Synthesis and antiproliferative activity of 2,6-Dibenzylamino-3,5- dicyanopyridines on human cancer cell lines

Article abstract of DOI:10.1016/j.ejmech.2005.07.005

A new series of 2,6-dibenzylamino-3,5-dicyanopyridines were synthesized and evaluated for their in vitro anticancer activity toward cell lines of nine different types of human cancer. Some of newly prepared compounds demonstrated remarkable anticancer act

Full text of DOI:10.1016/j.ejmech.2005.07.005

European Journal of Medicinal Chemistry 40 (2005) 1365–1372
www.elsevier.com/locate/ejmech
Short communication
Synthesis and antiproliferative activity
of 2,6-Dibenzylamino-3,5-dicyanopyridines on human cancer cell lines
Maria Teresa Cocco *, Cenzo Congiu, Valentina Lilliu, Valentina Onnis
Dipartimento di Tossicologia, Università degli Studi di Cagliari, Via Ospedale 72-09124 Cagliari, Italy
Received 26 May 2005; revised and accepted 18 July 2005
Available online 30 August 2005
Abstract
A new series of 2,6-dibenzylamino-3,5-dicyanopyridines were synthesized and evaluated for their in vitro anticancer activity toward cell
lines of nine different types of human cancer. Some of newly prepared compounds demonstrated remarkable anticancer activity against most
of the tested subpanel tumor cell lines.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Pyridines; Dicyanopyridines; Anticancer activity; Cytostatic activity
1. Introduction
ity. Thus 2-amino-6-[(2-aminophenyl)thio]-4-(2-furyl)
pyridine-3,5-dicarbonitrile has been reported as lead com-
pound that mimics the dominant negative host-encoded prion
protein (PrPC) mutants, which inhibit an abnormal isoform
(PrPSc) formation [7]. 2-Guanadino-3,5-dicyanopyridines
present moderate in vitro cytotoxic activity against P-388,
A-549, HT-29 and MEL-28 tumoral cell lines as well as they
are very potent stimulator of the release of histamine [3]. Some
recent patents can be found on 2-thio-3,5-dicyano-4-aryl-6-
aminopyrimidines as selective adenosine receptor ligands and
for treatment of cancer [8], and on 3,5-dicyanopyridine deriva-
tives exhibiting an excellent effect to open the maxi-K chan-
nel [9].
On the basis of above observations it is very attractive the
development of 3,5-dicyanopyridine derivatives as potential
antitumor agents. Continuing our interest in pyridine deriva-
tives endowed with anticancer activity [10,11], we designed
and synthesized a novel series 4-aryl or -heteroaryl-2,6-
dibenzylamino-3,5-dicyanopyridines to evaluate their anti-
cancer properties.
The pyridine scaffold is a widespread structural motif that
can be found in many natural products and in several phar-
macologically interesting compounds. Therefore the synthe-
sis of pyridine derivatives, aiming to develop new drugs, is
an active research area. Recently several researchers became
interested to cyanopyridine derivatives. 2-Cyanopyridylureas
derivatives have been claimed for their properties in treating
hyper-proliferative and angiogenesis disorders [1]. The
3-cyano-2,6-dihydropyridine is a potent inhibitor of dihydrou-
racil dehydrogenase and its coadministration with
1-ethoxymethyl-5-fluorouracil enhances the antitumor effect
[2].
3,5-Dicyanopyridines derivatives have been described as
intermediates in the synthesis of pyrido[2,3-d]pyrimidines as
antihistaminic agents [3], pyridothieno- and pyridodithieno-
triazines endowed with antihistaminic and cytotoxic activity
[4], 3,4,6-triazabenz[d,e]anthracene and 3,4,6,9-tetrazabenz
[d,e]anthracene that are DNA intercalating agents [5] and
acyclo-3-deazapyrimidine S-nucleosides that are active
toward HIV [6].
2. Chemistry
Besides their importance as synthetic intermediates 3,5-
dicyanopyridines derivatives are endowed of biological activ-
The target 2,6-dibenzylamino-3,5-dicyanopyridines 3-19
(Table 1) were synthesized as shown in Scheme 1. 4-Aryl-
2,6-diamino-3,5-dicyanopyridines are generally prepared
starting from the corresponding 2-alkoxy-6-amino-3,5-
* Corresponding author. Tel.: +39 0706758645; fax: +39 0706758612.
E-mail addresses: tcocco@unica.it (M.T. Cocco), ccongiu@unica.it
(C. Congiu), vonnis@unica.it (V. Onnis).
