Synthesis of Some Polysubstituted Nicotinonitriles and Derived Pyrido[2,3-d]pyrimidines as in Vitro Cytotoxic and Antimicrobial Candidates

Article abstract of DOI:10.1155/2016/2135893

The synthesis of polysubstituted pyridines, in addition to some derived pyrido[2,3-d]pyrimidine ring systems supported with chemotherapeutically active functionalities, is described. They were evaluated for their in vitro cytotoxic effects against three d

Full text of DOI:10.1155/2016/2135893

Hindawi Publishing Corporation
Journal of Chemistry
Volume 2016, Article ID 2135893, 12 pages
http://dx.doi.org/10.1155/2016/2135893
Research Article
Synthesis of Some Polysubstituted Nicotinonitriles
and Derived Pyrido[2,3-d]pyrimidines as In Vitro Cytotoxic
and Antimicrobial Candidates
Hassan M. Faidallah,1,2 Sherif A. F. Rostom,3,4 and Khalid A. Khan1
1 Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
2Department of Chemistry, Faculty of Science, University of Alexandria, Alexandria, Egypt
3Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260,
Jeddah 21589, Saudi Arabia
4Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521, Egypt
Correspondence should be addressed to Hassan M. Faidallah; hfaidallahm@hotmail.com
Received 8 February 2016; Revised 20 April 2016; Accepted 21 April 2016
Academic Editor: Artur M. S. Silva
Copyright © 2016 Hassan M. Faidallah et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
e synthesis of polysubstituted pyridines, in addition to some derived pyrido[2,3-d]pyrimidine ring systems supported with
chemotherapeutically active functionalities, is described. ey were evaluated for their in vitro cytotoxic effects against three
different human tumor cell lines (human colon carcinoma HT29, hepatocellular carcinoma Hep-G2, and Caucasian breast
adenocarcinoma MCF7). Nine compounds displayed variable cytotoxic potential, among which alkylthio analogs 33, 34, and 37
emerged as the most active members, being almost twice as active as doxorubicin against the colon carcinoma HT29 cell line.
In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect
on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line. Meanwhile, nineteen compounds were
able to exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacteria, together with moderate
antifungal activities. e pyrido[2,3-d]pyrimidine-2(1H)-thione 30 together with its alkylthio derivatives 33 and 34 stemmed as
the most active antimicrobial members being equipotent to ampicillin against S. aureus, E. coli, and P. aeruginosa, together with a
noticeable antifungal activity against C. albicans. Compounds 33 and 34 could be considered as a promising template for possible
dual antimicrobial-anticancer candidates.
1. Introduction
experience immunotherapy, organ transplantation, carcino-
mas, or AIDS [5]. erefore, there is a genuine demand for
finding out new agents endowed with antimicrobial activity
that acts with nonconventional mechanisms. Patients who
undergo cancer chemotherapy are clearly liable to microbial
infections owing to the subsidiary reduction of immunity.
Concomitant use of several drugs for treating cancer joined
with microbial invasions might cause additional complica-
tions in patients with malfunctioned kidneys. Consequently,
the idea of administering one drug with dual action could
be beneficial from both curative and economic viewpoints.
In recent years, cyanopyridines and some of their fused-
ring systems such as quinolines and pyridopyrimidines have
Over the past two decades, several reports have discussed the
growing number of cancerous diseases and the prominent
role displayed by chemotherapy as the most fruitful treatment
for many disseminated types of tumors. is has resulted in
the activation of much research targeting the discovery of
prominent lead structures that would be beneficial in styling
novel antitumor chemotherapeutic agents [1–3]. Additionally,
the dilemma of multi-drug resistant (MDR) bacteria arising
from the misuse of traditional antimicrobial agents has
been extensively studied [4]. In addition, mycotic invasions
became a complicated problem especially in patients who

2
Journal of Chemistry
been under focus as potential chemotherapeutic candidates
because of their reported distinctive antimicrobial [6–8],
antitubercular [9], and antiviral [10] activities, in addition to
the well documented antineoplastic [11], antiproliferative [12],
and cytotoxic [13] potentials. Furthermore, several pyridine-
containing compounds were reported to possess dual anti-
cancer and antimicrobial activities [14–17].
During our current interest in exploring new lead struc-
tures that possess chemotherapeutic potential, much atten-
tion was focused on the antimicrobial and anticancer activ-
ities of some pyridine derivatives [18–24], particularly those
possessing the 4,6-disubstituted-2-aminopyridin-3-carboni-
trile (nicotinonitriles) scaffold which revealed favourable
broad spectrum anticancer and/or antimicrobial efficacy.
e collected results persuaded the design and synthesis
of novel polysubstituted nicotinonitriles and some derived
pyrido[2,3-d]pyrimidines incorporating different functional
groups that are claimed to account for the bioactivity of
related antitumor and antimicrobial candidates. Additionally,
the substituents encountered in these derivatives were chosen
so as to affect the electronic environment of the compounds,
which would synergistically influence the targeted biological
activities, in an attempt to obtain novel candidates with both
anticancer and antimicrobial potentials.
C -H). 13C NMR (ꢁ, ppm): 91.4 (CH ), 113.4, 115.6, 117.5 (CN),
5
2
120.3, 124.8, 126.6, 127.1, 128.4, 129.5, 139.9, 142.5, 148.1, 150.6,
161.5, 165.7 (Ar-C). MS: m/z 315 (M+, 100), 303 (21), 289 (82),
277 (14), 273 (76), 272 (42), 227 (55), 84 (58), 75 (28). Anal.%
Calcd for C H N O (315.33): C, 72.37; H, 4.16; N, 13.33.
19 13
3
2
Found: C, 72.25; H, 4.20; N, 13.21.
(2) 2-Amino-6-phenyl-4-(2-thienyl)nicotinonitrile (2). Yield:
(1.8 g, 54%); m.p.: 190–192∘C; IR (cm−1, KBr): 3167, 3387
1
(NH ), 2228 (CN). H NMR (ꢁ, ppm): 6.10 (s, 2H, NH ),
2
2
7.02–8.11 (m, 9H, 8 Ar-H + pyridine C -H). 13C NMR (ꢁ,
5
ppm): 90.6, 110.0, 118.1 (CN), 122.3, 125.2, 126.8, 127.1, 128.3,
129.6, 139.7, 142.6, 150.7, 162.2, 165.4 (Ar-C). Anal.% Calcd for
C H N S (277.35): C, 69.29; H, 4.00; N, 15.15. Found: C,
16 11
3
69.21; H, 4.40; N, 15.21.
(3) 2-Amino-4-(benzo[d][1,3]dioxol-5-yl)-6-(4-bromophe-
nyl)nicotinonitrile (3). Yield: (1.9 g, 68%); m.p.: 198–200∘C;
IR (cm−1, KBr): 3175, 3358 (NH ), 2223 (CN). H NMR
1
2
(ꢁ, ppm): 6.03 (s, 2H, CH ), 6.21 (s, 2H, NH ), 7.68–7.87
2
2
(m, 8H, 7 Ar-H + pyridine C -H). 13C NMR (ꢁ, ppm): 91.2
5
(CH ), 113.6, 115.4, 117.8 (CN), 121.6, 125.5, 126.3, 127.0, 128.2,
2
129.2, 139.4, 142.2, 148.0, 150.5, 162.5, 164.9 (Ar-C). Anal.%
Calcd for C H BrN O (394.23): C, 57.89; H, 3.07; N, 10.66.
19 12
3
2
Found: C, 57.82; H, 3.03; N, 10.61.
2. Experimental
(4) 2-Amino-6-(4-bromophenyl)-4-(2-thienyl)nicotinonitrile
(4). Yield: (2.02 g, 73%); m.p.: 200–202∘C; IR (cm−1, KBr):
2.1. Chemistry. Melting points were determined on a Gal-
lenkamp melting point apparatus and are uncorrected. e
infrared (IR) spectra were recorded on Shimadzu FT-IR
8400S infrared spectrophotometer using the KBr pellet tech-
nique. 1H and 13C NMR spectra were recorded on a Bruker
DPX-400 FT NMR spectrometer using Tetramethylsilane
3416, 3198 (NH ), 2220 (CN). 1H NMR (ꢁ, ppm): 6.18 (s, 2H,
2
NH ), 7.05–8.01 (m, 8H, 7 Ar-H + pyridine C -H). 13C NMR
2
5
(ꢁ, ppm): 91.4, 110.9, 118.0 (CN), 122.2, 125.3, 126.4, 127.2, 128.4,
129.3, 139.8, 142.7, 151.2, 162.8, 165.5 (Ar-C). Anal.% Calcd for
C H BrN S (356.24): C, 53.95; H, 2.83; N, 11.80. Found: C,
16 10
3
53.88; H, 2.79; N, 11.85.
(TMS) as the internal standard and DMSO-ꢀ6 as a solvent
(Chemical shifs in ꢁ, ppm). Splitting patterns were desig-
nated as follows: s: singlet; t: triplet; q: quartet; m: multiplet.
e Electron Impact Ionization Mass Spectra were recorded
on a Nermag R10-10C at 70 ev. 1000 Ex spectrometer. Elemen-
tal analyses were performed on a 2400 Perkin Elmer Series
2 analyzer and the values found were within 0.4% of the
theoretical values. Follow-up of the reactions and checking
the homogeneity of the compounds were made by TLC on
silica gel-protected aluminum sheets (Type 60 F254, Merck)
and the spots were detected by exposure to UV-lamp at ꢂ 254.
(5) 2-Amino-6-(4-methoxyphenyl)-4-(2-thienyl)nicotinoni-
trile (5). Yield: (1.63 g, 62%); m.p.: 160–162∘C; IR (cm−1,
KBr): 3172, 3382 (NH ), 2228 (CN). 1H NMR (ꢁ, ppm): 3.75
2
(s, 3H, OCH ), 6.18 (s, 2H, NH ), 7.10–8.12 (m, 8H, 7 Ar-H +
3
2
pyridine C -H). 13C NMR (ꢁ, ppm): 56.1 (OCH ), 97.9, 110.0,
5
3
117.8 (CN), 122.1, 125.4, 126.4, 127.2, 128.1, 129.2, 138.9, 142.2,
151.8, 161.2, 165.4 (Ar-C). MS: m/z 307 (M+, 100), 225 (18),
281 (85), 265 (78), 182 (63), 84 (15), 83 (12), 75 (34). Anal.%
Calcd for C H N OS (307.37): C, 66.43; H, 4.26; N, 13.67.
17 13
3
Found: C, 66.08; H, 3.90; N, 13.34.
2.1.1. 2-Amino-4,6-disubstituted Nicotinonitriles (1–5). A
one-pot mixture of the appropriate aromatic aldehyde
(10 mmol), the ketone (10 mmol), malononitrile (0.66 g,
10 mmol), and ammonium acetate (6.2 g, 80 mmol) in abso-
lute ethanol (50 mL) was refluxed for 4-5 h. e reaction
mixture was allowed to attain room temperature and the
resulting precipitate was filtered, washed with water, dried,
and recrystallized from ethanol.
2.1.2. 5,7-Disubstituted Pyrido[2,3-d]pyrimidine-4(3H)-ones
(6–10). A mixture of the appropriate nicotinonitriles 1–5
(10 mmol) and formic acid (5 mL) was heated in a boiling
water bath for 30 min. Afer being cooled to room temper-
ature, the reaction mixture was poured onto ice-cold water;
the precipitated solid product was filtered, washed with water,
dried, and recrystallized from the appropriate solvent.
(1) 2-Amino-4-(benzo[d][1,3]dioxol-5-yl)-6-phenylnicotinoni-
(1) 5-(Benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-d]pyrim-
idine-4(3H)-one (6). Yield: (1.8 g, 59%; methanol); m.p.: 168–
170∘C; IR (cm−1, KBr): 3285 (NH), 1648 (C=O). 1H NMR (ꢁ,
trile (1). Yield: (1.7 g, 67%); m.p.: 162–164∘C; IR (cm−1, KBr):
3149, 3354 (NH ), 2218 (CN). 1H NMR (ꢁ, ppm): 5.98 (s, 2H,
2
CH ), 6.17 (s, 2H, NH ), 6.74–7.88 (m, 9H, 8 Ar-H + pyridine
ppm): 6.18 (s, 2H, CH ); 6.84–7.95 (m, 10 H, Ar-H); 8.16 (s,
2
2
2

