149(
,
,
C NHCH2 CH2
C
CH2
N
.
+
NH
+
NH
C NH CH3
N
N
4.05%), 135 (
, 26.37%), 134 (
C NH CH2
N
N

12
S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
NH
C NH CHC CH2
NH
C NH
N
N
+
+
+
8.60%),
(
160
(
,
18.67%),
157
8.13%), 122 (
, 3.97%), 106 (
, 13.26%), 105
21.49%), 96
C
N
NH
H
N
N
.
.
+
NH
+
+
,
14.00%), 148
(
,
N
+
N
C NH CH2 CH2
(
(
,
50.75%), 97
(
,
H2N (CH )
2 3 N
N CH3
CN
N C NH (CH2)3CH2
N
+
,
N
47.32%),
142
(
13.68%),
141
H N (CH )
2 3N
2
.
+
, 100%). Anal. calcd. for C14H18N8 C 56.37; H
N C NH (CH2)2C CH2
+
+
H
(
, 9.36%), 127(
, 14.73%), 126
H2C H2C N
N CH3
H3C N
N (CH2)2CH2
6.04; N 37.58%. Found C 56.39; H 6.01; N 37.60%.
.
+
+
N
H C C N
(
, 32.96%), 111 (
, 14.32%), 104
2
4.3.4. N-{3-[4-(2-Pyridineimidoylamino-propyl)-piperazin-1-yl]-
propyl}-pyridine-2-carboxamidine (5d)
H2C C N
N CH3
H
H
.
Yield 78%. mp 138e140 ꢃC. IR (KBr) nmax: 3436 & 3279 (NH), 1678
.
+
+
(eCaNe), 1587, 1568 & 1520 (Ar) cmꢁ1. 1H NMR (500 MHz; DMSO-
+
(
, 44.10%), 78 (
, 5.77%), 77 (
, 21.97%), 56
N
N
CN
N
d6 þ D2O) (ppm): 1.056e1.084 (t, 2H, J ¼ 7 Hz, CH2),1.672e1.790 (m,
d
.
4H, 2 ꢀ CH2), 2.362e2.386 (t, 8H, J ¼ 6 Hz, 4 ꢀ CH2), 3.334e3.392 (m,
6H, 3 ꢀ CH2), 7.545e7.568 (t, 2H, J ¼ 6 Hz, Ar), 7.957e8.017 (m, 4H, Ar),
(
, 100%). Anal. Calcd. for C22H32N8 C 64.70; H 7.84;
+
NH CH N CH2
8.604e8.613 (d, 2H, J ¼ 4.5 Hz, Ar). 13C NMR (125 MHz, DMSO-d6)
d:
N 27.45%. Found C 64.72; H 7.80; N 27.43%.
25.30 (CH2), 52.26 (CH2), 56.15 (CH2), 121.80 (py), 125.60 (py), 137.87
(py), 148.43 (py), 149.45 (py) and 164.24 (amidine). GCeMS m/z 408
4.3.6. N-{3-[4-(3-Pyrazinimidoylamino-propyl)-piperazin-1-yl]-
propyl}-pyrazine-2-carboxamidine (5f)
.
+
NH
(Mþ, 6.64%), 329 (
, 7.62%),
Yield 85%. mp 153e155 ꢃC. IR (KBr) nmax: 3446 & 3311 (NH),1649
C NH (CH2)3 N
N (CH2)3
N C NH
(eCaNe), 1595, 1525 & 1469 (Ar) cmꢁ1. 1H NMR (500 MHz; D2O)
N
d
(ppm): 1.736e1.796 (m, 4H, 2 ꢀ CH2), 1.943e2.751 (m, 12H,
NH
251
(
,
14.47%),
210
209
N C NH
N
N (CH2)3
C
+
NH (CH2)3
6 ꢀ CH2), 3.218e3.245 (t, 4H, J ¼ 7 Hz, 2 ꢀ CH2), 8.567e8.606 (m,
4H, pyrazine), 8.953e8.955 (d, 2H, J ¼ 1.0 Hz, pyrazine). 13C NMR
+
(
,
11.08%),
(125 MHz, DMSO-d6)
d: 27.87 (CH2), 53.57 (CH2), 56.61 (CH2),
H2C (CH2)2N
N (CH2)3 N C NH
140.44 (pyra), 143.65 (pyra), 144.70 (pyra), 145.07 (pyra), 147.52
.