0223-5234/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2005.07.005

1366
M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
Table 1
followed by intramolecular cyclization with loss of ammo-
nia.Although 3-amino-3-benzylaminopropenenitrile 2 is isol-
able, as previously reported by us [15], recovering 2 from
reaction mixture and reacting it with aldehydes in a separate
vessel does not improve 3,5-dicyanopyridine 3-19 yields. All
the newly synthesized compounds gave corrected analytical
data. The IR and NMR spectral data were consistent with the
assigned structure.
Structure for 2,6-Dibenzylamino-3,5-dicyanopyridines 3-19
Compd.
3
X
4-OMePh
3-OMePh
2,5-(OMe)2Ph
3,4,5-(OMe)3Ph
4-ClPh
4
5
6
7
8
2-ClPh
9
2,4-Cl2Ph
2,6-Cl2Ph
3-OHPh
10
11
12
13
14
15
16
17
18
19
3. Pharmacology
4-OHPh
3-OH,4-OMePh
2-Pyridyl
3-Pyridyl
4-Pyridyl
2-Thienyl
2-Furyl
Evaluation of anticancer activity on dicyanopyridines was
performed at National Cancer Institute (NCI). First dicyan-
opyridines 3-19 have been evaluated for their cytotoxicity in
primary 3-cell line test at 10−4 M concentration. The cell lines
adopted in this screen were NCI-H460 (Lung), MCF7 (Breast)
and SF-268 (CNS) due to their being good predictor of anti-
cancer activity (Table 2).
For NCI criteria, compounds, which reduce the growth of
any one of the cell lines to approximately 32% or less, are
considered active and passed on for evaluation in the full panel
of cell lines over a 5-log dose range. Dicyanopyridines 7, 8,
9, 11, 12, that meet these criteria, were evaluated for their
anticancer activity following the known in vitro disease-
oriented antitumor screening program, which is based upon
use of multiple panel of about 60 human tumor cell lines
[16,17]. Each compound is tested at minimum of five con-
centrations at 10-fold dilution against every cell line in the
panel. A 48 h continuous drug exposure protocol is used and
a sulforhodamine B (SRB) protein assay is used to estimate
cell viability or growth [18]. The anticancer activity of each
compound is deduced from dose–response curves and is pre-
sented in three different Tables according to the data pro-
vided by NCI [19]. The response parameters GI50, TGI and
2-(5-(Me)furyl)
Table 2
Antiproliferative activity of compounds 3-19 at 10−4 M concentration
expressed in growth percentage
Scheme 1
.
Synthetic pathway to 3,5-dicyanopyridines 3-19, i: Benzyla-
mine, MeCN, r.t. 24 h; ii: X-CHO, reflux, 3 h.
Compound
Cell lines
dicyanopyridine or 2-chloro-6-amino-3,5-dicyanopyridine
derivatives [12,13] or by reaction of arylidenemalononitrile
with an imidate in the presence of ammonium acetate [14].
In this paper we developed an alternative and simple syn-
thetic pathway to obtain 4-aryl-2,6-dialkylamino-3,5-
dicyanopyridines.
MCF7
100
100
86
NCI-H460
106
96
SF-268
111
98
3
4
5
93
113
90
6
78
91
7
55
4
80
8
71
5
69
2,6-Dibenzylamino-3,5-dicyanopyridines 3-19 were ob-
tained through an one-pot, two-step reaction starting from
the easily available 3-amino-3-ethoxypropenenitrile 1. This
last was first treated with benzylamine in EtOH solution to
give 3-amino-3-benzylaminopropenenitrile 2, and then with
the appropriate aryl or heteroarylaldehyde. The reaction mix-
ture was heated at reflux and, after 3 h, 2,6-dibenzylamino-
3,5-dicyanopyridines 3-19 were obtained in good to excel-
lent yields. The pathway to formation of 3,5-dicyanopyridine
ring was initiated by condensation of two molecules of
3-amino-3-benzylaminopropenenitrile 2 with the aldehyde,
9
76
26
64
10
11
12
13
14
15
16
17
18
19
81
76
98
77
2
110
102
95
75
18
67
78
107
104
100
105
87
104
108
107
105
95
118
113
108
113
95
114
103
116

M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
1367
LC50 (Tables 3–5) refer to the drug concentration that pro-
duced 50% inhibition, total growth inhibition and 50% cyto-
toxicity, respectively, and are expressed in 10−5 molar con-
centrations. Moreover Table 3 shows full panel mean-graph
midpoint values (MG-MID) that are the average of GI50 val-
ues of all cell lines.
