Novel synthesis of nicotinamide derivatives of cytotoxic properties

Article abstract of DOI:10.1016/j.bmc.2006.02.031

A variety of 2-substituted-4,6-diaryl-3-pyridinecarboxamides 5 were synthesized through aromatic nucleophilic substitution reaction of secondary amines with 2-bromo analogues 4. The latter were obtained via bromination of 2-cyano-3,5-diaryl-5-oxo-N-substi

Full text of DOI:10.1016/j.bmc.2006.02.031

Bioorganic & Medicinal Chemistry 14 (2006) 4466–4476
Novel synthesis of nicotinamide derivatives of cytotoxic properties
Adel S. Girgis,a,* Hanaa M. Hosnia and Flora F. Barsoumb
aPesticide Chemistry Department, National Research Centre, Dokki, 12622 Cairo, Egypt
bPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Received 20 November 2005; revised 15 February 2006; accepted 15 February 2006
Available online 9 March 2006
Abstract—A variety of 2-substituted-4,6-diaryl-3-pyridinecarboxamides 5 were synthesized through aromatic nucleophilic substitu-
tion reaction of secondary amines with 2-bromo analogues 4. The latter were obtained via bromination of 2-cyano-3,5-diaryl-5-oxo-
N-substituted pentamides 3 in glacial acetic acid. Moreover, pentamide derivatives 3 were prepared through base-catalyzed Michael
addition of cyanacetanilides 2 with 1,3-diaryl-2-propen-1-ones 1. Otherwise, reaction of 2-bromo-3-pyridinecarboxamides 4 with
primary aromatic amines in refluxing pyridine afforded the corresponding 2-(arylamino)-3-pyridinecarboxamides 6 besides the unex-
pected 2-unsubstituted amino analogues 7. Antitumor properties of the synthesized pyridinecarboxamides utilizing 59 different
human tumor cell lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate as well
as kidney, were screened. Many of the tested compounds show considerable in vitro antitumor properties especially 5c and 7a, which
reveal moderate activities against most of the used human tumor cell lines. It has also been achieved that, all the tested nicotinamide
derivatives reveal promising antitumor properties against MDA-MB-231/ATCC (breast cancer).
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Many publications stated that, treatment with nicotin-
amide prevents or delays insulin-deficient diabetes
(anti-diabetogenic activity) via improving beta cell func-
tion.14–20 It has also been achieved that, nicotinamide is
an oral antimicrobial active agent for co-infection with
Mycobacterium tuberculosis and human immunodefi-
ciency virus (HIV) which is responsible for one-third of
all deaths due to acquired immunodeficiency syndrome
(AIDS).21 Numerous pharmacological compositions
containing nicotinamide were appeared to be effective
as antidandruff, anti-itching, hair growth promoting,
gray hair preventing, increasing hair elasticity, and treat-
ing acne, fine lines as well as age spots.22–31
Nicotinamide (3-pyridinecarboxamide) ‘vitamin B3’ has
been mainly used to treat pellagra in past clinical prac-
tice1 and is an effective agent in treating some neurode-
generative diseases ‘for example, Alzheimer’s disease,
Parkinson’s disease and other cognitive disorders of
mammals’.2 It has been also reported to be a potential
therapeutic agent for the treatment of retinal degenera-
tion.3 Other publications mentioned that, nicotinamide
and its derivatives both prevent and reverse neuronal
and vascular cell injury.4–6 Moreover, nicotinamide is
helpful in the maintenance of cellular energy balance
during ischemia and reperfusion conditions (e.g., cardi-
ac, testicles, and liver injury). Many administrations of
nicotinamide suggested the therapeutic interest in
preventing the development of stroke by rescuing the
still viable but injured cells and partially preventing
infarction.7–9 Others were used for treating acquired
immunodeficiency syndrome (AIDS),10,11 inflammatory,
allergic, and respiratory diseases.12,13
Moreover, various substituted nicotinamides were used
as fungicides,32,33 pesticides34–39 or for treatment of
benign prostatic hyperplasia.40 Nikethamide ‘N,N-dieth-
yl-3-pyridinecarboxamide’ is a well-known drug used as
respiratory analeptic.1
In continuation of our previous work directed toward
construction of heterocycles bearing natural product
residue related compounds of anticipated biological
and/or pharmacological properties,41,42 it is intended
in the present work to investigate the synthesis of novel
3-pyridinecarboxamide derivatives utilizing easily acces-
sible starting materials and facile synthetic approaches.
The interest for the synthesis of these analogues is also
Keywords: 2-Propen-1-ones; Cyanacetanilides; 3-Pyridinecarboxa-
mides; Michael reaction; Aromatic nucleophilic substitution.
*
Corresponding author. Tel.: +202 2352405; fax: +202 3370931;
e-mail: girgisas10@yahoo.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.031

A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
4467
originated due to activities associated with the structure
of 2-(substituted amino)-3-pyridinecarboxamide deriva-
tives as antitumor43–45 in addition to inhibitory
properties against the proliferation of human immuno-
deficiency virus (HIV) as well as hepatitis B virus
(HBV) and hepatitis C virus (HCV).46 The antitumor
properties of the newly synthesized 3-pyridinecarboxa-
mide derivatives against a variety of human tumor cell
lines will be also screened.
pyridine gave two products which were separated and
purified by silica gel 60G F254 TLC. The structures of
which were established to be 2-(arylamino)nicotina-
mides 6a–i and the unexpected 2-(unsubstituted
amino)nicotinamide analogues 7a–c based on spectro-
1
scopic (IR, H NMR, and MS) and elemental analyses
data (Scheme 2).
Formation of 7 probably took place through imino-
form isomerization originated from the primary aromat-
ic amines under the used basic reaction conditions
(refluxing pyridine), which via hydrolysis ‘due to
unavoidable moisture’ liberated ammonia. The latter
due to aromatic nucleophilic substitution with the used
starting 2-bromo-3-pyridinecarboxamides 4 gave finally,
the 2-amino-3-pyridinecarboxamides 7. It has been no-
ticed that, the yields of 7a–c (39–30%) were greater in
case of using aromatic amine substituted with deactivat-
ing moiety ‘p-chloroaniline’ compared with the cases,
when the amines were substituted with electron-donat-
ing or-activating functions (p-toluidine ‘yields of 7a–c
are 16–15%’ and p-anisidine ‘yields of 7a and b are
11–10%’). This observation supports the role of substi-
tuent attached with the used aromatic amine in deriving
the reaction mechanistic route toward the unexpected
product 7 formation, which coincides with the role of
substitution favoring the imino-form process isomeriza-
tion during the reaction course. Eventually, it could be
concluded that, the opportunity of 7 formation under
the described basic reaction conditions appeared greater
when the used primary aromatic amine substituted with
deactivating moieties. Formation of 7 during the reac-
tion course seems similar to what was previously report-
ed about the yielding of 2-amino-3-pyridinecarbonitrile
derivatives through the reaction of 2-bromo analogues
with a-primary amino acid esters (glycine or alanine
esters) in refluxing pyridine.41 It was assumed in the
2. Results and discussion
2.1. Chemistry
Reaction of 1,3-diaryl-2-propen-1-ones 1a and b with
cyanacetamides 2a and b in refluxing ethanol in the
presence of a catalytic amount of morpholine as a basic
catalyst afforded the corresponding 2-cyano-3,5-diaryl-
5-oxo-N-substituted pentamides 3a–c in good yields.