+
(pyra) and 164.18 (amidine). GCeMS m/z 410 (Mþ, 4.69%), 163
(
(
(
,
67.82%),
208
N C NH
H2C C CH2
H
N
N
N (CH2)3
NH
NH
C NHCH2 CH2
N
N
+
+
(
,
11.18%), 149
(
,
C NH (CH2)2CH2
+
N
N
,
38.45%),
183
CH
N (CH2)3N C NH
CH2
CH2
C
2
.
+
NH
C NH
N
NH
C NHCH3
N
+
.
70.70%), 136
21.54%), 105 (
(
,
9.87%), 121 (
,
NH
+
+
,11.08%),161 (
,
CH CH2 N
N (CH2)3 NH
NH
N
C NH CH2 CH CH2
N
N
.
+
.
+
NH
N
+
N
C
N
+
, 3.37%), 78 (
, 100%). Anal. Calcd.
45.48%), 134 (
, 39.26%), 105 (
, 10.73%).
C NH CH2
N
C
N
N
N
Anal. Calcd. for C22H32N8 C 64.70; H 7.84; N 27.45%. Found C 64.73; H
7.80; N 27.46%.
for C20H30N10 C 58.53; H 7.31; N 34.14%. Found C 58.55; H 7.33; N
34.10%.
4.3.5. N-{3-[4-(3-Isonicotinimidoylamino-propyl)-piperazin-1-yl]-
propyl}-isonicotinamidine (5e)
4.3.7. N-(40-Pyridinimidoylamino-biphenyl-4-yl)-pyridine-2-
carboxamidine (5g)
Yield 82%. mp 159e160 ꢃC.IR(KBr)nmax: 3421 (NH),1661 (eCaNe),
Yield 74%. mp 232e234 ꢃC. IR (KBr) nmax: 3467 & 3356 (NH),1639
1541 (Ar) cmꢁ1.1H NMR (500 MHz; DMSO-d6)
d (ppm): 1.723e1.749 (t,
(eCaNe), 1561 & 1492 (Ar) cmꢁ1 1H NMR (500 MHz; DMSO-d6)
.
4H, J ¼ 6.5 Hz, 2 ꢀ CH2), 2.042e2.373 (m,12H, 6 ꢀ CH2), 3.123e3.147 (t,
d
(ppm): 4.994 (s, 4H, 4 ꢀ NH, exch), 6.571e6.588 (d, 4H, J ¼ 8.5 Hz,
4H, J ¼ 6 Hz, 2 ꢀ CH2), 7.705e7.714 (d, 4H, J ¼ 4.5 Hz, py), 8.595e8.607
Ar), 7.192e7.209 (d, 4H, J ¼ 8.5 Hz, Ar), 7.746e7.774 (m, 2H, py),
(d, 4H, J ¼ 6 Hz, py). 13C NMR (125 MHz, DMSO-d6)
d: 28.92 (CH2),
8.058e8.079 (m, 4H, py), 8.768e8.780 (m, 2H, py). 13C NMR
53.48 (CH2), 56.62 (CH2), 121.52 (py), 144.64 (py), 150.14 (py) and
(125 MHz, DMSO-d6) d: 114.37 (Ph), 117.38 (Ph), 126.02 (py), 127.66
(py),128.93 (Ph),132.67 (Ph),137.75 (py),146.77 (py) 151.07 (py) and
164.08 (amidine). GCeMS m/z 408 (Mþ, 1.75%), 287
.