Journal of Chemistry
3
1H, NH). 13C NMR (ꢁ, ppm): 91.8 (CH ), 113.5, 116.5, 118.2,
NMR (ꢁ, ppm): 2.35 (s, 3H, CH ), 6.18 (s, 2H, CH ); 6.74–
2
3
2
120.1, 120.2, 122.8, 125.3, 127.5, 131.4, 142.6, 144.2, 148.1, 151.9,
157.6, 164.3, 173.7 (Ar-C), 170.5 (CO). MS: m/z 343 (M+, 90),
342 (100), 331 (32), 326 (20), 314 (18), 297 (44), 284 (53), 206
(10), 83 (12), 75 (22). Anal.% Calcd for C H N O (343.34):
7.88 (m, 9H, Ar-H); 8.16 (s, 1H, NH). 13C NMR (ꢁ, ppm): 20.9
(CH ), 90.9 (CH ), 113.4, 116.2, 118.1, 120.0, 121.2, 122.7, 125.4,
3
2
127.3, 131.2, 142.5, 144.1, 148.1, 151.8, 157.3, 164.1, 173.0 (Ar-C),
170.1 (CO). Anal.% Calcd for C H N O (357.37): C, 70.58;
20 13
3
3
21 15
3
3
C, 69.97; H, 3.82; N, 12.24. Found: C, 70.02; H, 3.77; N, 12.31.
H, 4.23; N, 11.76. Found: C, 70.47; H, 4.19; N, 11.85.
(2) 7-Phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-4(3H)-one
(2) 2-Methyl-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-
4(3H)-one (12). Yield: (2.11 g, 62%, ethanol); m.p.: 228–230∘C;
IR (cm−1, KBr): 3270 (NH), 1654 (C=O). 1H NMR (ꢁ; ppm):
(7). Yield: (1.9 g, 62%, ethanol); m.p.: 160–162∘C; IR (cm−1,
1
KBr): 3292 (NH), 1656 (C=O). H NMR (ꢁ, ppm): 6.76–
8.02 (m, 10H, Ar-H); 8.16 (s, 1H, NH). 13C NMR (ꢁ, ppm):
117.2, 121.6, 122.2, 122.8, 125.3, 125.4, 127.5, 138.4, 142.6, 144.3,
151.9, 157.8, 164.5, 173.4 (Ar-C), 170.2 (CO). Anal.% Calcd for
C H N OS (305.36): C, 66.87; H, 3.63; N, 13.76. Found: C,
2.28 (s, 3H, CH ), 7.25–8.09 (m, 9H, Ar-H); 8.14 (s, 1H, NH).
3
13C NMR (ꢁ, ppm): 21.1 (CH ), 117.5, 121.4, 122.2, 122.8, 125.5,
3
125.6, 127.1, 138.5, 142.4, 144.5, 151.8, 157.7, 164.2, 173.4 (Ar-C),
168.8 (CO). MS: m/z 319 (M+, 82), 318 (100), 304 (78), 302 (35),
236 (12), 206 (14), 132 (28), 84 (43), 83 (12), 75 (21). Anal.%
Calcd for C H N OS (319.38): C, 67.69; H, 4.10; N, 13.16.
17 11
3
66.80; H, 3.65; N, 13.81.
18 13
3
(3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)pyrido[2,
3-d]pyrimidine-4(3H)-one (8). Yield: (2.3 g, 72%, ethanol);
m.p.: 110–112∘C; IR (cm−1, KBr): 3320 (NH), 1652 (C=O). 1H
Found: C, 67.54; H, 4.02; N, 13.11.
(3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-2-meth-
NMR (ꢁ, ppm): 6.11 (s, 2H, CH ); 6.80–7.98 (m, 9H, Ar-H);
ylpyrido[2,3-d]pyrimidine-4(3H)-one (13). Yield: (2.10 g, 64%,
2
8.21 (s, 1H, NH). 13C NMR (ꢁ, ppm): 91.5 (CH ), 113.2, 116.7,
ethanol); m.p.: 220–222∘C; IR (cm−1, KBr): 3265 (NH), 1651
2
1
119.3, 120.1, 121.7, 122.8, 125.5, 127.2, 131.6, 142.3, 144.0, 148.2,
151.7, 157.3, 164.0, 173.6 (Ar-C), 170.2 (CO). Anal.% Calcd for
C H BrN O (422.24): C, 56.89; H, 2.86; N, 9.95. Found: C,
(C=O). H NMR (ꢁ, ppm): 2.32 (s, 3H, CH ), 6.16 (s, 2H,
3
CH ); 6.82–7.96 (m, 8H, Ar-H); 8.23 (s, 1H, NH). 13C NMR (ꢁ,
2
20 12
3
3
ppm): 20.2 (CH ), 91.3 (CH ), 113.5, 116.3, 118.2, 119.9, 120.0,
3
2
56.82; H, 2.79; N, 9.91.
122.1, 125.4, 127.6, 131.4, 142.6, 144.2, 152.8, 148.1, 157.9, 163.4,
171.7 (Ar-C), 173.2 (CO). Anal.% Calcd for C H BrN O
21 14
3
3
(4) 7-(4-Bromophenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimidine-
4(3H)-one (9). Yield: (2.0 g, 70%, methanol); m.p.: 128–130∘C;
(436.27): C, 57.82; H, 3.23; N, 9.63. Found: C, 57.91; H, 3.17;
N, 9.75.
IR (cm−1, KBr): 3298 (NH), 1650 (C=O). 1H NMR (ꢁ, ppm):
6.72–7.69 (m, 9H, Ar-H); 8.22 (s, 1H, NH). 13C NMR (ꢁ,
ppm): 118.5, 120.6, 121.7, 122.8, 125.3, 127.5, 129.4, 132.3, 138.4,
142.3, 147.3, 157.8, 162.5, 173.1 (Ar-C), 170.3 (CO). Anal.%
Calcd for C H BrN OS (384.25): C, 53.14; H, 2.62; N, 10.94.
(4) 7-(4-Bromophenyl)-2-methyl-5-(2-thienyl)pyrido[2,3-
d]pyrimidine-4(3H)-one (14). Yield: (2.0 g, 63%, ethanol);
m.p.: 248–250∘C; IR (cm−1, KBr): 3270 (NH), 1652 (C=O).
1H NMR (ꢁ, ppm): 2.35 (s, 3H, CH ), 6.68–7.77 (m, 8H,
17 10
3
3
Ar-H); 8.25 (s, 1H, NH). 13C NMR (ꢁ, ppm): 21.0 (CH ),
Found: C, 53.03; H, 2.60; N, 11.03.
3
118.8, 120.5, 121.8, 122.9, 125.2, 127.4, 129.3, 132.2, 138.3, 142.2,
147.1, 157.7, 163.1, 172.9 (Ar-C), 170.2 (CO). Anal.% Calcd for
C H BrN OS (398.28): C, 54.28; H, 3.04; N, 10.55. Found:
(5) 7-(4-Methoxyphenyl)-5-(2-thienyl)pyrido[2,3-d]pyrimi-
dine-4(3H)-one (10). Yield: (1.8 g, 66%, ethanol); m.p.: 166–
168∘C; IR (cm−1, KBr): 3304 (NH), 1654 (C=O). 1H NMR (ꢁ,
18 12
3
C, 54.17; H, 3.00; N, 10.61.
ppm): 3.82 (s, 3H, OCH ); 6.92–8.01 (m, 9H, Ar-H); 8.02 (s,
3
1H, NH). 13C NMR (ꢁ, ppm): 56.1 (OCH ), 114.5, 118.5, 120.7,
(5) 2-Methyl-7-(4-methoxyphenyl)-5-(2-thienyl)pyrido[2,3-
3
122.8, 125.5, 127.4, 128.1, 132.2, 142.4, 147.5, 157.7, 160.6, 163.8,
173.6 (Ar-C), 170.2 (CO). MS: m/z 335 (M+, 89), 334 (100),
318 (62), 304 (48), 252 (31), 206 (14), 132 (22), 83 (16), 75 (21).
Anal.% Calcd for C H N O S (335.38): C, 64.46; H, 3.91; N,
d]pyrimidine-4(3H)-one (15). Yield: (2.2 g, 65%, ethanol);
m.p.: 200–202∘C; IR (cm−1, KBr): 3259 (NH), 1653 (C=O).
1H NMR (ꢁ, ppm): 2.36 (s, 3H, CH ), 3.81 (s, 3H, OCH );
3
3
6.81–8.00 (m, 8H, Ar-H); 8.22 (s, 1H, NH). 13C NMR (ꢁ,
18 13
3
2
12.53. Found: C, 64.61; H, 3.59; N, 12.44.
ppm): 20.8 (CH ), 56.1 (OCH ), 91.9 (CH ), 113.5, 116.6,
3
3
2
118.8, 120.41, 120.6, 122.7, 125.5, 127.3, 131.3, 142.2, 144.7, 148.4,
160.1, 157.3, 164.3, 173.3 (Ar-C), 170.2 (CO). Anal.% Calcd for
C H N O S (349.41): C, 65.31; H, 4.33; N, 12.03. Found: C,
2.1.3. 2-Methyl-5,7-disubstituted-pyrido[2,3-d]pyrimidine-
4(3H)-ones (11–15). A mixture of the starting nicotinonitriles
1–5 (10 mmol), acetic anhydride (5 mL), and conc. H SO
(0.5 mL) was heated in a boiling water bath for 10 min and
then cooled. e reaction mixture was poured onto ice-cold
water, treated with 20% NaOH solution until it acquires pH
of 11-12. e crude solid product was filtered, dried, and
recrystallized from the appropriate solvent.
19 15
3
2
65.25; H, 4.29; N, 12.13.
2
4
2.1.4. 4-Imino-3-phenyl-5,7-disubstituted-dihydropyrido[2,3-
d]pyrimidine-2(1H)-thiones (16–20). To a solution of the
appropriate nicotinonitriles 1–5 (10 mmol) in pyridine
(15 mL), phenyl isothiocyanate (0.15 g, 15 mmol) was added,
and the mixture was refluxed for 4 h. Afer cooling, the solid
product thus formed was filtered, washed thoroughly with
water, dried, and recrystallized from the appropriate solvent.
(1) 5-(Benzo[d][1,3]dioxol-5-yl)-2-methyl-7-phenylpyrido[2,3-
d]pyrimidine-4(3H)-one (11). Yield: (2.42 g, 60%, methanol);
m.p.: 238–240∘C; IR (cm−1, KBr): 3277 (NH), 1656 (C=O). 1H