The structure of the latter was established through
1
spectroscopic (IR and H NMR) as well as elemental
analyses data.
Addition of bromine to pentamides 3a–c in glacial acetic
acid at 60–70 ꢁC gave directly the 2-bromo-4,6-diaryl-N-
substituted-3-pyridinecarboxamides 4a–c in 82–77%
yield. The structure of 4a–c was deduced through spec-
1
troscopic (IR, H NMR, and MS) and elemental analy-
ses data (Scheme 1).
Reaction of 2-bromonicotinamides 4a–c with secondary
amines ‘piperidine and morpholine’ in refluxing tetrahy-
drofuran afforded smoothly, the 2-substituted nicotin-
amide derivatives 5a–f in good yields. On the other
hand, reaction of 4a–c with aromatic amines ‘aniline,
p-toluidine, p-anisidine, and p-chloroaniline’ in refluxing
R
O
Ph
O
H
N
EtOH, morpholine
R'
CN
H
+
N
O
Ph
R
O
R'
CN
(1)
(2)
(3)
Br2, AcOH
O
R
N
R'
N
H
Ph
Br
(4)
1a; R = 4-ClC6H4
1b; R = 4-H3CC6H4
2a; R = Ph
3a, 4a; R = 4-ClC6H4, R' = Ph
3b, 4b; R = 4-ClC6H4, R' = 4-H3CC6H4
3c, 4c; R = 4-H3CC6H4, R' = Ph
2b; R = 4-H3CC6H4
Scheme 1.

4468
A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
O
R
R'
X
X
NH
N
H
THF
Ph
N
N
(5)
(4)
R
O
R
O
R2NH2
R'
R'
N
N
H
H
+
Ph
pyridine
NHR2
Ph
N
N
NH2
(6)
(7)
5a; R = 4-ClC6H4, R' = Ph, X = CH2
5b; R = 4-ClC6H4, R' = Ph, X = O
6a; R = 4-ClC6H4, R' = R2 = Ph
6b; R = 4-ClC6H4, R' = Ph, R2 = 4-H3CC6H4
6c; R = 4-ClC6H4, R' = Ph, R2 = 4-H3COC6H4
6d; R = R2 = 4-ClC6H4, R' = Ph
6e; R = 4-ClC6H4, R' = R2 = 4-H3CC6H4
6f; R = 4-ClC6H4, R' = 4-H3CC6H4, R2 = 4-H3COC6H4
6g; R = R2 = 4-ClC6H4, R' = 4-H3CC6H4
6h; R = R2 = 4-H3CC6H4, R' = Ph
5c; R = 4-ClC6H4, R' = 4-H3CC6H4, X = CH2
5d; R = 4-ClC6H4, R' = 4-H3CC6H4, X = O
5e; R = 4-H3CC6H4, R' = Ph, X = CH2
5f; R = 4-H3CC6H4, R' = Ph, X = O
7a; R = 4-ClC6H4, R' = Ph
7b; R = 4-ClC6H4, R' = 4-H3CC6H4
7c; R = 4-H3CC6H4, R' = Ph
6i; R = 4-H3CC6H4, R' = Ph, R2 = 4-ClC6H4
Scheme 2.
latter reaction that, the mechanistic pathway proceeded
analogously to the famous ninhydrin reaction with a-
amino acids,47 where the amino acids isomerized to
the corresponding imino-acid forms under the effect of
applied reaction conditions. Then, upon hydrolysis,
due to unavoidable moisture, ammonia was liberated
which in turn interacted with 2-bromo-3-pyridinecar-
bonitriles giving the 2-amino derivatives. Another
observation was also reported about the formation of
2-aminonicotinate esters through the reaction of 2-
bromonicotinates with primary aromatic amines under
similar reaction conditions.42
Other reports assumed that the potent antitumoral
nicotinamide properties were attributed to the primary
mechanism of action regulated by inhibition at the gene
transcription level.51
Antitumor activity screening for the synthesized nicoti-
namides utilizing 59 different human tumor cell lines,
representing leukemia, melanoma, and cancers of the
lung, colon, brain, ovary, breast, prostate as well as kid-
ney, was carried out according to the previously report-
ed standard procedure.52–54 The obtained results
(Table 1) represent concentrations of the used investi-
gated compounds resulting in growth inhibition of
50% (GI50) for the tested human tumor cell lines.
2.2. Antitumor activity
Nicotinamides are a well-known class of drugs that con-
tain many analogues having radio- and chemosensitizing
properties. Particularly, the N-substituted derivatives
which distinguished because they are: (i) susceptible to
radiolysis, (ii) induce cytotoxicity by apoptosis but not
necrosis, (iii) inhibit cell proliferation, (iv) activate poly
adenosine diphosphate ribosyl transferase, and (v) have
a much-reduced effect on microregional tumor blood
perfusion.48,49 Multiple mechanistic properties were pre-
sumed explaining the well-established activities for
example, they are known to affect blood flow, inhibit
DNA repair, and enhance DNA damage induction.50
From the in vitro observed data it has been noticed that,
2-(unsubstituted amino)-3-pyridinecarboxamides 7a and
b reveal better antitumor properties compared with the
corresponding 2-arylamino-3-pyridinecarboxamides 6.