+
NH
.
(
,
4.40%),
3.14%),
4.42%),
246
210
184
163.96 (amidine). GCeMS m/z 392 (Mþ, 1.56%), 104 (
,
N
C NH (CH2)3N
N CH2 CH CH2
+
CN
N
NH
(
,
+
N
+
N
C NH (CH2)3
N
N
9.36%), 78 (
, 72.94%). Anal. Calcd. for C24H20N6 C 73.46; H 5.10;
.
+
(
,
N 21.42%. Found C 73.42; H 5.08; N 21.40%.
HN HC HN (CH2)3 N
N CH2 CH CH2
.
+
4.3.8. N-(40-Isonicotinimidoylamino-biphenyl-4-yl)-
isonicotinamidine (5h)
(
,
27.06%),
162
HN HCHN (CH2)3N
N CH3
, 8.19%), 161
C NH (CH2)2CH2
.
Yield 76%. mp 238e240 ꢃC. IR (KBr) nmax: 3469 & 3367 (NH),
NH
NH
1614 (eCaNe), 1546 & 1494 (Ar) cmꢁ1. 1H NMR (500 MHz; DMSO-
+
+
(
(
,
N
N
C NHCH2C CH2
H
d6)
d
(ppm): 5.329 (s, 2H, 2 ꢀ NH, exch), 6.739e6.816 (m, 6H, Ar),

S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
13
7.062e7.075 (d, 2H, J ¼ 6.5 Hz, Ar), 7.362e7.378 (t, 2H, Ar), 7.510e
10% fetal bovine serum, 100
m
g/ml streptomycin and 100 units/ml
7.527 (t, 2H, Ar), 7.671e7.687 (t, 2H, Ar), 8.092 (bs, 2H, 2 ꢀ NH,
penicillin) in a carbon dioxide incubator (37 ꢃC, 5% CO2, 90% RH). All
cell culture reagents were from GIBCO (Invitrogen, USA). Penicillin,
streptomycin, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H
tetrazolium bromide), cell culture grade DMSO, 5-fluorouracil (5-
FU), cyclophosphamide and actidione (cycloheximide) were from
Himedia (Mumbai, India).
exch), 8.851e8.857 (d, 2H, J ¼ 3 Hz, Ar). 13C NMR (125 MHz, DMSO-
d6) d: 114.36 (Ph), 116.72 (Ph), 119.40 (Ph), 125.87 (py), 128.79 (Ph),
146.81 (py),150.70 (py) and 164.23 (amidine). GCeMS m/z 392 (Mþ,
.
+
NH
C
3.48%),
197
(
,
10.49%),
181
H
N
N
MTTassay was carried out as described in literature [41]. In brief,
5 ꢀ 103 cells in 200
ml of medium were seeded in 96-well plates
(Griener, Germany). Serial dilutions of compound initially ranging
+
+
(
N
,
2.02%), 105
(
N
, 40.84%), 79
C NH
C N
from 0 to 100 mM in DMSO were added to the monolayer. The final
.
+
.
+
DMSO concentration for all dilutions was 0.1% which was used as
vehicle control. The cultures were assayed after 24 h by the addition
(
, 30.13%), 76 (
, 44.82%). Anal. Calcd. for C24H20N6 C
N
of 50
MTT-containing medium was aspirated and 200
(Himedia, Mumbai, India) and 25 l of Sorensen glycine buffer
m
l of 5 mg/ml MTT and incubating for another 4 h at 37 ꢃC. The
73.46; H 5.10; N 21.42%. Found C 73.40; H 5.08; N 21.41%.
ml of DMSO
m
4.3.9. N-(40-Pyrazinimidoylamino-biphenyl-4-yl)-pyrazine-2-
(0.1 M glycine and 0.1 M NaCl, pH 10.5) were added to lyse the cells
and solubilize the water insoluble formazone. Absorbance of the
lysates was determined on a Fluostar optima (BMG Labtech,
Germany) microplate reader at 570 nm.
