Synthesis and antitumour activity of certain pyrido[2,3-d ] pyrimidine and 1,8-naphthyridine derivatives

Article abstract of DOI:10.3184/174751914X13910886393992

In an effort to establish new candidates with improved anticancer activity, we report here the synthesis of various series of 2,4,5,7-tetrasubstituted pyrido[2,3-d ]pyrimidines and their related isosteres substituted 1,8-naphthyridines. The cytotoxic acti

Full text of DOI:10.3184/174751914X13910886393992

JOURNAL OF CHEMICAL RESEARCH 2014
VOL. 38 MARCH, 147–153
RESEARCH PAPER 147
Synthesis and antitumour activity of certain pyrido[2,3-d] pyrimidine and
1,8-naphthyridine derivatives
Afaf K. Elansary, Ashraf A. Moneer, Hanan H. Kadry* and Ehab M. Gedawy
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt
In an effort to establish new candidates with improved anticancer activity, we report here the synthesis of various series of
2,4,5,7-tetrasubstituted pyrido[2,3-d]pyrimidines and their related isosteres substituted 1,8-naphthyridines. The cytotoxic activity
of the newly synthesised compounds against human breast cancer cell line, MCF7 was investigated. Most of the tested compounds
exploited potent to moderate growth inhibitory activity, in particular 7-(4-chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]pyrimidin-
4-amine exhibited superior potency to the reference drug doxorubicin (IC50 =7.54 and 8.48 µM respectively).
Keywords: synthesis, pyrido[2,3-d]pyrimidine, naphthyridine, inhibitor, substituent effect, antitumour
The pyrido[2,3-d]pyrimidine ring system are an interesting
class of heterocycles because of several biological activities
associated with this scaffold such as antibacterial,1 anti-
inflammatory,2 antihypertensive,3 diuretic,4 antihistaminic
Results and discussion
The synthetic approaches adopted to obtain the target
compounds are outlined in Schemes 1–3. Compounds 2a–d
were synthesised by one-pot reaction of the corresponding
cycloalkanones 1a–d, appropriate aromatic aldehydes, ethyl
cyanoacetate and ammonium acetate following a procedure
previously reported by Fathalla et al.22 in 55–68% yield.
The formation of compounds 2a–d was confirmed by IR
spectra that showed C≡N band at υ 2200 cm–1 as well as NH
bands at υ 3300–3150 cm–1, in addition to C=O absorption
at υ 1660–1650 cm–1. The1H NMR spectra of compounds
2a–d showed an exchangeable singlet signal at δ 12.504–
12.740 ppm corresponding to the NH protons. Chlorination of
3-cyanopyridones 2a–d with phosphorus oxychloride/N,N-
dimethylaniline mixture gave the corresponding 2-chloro
derivatives, 3a–d, which were confirmed by IR spectra that
showed the disappearance of C=O absorption. The latter
compounds were reacted with guanidine base, after liberation
from its hydrochloride salt, in pyridine to afford the substituted
2,4-diaminopyrido[2,3-d]pyrimidines 4a–d. The structures of
compounds 4a–d were established on the basis of elemental
analyses and spectral data. The IR spectra of compounds 4a–d
showed no absorption band for C≡N group, meanwhile revealed
characteristic absorption bands at 3400–3180 cm–1 indicating
5
and more specifically as cytotoxic agents.6 Their cytotoxic
activities might be attributed to inhibition of several enzymes
such as tyrosine kinases,7–9 cyclin-dependant kinase (CDK)10,11
and dihydrofolate reductase (DHFR).12 In a clinical study,
The second generation dihydrofolate reductase inhibitor
piritrexim (PTX), 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-
methylpyrido[2,3-d]pyrimidine, has been developed as a potent
small-molecule inhibitor that is effective against cancer cells
resistant to methotrexate.13 Piritrexim was active against a broad
range of tumours, with evidence of high tissue penetration due to
its lipophilicity.14 Also, lometrexol (5,10-dideazatetrahydrofolic
acid) was a new antifolate. One of the most promising preclinical
features of lometrexol in animal models was its significant
activity against a broad panel of solid tumours.15 (Fig. 1).