4
Journal of Chemistry
(1) 5-(Benzo[d][1,3]dioxol-5-yl)-4-imino-3,7-diphenyl-3,4-
dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (16). Yield:
(3.2 g, 82%, methanol); m.p.: 178–180∘C; IR (cm−1, KBr):
2.1.5. N-((3-Cyano-4,6-disubstituted-pyridin-2-yl)thiocar-
bamoyl)benzamides (21–23). To a solution of the appropri-
ate nicotinonitriles 1–5 (20 mmol) in dry acetone (30 mL),
a solution of benzoyl isothiocyanate (1.65 g, 20 mmol) in
dry acetone (10 mL) was added. e resulting mixture was
heated under reflux for 10 h. e reaction mixture was
concentrated and lef overnight at room temperature. e
1
3328 (NH), 1635 (C=N), 1204 (C=S). H NMR (ꢁ, ppm):
6.12 (s, 2H, CH ), 6.75 (s, 1H, C=NH); 6.78–7.99 (m, 15H,
2
14 Ar-H + NH). 13C NMR (ꢁ, ppm): 90.9 (CH ), 108.1,
2
109.8, 113.5, 115.6, 120.2, 121.3, 124.5, 125.3, 127.1, 127.5, 128.8,
129.0, 131.4, 139.5, 139.7, 148.3, 149.0, 160.0 (Ar-C), 163.6
(C=NH), 179.8 (CS). Anal.% Calcd for C H N O S
separated crystalline product was filtered, washed with Et O,
2
and recrystallized from the appropriate solvent.
26 18
4
2
(450.52): C, 69.32; H, 4.03; N, 12.44. Found: C, 69.24; H, 3.99;
(1) N-((3-Cyano-6-phenyl-4-(2-thienyl)pyridin-2-yl)carbamo-
thioyl)benzamide (21). Yield: (3.7 g, 85%, methanol); m.p.:
126–128∘C; IR (cm−1, KBr): 3433 (NH), 2215 (CN), 1661
(C=O), 1166 (C=S). 1H NMR (ꢁ, ppm): 6.39 (s, 1H, CSNH);
7.10–8.14 (m, 14H, Ar-H); 8.43 (s, 1H, CONH). 13C NMR (ꢁ,
ppm): 96.4, 111.2, 118.0 (CN), 122.4, 125.5, 126.3, 127.2, 127.4,
128.7, 129.0, 132.0, 133.3, 139.9, 142.3, 151.7, 161.8, 165.2 (Ar-C),
169.2 (CO), 180.1 (CS). MS: m/z 440 (M+, 100), 414 (76), 333
(42), 311 (19), 252 (31), 206 (14), 105 (66), 83 (18), 75 (27).
Anal.% Calcd for C H N OS (440.54): C, 65.43; H, 3.66;
N, 12.40.
(2) 4-Imino-3,7-diphenyl-5-(2-thienyl)-3,4-dihydropyrido[2,
3-d]pyrimidine-2(1H)-thione (17). Yield: (3.89 g, 82%, meth-
anol); m.p.: 168–170∘C; IR (cm−1, KBr): 3287 (NH), 1639
(C=N), 1198 (C=S). 1H NMR (ꢁ, ppm): 6.86 (s, 1H, C=NH);
6.78–7.80 (m, 15H, 14 Ar-H + NH). 13C NMR (ꢁ, ppm):
109.2, 110.6, 121.5, 122.8, 124.3, 125.9, 127.1, 127.3, 127.6,
129.1, 132.3, 139.9, 142.4, 144.9, 149.7, 158.1, 162.1 (Ar-C),
164.7 (C=NH), 180.3 (C=S). Anal.% Calcd for C H N S
24 16
4
2
23 16
4
2
N, 12.72. Found: C, 65.23; H, 3.96; N, 12.99.
(412.53): C, 66.97; H, 3.91; N, 13.58. Found: C, 66.90; H, 3.88;
N, 13.65.
(2) N-((4-(Benzo[d][1,3]dioxol-5-yl)-6-(4-bromophenyl)-
3-cyanopyridin-2-yl)carbamothioyl)benzamide (22). Yield:
(4.5 g, 82%, acetic acid); m.p.: 198–200∘C; IR (cm−1, KBr):
3425 (NH), 2226 (CN), 1665 (C=O), 1174 (C=S). 1H NMR (ꢁ,
(3) 5-(Benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-4-imino-
3-phenyl-3,4-dihydropyrido[2,3-d]pyrimidine-2(1H)-thione
(18). Yield: (3.3 g, 78%, ethanol); m.p.: 198–200∘C; IR (cm−1,
1
ppm,): 6.02 (s, 2H, CH ), 6.35 (s, 1H, CSNH); 6.63–7.89 (m,
KBr): 3321 (NH), 1637 (C=N), 1203 (C=S). H NMR (ꢁ,
2
13H, Ar-H); 8.48 (s, 1H, CONH). 13C NMR (ꢁ, ppm): 91.7
ppm): 5.98 (s, 2H, CH ), 6.78 (s, 1H, C=NH); 6.88–8.12
2
(m, 14H, 13 Ar-H + NH). 13C NMR (ꢁ, ppm): 91.3 (CH ),
(CH ), 95.4, 113.0, 115.5, 117.7 (CN), 120.2, 122.3, 125.5, 126.6,
2
2
127.1, 127.3, 128.8, 129.2, 131.6, 133.4, 139.9, 142.4, 148.0, 151.7,
162.7, 165.2 (Ar-C), 168.1 (CO), 179.9 (CS). Anal.% Calcd for
C H BrN O S (557.42): C, 58.18; H, 3.07; N, 10.05. Found:
108.6, 109.8, 113.8, 116.1, 120.4, 124.8, 125.5, 127.1, 127.4,
128.8, 129.3, 131.6, 139.5, 140.0, 148.1, 149.2, 158.2, 160.8
(Ar-C), 164.4 (C=NH), 181.2 (C=S). MS: m/z 530 (M+,
74), 529 (100), 449 (80), 369 (29), 361 (17), 330 (25), 132
(26), 84 (30), 75 (22). Anal.% Calcd for C H BrN O S
27 17
4
3
C, 58.11; H, 3.09; N, 10.15.
26 17
4
2
(3) N-((6-(4-Bromophenyl)-3-cyano-4-(2-thienyl)pyridin-2-
yl)carbamothioyl)benzamide (23). Yield: (4.1 g, 79%, acetic
acid); m.p.: 210–212∘C; IR (cm−1, KBr): 3422 (NH), 2218
(529.41): C, 58.99; H, 3.24; N, 10.58. Found: C, 58.91; H, 3.26;
N, 10.45.
1
(CN), 1662 (C=O), 1170 (C=S). H NMR (ꢁ, ppm): 6.28 (s,
(4) 7-(4-Bromophenyl)-4-imino-3-phenyl-5-(2-thienyl)-3,4-
dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (19). Yield:
(3.4 g, 76%, ethanol); m.p.: 180–182∘C; IR (cm−1, KBr): 3284
1H, CSNH); 7.02–8.32 (m, 12H, Ar-H); 8.52 (s, 1H, CONH).
13C NMR (ꢁ, ppm): 94.4, 111.0, 118.1 (CN), 122.8, 125.3,
126.5, 127.3, 127.5, 128.6, 129.1, 131.9, 133.5, 139.8, 142.6, 151.5,
161.9, 165.4 (Ar-C), 169.3 (CO), 180.4 (CS). Anal.% Calcd for
C H BrN OS (519.44): C, 55.50; H, 2.91; N, 10.79. Found:
1
(NH), 1642 (C=N), 1195 (C=S). H NMR (ꢁ, ppm): 6.81 (s,
1H, C=NH); 7.34–7.82 (m, 14H, 13 Ar-H + NH). 13C NMR (ꢁ,
ppm): 108.4, 110.9, 121.6, 122.8, 124.5, 125.5, 126.8, 127.2, 127.4,
128.9, 132.4, 139.8, 142.5, 144.4, 149.0, 158.3, 160.0 (Ar-C),
164.6 (C=NH), 179.3 (C=S). Anal.% Calcd for C H BrN S
24 15
4
2
C, 55.14; H, 3.19; N, 11.01.
23 15
4
2
(491.43): C, 56.21; H, 3.08; N, 11.40. Found: C, 56.12; H, 3.06;
N, 11.51.
2.1.6. 4-Amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-
2(1H)-ones (24–27). A mixture of the appropriate nicotin-
onitriles 1–5 (10 mmol) and urea (0.6 g, 10 mmol) was fused at
260–300∘C using a sand bath for 1 h. e reaction mixture was
allowed to attain room temperature; the crude solid product
was treated with water, then rubbed with ethanol, filtered, and
(5) 4-Imino-7-(4-methoxyphenyl)-3-phenyl-5-(2-thienyl)-3,4-
dihydropyrido[2,3-d]pyrimidine-2(1H)-thione (20). Yield:
(2.3 g, 73%, DMF); m.p.: 190–192∘C; IR (cm−1, KBr): 3312
1
(NH), 1639 (C=N), 1188 (C=S). H NMR (ꢁ, ppm): 3.73 (s,
recrystallized from DMF/H O.
2
3H, OCH ), 6.83 (s, 1H, C=NH); 6.79–7.94 (m, 13H, 12 Ar-H
3
+ NH). 13C NMR (ꢁ, ppm): 56.0 (OCH ), 106.1, 114.6, 120.2,
(1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-
d]pyrimidine-2(1H)-one (24). Yield: (2.4 g, 71%); m.p.: 278–
280∘C; IR (cm−1, KBr): 3280, 3323, 3435 (NH & NH ),
3
121.6, 122.8, 124.5, 125.5, 126.8, 127.2, 127.4, 128.9, 132.4, 142.5,
144.4, 149.0, 158.3, 160.0 (Ar-C), 164.8 (C=NH), 179.6 (C=S).
Anal.% Calcd for C H N OS (422.56): C, 65.14; H, 4.10;
2
1
1660 (C=O). H NMR (ꢁ, ppm): 5.98 (s, 2H, CH ), 6.52 (s,
24 18
4
2
2
2H, NH ), 6.74–8.13 (m, 10H, 9 Ar-H + NH). 13C NMR
N, 12.66. Found: C, 65.09; H, 4.17; N, 12.56.
2