carboxamidine (5i)
Yield 76%. mp > 300 ꢃC. IR (KBr) nmax: 3500 & 3384 (NH),1624 (e
CaNe), 1596, 1558 & 1490 (Ar) cmꢁ1. 1H NMR (500 MHz; DMSO-d6)
d
(ppm): 6.715 (bs, 4H, 4 ꢀ NH, exch), 7.015e7.064 (q, 4H, J ¼ 6.5 Hz,
The percentage inhibition was calculated as
Mean OD of vehicle treated cellsðnegative controlÞ ꢁ Mean OD of treated cells ꢀ 100
Mean OD of vehicle treated cellsðnegative controlÞ
16 Hz, Ar), 7.668e7.684 (t, 4H, Ar), 8.727e8.730 (d, 2H, J ¼ 1.5 Hz,
pyrazine), 8.820e8.830 (t, 2H, J ¼ 2.5 Hz, pyrazine), 9.492e9.500 (d,
The IC50 values were calculated using graph pad prism, version
5.02 software (Graph Pad Software Inc., CA, USA).
2H, J ¼ 4 Hz, pyrazine). 13C NMR (125 MHz, DMSO-d6)
d: 114.34 (Ph),
115.73 (Ph), 125.97 (Ph), 129.82 (Ph), 140.87 (pyra), 145.68 (pyra),
148.07 (pyra) and 148.51 (pyra), 164.33 (amidine). Anal. Calcd. for
C22H18N8 C 67.00; H 4.56; N 28.42%. Found C 67.03; H 4.52; N
28.43%.
Acknowledgments
We are thankful to technical staff of the Chemistry Department,
I.I.T. Roorkee, for spectroscopic studies and elemental analysis.
Thanks also due to Head I.I.C. for providing NMR facility. Ms. Surbhi
Arya (SRF) is thankful to CSIR New Delhi for financial assistance.
4.4. Anti-inflammatory activity
Paw oedema inhibition test was used on albino rats of Charles
Foster by adopting the method of Winter et al. [40]. Groups of five
animals of both sexes (body weight 120e160 g), excluding pregnant
females, were given a dose of test compound. Thirty minute later,
0.20 mL of 1% freshly prepared carrageenan suspension in 0.9% NaCl
solution was injected subcutaneously into the planter aponeurosis
of the hind paw and the volume was measured by a water ple-
thysmometer apparatus and then measured again 1e3 h later. The
mean increase of paw volume at each interval was compared with
that of control group (five rats treated with carrageenan but not
with test compound) at the same intervals and percent inhibition
value calculated by the formula given below.
References
[1] S.M. Sondhi, M. Dinodia, J. Singh, R. Rani, Curr. Bioactive Compd. 3 (2007)
91e108.
[2] A. Bentolini, A. Ottani, M. Sandrini, Curr. Med. Chem. 9 (2002) 1033e1043.
[3] L.S. Simon, Arthritis Care Res. 45 (2001) 209e215.
[4] P. Moutevelis-Minakakis, E. Papavassilopoulou, G. Michas, K. Georgikopoulou,
M.-E. Ragoussi, N. Neophytou, P. Zoumpoulakis, T. Mavromoustakos,
D. Hadjipavlou-Litina, Bioorg. Med. Chem. 19 (2011) 2888e2902.
[5] Y. Marrero-Ponce, D. Siverio-Mota, M. Galvez-Llompart, M.C. Recio,
R.M. Giner, R. Garcia-Domenech, F. Torrens, V.J. Aran, M.L. Cordero-Maldo-
nado, C.V. Esguera, P.A.M. de Witte, A.D. Crawford, Eur. J. Med. Chem. 46
(2011) 5736e5753.