methoxy substituents. Furthermore the introduction of a
4-methoxy group on 11 leads to compound 13 that showed
low activity. Significant antiproliferative activity is induced
by a 2-chlorine atom on phenyl moiety (compound 8). The
displacement of chlorine from 2-position to 4-position (com-
pound 7) on phenyl ring seems do not affect activity. As mat-
ter of fact compounds 7 and 8 showed similar MG-MID val-
ues (4.71 and 4.75 respectively). The introduction of a second
chlorine atom produces variable effects: the 2,4-dichloro
derivative 9 shows potent and selective activity against some
cell lines, in contrast the 2,6-dichloro derivative 10 is inac-
tive.
A COMPARE [20] analysis was performed with the more
active compounds to investigate whether it resemble antican-
cer drugs of the NCI standard agent database and to probably
predict its mechanism of action. The COMPARE algorithm
was developed to determine the degree of similarity of mean
graph fingerprints obtained from the in vitro anticancer screen
with patterns of activity of standard agents. The hypothesis is
that, if the data pattern of a compound correlates well with
the data pattern of compounds belonging to the standard agent
database, the compound of interest may have the same mecha-
nism of action as those agents with known mechanism. A
correlation coefficient of 0.55– 0.6 is considered the lowest
correlation that suggests a relationship with another com-
pound [21]. Using GI50 values of dicyanopyridines 7, 8, 9,
11, 12 as seed, COMPARE analysis showed correlation coef-
ficients with the standard agents below 0.55. These results
illustrate that the mechanism of action of the novel dicyan-
opyridines may differ from that of the standard antitumor
drugs. Therefore, their antitumoral activity may be caused by
a new and unknown mechanism.
4. Results and discussion
Evaluation of the data reported in Table 3 revealed that
dicyanopyridines 7, 8, 9, 11, 12 showed appreciable antican-
cer activity against most of the tested subpanel tumor cell
lines. From the analysis of GI50 data we can evince that all
these dicyanopyridines exhibited cytostatic activity at 10−5
M and in some cases at 10−9 M concentration.
Dicyanopyridine 11 proved to be the most active member
within the series, showing potent and broad spectrum of anti-
tumoral activity (MG-MID value –5.12). Compound 11
showed an in vitro chemosentitive profile toward 33 different
cancer cell lines with GI50 values lying in the micromolar
concentration range (GI50 values between 0.168·10−5 M and
0.779·10−5 M). Compound 11 displayed some selectivity on
renal cancer subpanel at GI50 (values between 0.201·10−5
and 1.38·10−5 M), TGI (values between 0.577·10−5 M and
8.76·10−5 M) and LC50 levels (values between 2.54·10−5
M
M
and 8.83·10−5 M). Furthermore the growth of all leukemia
cell lines was inhibited by compound 11 at micromolar con-
centrations (GI50 values between 0.209·10−5 M and 0.338·10−5
M). Although less potent when compared to 11, compounds
7, 8 and 12 showed activity against most of the tested cell
lines (MG-MID values 4.71, 4.75 and 4.62 respectively). In
particular compound 12 displayed high antiproliferative activ-
ity against HOP-92 non-small cell lung cancer cell line (GI50
value 0.0315·10−5 M and TGI value 0.798·10−5 M). Com-
pound 9 selectively exhibited high potency against leukemia
HL-60(TB) cell line (GI50 < 0.0001 10−5 M), HOP92 cell line
(GI50 0.112·10−5 M), CNS cancer SF-268 cell line
(GI50 < 0.0001 10−5 M), leukemia SR cell line (GI50
0.207·10−6 M), and MCF7 of breast cancer (GI50 0.120·10−5
M). Furthermore compound 9 inhibited totally the growth of
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Stuart Scientific Melt-
ing point SMP1 and are uncorrected. IR spectra were recorded
on Nujol mulls between salt plates, unless otherwise indi-
cated, in BrukerVector 22 spectrophotometer. 1H-NMR spec-
tra were recorded, in DMSO-d6 solution, on a Varian Unity
300 spectrometer. The chemical shift are reported in part per
million (d, ppm) downfield from tetramethylsilane, which was
used as internal standard Elemental analyses were carried out
with a Carlo Erba model 1106 Elemental Analyzer and the
values found were within 0.4% of theoretical values.
SF-268 and SR cell lines at 0.00175.10−5 M and 2.18.10−5
concentration respectively.
M
An aryl substituent at 4-positon of 3,5-dicyanopyridines
appears to favourably modulate antiproliferative activity.