On the other hand, naphthyridine derivatives continued to be
of great interest due to the wide spectrum of their physiology
such as anti-inflammatory,16 anti-aggressive,17 and antitumour
activities.18,19 Several synthetic approaches have been
developed to synthesise the 1,8-naphthyridine derivatives,20
but due to their great importance, the development of new
synthetic methods remain an active research area.Based on
the above finding, coupled with our efforts to synthesise novel
fused pyridines acting as antitumour agents,21 we decided to
prepare and evaluate certain new pyrido[2,3-d]pyrimidines
and their bioisosteric analogue 1,8-naphthyridine derivatives,
structurally-related to the aforementioned anticancer agents
(Fig. 1). Also the effect of different substituent at positions
2,4,5, and 7 to substantiate the possible influence of such
substitution on the in vitro antitumour activities against human
breast adenocarcinoma MCF-7 cell line.
1
the presence of NH2 functionalities. Furthermore, their H
NMR spectra revealed the appearance of singlet signals at
δ6.418–7.158and11.890–12.026 ppmcorrespondingtotwoNH2
groups. Scheme 2 illustrates the synthesis of several substituted
pyrido[2,3-d]pyrimidines 6–12 starting from the 2-amino-
4,6-disubstituted pyridine-3-carbonitriles 5a,b. Compounds
5a,b were synthesised by applying a one-pot reaction of the
respective substituted acetophenone with appropriate aromatic
aldehydes and malononitrile in the presence of ammonium
COOH
H
NH
OCH3
CH3 NH2
HOOC
O
OH
N
N
N
N
N
NH2
N
NH2
H
OCH3
Piritrexim
Lometrexol
Fig. 1 Some drugs containing pyrido[2,3-d]pyrimidine currently in clinical development.
* Correspondent. E-mail: hanankadry2005@yahoo.com
JCR1302284_FINAL.indd 147
04/03/2014 09:53:26

148 JOURNAL OF CHEMICAL RESEARCH 2014
O
OC2H5
NC
ArCHO
CH2R1
+
+
R
O
1a-d
1a R,R1= -(CH2)4
1b R,R1= -(CH2)4
1c R,R1= -(CH2)5
1d R,R1= -(CH2)6
NO2
NH2
i
CN
Ar
Ar
R1
R
CN
N
N
R1
R
CN
Cl
ii
12
Cl
N
H
O
NO2
N
i
2a-d
3a-d
NO2
NH2
CN
iii
ii
a
b
c
d
R,R1=-(CH2)4- , Ar = 3-NO2C6H4
R,R1=-(CH2)4- , Ar = 2-NO2C6H4
R,R1=-(CH2)5- , Ar = 2-NO2C6H4
R,R1=-(CH2)6- , Ar = 2-NO2C6H4
N
NH2
CN
O
Cl
Ar NH2
N
5 a
R1
N
N
H
Cl
R
N
N
NH2
13
4a-d
Scheme 1 Reagent and conditions: (i) CH COONH , n-butanol, reflux
8 h; (ii) POCL dimethylaniline, reflux 10 h; (i3ii) guani4dine HCL, NaOC2H5,
pyridine, reflu3x 5 h.
Scheme 3 Reagent and condition: (i) benzylidenemalononitrile, piperidine,
dioxane, reflux 3 h; (ii) ethyl cyanoacetate, triethylamine, ethanol, reflux 3 h.
Ar NH2
ii
N
O
Ar1
N
N
Ar1
CH3
ArCHO
+
6a,b
Ie,f
1e Ar1=4-ClC6H4
1f Ar1=3-NO2C6H4
Ar= 3-NO2C6H4,
S
Ar NH2
N
iii
i
Ar1
N
N
O
H
7a,b
Ar
Ar NH2
N
CN
Ar1
N
NH2
iv
Ar1
N
N
S
H
8 a,b
5a,b
Ar NH2
N
v
Ar1
N
N
S
NH2
9 a,b
a Ar= 3-NO2C6H4 Ar1=4-ClC6H4
b Ar = C4H3S
Ar1=3-NO2C6H4
Ar
vi
NH
Ar1
N
N
H
S
10 a,b
Ar NH2
N
C6H5
S
vii
Ar1
N
N
11 a,b
Scheme 2 Reagent and condition: (i) malononitrile, CH3COONH4, n-butanol, reflux 8 h; (ii) formamide, oil bath, reflux 15 h; (iii) urea, oil bath, reflux 4 h;
(iv) thiourea, oil bath, reflux 4 h; (v) guanidine HCL methanol, reflux 6 h; (vi) CS2, pyridine, reflux 15 h; (vii) PhCNS, DMF, TEA, reflux 10 h.