[6] T. Farida, P. Roberto, F. Gabriele, B. Alessandra, F. Paola, O. Fulvia, F. Milo,
C. Paola, J. Med. Chem. 55 (2012) 2112e2124.
[7] L. Wen-Shan, W. Chie-Hong, K. Shengkai, C.T. Tzu, C.J. Ya, Y. Ching-Fa,
V.M. Shivaji, J. Shu-Chuan, J. Med. Chem. 55 (2012) 1583e1592.
[8] C. Lonez, A. Legat, M. Vandenbranden, J.-M. Ruysschaert, Cell. Mol. Life Sci. 65
(2008) 620e630.
[9] A. Giordani, S. Mandelli, S. Zanzola, F. Tarchino, G. Caselli, T.S. Fiorentino, S.
Mazzari, F. Makovec, L.C. Rovati, PCT Int. Appl. WO 2008014815 2008 Chem.
Abstr.148 (2008) 239229.
% anti ꢁ inflammatory activity ¼ ½1 ꢁ Dt=Dcꢄ ꢀ 100
Dt and Dc are paw volumes of oedema in tested and control groups,
respectively.
[10] V.A. Glushkov, L.V. Anikina, Y.B. Vikharev, E.V. Feshina, Y.V. Shklyaev, Pharm.
Chem. J. 39 (2005) 533e536.
4.5. In vitro cytotoxicity against human cancer cell lines
[11] C. Franca da Silva, M.M. Batista, D.G.J. Batista, E. Mello de Souza, P. Bernardino
da Silva, G. Melo de Oliveira, A. Meuser, A.R. Shareef, D.W. Boykin, C.S. Maria
de Nazare, Antimicrob. Agents Chemother. 52 (2008) 3307e3314.
[12] L. Hu, R.K. Arafa, M.A. Ismail, A. Patel, M. Munde, W.D. Wilson, T. Wenzler,
R. Brun, D.W. Boykin, Bioorg. Med. Chem. 17 (2009) 6651e6658.
[13] G. Madhusudhan, V. Balraju, T. Rajesh, B.V. Narayana, R.N. Reddy, Indian J.
Chem. Sec. B 48B (2009) 569e573.
Human breast (T47D), lung (NCI H-522), colon (HCT-15), ovary
(PA-1) and liver (HepG2) cancer cell lines were obtained from
National Center for Cell Science (NCCS), Pune, India. Cells were
grown in tissue culture flask in complete growth medium (RPMI-
1640 medium with 2 mM glutamine, pH 7.4 supplemented with

14
S. Arya et al. / European Journal of Medicinal Chemistry 59 (2013) 7e14
[14] M.A. Ismail, R.K. Arafa, R. Brun, T. Wenzler, Y. Miao, W.D. Wilson, C. Generaux,
A. Bridges, J.E. Hall, D.W. Boykin, J. Med. Chem. 49 (2006) 5324e5332.
[15] M. Calas, M. Ouattara, G. Piquet, Z. Ziora, Y. Bordat, M.L. Ancelin, R. Escale,
H. Vial, J. Med. Chem. 50 (2007) 6307e6315.
[16] F.S. Han, I.H. Osajima, M. Cheung, H. Tokuyama, T. Fukuyama, Chem. Eur. J. 13
(2007) 3026e3038.
[17] W. Li, Z. Wang, V. Gududuru, B. Zbytek, A.T. Slominski, J.T. Dalton, D.D. Miller,
Anticancer Res. 27 (2007) 883e888.
[18] K. Bielawski, A. Bielawska, Chem. Med. Chem. 3 (2008) 536e542.
[19] S.M. Sebti, S. Chellappan, N.J. Lawrence, PCT Int. Appl. WO 2007062222, Chem.
Abstr. 147 (2007) 31092.
[26] K. Koiwai, M. Isozaki, N. Sakaguchi, K. Kono, JP Patent 2005008623, Chem.