Moreover, 2-(1-piperidinyl)-N-(4-methylphenyl)-3-pyri-
dinecarboxamide 5c exhibits enhanced antitumor prop-
erties than the corresponding tested analogues 5a, b,
and d. It has also been noticed that, all the tested com-
pounds show considerable antitumor activities against
MDA-MB-231/ATCC (breast cancer). Generally,
compounds 5c and 7a exhibit moderate antitumor prop-
erties against most of the tested human tumor cell lines

A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
4469
Table 1. Concentrations resulting in growth inhibition of 50% (GI50, mg/l) of in vitro human tumor cell lines
Panel/cell line
Compound
5a
5b
5c
5d
6a
6c
6d
6f
6g
7a
7b
Leukemia
CCRF-CEM
HL-60(TB)
K-562
>46.80
>46.80
NT
>46.99
1.71
3.40
18.75
>48.40
>48.40
>48.40
>48.40
21.13
>47.60
3.22
>50.60
14.59
30.49
1.84
16.90
>52.00
>52.00
NT
>52.44
>52.44
>52.44
>52.44
NT
8.55
2.90
4.00
3.82
4.59
1.10
3.77
10.79
6.81
7.30
10.42
10.66
4.65
0.17
1.37
0.67
2.07
12.52
0.34
MOLT-4
RPMI-8226
SR
>46.80
>46.80
1.51
2.41
4.59
>52.00
NT
NT
12.11
>47.60
NT
17.95
10.57
0.44
10.42
0.65
NT
NT
2.88
NT
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
16.23
>46.99
28.32
>46.99
8.95
8.00
21.53
8.00
9.62
12.39
6.65
6.50
7.82
6.81
>48.40
>48.40
44.14
>47.60
>47.60
>47.60
>47.60
43.41
>50.60
>50.60
>50.60
>50.60
>50.60
>50.60
>50.60
NT
37.84
39.62
36.98
19.86
>51.04
19.40
22.28
NT
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
NT
>52.44
>52.44
>52.44
>52.44
>52.44
>52.44
NT
12.64
10.76
17.86
6.34
3.86
2.80
24.37
3.86
HOP-92
>48.40
>48.40
31.98
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
>46.99
21.98
>46.99
10.52
3.65
10.52
8.16
12.79
3.44
>47.60
>47.60
>47.60
20.78
15.30
>48.40
6.68
11.01
9.82
7.11
12.50
3.95
4.04
NT
>52.00
NT
>52.44
>50.60
18.53
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
>46.80
>46.80
19.96
>46.99
>46.99
4.09
11.30
25.88
4.60
>48.40
>48.40
13.64
>47.60
>47.60
0.85
9.42
>50.60
12.42
7.21
34.51
5.34
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
>52.44
>52.44
21.36
7.45
14.19
5.91
4.86
12.21
1.89
>46.80
>46.80
>46.80
>46.80
8.17
2.47
9.62
5.93
24.82
45.17
>47.60
12.52
20.61
14.72
5.73
>52.44
>52.44
>52.44
2.63
7.62
2.38
2.49
>50.60
>50.60
>50.60
10.76
11.01
21.47
KM12
SW-620
>46.99
>46.99
15.96
15.60
>48.40
>48.40
>47.60
>47.60
14.72
27.41
4.24
13.09
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
>46.80
>46.80
17.39
>46.99
>46.99
20.05
8.57
8.57
6.21
7.64
8.00
5.66
>48.40
>48.40
16.40
>47.60
>47.60
>47.60
>47.60
>47.60
>47.60
>50.60
>50.60
>50.60
>50.60
NT
8.09
13.74
>51.04
38.72
6.00
>52.00
>52.00
>52.00
>52.00
NT
>52.44
>52.44
>52.44
>52.44
NT
20.51
14.52
14.52
24.09
8.75
20.27
2.67
11.66
27.98
20.27
2.86
>46.80
>46.80
>46.80
>46.99
11.80
27.04
>48.40
15.30
20.65
>50.60
26.79
>52.00
>52.44
8.95
Melanoma
LOXIMVI
MALME-3M
M14
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.99
14.19
5.93
6.21
>48.40
7.50
43.13
>47.60
43.41
23.85
>50.60
>50.60
>50.60
>50.60
43.07
23.87
>51.04
14.39
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
>52.00
>52.44
>52.44
26.28
10.28
8.95
8.75
4.75
5.21
>46.99
>46.99
>46.99
27.04
8.38
4.43
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
13.58
29.72
>48.20
>48.20
10.79
>48.40
>48.40
>48.40
>48.40
24.26
>47.60
>47.60
>47.60
>47.60
>47.60
>51.04
12.53
>52.44
9.54
0.41
18.28
25.23
7.80
22.23
26.72
5.46
>50.60
>50.60
>50.60
>51.04
40.54
29.37
>46.99
8.95
>52.44
>52.44
15.92
5.91
14.69
7.36
Ovarian cancer
IGROV1
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
40.93
>46.99
17.06
9.40
7.82
>48.40
>48.40
>48.40
>48.40
>48.40
>48.40
>47.60
>47.60
>47.60
>47.60
>47.60
>47.60
>50.60
35.82
34.21
>51.04
27.41
10.18
>52.00
NT
>52.44
1.95
NT
7.98
7.62
11.40
3.36
7.36
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
8.00
>52.00
>52.00
>52.00
NT
11.53
35.64
28.97
>39.99
>46.99
>46.99
>46.99
11.83
6.97
14.22
>50.60
>50.60
44.07
>51.04
>51.04
12.53
>52.44
>52.44
NT
>41.39
>41.39
>41.39
Renal cancer
786-0
A498
14.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
>46.80
9.82
>46.99
31.05
>46.99
9.38
3.83
8.77
6.07
15.96
8.77
7.47
7.13
6.81
11.61
>48.40
>48.40
22.12
5.95
12.23
>47.60
>47.60
>47.60
5.72
>50.60
>50.60
>50.60
>50.60
>50.60
>50.60
>50.60
>50.60
>51.04
32.20
17.70
28.05
9.08
>52.00
>52.00
>52.00
>52.00
NT
>52.44
0.81
6.95
39.08
9.16
3.07
>41.39
2.93
ACHN
CAKI-1
RXF-393
SN12C
TK-10
>52.44
4.67
NT
11.53
6.05
6.87
3.00
>46.99
8.95
3.10
>48.40
10.35
5.56
>47.60
>47.60
>47.60
36.98
11.17
21.77
>52.00
>52.00
>52.00
>52.44
14.44
>52.44
10.28
8.75
7.11
3.36
4.14
UO-31
2.61
Prostate cancer
PC-3
Du-145
>46.80
>46.80
8.75
>46.99
7.30
11.83
>48.40
>48.40
38.69
>47.60
33.43
>50.60
11.17
>51.04
>52.00
>52.00
>52.44
>52.44
5.03
19.58
1.89
2.38
(continued on next page)

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A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
Table 1 (continued)
Panel/cell line
Compound
5a
5b
5c
5d
6a
6c
6d
6f
6g
7a
7b
Breast cancer
MCF7
>46.80
>46.80
30.92
26.43
>46.99
8.17
13.58
>48.40
>48.40
9.01
>47.60
>47.60
44.42
NT
>50.60
49.45
NT
NT
>52.00
4.63
NT
7.80
9.37
9.59
2.17
4.64
9.05
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
6.50
6.07
44.45
>52.44
17.37
0.47
9.95
17.70
25.58
>51.04
6.73
>46.80
>46.80
30.92
>46.99
23.55
13.90
15.60
12.97
11.56
>48.40
>48.40
>48.40
>48.40
>47.60
>47.60
>47.60
>47.60
>50.60
>50.60
>50.60
>50.60
>52.00
NT
39.99
8.95
>41.39
2.22
8.26
MDA-MB-435
BT-549
T-47D
NT
>52.44
20.40
>46.99
>46.99
>52.00
>52.00
13.86
13.55
>46.80
3.69
NT, Not tested.