Journal of Chemistry
5
(ꢁ, ppm): 93.5 (CH ), 113.7, 115.8, 120.6, 124.4, 126.5, 127.2,
165.4 (Ar-C), 182.3 (CS). MS: m/z 336 (M+, 74), 320 (100),
303 (72), 220 (31), 132 (15), 83 (11), 75 (25). Anal.% Calcd for
C H N S (336.43): C, 60.69; H, 3.60; N, 16.65. Found: C,
2
128.8, 129.0, 129.3, 139.8, 142.4, 148.1, 150.8, 158.5, 160.4, 164.4
(Ar-C), 166.2 (CO). Anal.% Calcd for C H N O (358.36):
20 14
4
3
17 12
4
2
C, 67.03; H, 3.94; N, 15.63. Found: C, 67.10; H, 3.89; N, 15.49.
60.62; H, 3.57; N, 16.78.
(2) 4-Amino-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-
(3) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophe-
2(1H)-one (25). Yield: (2.1 g, 75%); m.p.: 280–282∘C; IR (cm−1,
nyl)pyrido[2,3-d]pyrimidine-2(1H)-thione (30). Yield: (2.6 g,
73%, ethanol); m.p.: 230–232∘C; IR (cm−1, KBr): 3288, 3346,
KBr): 3276, 3336, 34659 (NH & NH ), 1665 (C=O). 1H NMR
2
1
3438 (NH & NH ), 1173 (C=S). H NMR (ꢁ, ppm): 5.96 (s,
(ꢁ, ppm): 6.44 (s, 2H, NH ), 6.82–8.15 (m, 10H, 9 Ar-H +
2
2
NH). 13C NMR (ꢁ, ppm): 109.6, 110.0, 122.2, 125.5, 126.5, 127.1,
128.1, 129.4, 139.7, 142.3, 151.2, 159.1, 162.5, 165.1 (Ar-C), 166.4
(CO). Anal.% Calcd for C H N OS (320.37): C, 63.73; H,
2H, CH ), 6.59 (s, 2H, NH ), 6.84–8.03 (m, 9H, 8 Ar-H
2
2
+ NH). 13C NMR (ꢁ, ppm): 92.1 (CH ), 113.5, 115.4, 120.1,
2
124.6, 126.4, 127.1, 128.8, 129.1, 129.5, 139.7, 142.7, 148.0, 151.0,
159.3, 160.5, 164.9 (Ar-C), 181.4 (CS). Anal.% Calcd for
C H BrN O S (453.31): C, 52.99; H, 2.89; N, 12.36. Found:
17 12
4
3.78; N, 17.49. Found: C, 63.81; H, 3.67; N, 17.57.
20 13
4
2
(3) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophe-
C, 52.93; H, 2.87; N, 12.47.
nyl)pyrido[2,3-d]pyrimidine-2(1H)-one (26). Yield: (2.2 g,
75%); m.p.: 260–262∘C; IR (cm−1, KBr): 3271, 3326, 3460
(4) 4-Amino-7-(4-bromophenyl)-5-(2-thienyl)pyrido[2,3-
1
d]pyrimidine-2(1H)-thione (31). Yield: (1.7 g, 52%, ethanol);
(NH & NH ), 1667 (C=O). H NMR (ꢁ, ppm): 6.10 (s, 2H,
2
m.p.: 243–245∘C; IR (cm−1, KBr): 3279, 3329, 3460 (NH &
CH ), 6.48 (s, 2H, NH ), 6.84–8.13 (m, 9H, 8 Ar-H + NH).
2
2
1
13C NMR (ꢁ, ppm): 92.6 (CH ), 113.5, 115.6, 122.1, 125.5, 126.4,
2
2
NH ), 1177 (C=S). H NMR (ꢁ, ppm): 6.49 (s, 2H, NH ),
2
6.67–8.11 (m, 9H, 8 Ar-H + NH). 13C NMR (ꢁ, ppm): 109.3,
110.7, 122.1, 125.5, 127.1, 127.6, 127.9, 129.4, 139.6, 143.2, 150.7,
158.1, 162.0, 165.5 (Ar-C), 182.7 (CS). Anal.% Calcd for
C H BrN S (415.33): C, 49.16; H, 2.67; N, 13.49. Found: C,
127.2, 128.8, 129.0, 129.4, 139.6, 142.6, 148.1, 150.9, 157.8, 160.2,
164.8 (Ar-C). 166.2 (CO). Anal.% Calcd for C H BrN O
20 13
4
3
(437.25): C, 54.94; H, 3.00; N, 12.81. Found: C, 54.91; H, 2.97;
N, 12.89.
17 11
4
2
49.12; H, 2.64; N, 13.38.
(4) 4-Amino-7-(4-bromophenyl)-5-(2-thienyl)pyrido[2,3-
(5) 4-Amino-7-(4-methoxyphenyl)-5-(2-thienyl)pyrido[2,3-
d]pyrimidine-2(1H)-thione (32). Yield: (2.6 g, 71%, ethanol);
m.p.: 298–300∘C; IR (cm−1, KBr): 3277, 3344, 3431 (NH &
d]pyrimidine-2(1H)-one (27). Yield: (2.1 g, 73%); m.p.:
250–252∘C; IR (cm−1, KBr): 3284, 3346, 3455 (NH & NH ),
2
1664 (C=O). 1H NMR (ꢁ, ppm): 6.41 (s, 2H, NH ); 6.97–8.01
1
2
NH ), 1168 (C=S). H NMR (ꢁ, ppm): 2.89 (s, 3H, OCH ),
(m, 8H, Ar-H); 8.02 (s, 1H, NH). 13C NMR (ꢁ, ppm): 108.1,
110.3, 122.7, 125.2, 126.7, 127.3, 127.4, 129.0, 139.3, 142.6, 144.5,
158.4, 159.3, 166.1 (Ar-C), 162.3 (CO). Anal.% Calcd for
C H BrN OS (399.27): C, 51.14; H, 2.78; N, 14.03. Found:
2
3
6.75 (s, 2H, NH ); 6.94–8.27 (m, 9H, 8 Ar-H + NH). 13C
2
NMR (ꢁ, ppm): 56.1 (OCH ) 108.8, 110.6, 114.6, 122.9, 125.3,
3
127.1, 128.2, 129.0, 139.7, 142.2, 144.6, 157.8, 158.7, 164.5 (Ar-C),
183.3 (CS). Anal.% Calcd for C H N OS (366.46): C,
17 11
4
18 14
4
2
C, 51.05; H, 2.74; N, 14.07.
59.00; H, 3.85; N, 15.29. Found: C, 58.86; H, 3.83; N, 15.17.
2.1.7. 4-Amino-5,7-disubstituted-pyrido[2,3-d]pyrimidine-
2(1H)-thiones (28–32). A mixture of the appropriate nicoti-
nonitriles 1–5 (10 mmol) and thiourea (0.8 g, 10 mmol) was
fused at 260–300∘C using a sand bath for 1.5 h. e reaction
mixture was worked up as described under compounds 24–
27 and recrystallized from the appropriate solvent.
2.1.8.2-Alkylthio-4-amino-5,7-disubstituted-pyrido[2,3-d]pyr-
imidines (33–37). To a stirred solution of proper thiones
30–32 (10 mmol) in 1N NaOH (5 mL) and ethanol (2 mL),
the selected alkyl iodide (12 mmol) was added. e reaction
mixture was stirred at room temperature for 2-3 h and the
precipitated product was filtered, washed with cold ethanol,
dried, and recrystallized from proper solvent.
(1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-phenylpyrido[2,3-
d]pyrimidine-2(1H)-thione (28). Yield: (2.1 g, 67%, ethanol);
m.p.: 220–222∘C; IR (cm−1, KBr): 3272, 3345, 3462 (NH &
NH ), 1179 (C=S). 1H NMR (ꢁ, ppm): 5.98 (s, 2H, CH ), 6.52
(1) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophenyl)-
2-(methylthio)pyrido[2,3-d]pyrimidine (33). Yield: (3.5 g,
76%, methanol); m.p. 240–242∘C; IR (cm−1, KBr): 3365,
3435 (NH ). 1H NMR (ꢁ, ppm): 2.49 (s, 3H, SCH ); 6.41 (s,
2
2
(s, 2H, NH ), 6.74–8.13 (m, 10H, 9 Ar-H + NH). 13C NMR (ꢁ,
2
ppm): 91.4 (CH ), 113.4, 115.6, 120.3, 124.8, 126.6, 127.1, 128.4,
2
2
3
129.0, 129.5, 139.9, 142.5, 148.1, 150.6, 158.3, 160.5, 164.7 (Ar-C),
182.4 (CS). Anal.% Calcd for C H N O S (374.42): C, 64.16;
2H, CH ); 6.92–8.06 (m, 10H, 8 Ar-H + NH ). 13C NMR
2
2
(ꢁ, ppm): 19.4 (SCH ), 91.4 (CH ), 102.1, 113.8, 115.7, 120.1,
20 14
4
2
3
2
H, 3.77; N, 14.96. Found: C, 64.09; H, 3.76; N, 14.90.
120.2, 121.7, 127.4, 129.1, 131.2, 132.1, 138.9, 148.1, 149.3, 157.8,
158.2, 167.1, 167.7 (Ar-C). Anal.% Calcd for C H BrN O S
21 15
4
2
(2) 4-Amino-7-phenyl-5-(2-thienyl)pyrido[2,3-d]pyrimidine-
(467.34): C, 53.97; H, 3.24; N, 11.99. Found: C, 53.87; H, 3.19;
2(1H)-thione (29). Yield: (2.2 g, 52%, methanol); m.p.: 198–
N, 12.14.
200∘C; IR (cm−1, KBr): 3277, 3325, 3438 (NH & NH ), 1166
2
(C=S). 1H NMR (ꢁ, ppm): 6.44 (s, 2H, NH ), 6.82–8.15 (m,
(2) 4-Amino-5-(benzo[d][1,3]dioxol-5-yl)-7-(4-bromophe-
nyl)-2-(ethylthio)pyrido[2,3-d]pyrimidine (34). Yield: (2.3 g,
59%, ethanol); m.p.: 176–178∘C; IR (cm−1, KBr): 3288, 3429
2
10H, 9 Ar-H + NH). 13C NMR (ꢁ, ppm): 108.6, 110.0, 122.2,
125.5, 126.8, 127.1, 128.3, 129.6, 139.7, 142.6, 150.7, 158.2, 162.2,