Abstr. 142 (2005) 120561.
[27] G. Brasche, S.L. Buchwald, Angew. Chem. Int. Ed. 47 (2008) 1932e1943.
[28] S.M. Sondhi, J. Singh, S.K. Agarwal, A.K. Saxena, P. Roy, Med. Chem. Res. 21
(2012) 91e99.
[29] S.M. Sondhi, R. Rani, J. Singh, P. Roy, S.K. Agarwal, A.K. Saxena, Bioorg. Med.
Chem. Lett. 20 (2010) 2306e2310.
[30] S.M. Sondhi, J. Singh, R. Rani, P.P. Gupta, S.K. Agarwal, A.K. Saxena, Eur. J. Med.
Chem. 45 (2010) 555e563.
[31] S.M. Sondhi, S. Arya, R. Rani, N. Kumar, P. Roy, Med. Chem. Res. 21 (2012)
3620e3628.
[20] C. Marzano, S.M. Sbovata, F. Bettio, R.A. Michelin, R. Seraglio, T. Kiss, A. Venzo,
R. Bertani, J. Biol. Inorg. Chem. 12 (2007) 477e493.
[21] K. Bielawski, A. Bielawska, K. Sosnowska, W. Miltyk, K. Winnicka, J. Palka,
Biochem. Pharmacol. 72 (2006) 320e321.
[22] P. Sienkiewicz, K. Bielawski, A. Bielawska, J. Palka, J. Environ. Toxicol.
Pharmacol. 20 (2005) 118e124.
[23] S. Sasaki, J. Fukushima, H. Arai, K. Kusakabe, K. Hamajima, N. Ishii, F. Hirahara,
K. Okuda, S. Kawamoto, J.M. Ruysschaert, M. Vandenbranden, B. Wahren,
K. Okuda, Eur. J. Immunol. 27 (1997) 3121e3129.
[24] A. Panico, P. Vinici, M. Inert, V. Cardile, B. Gentile, G. Ronsisvalle, Il Farmaco 57
(2002) 671e675.
[25] M.J. Rudolph, C.R. Illig, N.L. Subasinghe, K.J. Wilson, J.B. Hoffman, T. Randle,
D. Green, C.T. Molloy, R.M. Soll, F. Lewandowski, M. Zhang, R. Bone,
J.C. Spurlino, I.C. Deckman, C. Manthey, C. Sharp, D. Maguire, B.L. Grasberger,
R.L. Desjarlais, Z. Zhou, Bioorg. Med. Chem. Lett. 12 (2002) 491e495.
[32] J.C. Gravis, A. Taurins, Can. J. Chem. 39 (1961) 761e764.
[33] J. Wang, F. Xu, T. Cai, Q. Shen, Org. Lett. 10 (2008) 445e448.
[34] I. Bae, H. Han, S. Chang, J. Am. Chem. Soc. 127 (2005) 2038e2039.
[35] K. Nadrah, M. Sollner Dolenc, Synlett 18 (2007) 1257e1258.
[36] L. Benati, G. Bencivenni, R. Leardini, D. Nanni, M. Minozzi, P. Spagnolo,
R. Scialpi, G. Zanardi, Org. Lett. 8 (2006) 2499e2502.
[37] S.M. Sondhi, M. Dinodia, A. Kumar, Bioorg. Med. Chem. 14 (2006) 4657e
4663.
[38] Y. Zhang, K.A. DeKorver, A.G. Lohse, Y.S. Zhang, J. Huang, R.P. Hsung, Org. Lett.
11 (2009) 899e902.
[39] L.K. Thompson, C.J. Matthews, L. Zhao, C. Wilson, M.A. Leech, J.A.K. Howard,
J. Chem. Soc. Dalton Trans. 15 (2001) 2258e2262.
[40] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. 111 (1962)
544e547.
[41] T. Mosmann, J. Immunol. Methods 65 (1983) 55e63.