Replacement of phenyl ring with an heterocyclic ring leads
to a loss of activity. The presence of functionality as 4-chlorine
atom or hydroxyl group in 3 or 4 position of phenyl ring has
an enhancing effect on anticancer potency. Thus, 2,6-
bis(benzylamino)-4-(3-hydroxyphenyl)pyridine-3,5-
5.2. General Procedure for the Synthesis
of 3,5-Dicyanopyridines (3-19)
dicarbonitrile (11) is the most active member within the series.
The displacement of hydroxy group from 3-position to
4-position on phenyl ring leads to 12 that retains antitumoral
activity on the same cell lines but at higher concentrations.
Methylation of hydroxy groups is detrimental for the activ-
ity, as well as the introduction on phenyl ring of two or more
Benzylamine (0.54 g, 5 mmol) was added to a solution of
3-amino-3-ethoxypropenenitrile 1 (0.55 g, 5 mmol) in anhy-
drous acetonitrile (10 mL). The resulting solution was kept at
room temperature for 24 hours and then the appropriate alde-

1368
M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
Table 3
GI50 values of compounds 7-9, 11, 12
Compd
7
8
9
11
12
Panel/cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
2.82
-
-
1.52
-
-
1.02
< 0.001
> 10
0.338
-
-
2.74
-
-
MOLT-4
RPMI-8226
SR
3.43
1.89
1.33
2.68
4.86
1.08
3.46
3.35
0.207
0.330
0.311
0.209
2.04
2.19
0.709
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
2.83
2.73
1.61
-
1.06
2.28
1.91
1.02
2.09
2.13
2.43
0.447
0.357
0.239
1.75
4.06
0.499
1.26
> 10
> 10
> 10
0.112
> 10
> 10
> 10
> 10
> 10
1.33
1.16
1.05
0.251
0.291
1.10
2.21
1.49
0.589
3.41
4.85
1.73
0.0315
1.43
4.53
3.23
2.12
2.02
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
4.69
7.11
1.58
1.88
2.23
2.52
1.62
7.40
> 10
1.63
> 10
7.34
4.70
1.89
> 10
> 10
> 10
> 10
> 10
> 10
7.44
3.67
9.56
3.63
3.95
1.45
2.96
6.02
1.88
3.61
0.448
0.433
4.13
0.403
0.449
HT29
KM12
SW-620
CNS Cancer
SF-268
1.43
1.42
2.59
3.16
2.21
1.24
1.26
1.26
1.79
2.34
1.03
0.246
< 0.001
> 10
> 10
> 10
2.61
1.95
2.73
2.71
1.31
5.05
6.78
1.92
SF-295
SF-539
SNB-19
SNB-75
0.292
0.518
1.71
3.94
0.402
U251
> 10
Melanoma
LOX IMVI
MALME-3 M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal Cancer
786-0
1.24
1.44
2.00
1.20
2.59
2.59
6.79
1.47
0.475
2.10
2.32
2.29
4.95
5.12
> 10
1.24
> 10
> 10
> 10
> 10
>10
> 10
> 10
> 10
0.296
1.01
0.665
0.623
6.10
0.168
> 10
0.21
1.67
2.95
2.99
4.11
6.67
1.53
4.32
2.15
1.35
1.47
0.957
2.42
1.90
3.32
0.508
1.84
0.503
2.68
1.14
6.27
3.56
> 10
3.47
9.38
> 10
> 10
1.97
0.331
2.36
2.13
1.92
0.779
2.35
2.96
2.77
6.03
5.81
3.45
2.00
1.04
1.34
2.01
2.66
1.61
1.44
3.19
1.34
1.92
0.573
1.46
2.46
0.453
2.23
3.81
> 10
> 10
> 10
> 10
7.07
> 10
> 10
> 10
1.30
2.30
2.49
1.69
1.07
3.91
0.598
2.26
1.86
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
0.201
0.263
0.277
1.38
0.231
0.411
0.612
Prostate Cancer
PC-3
1.79
1.77
1.20
2.15
5.58
>10
0.466
1.28
2.56
3.24
DU-145
Breast Cancer
MCF7
1.38
1.98
1.42
1.56
1.57
1.96
2.73
–4.71
2.00
3.71
0.576
1.13
2.18
1.87
6.73
–4.75
0.120
> 10
> 10
8.57
> 10
-
1.27
1.38
3.97
4.70
0.268
2.29
2.52
3.28
2.38
–4.62
NCI/ADR-RES
MDA-MB-231/ ATCC
HS 578T
MDA-MB-435
BT-549
0.223
0.376
0.291
1.01
0.403
–5.12
T-47D
7.23
–4.28
a
MG-MID
a Full panel mean-graph mid point (log GI50).