(Figs. 1 and 2). It has also been noticed that, compound
7b reveals considerable antitumor properties among all
the tested cancer cell lines except OVCAR-5, OVCAR-
8, SK-OV-3 (ovarian cancer), A498 (renal cancer), and
HS 578T (breast cancer). Similarly, compound 6d re-
veals moderate antitumor activities against all the tested
cancer cell lines except, NCI-H226 (non-small cell lung
cancer), SF-539 (CNS cancer), MALME-3M, SK-
MEL-2, SK-MEL-5 (melanoma), IGROV1, OVCAR-
5, OVCAR-8 (ovarian cancer), 786-0 (renal cancer),
Du-145 (prostate cancer), and BT-549 (breast cancer).
It has also been noticed that, compound 7b seems to
be the most active prepared nicotinamide derivative
against all the tested leukemia, colon, and breast cancer
cell lines considering the observed effective low GI50
concentrations. However, compound 5c reveals the best
detected antitumor affinities against most of the tested
CNS and ovarian cancer cell lines. In addition, few pre-
pared compounds show remarkable antitumor GI50
properties against the tested human tumor cell lines at
low concentrations (<1.0 mg/l) particularly, 5b
(GI50 = 0.65 mg/l against SR), 6a (GI50 = 0.34,
0.85 mg/l against MOLT-4 and HCT-116, respectively),
6d (GI50 = 0.44 mg/l against RPMI-8226), 6g
(GI50 = 0.41, 0.81, and 0.47 mg/l against SK-MEL-5,
A498, and HS 578T, respectively), and 7b (GI50 = 0.17,
0.67 mg/l against HL-60(TB) and MOLT-4, respective-
ly). From all the above data, especially those observed
through compounds 7a and b, it could be concluded
that, 2-amino-N-arylnicotinamide analogues may be a
hint for determining a highly effective broad spectrum
antitumor agent.
Figure 1. Dose–response curves for the tested human tumor cell lines of compound 5c.

A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
4471
Figure 2. Dose–response curves for the tested human tumor cell lines of compound 7a.
3. Experimental
165–167 ꢁC, yield 80%. IR: mmax/cmÀ1 3299 (NH), 2251
(C„N), 1687, 1658 (C@O), 1600, 1548 (C@C). 1H
NMR (CDCl3): d 3.57 (dd, 1H, upfield H of CH2CH,
J = 5.1, 17.7 Hz), 3.69 (dd, 1H, downfield H of CH2CH,
J = 8.1, 17.7 Hz), 4.23–4.29 (m, 2H, CH2CH +
CH2CHCH), 7.10–7.97 (m, 15H, 14 arom. H + NH).
Anal. Calcd for C24H19ClN2O2 (402.863): C, 71.55; H,
4.75; N, 6.96. Found: C, 71.68; H, 4.89; N, 7.22.
Melting points are uncorrected and recorded on an Elec-
trothermal 9100 digital melting point apparatus. IR
spectra were recorded (KBr) on a Bruker Vector 22
1
spectrophotometer. H NMR spectra were recorded on
a Varian MERCURY 300 (300 MHz). Mass spectra
were recorded on a Finnigan SSQ 7000 spectrometer
(EI 70 eV). The starting compounds 1a and b,55 2a
and b56 were prepared according to the previously
reported procedures.
3.1.2. 3-(4-Chlorophenyl)-2-cyano-N-(4-methylphenyl)-5-
oxo-5-phenylpentamide (3b). Reaction time 15 h, color-
less crystals from n-butanol, mp 201–203 ꢁC, yield
74%. IR: mmax/cmÀ1 3319 (NH), 2250 (C„N), 1684
(C@O), 1606, 1542 (C@C). 1H NMR (DMSO-d6): d
2.26 (s, 3H, CH3), 3.45 (dd, 1H, upfield H of CH2CH,
J = 3.9, 17.4 Hz), 3.82 (dd, 1H, downfield H of CH2CH,
J = 9.9, 17.7 Hz), 4.05–4.15 (m, 1H, CH2CH), 4.36 (d,
1H, CH2CHCH, J = 8.4 Hz), 7.09–7.95 (m, 13H, arom.
H), 10.38 (s, 1H, NH). Anal. Calcd for C25H21ClN2O2
(416.893): C, 72.02; H, 5.08; N, 6.72. Found: C, 72.22;
H, 5.27; N, 6.98.
3.1. Synthesis of 2-cyano-3,5-diaryl-5-oxo-N-substituted
pentamides (3a–c)
A mixture of equimolar amounts of 1a and b (10 mmol)
and the corresponding 2a and b in absolute ethanol
(20 ml) containing morpholine (3–5 drops) was boiled
under reflux for the appropriate time. The solid separat-
ed, while refluxing, was collected and crystallized from a
suitable solvent affording the corresponding 3b and c. In
case of 3a, the reaction mixture was evaporated untill
dryness under reduced pressure and the remaining resi-
due was triturated with methanol (5 ml). So, the separat-
ed solid was collected and crystallized from a suitable
solvent giving 3a.
3.1.3.
2-Cyano-3-(4-methylphenyl)-5-oxo-5-phenyl-N-
phenylpentamide (3c). Reaction time 20 h, colorless crys-
tals from n-butanol, mp 194–196 ꢁC, yield 68%. IR:
m
max/cmÀ1 3322 (NH), 2254 (C„N), 1691 (C@O),
1606, 1551 (C@C). 1H NMR (DMSO-d6): d 2.24 (s,
3H, CH3), 3.39 (dd, 1H, upfield H of CH2CH, J = 3.9,
17.1 Hz), 3.77 (dd, 1H, downfield H of CH2CH,
J = 9.3, 17.4 Hz), 3.99–4.07 (m, 1H, CH2CH), 4.34 (d,
3.1.1. 3-(4-Chlorophenyl)-2-cyano-5-oxo-5-phenyl-N-phe-
nylpentamide (3a). Reaction time 18 h, colorless crystals
from ethyl acetate–n-hexane mixture as 1:4, v/v, mp

4472
A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
1H, CH2CHCH, J = 8.7 Hz), 7.09–7.89 (m, 14H, arom.