JCR1302284_FINAL.indd 148
04/03/2014 09:53:27

JOURNAL OF CHEMICAL RESEARCH 2014 149
Fig. 2 IC50 in µM of the synthesised compounds and doxorubicin against human breast adenocarcinoma cell line (MCF7).
1
acetate. The IR spectra of 5a,b revealed the appearance of
absorption bands at 3362–3140 cm–1 corresponding to NH2 and
The H NMR spectra of compounds 12 and 13 displayed a
singlet signal at δ 6.95 ppm corresponding to C-6 proton of
naphthyridine. Both the analytical and spectral data (IR, H
NMR 13C NMR and MS) of the newly synthesised compounds
1
absorption bands at 2203 cm–1 attributed to CN group. The H
1
NMR spectra was consistent with the proposed structure.
Accordingly, treatment of 5a,b with excess formamide
produced 4-amino-5,7-disubstitutedpyrido[2,3-d]pyrimidines
6a,b following reported reaction conditions.23 The IR spectra
proved as useful in tracing the disappearance of the C≡N
stretching absorption of the parent compound 5a,b. Whereas
the 1H NMR spectra of 6a and 6b displayed signals at δ 10.02
and 10.442 ppm respectively corresponding to NH2 group.
Furthermore, the mass spectrum of 6a displayed molecular ion
peaks at m/z 377 and 379 corresponding to (M+) and (M+2)
in a ratio 3:1. On the other hand, reacting compounds 5a,b
with urea or thiourea afforded 7a,b and 8a,b respectively,
following the reaction conditions reported for the preparation
of related compounds.23 The structures of compounds 7a,b
were in full agreement with the proposed structures.
In vitro anticancer screening
The newly synthesised compounds were evaluated for their
in vitro cytotoxic activity against human breast cancer cell
line, MCF-7. Doxorubicin, which is one of the most effective
anticancer agents, was used as the reference drug in this study.
The IC50 (dose of the compound which caused a 50% reduction
of survival values) are shown in Table 1 and represented
graphically in Fig. 2. From the results in Table 1, it is evident
that all the tested compounds displayed potent to moderate
growth inhibitory activity, in particular compounds 4c and 6a
(IC50 =7.70 and 7.54 µM respectively) were found to be more
potent and efficacious than doxorubicin (IC50 values 8.48 µM).
Further studies are recommended to explore the mechanism of
action as well as the effect of substitution on the anticancer effect
of these compounds. We hope that the synthesised compounds
serve as lead chemical entities for further modification to
render them clinically useful drug agents.
1
and 8a,b were confirmed by IR and H NMR spectra. The
IR spectra clearly showed the disappearance of the C≡N
stretching absorption of the parent compound 5a,b and the
appearance of C=O (in compounds 7a,b) or C=S (in compounds
8a,b) stretching signals together with NH2 absorption band.
1
The H NMR spectra of compounds 7a,b and 8a,b revealed
two D2O exchangeable singlet signals at δ 6.51–7.04 and δ
11.18–11.32 ppm corresponding to NH2 and NH, respectively.