M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
1369
Table 4
TGI values of compounds 7-9, 11, 12
Compd
7
8
9
11
12
Panel/cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
> 10
-
-
> 10
9.71
5.28
4.55
-
-
> 10
> 10
3.28
2.85
> 10
> 10
> 10
> 10
2.18
> 10
-
-
> 10
2.28
0.748
> 10
-
-
> 10
> 10
9.17
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
> 10
7.90
3.12
-
3.12
8.00
8.69
2.87
5.75
> 10
8.53
1.80
2.55
1.45
> 10
> 10
3.01
5.50
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
6.35
4.50
2.31
1.89
0.786
3.74
8.62
3.14
3.30
> 10
> 10
9.30
0.798
6.71
> 10
> 10
5.38
7.51
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
> 10
> 10
4.13
> 10
> 10
> 10
9.69
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1.61
3.37
> 10
> 10
6.82
> 10
> 10
3.41
>10
> 10
5.97
> 10
HT29
KM12
SW-620
CNS Cancer
SF-268
3.56
3.44
6.81
> 10
7.23
2.69
5.85
7.62
4.05
> 10
4.88
1.03
0.00175
> 10
> 10
> 10
> 10
4.65
1.32
2.21
> 10
> 10
1.54
> 10
> 10
3.74
> 10
> 10
5.45
SF-295
SF-539
SNB-19
SNB-75
U251
> 10
Melanoma
LOX IMVI
MALME-3 M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal Cancer
786-0
2.93
5.45
9.98
1.89
> 10
> 10
> 10
3.35
3.59
9.02
> 10
9.34
> 10
> 10
> 10
6.02
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
1.15
3.25
> 10
2.30
> 10
0.432
> 10
0.704
4.14
6.85
> 10
9.07
> 10
3.43
> 10
> 10
3.24
3.71
> 10
7.61
5.72
> 10
4.11
4.97
4.53
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
4.11
1.94
>10
4.80
5.22
4.65
7.48
8.74
>10
>10
>10
>10
3.86
2.64
3.28
4.67
7.41
3.83
3.28
> 10
3.63
4.69
6.28
> 10
> 10
2.51
6.60
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
2.76
0.577
1.17
1.94
3.49
0.669
1.74
8.76
> 10
6.38
5.71
4.48
> 10
5.32
5.94
6.85
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
4.87
5.57
> 10
8.86
> 10
> 10
2.88
3.44
> 10
> 10
DU-145
Breast Cancer
MCF7
8.31
5.99
3.32
4.04
4.36
6.04
> 10
> 10
> 10
3.38
4.30
> 10
5.39
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
8.84
4.80
0.734
2.07
0.997
3.58
> 10
> 10
> 10
3.30
> 10
6.98
> 10
> 10
NCI/ADR-RES
MDA-MB-231/ ATCC
HS 578T
MDA-MB-435
BT-549
T-47D

1370
M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
Table 5
LC50 values of compounds 7-9, 11, 12
Compd
7
8
9
11
12
Panel/cell line
Leukemia
CCRF-CEM
HL-60(TB)
K-562
> 10
-
-
> 10
> 10
> 10
> 10
-
-
> 10
> 10
9.96
8.04
> 10
> 10
> 10
> 10
> 10
> 10
-
-
> 10
> 10
> 10
> 10
-
-
> 10
> 10
> 10
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
> 10
> 10
6.04
-
9.21
> 10
> 10
8.11
> 10
> 10
> 10
5.33
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
506
7.73
5.84
> 10
> 10
6.61
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322 M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
4.01
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
HT29
KM12
SW-620
CNS Cancer
SF-268
8.91
8.34
> 10
> 10
> 10
5.82
> 10
> 10
9.14
> 10
> 10
6.98
> 10
> 10
> 10
> 10
> 10
> 10
> 10
6.04
8.30
> 10
> 10
4.89
> 10
> 10
> 10
> 10
> 10
> 10
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3 M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal Cancer
786-0
6.88
> 10
> 10
6.06
> 10
> 10
> 10
7.62
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
4.03
> 10
> 10
7.76
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
7.70
> 10
> 10
7.80
9.33
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
8.58
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
7.47
6.75
8.05
> 10
> 10
9.12
7.49
> 10
9.82
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
5.83
2.54
4.34
> 10
8.83
2.89
6.18
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
> 10
> 10
> 10
> 10
> 10
> 10
> 10
9.20
> 0
> 10
DU-145
Breast Cancer
MCF7
> 10
> 10
7.76
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
5.03
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
NCI/ADR-RES
MDA-MB-231/ ATCC
HS 578T
MDA-MB-435
BT-549
T-47D

M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
1371
hyde (2.5 mmol) was added. The resulting mixture was
refluxed for 3 h. After cooling, the formed precipitate was
filtered off and washed with diethyl ether.