6
Journal of Chemistry
(NH ). 1H NMR (ꢁ, ppm): 1.24 (t, J = 6 Hz, 3H, CH ); 2.88
cells were incubated either alone (negative control) or with
different concentrations of the test compounds to give a final
concentration of 100-50-25-12.5-6.25-3.125-1.56-0.78 ꢃg/mL.
DMSO was employed as a vehicle for dissolution of the tested
compounds and its final concentration on the cells was less
than 0.2%. Cells were suspended in RPMI 1640 medium
(for HepG2 and HT29 cell lines) and DMEM (for MCF 7
cell line), 1% antibiotic-antimycotic mixture (10,000 IU/mL
Penicillin Potassium, 10,000 ꢃg/mL Streptomycin Sulphate,
and 25 ꢃg/mL Amphotericin B), and 1% L-Glutamine in
2
3
(q, J = 6 Hz, SCH ), 6.41 (s, 2H, CH ); 6.92–8.06 (m, 10H, 8
2
2
Ar-H + NH ). 13C NMR (ꢁ, ppm): 16.1 (CH ), 28.1 (SCH ),
2
3
2
91.4 (CH ), 102.1, 113.8, 115.7, 120.1, 120.2, 121.7, 127.4, 129.1,
2
131.2, 132.1, 138.9, 148.1, 149.3, 157.8, 158.2, 167.1, 167.7 (Ar-C).
Anal.% Calcd for C H BrN O S (481.36): C, 54.89; H, 3.56;
22 17
4
2
N, 11.64. Found: C, 54.79; H, 3.53; N, 11.72.
(3) 4-Amino-7-(4-bromophenyl)-2-(methylthio)-5-(2-thi-
enyl)pyrido[2,3-d]pyrimidine (35). Yield: (2.3 g, 66%,
ethanol); m.p.: 185–187∘C; IR (cm−1, KBr): 3391, 3452 (NH ).
96-well flat bottom microplate at 37∘C under 5% CO .
2
2
1H NMR (ꢁ, ppm): 2.42 (s, 3H, SCH ); 6.55 (s, 2H, NH )
Afer 24 h of incubation, the medium was aspirated and
40 ꢃL of MTT salt (2.5 ꢃg/mL) was added to each well and
3
2
7.10–7.89 (m, 8H, Ar-H). 13C NMR (ꢁ, ppm): 19.1 (SCH ),
3
incubated for further 4 h at 37∘C under 5% CO . To stop
100.6, 120.9, 121.7, 122.8, 125.3, 127.4, 129.2, 129.5, 132.4, 138.6,
142.7, 145.0, 159.6, 158.7, 167.1, 167.6 (Ar-C). Anal.% Calcd for
C H BrN S (429.36): C, 50.35; H, 3.05; N, 13.05. Found:
2
the reaction and dissolve the formed crystals, 200 ꢃL of
10% sodium dodecyl sulphate (SDS) in deionized water was
added to each well and incubated overnight at 37∘C. e
absorbance was then measured using a microplate multiwell
reader (Bio-Rad Laboratories Inc., model 3350, Hercules,
California, USA) at 595 nm and a reference wavelength of
620 nm. A statistical significance was tested between samples
and negative control (cells with vehicle) using independent
t-test by SPSS 11 program. e results are presented in
18 13
4
2
C, 50.41; H, 3.00; N, 13.18.
(4) 4-Amino-7-(4-methoxyphenyl)-2-(methylthio)-5-(2-thi-
enyl)pyrido[2,3-d]pyrimidine (36). Yield: (2.6 g, 61%,
DMF/H O); m.p.: 238–240∘C; IR (cm−1, KBr): 3385, 3447
2
1
(NH ). H NMR (ꢁ, ppm): 2.45 (s, 3H, SCH ), 3.82 (s, 3H,
2
3
OCH ), 6.62 (s, 2H, NH ); 6.74–8.12 (m, 8H, Ar-H). 13C
3
2
NMR (ꢁ, ppm): 56.1 (OCH ), 108.7, 110.4, 114.4, 122.3, 125.5,
3
Table 1 as LC (ꢃM) which is the lethal concentration of
50
127.1, 128.3, 129.2, 139.6, 142.2, 144.8, 157.8, 158.3, 167.3, 167.8
(Ar-C). MS: m/z 380 (M+, 100), 364 (70), 333 (82), 317 (54),
286 (22), 234 (30), 132 (26), 83 (16), 75 (24). Anal.% Calcd for
C H N OS (380.49): C, 59.98; H, 4.24; N, 14.73. Found: C,
the compound which causes death of 50% of the cells in
24 h.
19 16
4
2
59.91; H, 4.20; N, 14.84.
2.2.2. In Vitro Antibacterial and Antifungal Activities. All of
the newly synthesized compounds were evaluated for their
in vitro antibacterial activity against Staphylococcus aureus
(ATCC 6538), Bacillus subtilis (ATCC 6633), and Micrococcus
luteus (ATCC 21881) as examples of Gram-positive bacteria
and Escherichia coli (ATCC 25922), Pseudomonas aeruginosa
(ATCC 27853), and Klebsiella pneumonia (clinical isolate) as
examples of Gram-negative bacteria. ey were also evalu-
ated for their in vitro antifungal potential against Candida
albicans (ATCC 10231) and Aspergillus niger (recultured)
fungal strains. Each 100 mL of sterile molten agar (at 45∘C)
received 1 mL of 6 h-broth culture and then the seeded
agar was poured into sterile Petri dishes. Cups (8 mm in
diameter) were cut in the agar. Each cup received 0.1 mL of
the 1 mg/mL solution of the test compounds. e plates were
then incubated at 37∘C for 24 h or, in case of C. albicans,
for 48 h. A control using DMSO without the test compound
was included for each organism. Ampicillin trihydrate and
Clotrimazole were used as reference drugs. e results were
recorded for each tested compound as the average diameter of
inhibition zones (IZ) of bacterial or fungal growth around the
discs in mm. e minimal inhibitory concentrations (MIC) of
the most active compounds were measured using the twofold
serial broth dilution method [27]. e test organisms were
grown in their suitable broth: 24 h for bacteria and 48 h for
fungi at 37∘C. Twofold serial dilutions of solutions of the test
(5)
4-Amino-2-(ethylthio)-7-(4-methoxyphenyl)-5-(2-thi-
enyl)pyrido[2,3-d]pyrimidine (37). Yield: (2.1 g, 61%,
DMF/H O); m.p.: 238–240∘C; IR (cm−1, KBr): 3378, 3425
2
(NH ). 1H NMR (ꢁ, ppm): 1.28 (t, J = 6 Hz, 3H, CH ); 2.85
2
3
(q, J = 6 Hz, 2H, SCH ), 3.81 (s, 3H, OCH ), 6.60 (s, 2H,
2
3
NH ); 6.75–8.04 (m, 8H, Ar-H). 13C NMR (ꢁ, ppm): 15.7
2
(CH ), 28.0 (SCH ), 56.0 (OCH ), 108.4, 110.1, 114.5, 122.2,
3
2
3
125.5, 127.0, 128.2, 129.1, 139.9, 142.5, 144.7, 157.7, 158.2, 167.0,
167.6 (Ar-C). Anal.% Calcd for C H N OS (394.51): C,
20 18
4
2
60.89; H, 4.60; N, 14.20. Found: C, 60.79; H, 4.57; N, 14.34.
2.2. Biological Activity
2.2.1. In Vitro MTT Cytotoxicity Assay. e synthesized
compounds were investigated for their in vitro cytotoxic effect
via the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) method [25, 26] against a panel
of three human tumor cell lines, namely, Caucasian breast
adenocarcinoma MCF7, hepatocellular carcinoma HepG2,
and colon carcinoma HT29 and a normal nontransformed
human foreskin fibroblast Hs27 cell line. e procedures
were done in a sterile area using a laminar flow cabinet
biosafety class II level (Baker, SG403INT, Stanford, ME,
USA). Cells were batch-cultured for 10 days and then seeded
at concentration of 10 × 103 cells/well in fresh complete
compounds were prepared [28] using 200, 100, 50, 25, 12.5,
and 6.25 ꢃg/mL. e tubes were then inoculated with the test
organisms; each 5 mL received 0.1 mL of the above inoculum
and was incubated at 37∘C for 48 h. en, the tubes were
observed for the presence or absence of microbial growth.
growth medium in 96-well microtiter plastic plates at 37∘C
for 24 h under 5% CO using a water jacketed carbon dioxide
2
incubator (Sheldon, TC2323, Cornelius, OR, USA). Media
was aspirated, fresh medium (without serum) was added, and