H), 10.46 (s, 1H, NH). Anal. Calcd for C25H22N2O2
(382.45): C, 78.51; H, 5.80; N, 7.33. Found: C, 78.58;
H, 5.84; N, 7.15.
3.3.1. 4-(4-Chlorophenyl)-6-phenyl-N-phenyl-2-(1-pipe-
ridinyl)-3-pyridinecarboxamide (5a). Reaction time
40 h, colorless crystals purified by silica gel TLC using
chloroform–light petroleum (60–80 ꢁC) mixture as 3:1,
v/v, for elution, mp 194–196 ꢁC, yield 69%. IR: mmax
/
3.2. Synthesis of 2-bromo-4, 6-diaryl-N-substituted-3-
pyridinecarboxamides (4a–c)
cmÀ1 3232 (NH), 1643 (C@O), 1597, 1540 (C@N,
1
C@C). H NMR (CDCl3): d 1.60–1.72 (br m, 6H, pip.
3 CH2), 3.47 (br s, 4H, pip. 2 NCH2), 7.11–8.09 (m,
15H, 14 arom. H+pyr. H-5), 8.65 (br s, 1H, NH). MS:
m/z (%) 469 [(M+2), 3], 467 (M, 7), 376 (60), 375
(100), 347 (9). Anal. Calcd for C29H26ClN3O
(467.973): C, 74.43; H, 5.60; N, 8.98. Found: C, 74.34;
H, 5.57; N, 8.87.
To a solution of the appropriate 3a–c (5 mmol) in gla-
cial acetic acid (15 ml), heated at 60–70 ꢁC, a solution
of bromine (5.5 mmol) in glacial acetic acid (5 ml) was
added dropwise while stirring, at such a rate maintain-
ing the same temperature within 1/4 h period. After
complete addition, stirring was continued for 3 h at
the same temperature. The separated solid was collect-
ed and purified on silica gel (60G F254) TLC using
chloroform for elution giving 4a–c as colorless
crystals.
3.3.2. 4-(4-Chlorophenyl)-2-(4-morpholinyl)-6-phenyl-N-
phenyl-3-pyridinecarboxamide (5b). Reaction time 35 h,
colorless crystals purified by silica gel TLC using chloro-
form–light petroleum (60–80 ꢁC) mixture as 5:1, v/v, for
elution, mp 223–225 ꢁC, yield 77%. IR: mmax/cmÀ1 3378
(NH), 1666 (C@O), 1594, 1538 (C@N, C@C). 1H
3.2.1. 2-Bromo-4-(4-chlorophenyl)-6-phenyl-N-phenyl-3-
pyridinecarboxamide (4a). Mp 237–239 ꢁC, yield 82%.
IR: mmax/cmÀ1 3273, 3246 (NH), 1650 (C@O), 1600,
NMR (CDCl3):
d
3.42 (t, 4H, mor. 2NCH2,
J = 4.2 Hz), 3.72 (t, 4H, mor. 2OCH2, J = 4.2 Hz),
7.06–8.00 (m, 16H, 14 arom. H+pyr. H-5+NH). MS:
m/z (%) 471 [(M+2), 4], 469 (M, 9), 378 (42), 377
(100), 349 (11). Anal. Calcd for C28H24ClN3O2
(469.943): C, 71.56; H, 5.15; N, 8.94. Found: C, 71.35;
H, 4.88; N, 9.02.
1
1547 (C@N, C@C). H NMR (DMSO-d6): d 7.10–7.68
(m, 12H, arom. H), 8.08 (s, 1H, pyr. H-5), 8.16-8.19
(m, 2H, arom. H), 10.62 (s, 1H, NH). MS: m/z (%)
466 [(M+4), 2], 464 [(M+2), 8], 462 (M, 6), 371 (13),
370 (62). Anal. Calcd for C24H16BrClN2O (463.752):
C, 62.15; H, 3.48; N, 6.04. Found: C, 62.27; H, 3.57;
N, 6.08.
3.3.3. 4-(4-Chlorophenyl)-N-(4-methylphenyl)-6-phenyl-
2-(1-piperidinyl)-3-pyridinecarboxamide (5c). Reaction
time 50 h, colorless crystals purified by silica gel TLC
using chloroform for elution, mp 222–224 ꢁC, yield
67%. IR: mmax/cmÀ1 3224 (NH), 1640 (C@O), 1596,
3.2.2. 2-Bromo-4-(4-chlorophenyl)-N-(4-methylphenyl)-6-
phenyl-3-pyridinecarboxamide (4b). Mp 263–265 ꢁC,
yield 80%. IR: mmax/cmÀ1 3280 (NH), 1653 (C@O),
1602, 1529 (C@N, C@C). 1H NMR (DMSO-d6): d
2.26 (s, 3H, CH3), 7.10–7.67 (m, 11H, arom. H), 8.09
(s, 1H, pyr. H-5), 8.16-8.19 (m, 2H, arom. H), 10.56
(s, 1H, NH). MS: m/z (%) 480 [(M+4), 8], 478 [(M+2),
26], 476 (M, 20), 372 (100), 371 (16), 370 (75). Anal.
Calcd for C25H18BrClN2O (477.772): C, 62.84; H,
3.80; N, 5.86. Found: C, 62.65; H, 3.69; N, 5.71.
1
1540 (C@N, C@C). H NMR (CDCl3): d 1.52–1.62 (br
m, 6H, pip. 3 CH2), 2.25 (s, 3H, CH3), 3.37 (br s, 4H,
pip. 2 NCH2), 7.04–8.01 (m, 14H, 13 arom. H+pyr. H-
5), 8.35 (br s, 1H, NH). MS: m/z (%) 483 [(M+2), 2],
481 (M, 2), 376 (33), 375 (100), 347 (5). Anal. Calcd
for C30H28ClN3O (481.993): C, 74.75; H, 5.85; N, 8.72.
Found: C, 74.79; H, 5.88; N, 8.82.
3.2.3. 2-Bromo-4-(4-methylphenyl)-6-phenyl-N-phenyl-3-
pyridinecarboxamide (4c). Mp 258–260 ꢁC, yield 77%.
IR: mmax/cmÀ1 3270, 3242 (NH), 1651 (C@O), 1602,
3.3.4. 4-(4-Chlorophenyl)-N-(4-methylphenyl)-2-(4-mor-
pholinyl)-6-phenyl-3-pyridinecarboxamide (5d). Reaction
time 50 h, colorless crystals purified by silica gel TLC
using chloroform for elution, mp 231–233 ꢁC, yield
75%. IR: mmax/cmÀ1 3219 (NH), 1641 (C@O), 1596,
1
1550 (C@N, C@C). H NMR (DMSO-d6): d 2.32 (s,
3H, CH3), 7.07–7.58 (m, 12H, arom. H), 8.04 (s, 1H,
pyr. H-5), 8.15–8.18 (m, 2H, arom. H), 10.67 (s, 1H,
NH). MS: m/z (%) 444 [(M+2), 14], 442 (M, 14), 351
(22), 350 (100). Anal. Calcd for C25H19BrN2O
(443.329): C, 67.73; H, 4.32; N, 6.32. Found: C, 67.47;
H, 4.19; N, 6.25.