5.2.8. 2,6-Bis(benzylamino)-4-(2,6-dichlorophenyl)pyri-
dine-3,5-dicarbonitrile (10)
Yield 87%. M.p. 220 °C. Anal. (C27H191N5Cl2) C, H, N. IR
(Nujol): 3373, 2204, 1626, 1581 cm−1. H-NMR (DMSO-
d6) d: 4.54 (d, J = 5.8 Hz, 4H, CH2), 7.10 (m, 10H, benzyl),
7.26-7.66 (m, 3H, aryl), 8.40 (t, J = 5.8 Hz, 2H, NH).
5.2.1. 2,6-Bis(benzylamino)-4-(4-methoxyophenyl)pyridine-
3,5-dicarbonitrile (3)
Yield 88%. M.p. 245 °C. Anal. (C28H23N5O) C, H, N. IR
1
(Nujol): 3376, 3334, 2195, 1588 cm−1. H-NMR (DMSO-
5.2.9. 2,6-Bis(benzylamino)-4-(3-hydroxyphenyl)pyridine-
3,5-dicarbonitrile(11)
Yield 63%. M.p. 215 °C. Anal. (C27H21N5O) C, H, N. IR
(Nujol): 3348, 2200, 1566 cm−1. 1H-NMR (DMSO-d6) d: 4.47
(d, J = 5.8 Hz, 4H, CH2), 6.82, 7.25 (m, 4H, aryl), 7.16 (s,
10H, benzyl), 8.10 (t, J = 5.8 Hz, 2H, NH), 9.73 (s, 1H, OH).
d6): 3.78 (s, 3H, OCH3), 4.47 (d, J = 5.8 Hz, 4H, CH2), 7.03
(d, J = 8.8 Hz, 2H, aryl), 7.40 (d, J = 8.4 Hz, 2H, Aryl), 7.17
(m, 10H, benzyl), 8.08 (t, J = 5.8 Hz, 2H, NH).
5.2.2. 2,6-Bis(benzylamino)-4-(3-methoxyphenyl)pyridine-
3,5-dicarbonitrile (4)
Yield 82%. M.p. 210 °C. Anal. (C28H23N5O) C, H, N. IR
5.2.10. 2,6-Bis(benzylamino)-4-(4-hydroxyphenyl)pyridine-
3,5-dicarbonitrile (12)
1
(Nujol): 3347, 3302, 2219, 2202, 1595 cm−1. H-NMR
(DMSO-d6): 3.75 (s, 3H, OCH3), 4.48 (d, J = 5.8 Hz, 4H,
CH2), 7.02, 7.19, 7.34 (m, 14H, aryl and benzyl), 8.13 (t,
J = 5.8 Hz, 2H, NH).
Yield 68%. M.p. 260 °C. Anal. (C27H21N5O) C, H, N. IR
1
(Nujol): 3496, 3318, 2201, 1628, 1592 cm−1. H-NMR
(DMSO-d6) d: 4.47 (d, J = 5.8 Hz, 4H, CH2), 6.82 (d,
J = 8.4 Hz, 2H, aryl), 7.16 (s, 10H, benzyl), 7.28 (d, J = 8.4 Hz,
2H, Aryl), 8.05 (t, J = 5.8 Hz, 2H, NH), 10.04 (s, 1H, OH).
5.2.3. 2,6-Bis(benzylamino)-4-(2,5-dimethoxyphenyl)pyri-
dine-3,5-dicarbonitrile (5)
Yield 75%. M.p. 235 °C. Anal. (C29H251N5O2) C, H, N. IR
(Nujol): 3370, 3338, 2202, 1578 cm−1. H-NMR (DMSO-
d6) d: 3.68 (s, 6H, 2OCH3), 4.47 (d, J = 6.1 Hz, 4H, CH2),
6.84-7.18 (m, 13H, aryl and benzyl), 8.08 (t, J = 6.1 Hz, 2H,
NH).