Journal of Chemistry
7
Table 1: Cytotoxic effects LC (ꢃM)a of the active compounds on three human tumor and nontransformed cell lines and using the MTT
50
assay.
Human tumor cell lines
Nontransformed normal cell line
Compound
number
Colon carcinoma (HT29) Hepatocellular carcinoma (HePG2) Breast cancer (MCF 7)
Foreskin fibroblast (Hs27)
b
70.5
46.7
62.2
113.6
153.4
25.2
28.8
40.4
26.9
40.0
—
—
97.3
—
NTc
NT
NT
NT
NT
>200
>200
>200
>200
NT
21
22
23
30
32
33
34
36
98.9
—
107
111.3
64.6
70.1
97.7
71.2
3.0
—
123.0
6.4
7.9
22.8
8.91
4.0
37
Doxorubicind
aLC : lethal concentration of the compound which causes death of 50% of cells in 24 h; btotally inactive against this cell line; cNT: not tested; dDoxorubicin:
50
positive control cytotoxic agent.
3. Results and Discussion
revealed the CN absorption bands at 2226–2215 cm−1, in
addition to the C=O and C=S bands at 1665–1661 and
1174–1166 cm−1, respectively, whereas their 13C NMR spectra
exhibited two characteristic signals at ꢁ 168.1–169.3 and 179.9–
180.4 ppm for the CO and CS carbons, respectively.
3.1. Chemistry. e pathways adopted for the preparation
of the intermediate and target compounds are illustrated in
Scheme 1. Preparation of the key intermediates 2-amino-
4,6-disubstituted nicotinonitriles 1–5 was performed accord-
ing to Hantzsch-type synthesis via multicomponent one-
pot cyclocondensation of the appropriate aromatic aldehyde
(piperonal or thiophene-2-carbaldehyde) with the substi-
tuted acetophenone, malononitrile, and ammonium acetate
as a nitrogen source. e reaction passes through a two-
step procedure that involved the formation of chalcones
via Claisen-Schmidt condensation, followed by cycloconden-
sation with malononitrile and ammonium acetate. When
compounds 1–5 were heated with either formic acid or
acetic anhydride, the targeted 5,7-disubstituted pyrido[2,3-
d]pyrimidine-4(3H)-ones 6–10 and their 2-methyl analogs
11–15, respectively, were successfully obtained. e IR spectra
of the latter compounds showed the absence of the CN group
absorption and the appearance of new sharp absorption
bands at 1656–1648 cm−1 attributed to the newly formed
C=O groups at position 4, beside the NH absorption bands
at 3320–3259 cm−1. Meanwhile, the 1H-NMR spectra of
compounds 11–15 showed new singlets at ꢁ 2.28–2.36 ppm
Furthermore, direct condensation of the starting
compounds 1–4 with urea at 260–300∘C was attempted to
produce the target 4-amino-5,7-disubstituted-pyrido[2,3-
d]pyrimidine-2(1H)-ones 24–27. e newly introduced
C=O function was obvious both in the IR at 1667–1660 cm−1
(beside the lack of CN absorption) and in the 13C NMR
spectra at ꢁ 166.1–166.4 ppm. Analogously, when compounds
1–5 were directly fused with thiourea at 260–300∘C, the
corresponding
4-amino-5,7-disubstituted-pyrido[2,3-
d]pyrimidine-2(1H)-thiones 28–32 were obtained. eir
13C NMR spectra clearly revealed the signals attributed to
the C=S carbons at ꢁ 181.4–183.3 ppm. Finally, the synthesis
of the targeted alkylthio derivatives 33–37 was successfully
achieved via thioalkylation of the 2-thione derivatives 30–32
with either methyl or ethyl iodide in 1N NaOH medium. e
1H-NMR spectra of 33, 35, and 36 showed the methylthio
singlets at ꢁ 2.42–2.49 ppm, whereas the ethylthio derivatives
34 and 37 exhibited triplets at ꢁ 1.24–1.28 ppm and quartets
at ꢁ 2.85–2.88 ppm due to the ethyl CH and CH protons,
due to the newly introduced CH group, whereas their 13C
3
2
3
respectively.
NMR spectral data exhibited new singlets at ꢁ 20.2–21.1 ppm
due to the new CH group and the CO signals at ꢁ 168.8–
3
173.2 ppm.
3.2. Biological Evaluation
Reacting compounds 1–5 with the phenyl isothiocyanate
in pyridine medium led to the formation of the substituted
dihydropyrido[2,3-d]pyrimidine-2(1H)-thiones 16–20. eir
IR spectra showed new bands at 1642–1635 cm−1 and at
1204–1188 cm−1 corresponding to the C=N moiety and to
the C=S group, respectively. eir 13C NMR spectral data
were characterized by the presence of two signals at ꢁ 179.3–
181.2 and ꢁ 164.4–164.8 ppm for the C=S and C=NH groups,
respectively. However, condensing the starting compounds
2–4 with benzoyl isothiocyanate in acetone afforded the
corresponding thioureido derivatives 21–23. eir IR spectra
3.2.1. In Vitro MTT Cytotoxicity Assay. All the newly synthe-
sized compounds were evaluated for their in vitro cytotoxic
effect via the standard MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] method [25, 26] against
a panel of three human tumor cell lines, namely, colon
carcinoma HT29, hepatocellular carcinoma HePG2, and
Caucasian breast adenocarcinoma MCF7. e results are
presented in Table 1 as LC (ꢃM) which is the lethal
50
concentration of the compound that causes death of 50% of
the cells in 24 h.