1
1538 (C@N, C@C). H NMR (CDCl3): d 2.25 (s, 3H,
CH3), 3.43 (t, 4H, mor. 2NCH2, J = 4.2 Hz), 3.72 (t,
4H, mor. 2OCH2, J = 4.2 Hz), 7.04–7.99 (m, 15H, 13
arom. H+pyr. H-5+NH). MS: m/z (%) 485 [(M+2), 2],
483 (M, 5), 378 (16), 377 (47), 349 (7). Anal. Calcd for
C29H26ClN3O2 (483.973): C, 71.96; H, 5.42; N, 8.68.
Found: C, 72.10; H, 5.50; N, 8.77.
3.3. Reaction of 4 with secondary amines
A mixture of 4a–c (5 mmol) and the corresponding sec-
ondary amine (10 mmol) in tetrahydrofuran (25 ml) was
boiled under reflux for the appropriate time. The reac-
tion mixture was filtered while hot, to remove the sec-
ondary amine hydrobromide salt. Then, the mixture
was evaporated untill dryness under reduced pressure.
The formed solid upon triturating the residual material
with methanol (5 ml) was collected and purified on silica
gel (60G F254) TLC giving 5a–f.
3.3.5. 4-(4-Methylphenyl)-6-phenyl-N-phenyl-2-(1-pipe-
ridinyl)-3-pyridinecarboxamide (5e). Reaction time 60 h,
colorless crystals purified by silica gel TLC using chloro-
form for elution, mp 221–223 ꢁC, yield 71%. IR: mmax
/
cmÀ1 3379 (NH), 1679 (C@O), 1594, 1522 (C@N,
1
C@C). H NMR (CDCl3): d 1.64–1.74 (br m, 6H, pip.
3 CH2), 2.40 (s, 3H, CH3), 3.49 (br s, 4H, pip. 2
NCH2), 7.11–8.11 (m, 15H, 14 arom. H+pyr. H-5),
8.50 (br s, 1H, NH). MS: m/z (%) 447 (M, 3), 356

A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
4473
(23), 355 (100), 327 (5). Anal. Calcd for C30H29N3O
(447.55): C, 80.50; H, 6.53; N, 9.39. Found: C, 80.74;
H, 6.72; N, 9.61.
384 (7), 383 (12). Anal. Calcd for C31H24ClN3O2
(505.973): C, 73.58; H, 4.78; N, 8.30. Found: C, 73.73;
H, 4.95; N, 8.37.
3.3.6. 4-(4-Methylphenyl)-2-(4-morpholinyl)-6-phenyl-N-
phenyl-3-pyridinecarboxamide (5f). Reaction time 55 h,
colorless crystals purified by silica gel TLC using chloro-
3.4.4.
4-(4-Chlorophenyl)-2-[(4-chlorophenyl)amino]-6-
phenyl-N-phenyl-3-pyridinecarboxamide (6d). Reaction
time 75 h, pale yellow crystals purified by silica gel
TLC using chloroform–light petroleum (60–80 ꢁC) mix-
ture as 3:1, v/v, for elution, mp 209–211 ꢁC, yield 18%.
IR: mmax/cmÀ1 3417 (NH), 1660 (C@O), 1614, 1532
(C@N, C@C). 1H NMR (CDCl3): d 6.90 (br s, 1H,
NH), 7.07–8.08 (m, 19 H, 18 arom. H+pyr. H-5), 9.44
(br s, 1H, NH). MS: m/z (%) 513 [(M+4), 7], 511
[(M+2), 34], 509 (M, 45), 418 (69), 417 (100), 389 (10),
388 (8), 387 (11). Anal. Calcd for C30H21Cl2N3O
(510.40): C, 70.59; H, 4.15; N, 8.23. Found: C, 70.81;
H, 4.24; N, 8.37.
form for elution, mp 220–222 ꢁC, yield 76%. IR: mmax
/
cmÀ1 3377 (NH), 1666 (C@O), 1573, 1538 (C@N,
1
C@C). H NMR (CDCl3): d 2.40 (s, 3H, CH3), 3.55 (br
s, 4H, mor. 2NCH2), 3.84 (br s, 4H, mor. 2OCH2),
7.12–8.09 (m, 16H, 14 arom. H+pyr. H-5+NH). MS:
m/z (%) 449 (M, 8), 358 (27), 357 (100), 329 (4). Anal.
Calcd for C29H27N3O2 (449.53): C, 77.48; H, 6.06; N,
9.35. Found: C, 77.45; H, 6.01; N, 9.51.
3.4. Reaction of 4a–c with aromatic amines
A solution of 4a–c (5 mmol) and the corresponding pri-
mary aromatic amine (10 mmol) in pyridine (20 ml) was
boiled under reflux for the appropriate time. The solid
separated upon pouring the reaction mixture into water
(250 ml) and acidification with dil HCl (5%), was collect-
ed and washed with water. Then, it was purified on silica
gel (60G F254) TLC affording 6a–i and 7a–c.
3.4.5. 4-(4-Chlorophenyl)-N-(4-methylphenyl)-2-[(4-meth-
(6e).
ylphenyl)amino]-6-phenyl-3-pyridinecarboxamide
Reaction time 50 h, pale yellow crystals purified by silica
gel TLC using chloroform–light petroleum (60–80 ꢁC)
mixture as 3:1, v/v, for elution, mp 203–205 ꢁC, yield
40%. IR: mmax/cmÀ1 3414, 3323 (NH), 1659 (C@O),
1612, 1514 (C@N, C@C). 1H NMR (CDCl3): d 2.21
(s, 3H, CH3), 2.26 (s, 3H, CH3), 6.76 (br s, 1H, NH),
6.85–8.00 (m, 18 H, 17 arom. H+pyr. H-5), 9.17 (br s,
1H, NH). MS: m/z (%) 505 [(M+2), 18], 503 (M, 54),
398 (39), 397 (100), 369 (7), 368 (8), 367 (19). Anal.
Calcd for C32H26ClN3O (504.003): C, 76.25; H, 5.20;
N, 8.34. Found: C, 76.33; H, 5.31; N, 8.18.