5.2.11. 2,6-Bis(benzylamino)-4-(4-hydroxy-3-methoxyphe-
nyl)pyridine-3,5-dicarbonitrile(13)
Yield 60%. M.p. 220 °C. Anal. (C28H231N5O2) C, H, N. IR
(Nujol): 3403, 3309, 2211, 1587 cm−1. H-NMR (DMSO-
d6) d: 3.75 (s, 3H, OCH3), 4.47 (d, J = 5.8 Hz, 4H, CH2),
6.86, 7.05 (m, 3H, aryl), 7.16 (s, 10H, benzyl), 8.05 (t,
J = 5.8 Hz, 2H, NH), 9.48 (s, 1H, OH).
5.2.4. 2,6-Bis(benzylamino)-4-(3,4,5-trimethoxyphenyl)py-
ridine-3,5-dicarbonitrile (6)
Yield 80%. M.p. 200 °C. Anal. (C30H27N5O3) C, H, N. IR
(Nujol): 3320, 2203, 1573 cm−1. 1H-NMR (DMSO-d6) d: 3.69
(s, 3H, OCH3), 3.75 (s, 6H, OCH3), 4.48 (d, J 5.8 Hz, 4H,
CH2), 6.80 (s, 2H, aryl), 7.20 (s, 10H, benzyl), 8.11 (t,
J = 5.8 Hz, 2H, NH).
5.2.12. 2,6-Bis(benzylamino)-4-(2-pyridinyl)pyridine-3,5-
dicarbonitrile(14)
Yield 57% M.p. 220 °C. Anal. (C26H20N6) C, H, N. IR
(Nujol) 3326, 2200, 1597, 1577 cm−1. 1H-NMR (DMSO-d6)
d: 4.49 (d, J = 5.8 Hz, 4H, CH2), 7.18 (s, 10H, Benzyl), 7.51,
7.65, 7.96, 8.70 (m, 4H, pyridyl), 8.20 (t, J = 5.8 Hz, 2H, NH).
5.2.5. 2,6-Bis(benzylamino)-4-(4-chlorophenyl)pyridine-
3,5-dicarbonitrile (7)
Yield 93%. M.p. 220 °C. Anal. (C27H20N5Cl) C, H, N. IR
(Nujol): 3317, 3085, 3030, 2200, 2159, 1592, 1578 cm−1.
1H-NMR (DMSO-d6) d: 4.54 (d, J = 5.8 Hz, 4H, CH2), 7.16-
7.58 (m, 14H, aryl and benzyl), 8.05 (t, J = 5.8 Hz, 2H, NH).
5.2.13. 2,6-Bis(benzylamino)-4-(3-pyridinyl)pyridine-3,5-
dicarbonitrile (15)
Yield 68%. M.p. 250 °C. Anal. (C26H20N6) C, H, N. IR
1
(Nujol): 3350, 3311, 2215, 2198, 1581 cm−1. H-NMR
(DMSO-d6) d: 4.49 (m, 4H, CH2), 7.18 (m, 10H, benzyl),
7.54, 7.94, 8.68 (m, 4H, pyridyl), 8.24 (m, 2H, NH).
5.2.6. 2,6-Bis(benzylamino)-4-(2-chlorophenyl)pyridine-
3,5-dicarbonitrile (8)
5.2.14. 2,6-Bis(benzylamino)-4-(4-pyridinyl)pyridine-3,5-
dicarbonitrile (16)
Yield 90%. M.p. 210 °C. Anal. (C27H20N5Cl) C, H, N. IR
1
(Nujol): 3503, 3464, 3356, 2204, 1624 cm−1. H-NMR
Yield 70%. M.p. 235 °C. Anal. (C26H20N6) C, H, N. IR
(Nujol) 3475, 3328, 3032, 2202, 1632, 1596 cm−1. 1H-NMR
(DMSO-d6) d: 4.48 (d, J = 5.8 Hz, 4H, CH2), 7.17 (m, 10H,
benzyl), 7.49, 8.71 (m, 4H, pyridyl), 8.27 (t, J = 5.8 Hz, 2H,
NH).
(DMSO-d6) d: 4.50 (d, J = 5.8 Hz, 4H, CH2), 7.18-7.61 (m,
14H, aryl and benzyl), 8.12 (t, J = 5.8 Hz, 2H, 2NH).