8
Journal of Chemistry
R
O
N
R
O
N
R
NH
H
H
N
N
N
N
N
N
N
H
S
X
X
X
6: X = H; R = 3,4-(OCH2O)C6H3
7: X = H; R = 2-thienyl
11: X = H; R = 3,4-(OCH2O)C6H3
12: X = H; R = 2-thienyl
16: X = H; R = 3,4-(OCH2O)C6H3
17: X = H; R = 2-thienyl
8: X = Br; R = 3,4-(OCH2O)C6H3
9: X = Br; R = 2-thienyl
13: X = Br; R = 3,4-(OCH2O)C6H3
14: X = Br; R = 2-thienyl
18: X = Br; R = 3,4-(OCH2O)C6H3
19: X = Br; R = 2-thienyl
10: X = OCH3; R = 2-thienyl
15: X = OCH3; R = 2-thienyl
20: X = OCH3; R = 2-thienyl
iii
ii
i
iv
R
R
CN
CN
O
R
H
v
+
NH2
N
N
N
O
H
H
X
X
X
N
S
1: X = H; R = 3,4-(OCH2O)C6H3
2: X = H; R = 2-thienyl
O
X = H; Br; OCH3
R = 3,4-(OCH2O)C6H3; 2- thienyl
21: X = H; R = 2-thienyl
3: X = Br; R = 3,4-(OCH2O)C6H3
4: X = Br; R = 2-thienyl
22: X = Br; R = 3,4-(OCH2O)C6H3
23: X = Br; R = 2-thienyl
5: X = OCH3; R = 2-thienyl
vi
vii
R
NH2
N
R
NH2
N
R
NH2
N
viii
R2
O
N
N
H
N
N
S
S
N
N
H
X
X
X
28: X = H; R = 3,4-(OCH2O)C6H3
29: X = H; R = 2-thienyl
24: X = H; R = 3,4-(OCH2O)C6H3
25: X = H; R = 2-thienyl
33: X = Br; R = 3,4-(OCH2O)C6H3; R2 = Me
34: X = Br; R = 3,4-(OCH2O)C6H3; R2 = Et
35: X = Br; R = 2-thienyl; R2 = Me
30: X = Br; R = 3,4-(OCH2O)C6H3
31: X = Br; R = 2-thienyl
26: X = Br; R = 3,4-(OCH2O)C6H3
27: X = Br; R = 2-thienyl
36: X = OCH3; R = 2-thienyl; R2 = Me
37: X = OCH3; R = 2-thienyl; R2 = Et
32: X = OCH3; R = 2-thienyl
Scheme 1: Synthesis of the target compounds 1–37. Reagents and reaction conditions: i: malononitrile, NH OAc, EtOH, reflux, 4-5 h; ii:
4
HCOOH, heating, 30 min.; iii: Ac O, heating, 10 min.; iv: PhNCS, pyridine, reflux, 4 h; v: PhCONCS, acetone, reflux, 10 h; vi: urea, 260–
2
300∘C, 1 h; vii: thiourea, 260–300∘C, 1.5 h; viii: R X, 1N NaOH, ethanol, r.t., 2-3 h.
2
e obtained data revealed that the three tested human
tumor cell lines exhibited variable degree of sensitivity pro-
files towards nine of the tested compounds, namely, 21, 22, 23,
30, 32, 33, 34, 36, and 37, whereas the other twenty-six com-
pounds were either marginally active or even totally inactive.
Regarding the activity against the human colon carcinoma
HT29, this cell line proved to be very sensitive to all the
nine active compounds. In particular, it revealed distinctive
62.2 ꢃM, resp.) showed moderate cytotoxic potential against
the same cell line. Shifing to the hepatocellular carcinoma
HepG2, this cell line showed mild to weak sensitivity towards
seven of the tested analogs with LC range 64.6–111.3 ꢃM,
50
when compared to doxorubicin (LC 3.0 ꢃM). Among these,
50
the highest activity was displayed by compounds 33, 34, and
37 (LC 64.4, 70.1, and 71.2 ꢃM, resp.). On the other hand,
50
the human breast cancer MCF 7 emerged as the least sensitive
among the cell lines tested as its growth was affected by the
presence of only six test compounds. However, a remarkable
growth inhibition potential was shown by analogs 33, 34, and
37 as evidenced from their LC values (LC 6.4, 7.9, and
sensitivity towards compounds 33, 34, and 37 (LC 25.2,
50
28.8, and 26.9 ꢃM, resp.) even higher than doxorubicin (LC
50
40.0 ꢃM), the reference standard cytotoxic agent utilized in
this assay. Meanwhile, compounds 22 and 36 (LC 46.7 and
50
50
50
40.4 ꢃM, resp.) were nearly equipotent to doxorubicin (LC
8.91 ꢃM, resp.), which represents about 40–60% of the activity
50
40.0 ꢃM), whereas compounds 21 and 23 (LC 70.5 and
of doxorubicin (LC 4.0 ꢃM).
50
50

Journal of Chemistry
9
Further interpretation of the results revealed that com-
pounds 33, 34, 36, and 37 showed considerable broad spec-
trum cytotoxic activity against the three tested human tumor
cell lines. In particular, compounds 33, 34, and 37 proved
to be the most active members in this study with special
effectiveness against both the colon carcinoma HT29 (almost
of the nontransformed human foreskin fibroblast Hs27 cell
line (LC values > 200 ꢃM), whereas a lower differential
50
cytotoxic profile was shown by analog 36 against the tested
normal cell line (LC > 200 ꢃM), when compared with its
50
LC values against the human breast MCF7 and colon HT29
50
cancer cell lines (22.8 and 40.4 ꢃM, resp.) (Table 1).
twice as active as doxorubicin; LC 25.2, 28.8, and 26.9 versus
50
40 ꢃM, resp.) and human breast cancer MCF 7 (about 40–
3.2.3. In Vitro Antibacterial and Antifungal Activities. All
compounds were evaluated for their in vitro antibacterial
activity against Staphylococcus aureus (ATCC 6538), Bacillus
subtilis (ATCC 6633), and Micrococcus luteus (ATCC 21881)
as examples of Gram positive bacteria and Escherichia coli
(ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and
Klebsiella pneumonia (clinical isolate) as examples of Gram-
negative bacteria. ey were also evaluated for their in
vitro antifungal potential against Candida albicans (ATCC
10231) and Aspergillus niger (recultured) fungal strains. Agar-
diffusion method was used for determination of preliminary
antibacterial and antifungal activity. Ampicillin trihydrate
(antibiotic) and Clotrimazole (antifungal) were used as ref-
erence drugs [27]. Dimethylsulfoxide (DMSO) was used as
a blank and showed no antimicrobial activity. e results
were recorded for each tested compound as the average
diameter of inhibition zones (IZ) of bacterial or fungal
growth around the discs in mm. e minimum inhibitory
concentration (MIC, ꢃg/mL) was determined for compounds
that showed significant growth inhibition zones (≥13 mm)
using the twofold serial dilution method [29].
60% of the activity of doxorubicin; LC 6.4, 7.9, and 8.91
50
versus 4.0 ꢃM, resp.).
A close examination of the structures of the active
compounds showed that the nature of substituents (X and/or
R), together with ring entity (mono- or bicyclic), seemed to
influence the cytotoxic activity. In this context, compounds
substituted with the 4-bromo- or 4-methoxyphenyl moities
(X = Br and OCH ) together with the benzo[d][1,3]dioxol-
3
5-yl counterpart (22, 30, 33, and 34) were in favour of
better cytotoxic activity, when compared with their 2-thienyl
congeners (23, 32, 36, and 37), as revealed from their
LC values in Table 1. Moreover, the bicyclic pyrido[2,3-
50
d]pyrimidines proved to be more active than the monocyclic
nicotinonitriles. In this view, although the starting nicoti-
nonitriles 1–5 lacked cytotoxic efficacy, yet the thiocarbamoyl
benzamide derivatives 21–23 showed overall mild to mod-
erate activity, among which analog 22 (X = Br; R = 3,4-
(OCH O)C H ) was relatively the most active regarding both
2
6
3
potency and spectrum. Cyclization of the nicotinonitriles 1–
5 with different reagents yielded variably substituted bicyclic
pyrido[2,3-d]pyrimidines 6–10, 11–15, 16–20, and 24–27,
which were all inactive against the three tested cell lines.
However, isosteric replacement of 2-one functionality in
pyrido[2,3-d]pyrimidine-2(1H)-ones 24–27, with a 2-thione
group, yielded two weakly active analogs, namely, 30 (X = Br;
R = 3,4-(OCH O)C H ) and 32 (X = OCH ; R = 2-thienyl).
As revealed from MIC data recorded in Table 2, nineteen
of the newly synthesized compounds displayed variable
inhibitory effects on the growth of the tested Gram-positive
and Gram-negative microorganisms with pronounced activ-
ity against S. aureus and E. coli bacterial strains. In addition,
some members exhibited moderate antifungal activity against
C. albicans, whereas all the tested compounds lacked antifun-
gal activity against Aspergillus niger.
2
6
3
3
On the other hand, a remarkable improvement in both
cytotoxic spectrum and potency was observed by thioalky-
lation of the 2-thione function of compounds 28–32 either
with a methyl or ethyl group, where four potentially active
compounds 33, 34, 36, and 37 were obtained. Among these,
the analogs compounds 33 (X = Br; R = 3,4-(OCH O)C H ;
Among the tested Gram-positive bacterial strains, two
organisms, namely, S. aureus and B. subtilis, showed relatively
high sensitivity towards the tested compounds. Concerning
the activity against Gram-positive bacteria, compound 33
(MIC 6.25 ꢃg/mL) was equipotent to ampicillin against S.
aureus; meanwhile analogs 30, 34, and 36 (MIC 12.5 ꢃg/mL)
and 18, 20, 35, and 37 (MIC 25 ꢃg/mL) showed 50% and
25% of the activity of ampicillin, respectively. With regard
to the activity against B. subtilis, compounds 33 and 34
2
6
3
R = CH ), 34 (X = Br; R = 3,4-(OCH O)C H ; R = C H ),
2
3
2
6
3
2
2
5
and 37 (X = OCH ; R = 2-thienyl; R = C H ) stemmed as
3
2
2
5
the most active members in this study.
3.2.2. In Vitro Differential Cytotoxicity Effect. Owing to the
known common side effects ofen induced by chemothera-
peutic agents exemplified by the possible destruction of nor-
mal cells, it was thought worthwhile to evaluate the in vitro
differential cytotoxic effect of the most active analogs 33, 34,
36, and 37 on the nontransformed human foreskin fibroblast
Hs27 cell line, under the same previously mentioned MTT
assay experimental conditions.
(MIC 25 ꢃg/mL) exhibited half the activity of ampicillin (MIC
12.5 ꢃg/mL). M. luteus was the least sensitive Gram-positive
microorganism towards the majority of the tested com-
pounds, except analog 33 which showed 50% of the activity
of ampicillin (MIC 25 versus 12.5 ꢃg/mL). On the other hand,
investigation of anti-Gram-negative efficacy of the active
compounds revealed that three analogs 30, 33, and 34 were
able to produce a distinctive growth inhibitory profile against
Considering the obvious selective inhibitory potential
displayed by the analogs (33, 34, 36, and 37) on the growth
of the human breast MCF7 and colon HT29 cancer cell
lines, the results revealed that the potential cytotoxic activ-
E. coli being equipotent to ampicillin (MIC 6.25 ꢃg/mL),
whereas compounds 31, 36, and 37 (MIC 12.5 ꢃg/mL) were
50% less active than ampicillin against the same organism.
Moreover, the tested P. aeruginosa strain showed high sen-
sitivity towards compound 33 (MIC 12.5 ꢃg/mL) which was
equiactive with ampicillin, whereas the rest of the active
ity exhibited by 33, 34, and 37 (LC values range 6.4–
50
28.8 ꢃM) was confirmed by their clear differential cytotoxic
behavior, showing a marginal inhibitory effect on the growth