Whereas, 2,4-diamino-5,7-disubstituted pyrido[2,3-d]
pyrimidines 9a,b were obtained by reacting 5a,b with guanidine
hydrochloride in refluxing methanol in the presence of sodium
Experimental
Melting points are uncorrected and determined in one-end open
capillary tubes using Gallenkamp melting point apparatus MFB-595-
010M (Gallenkamp, London, UK). Microanalysis was carried out at
the Micro-analytical Unit, Faculty of Science, Cairo University and
the Regional Centre for Microbiology and Biotechnology, Al-Azhar
University. Analyses indicated were within ±0.4% of the theoretical
values. Infrared Spectra were recorded on Schimadzu FT-IR 8400S
spectrophotometer (Shimadzu, Kyoto, Japan), and expressed in
wavenumber (cm–1), using potassium bromide discs. The NMR spectra
were recorded on a Varian Gemini 200 MHz and Varian Mercury
VX-300 or Jeol-ECA500, 500 MHz Japan NMR spectrometer. 1H
NMR spectra were run at 300 MHz and 13C NMR spectra were run at
125 MHz in dimethylsulfoxide (DMSO-d6). Chemical shifts are quoted
in δ and were related to that of the solvents. Mass spectra were recorded
using Hewlett Packard Varian (Varian, Polo, USA) and Shimadzu gas
chromatograph mass spectrometer-QP 1000 EX (Shimadzu, Kyoto,
Japan). TLC was carried out using Art. DC-Plastikfolien, Kieselgel 60
F254 sheets (Merck, Darmstadt, Germany), the developing solvents
were chloroform: benzene: methanol [6.5: 3.5: 0.5] and the spots were
visualised at 366, 254 nm by UV Vilber Lourmat 77202 (Vilber, Marne
La Vallee, France). Compound 5a,24 benzylidenemalononitrile, 25 were
obtained according to reported procedure.
1
hydroxide. H NMR spectra of compounds 9a,b revealed the
appearance of two exchangeable singlet signal at δ 5.02,6.95
and δ 7.23–8.5 ppm corresponding to NH2 protons. Reacting
5a,b with carbon disulfide in refluxing pyridine afforded the
corresponding pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dithione
derivatives 10a,b, which were confirmed by spectral data. The
IR spectra proved as useful in tracing the disappearance of the
C≡N stretching absorption of the parent compound 5a,b and
the appearance of a C=S absorption. Finally, reaction of 5a,b
with phenyl isothiocyanate in refluxing dimethyl formamide
in the presence of triethylamine yielded the corresponding
4-amino-3-phenyl-5,7-disubstituted pyrido[2,3-d]pyrimidine-
2(3H)-thiones (11a,b) which were confirmed by spectral data.
One of the principal objectives of the present work was the
synthesis of substituted 1,8-naphthyridine derivatives12 and
13 (Scheme 3), thereby, reacting 5b with either benzylidene
malononitrile in refluxing dioxane in the presence of piperidine
or ethyl cyanoacetate in refluxing ethanol in the presence of
triethylamine afforded compounds 12 and 13 respectively.
JCR1302284_FINAL.indd 149
04/03/2014 09:53:33

150 JOURNAL OF CHEMICAL RESEARCH 2014
Table 1 In vitro cytotoxic activities of the synthesised compounds against MCF-7 cancer cell line
R2 R1
R2 R1
R2 R1
R3
R4
R3
R4
R3
R4
CN
R
C6H5
N
N
N
N
R
N
N
R
N
4–10
R
N
11
12 and 13
R4
Compound
R1
R2
R3
–
IC50/µMa
Doxorubicinb
–
–
–
–
8.48
44.34
9.63
7.70
9.69
4a
4b
4c
4d
6a
6b
7a
NH2
NH2
NH2
NH2
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
=S
3-NO2C6H4
2-NO2C6H4
2-NO2C6H4
2-NO2C6H4
3-NO2C6H4
C4H3S
3-NO2C6H4
C4H3S
3-NO2C6H4
C4H3S
3-NO2C6H4
C4H3S
3-NO2C6H4
C4H3S
3-NO2C6H4
C4H3S
3-NO2C6H4
3-NO2C6H4
–(CH2)4
–(CH2)4
–(CH2)5
–(CH2)6
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4-ClC6H4
3-NO2C6H4
4-ClC6H4
3-NO2C6H4
4-ClC6H4
3-NO2C6H4
4-ClC6H4
3-NO2C6H4
4-ClC6H4
3-NO2C6H4
4-ClC6H4
3-NO2C6H4
4-ClC6H4
4-ClC6H4
7.54
H
31.77
27.17
31.75
16.47
56.15
34.87
13.17
37.24
11.66
20.32
12.89
27.41
10.05
=O
=O
=S
7b
8a
8b
9a
9b
10a
10b
11a
11b
12
=S
NH2
NH2
=S
=S
=S
=S
C6H5
=O
=S
NH2
NH2
NH2
NH2
13
aThe values given are means of three experiments.
bDoxorubicin was used as reference drug.