3.4.1. 4-(4-Chlorophenyl)-6-phenyl-N-phenyl-2-(phenyla-
mino)-3-pyridinecarboxamide (6a). Reaction time 75 h,
pale yellow crystals purified by silica gel TLC using
chloroform–light petroleum (60–80 ꢁC) mixture as 3:1,
v/v, for elution, mp 200–202 ꢁC, yield 29%. IR: mmax
/
cmÀ1 3418 (NH), 1663 (C@O), 1621, 1529 (C@N,
C@C). 1H NMR (CDCl3): d 7.02-8.11 (m, 21H, 19
arom. H+pyr. H-5+NH), 9.42 (s, 1H, NH). MS: m/z
(%) 477 [(M+2), 19], 475 (M, 50), 384 (38), 383 (100),
355 (17), 354 (17), 353 (39). Anal. Calcd for
C30H22ClN3O (475.953): C, 75.70; H, 4.66; N, 8.83.
Found: C, 75.47; H, 4.48; N, 8.94.
3.4.6. 4-(4-Chlorophenyl)-2-[(4-methoxyphenyl)amino]-N-
(4-methylphenyl)-6-phenyl-3-pyridinecarboxamide (6f).
Reaction time 45 h, pale yellow crystals purified by silica
gel TLC using chloroform–light petroleum (60–80 ꢁC)
mixture as 20:1, v/v, for elution, mp 179–181 ꢁC, yield
71%. IR: mmax/cmÀ1 3371, 3275 (NH), 1635 (C@O),
1600, 1507 (C@N, C@C). 1H NMR (CDCl3): d 2.21
(s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.76 (br s, 1H, NH),
6.81–7.98 (m, 18H, 17 arom. H+pyr. H-5), 9.08 (br s,
1H, NH). MS: m/z (%) 521 [(M+2), 18], 519 (M, 70),
414 (31), 413 (48), 412 (100), 385 (3), 384 (4), 383 (6).
Anal. Calcd for C32H26ClN3O2 (520.003): C, 73.91; H,
5.04; N, 8.08. Found: C, 73.74; H, 4.96; N, 8.19.
3.4.2. 4-(4-Chlorophenyl)-2-[(4-methylphenyl)amino]-6-
phenyl-N-phenyl-3-pyridinecarboxamide (6b). Reaction
time 65 h, pale yellow crystals purified by silica gel
TLC using chloroform–light petroleum (60–80 ꢁC) mix-
ture as 3:1, v/v, for elution, mp 217–219 ꢁC, yield 37%.
IR: mmax/cmÀ1 3409, 3324 (NH), 1661 (C@O), 1614,
1
1519 (C@N, C@C). H NMR (CDCl3): d 2.36 (s, 3H,
CH3), 6.92 (br s, 1H, NH), 7.08–8.10 (m, 19 H, 18 arom.
H+pyr. H-5), 9.28 (br s, 1H, NH). MS: m/z (%) 491
[(M+2), 23], 489 (M, 54), 398 (50), 397 (100), 369 (18),
368 (17), 367 (34). Anal. Calcd for C31H24ClN3O
(489.973): C, 75.99; H, 4.94; N, 8.58. Found: C, 75.93;
H, 4.92; N, 8.53.
3.4.7. 4-(4-Chlorophenyl)-2-[(4-chlorophenyl)amino]-N-
(4-methylphenyl)-6-phenyl-3-pyridinecarboxamide (6g).
Reaction time 70 h, pale yellow crystals purified by silica
gel TLC using chloroform–light petroleum (60–80 ꢁC)
mixture as 3:1, v/v, for elution, mp 209–211 ꢁC, yield
14%. IR: mmax/cmÀ1 3421 (NH), 1658 (C@O), 1612,
1
1517 (C@N, C@C). H NMR (CDCl3): d 2.21 (s, 3H,
3.4.3. 4-(4-Chlorophenyl)-2-[(4-methoxyphenyl)amino]-6-
phenyl-N-phenyl-3-pyridinecarboxamide (6c). Reaction
time 60 h, pale yellow crystals purified by silica gel
TLC using chloroform–light petroleum (60–80 ꢁC) mix-
ture as 2:1, v/v, for elution, mp 193–195 ꢁC, yield 63%.
IR: mmax/cmÀ1 3369, 3250 (NH), 1635 (C@O), 1600,
CH3), 6.74 (br s, 1H, NH), 6.85–7.98 (m, 18 H, 17 arom.
H+pyr. H-5), 9.32 (br s, 1H, NH). MS: m/z (%) 527
[(M+4), 5], 525 [(M+2), 28], 523 (M, 54), 418 (50), 417
(100), 389 (6), 388 (4), 387 (8). Anal. Calcd for
C31H23Cl2N3O (524.42): C, 70.99; H, 4.42; N, 8.01.
Found: C, 70.73; H, 4.24; N, 7.97.
1
1508 (C@N, C@C). H NMR (CDCl3): d 3.73 (s, 3H,
OCH3), 6.80–7.98 (m, 20 H, 18 arom. H+pyr. H-
5+NH), 9.15 (br s, 1H, NH). MS: m/z (%) 507 [(M+2),
23], 505 (M, 59), 414 (42), 413 (65), 412 (100), 385 (5),
3.4.8. 4-(4-Methylphenyl)-2-[(4-methylphenyl)amino]-6-
phenyl-N-phenyl-3-pyridinecarboxamide (6h). Reaction
time 50 h, yellow crystals purified by silica gel TLC

4474
A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
using chloroform–light petroleum (60–80 ꢁC) mixture as
lung, colon, brain, ovary, breast, prostate as well as kid-
ney, was carried out according to the previously reported
standard procedure.52–54 The human tumor cell lines of
the cancer screening panel are grown in RPMI 1640
medium containing 5% fetal bovine serum and 2 mM
L-glutamine. For a typical screening experiment, cells
are inoculated into 96-well-microtiter plates in 100 ll at
plating densities ranging from 5000 to 40,000 cells/well
depending on the doubling time of individual cell lines.
After cell inoculation, the microtiter plates are incubated
at 37 ꢁC, 5% CO2, 95% air, and 100% relative humidity
for 24 h prior to addition of experimental test com-
pounds. After 24 h, two plates of each cell lines are fixed
in situ with trichloroacetic acid (TCA), to represent a
measurement of the cell population for each cell line at
the time of tested compound addition (time zero, Tz).
Experimental tested compounds are solubilized in
dimethylsulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the
time of the tested compound addition, an aliquot of fro-
zen concentrate is thawed and diluted to twice the desired
final maximum test concentration with complete medium
containing 50 lg/ml gentamicin. Additional four, 10-fold
or 1/2 log serial dilutions are made to provide a total of
five tested compound concentrations (10À4 to 10À8 M
concentrations) plus control. Aliquots of 100 ll of these
different tested compound dilutions are added to the
appropriate microtiter wells already containing 100 ll
of medium, resulting in the required final concentrations.