10
Journal of Chemistry
Table 2: Minimal inhibitory concentrations (MIC; ꢃg/mL) of the tested compounds.
Compound number
3
4
5
13
15
18
19
20
21
22
23
29
30
31
33
S. aureus
50
B. subtilis
50
M. luteus
E. coli
25
50
P. aeruginosa
100
—
K. pneumonia
C. albicans
—
a
—
—
—
—
50
100
100
100
200
100
100
100
100
100
100
100
50
—
200
100
—
—
—
—
—
100
—
100
—
—
—
100
50
50
12.5
25
100
25
50
—
6.25
50
100
50
100
25
200
100
100
50
100
100
25
—
200
100
—
200
50
50
100
25
—
—
50
25
50
50
100
50
50
100
12.5
50
6.25
12.5
25
12.5
25
6.25
—
50
100
50
100
50
6.25
12.5
6.25
6.25
25
12.5
12.5
6.25
—
200
100
50
100
25
200
12.5
25
50
25
100
12.5
—
100
200
50
50
—
50
100
200
100
100
50
100
—
—
100
25
25
100
50
50
25
34
35
36
37
100
200
100
50
12.5
—
—
12.5
—
Ampicillin
Clotrimazole
12.5
—
aInactive: MIC > 200 ꢀg/mL.
compounds revealed moderate to weak activity against the
same strain (MIC range 25–200 ꢃg/mL). Shifing to the
antifungal potential, ten compounds 18, 20, 29–31, and 33–
37 were able to display appreciable growth inhibitory activity
2-methyl-pyrido[2,3-d]pyrimidine-4(3H)-one derivatives 13
and 15 which were noticeably less active than their par-
ent compounds 3 and 5. Furthermore, reaction of starting
nicotinonitriles 1–5 with phenyl isothiocyanate gave rise
against C. albicans (MIC values 12.5–100 ꢃg/mL, resp.) when
compared with Clotrimazole (MIC 6.25 ꢃg/mL), the standard
antifungal agent utilized in this assay. Among these, analog
33 exhibited the best antifungal activity (MIC 12.5 ꢃg/mL)
that represents 50% of the standard. It is to be noted that all
the tested compounds lacked any antifungal activity against
Aspergillus niger.
to three active compounds 18–20, among which analog 18
(X =Br; R = 3,4-(OCH O)C H ) showed relatively better
2
6
3
antimicrobial spectrum and potential. Although cyclization
of nicotinonitriles 1–5 with urea into the corresponding
pyrido[2,3-d]pyrimidine-2(1H)-ones 24–27 led to total loss
of activity, yet isosteric replacement of the 2-one with a 2-
thione functionality yielded three significantly active broad
spectrum compounds 29–31, among which analog 30 (X
= Br; R = 3,4-(OCH O)C H ) was the most active being
Structurally, the nineteen active compounds represent
two variations: the substituted nicotinonitriles and the
bicyclic pyrido[2,3-d]pyrimidines. In general, relatively bet-
ter antibacterial potential and spectrum were mostly con-
fined to the derivatives comprising the bromine (X = Br)
and the benzo[d][1,3]dioxol-5-yl groups, when compared
2
6
3
equipotent to ampicillin against E. coli, together with a mild
antifungal activity. Finally, thioalkylation of the 2-thione
function of 28–32 afforded five methyl- or ethylthio deriva-
tives 33–37 with a great enhancement in both antimicrobial
spectrum and potential. In particular, compound 33 (X = Br;
R = 3,4-(OCH O)C H ; R = CH ) proved to be equipotent
with their methoxylated (X = OCH ) and 2-thienyl con-
3
geners. Besides, better antimicrobial activity was associated
with the bicyclic pyrido[2,3-d]pyrimidines when compared
with the monocyclic nicotinonitrile precursors (Table 2).
In this context, while the key nicotinonitrile precursors 3–
5 showed mild antimicrobial activity, yet condensation of
the 2-amino group with benzoyl isothiocyanate afforded
three thiocarbamoyl benzamide derivatives 21–23 which
revealed a slight improvement in the antimicrobial spectrum
although not in efficacy. Annelation of nicotinonitriles 1–5
into the pyrido[2,3-d]pyrimidine-4(3H)-ones 6–10 resulted
in complete abolishment of activity, whereas introducing a
2
6
3
2
3
to ampicillin against S. aureus, E. coli, and P. aeruginosa,
together with a noticeable antifungal activity against C.
albicans, whereas analog 34 (X = Br; R = 3,4-(OCH O)C H ;
2
6
3
R = C H ) was equiactive with ampicillin against E. coli.
2
2
5
4. Conclusions
irty-seven polysubstituted pyridines and some derived
bicyclic pyrido[2,3-d]pyrimidine ring systems supported
with various chemotherapeutically active functionalities were
successfully synthesized, characterized, and evaluated for
methyl group at C to the latter ring system furnished two
2

Journal of Chemistry
11
their biological activity as cytotoxic and/or antimicrobial
agents. Globally, the data obtained from both the cytotoxic
and antimicrobial assays suggested that higher biological
activities were confined mainly to compounds comprising the
4-bromo, 4-methoxy, benzo[d][1,3]dioxol-5-yl substituents,
and the bicyclic pyrido[2,3-d]pyrimidines rather than the
monocyclic nicotinonitriles.
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Regarding the cytotoxic activity, nine compounds were
able to show variable cytotoxic efficiency, among which
alkylthio analogs 33, 34, and 37 proved to be the most
active members with considerable broad spectrum cytotoxic
activity against the three tested human tumor cell lines and
particular effectiveness against both the colon carcinoma
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and 26.9 versus 40 ꢃM, resp.) and human breast cancer MCF
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50
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Competing Interests
[13] Y. Mao, W. Zhu, X. Kong et al., “Design, synthesis and biological
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e authors declare that there is no conflict of interests
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is project was funded by Saudi Basic Industries Corpora-
tion (SABIC) and the Deanship of Scientific Research (DSR),
King Abdulaziz University, Jeddah, under Grant no. S-1436-
130-3. e authors therefore acknowledge with thanks SABIC
and DSR for technical and financial support. Extendable
thanks are due to the staff members of the Bioassay-Cell
Culture Laboratory, National Research Centre (NRC), Cairo,
Egypt, for their efforts in performing the MTT cytotoxicity
assay.

12
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