5.2.7. 2,6-Bis(benzylamino)-4-(2,4-dichlorophenyl)pyri-
dine-3,5-dicarbonitrile (9)
Yield 82%. M.p. 225 °C. Anal. (C27H19N5Cl2) C, H, N. IR
1
(Nujol): 3335, 3285, 2210, 1657, 1580 cm−1. H-NMR
5.2.15. 2,6-Bis-benzylamino-4-thiophen-2-yl-pyridine-3,5-
dicarbonitrile (17)
(DMSO-d6) d: 4.62 (d, J = 5.8 Hz, 4H, CH2), 7.18 (s, 10H,
benzyl), 7.54 (m, 2H, aryl), 7.83 (s, 1H, aryl), 8.29 (d,
J = 5.8 Hz, 2H, NH).
Yield 57%. M.p. 215 °C. Anal. (C25H19N5S) C, H, N. IR
(Nujol): 3345, 2198, 1591 cm−1. 1H-NMR (DMSO-d6) d: 4.47

1372
M.T. Cocco et al. / European Journal of Medicinal Chemistry 40 (2005) 1365–1372
(d, J = 5.8 Hz, 4H, CH2), 7.06 (s, 10H, benzyl), 7.09, 7.44,
7.82 (m, 3H, thienyl), 8.17 (t, J = 5.8 Hz, 2H, NH).
evaluated and the assay results and dose–response param-
eters were calculated as previously described [19].
5.2.16. 2,6-Bis-benzylamino-4-furan-2-yl-pyridine-3,5-
dicarbonitrile(18)
Yield 61%. M.p. 240 °C. Anal. (C25H19N5O) C, H, N. IR
(Nujol) 3342, 3030, 2202, 1589 cm−1. 1H-NMR (DMSO-d6)
d: 4.46 (d, J = 4.5 Hz, 4H, CH2), 6.73, 7.20, 7.98 (m, 3H,
furyl), 7.15 (s, 10H, benzyl), 8.14 (t, J = 4.5 Hz, 2H, NH).
Acknowledgements
We thank the Antitumor Evaluation Branch and Biologi-
cal Testing Branch of the National Cancer Institute for per-
forming biological evaluations.
References
5.2.17. 2,6-Bis-benzylamino-4-(5-methyl-furan-2-yl)-pyri-
dine-3,5-dicarbonitrile (19)
[1] W.J. Scott, J. Dumas, S. Boyer, W. Lee, Y. Chen, B. Phillips, s. verna,
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(Nujol) 3348, 2210, 1592, 1569 cm−1. 1H-NMR (DMSO-d6)
d: 2.33 (s, 3H, CH3), 4.46 (d, J = 5.8 Hz, 4H, CH2), 6.36 (m,
1H, furyl), 7.16 (m, 11H, benzyl and furyl), 8.06 (t, J = 5.8 Hz,
2H, NH).
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5.3. Pharmacology
5.3.1. Primary anticancer assay
The compounds were tested by NCI in an in vitro three
cell line, one dose primary anticancer assay as a primary can-
cer screen. The three-cell line panel consists of the MCF7
(breast), NCI-H460 (lung) and SF-268 (CNS). Each cell line
is inoculated and preincubated on a microtriter plate. Test
agents are then added at single 10−4 M concentration and the
culture incubated for 48 h. End-point determinations are made
with alamar blue [22]. Results for each test agent are reported
as the percent of growth of the treated cells when compared
to untreated control cells. Compounds, which reduce the
growth of any one of the cell lines to approximately 32% or
less, are passed on for evaluation in the full panel of 60 cell
lines over a 5-log dose range.
5.3.2. Determination of GI50, TGI and LC50 values
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cancer types (leukemia, lung, colon, brain, melanoma, ova-
rian, renal, prostate and breast) formed the basis of this test.
The tumor cells were cultured in RPMI1640 medium supple-
mented with 5% foetal calf serum and 2 mM L-glutamine.
The tumor cells are inoculated over a series of standard
96-well microtrite plates in 100 mL of medium [16,17]. Den-
sity of inoculum depends on the type of tumor cell and from
its growth characteristics [23]. These cells are then preincu-
bated on the microtrite plate for 24 h before adding the com-
pounds. These were tested in DMSO solution at five different
concentrations (10−4, 10−5, 10−6, 10−7 and 10−8 M). After an
incubation of the chemical agent for 48 h with the tumor cell
lines a sulforhodamine B (SRB) protein assay was used to
estimate cell viability or growth. The cytotoxic effects are
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N.L. Anderson, J.K. Buolamwini, W.W. van Osdol, A.P. Monks,
D.A. Scudiero, E.A. Sausville, D.W. Zaharevitz, B. Bunow,
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C. Hose, J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich,
H. Campbell, J. Mayo, M. Boyd, J. Natl. Cancer Inst. 83 (1991)
757–766.