1:1, v/v, for elution, mp 174–176 ꢁC, yield 43%. IR: mmax
/
cmÀ1 3407, 3321 (NH), 1659 (C@O), 1614, 1520 (C@N,
1
C@C). H NMR (CDCl3): d 2.37 (s, 3H, CH3), 2.43 (s,
3H, CH3), 6.88 (br s, 1H, NH), 7.02–8.13 (m, 19 H, 18
arom. H+pyr. H-5), 9.46 (br s, 1H, NH). MS: m/z (%)
469 (M, 85), 378 (28), 377 (100), 349 (6), 348 (17), 347
(48). Anal. Calcd for C32H27N3O (469.56): C, 81.85;
H, 5.80; N, 8.95. Found: C, 81.79; H, 5.78; N, 8.82.
3.4.9. 2-[(4-Chlorophenyl)amino]-4-(4-methylphenyl)-6-
phenyl-N-phenyl-3-pyridinecarboxamide (6i). Reaction
time 60 h, pale yellow crystals purified by silica gel
TLC using chloroform–light petroleum (60–80 ꢁC) mix-
ture as 3:2, v/v, for elution, mp 185–187 ꢁC, yield 27%.
IR: mmax/cmÀ1 3411 (NH), 1658 (C@O), 1613, 1529
1
(C@N, C@C). H NMR (CDCl3): d 2.43 (s, 3H, CH3),
6.90 (s, 1H, NH), 7.02–8.10 (m, 19 H, 18 arom.
H+pyr. H-5), 9.62 (s, 1H, NH). MS: m/z (%) 491
[(M+2), 20], 489 (M, 36), 398 (42), 397 (78), 396 (100),
369 (6), 368 (10), 367 (13). Anal. Calcd for
C31H24ClN3O (489.973): C, 75.99; H, 4.94; N, 8.58.
Found: C, 76.18; H, 4.98; N, 8.66.
3.4.10. 2-Amino-4-(4-chlorophenyl)-6-phenyl-N-phenyl-3-
pyridinecarboxamide (7a). Colorless crystals, mp 198–
200 ꢁC, yield 20%, 15%, 11%, and 30% (during prepara-
tion of 6a, b, c, and d, respectively). IR: mmax/cmÀ1 3490,
3391, 3268 (NH2, NH), 1646 (C@O), 1599, 1544 (C@N,
C@C). 1H NMR (CDCl3): d 6.15 (br s, 2H, NH2), 6.97–
7.91 (m, 16H, 14 arom. H+pyr. H-5+NH). MS: m/z (%)
401 [(M+2), 10], 399 (M, 24), 308 (21), 307 (100). Anal.
Calcd for C24H18ClN3O (399.853): C, 72.09; H, 4.54; N,
10.51. Found: C, 72.34; H, 4.65; N, 10.70.
Following the tested compound addition, the plates are
incubated for an additional 48 h at 37 ꢁC, 5% CO2, 95%
air, and 100% relative humidity. For adherent cells, the
assay is terminated by the addition of cold TCA. Cells
are fixed in situ by the gentle addition of 50 ll of cold
50% (w/v) TCA (final concentration, 10% TCA) and
incubated for 60 min at 4 ꢁC. The supernatant is dis-
carded, and the plates are washed five times with tap
water and air-dried. Sulforhodamine B (SRB) solution
(100 ll) at 0.4% (w/v) in 1% acetic acid is added to each
well, and plates are incubated for 10 min at room tem-
perature. After staining, unbound dye is removed by
washing five times with 1% acetic acid and the plates
are air-dried. Bound stain is subsequently solubilized
with 10 mM trizma base, and the absorbance is read
on an automated plate readed at a wavelength of
515 nm. For cells suspension the methodology is the
same except that the assay is terminated by fixing settled
cells at the bottom of the wells by gently adding 50 ll of
80% TCA (final concentration, 16% TCA). Using the
seven absorbance measurements [time zero (Tz), control
growth (C) and test growth in the presence of the tested
compound at the five concentration levels (Ti)], the per-
centage growth is calculated at each of the tested com-
pound concentration levels.
3.4.11. 2-Amino-4-(4-chlorophenyl)-N-(4-methylphenyl)-
6-phenyl-3-pyridinecarboxamide (7b). Colorless crystals,
mp 177–179 ꢁC, yield 15%, 10%, and 39% (during prep-
aration of 6e,f, and g, respectively). IR: mmax/cmÀ1 3482,
3382, 3316 (NH2, NH), 1639 (C@O), 1594, 1515 (C@N,
1
C@C). H NMR (CDCl3): d 2.21 (s, 3H, CH3), 6.08 (br
s, 2H, NH2), 6.90–7.90 (m, 15H, 13 arom. H+pyr. H-
5+NH). MS: m/z (%) 415 [(M+2), 12], 413 (M, 40),
308 (20), 307 (100). Anal. Calcd for C25H20ClN3O
(413.883): C, 72.54; H, 4.87; N, 10.15. Found: C,
72.45; H, 4.86; N, 10.28.
3.4.12. 2-Amino-4-(4-methylphenyl)-6-phenyl-N-phenyl-
3-pyridinecarboxamide (7c). Colorless crystals, mp 201–
203 ꢁC, yield 16% and 36% (during preparation of 6h
and i, respectively). IR: mmax/cmÀ1 3491, 3386, 3267
(NH2, NH), 1643 (C@O), 1599, 1533 (C@N, C@C).
1H NMR (CDCl3): d 2.41 (s, 3H, CH3), 6.13 (s, 2H,
NH2), 6.96 (s, 1H, NH), 7.04–8.02 (m, 15H, 14 arom.
H+pyr. H-5). MS: m/z (%) 379 (M, 27), 288 (23), 287
(100). Anal. Calcd for C25H21N3O (379.44): C, 79.13;
H, 5.58; N, 11.07. Found: C, 78.96; H, 5.45; N, 11.01.
Percentage growth inhibition is calculated as [(Ti À Tz)/
(C À Tz)] · 100 for concentrations for which Ti PTz.
[(Ti À Tz)/Tz] · 100 for concentrations for which
Ti < Tz.
3.5. Antitumor activity
Antitumor activity screening for the synthesized nicoti-
namides utilizing 59 different human tumor cell lines,
representing leukemia, melanoma, and cancers of the
Growth inhibition of 50% (GI50) is calculated from
[(Ti À Tz)/(C À Tz)] · 100 = 50, which is the tested com-

A. S. Girgis et al. / Bioorg. Med. Chem. 14 (2006) 4466–4476
4475
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