Synthesis of 2-oxo-4,5,6-trisubstituted-1,2-dihydropyridine-3-
carbonitriles 2a–d; general procedure
4-(2-Nitrophenyl)-2-oxo-1,2,5,6,7,8,9,10-octahydrocycloocta[b]
pyridine-3-carbonitrile (2d): Crystallised from ethanol. Yield 68%;
m.p.>300 °C; IR νmax/cm–1: 3300 (NH), 3050 (CH aromatic), 2920, 2820
A mixture of appropriate cycloalkanones 1a–d (0.05 mol), appropriate
aromatic aldehyde (0.05 mol), ethyl cyanoacetate (5.65 mL, 0.05 mol)
and ammonium acetate (30.8 g, 0.4 mol) in butan-1-ol (150 mL) was
heated under reflux for 8 h. The separated solid was filtered, washed
with ethanol, dried and crystallised from appropriate solvent.
4-(3-Nitrophenyl)-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-
carbonitrile (2a): Crystallised from butan-1-ol. Yield 63%; m.p.
291–292°C; IR νmax/cm–1: 3280, 3150 (NH), 3080 (CH aromatic), 2949,
2910, 2866 (CH aliphatic), 2225 (C≡N), 1647(C=O), 1533, 1348 (NO2);
1H NMR (CDCl3) δ ppm: 1.59–1.72 (m, 4H, 2CH2), 2.04–2.12 (m, 2H,
CH2), 2.62–2.82 (m, 2H, CH2), 7.88–8.39 (m, 4H, ArH), 12.56 (s, 1H,
NH, exch. D2O). Anal. calcd for C16H13N3O3 (295.28): C, 65.07; H, 4.43;
N, 14.23; found: C, 64.99; H, 4.49; N, 14.13%.
1
(CH aliphatic), 2210 (C≡N), 1640 (C=O), 1540, 1340 (NO2); H NMR
(DMSO-d6) δ ppm: 1.15–1.20 (m, 4H, 2CH2), 1.37–1.41 (m, 4H, 2CH2),
1.65–1.70 (m, 2H, CH2), 2.16–2.24 (m, 4H, 2CH2), 2.76–2.80 (m, 2H,
CH2), 7.56 (d, 1H, J=7.8 Hz, H6 of ArH), 7.84 (t, 1H, J=8.1 Hz, H5 of
ArH), 7.96 (t, 1H, J=8.4 Hz, H4 of ArH), 8.33 (d, 1H, J=8.1 Hz, H3 of
ArH), 12.66 (s, 1H, NH, exch. D2O). Anal. calcd for C18H17N3O3 (323.32):
C, 66.86; H, 5.29; N, 12.99; found: C, 66.79; H, 5.10; N, 13.41%.
Synthesis of 2-chloro-4,5,6-trisubstituted pyridine-3-carbonitriles
(3a–d); general procedure
A mixture of the corresponding pyridones 2a–d (0.0075 mol), N,
N-dimethylaniline (10 mL), and phosphorous oxychloride (10 mL)
was heated under reflux for 10 h. The reaction mixture was cooled
and poured onto crushed ice. The formed solid was filtered, dried and
crystallised from ethanol.
2-Chloro-4-(3-nitrophenyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (3a): Yield 52%; m.p. 200–202 °C; IR νmax/cm–1: 3090 (CH
aromatic), 2960, 2935 (CH aliphatic), 2229 (C≡N), 1530, 1340 (NO2);
1H NMR (CDCl3) δ ppm: 1.76–1.91 (m, 4H, 2CH2), 2.33–2.62 (m, 2H,
CH2), 2.98–3.04 (m, 2H, CH2), 7.63–8.38 (m, 4H, ArH). Anal. calcd for
C16H12ClN3O2 (313.73): C, 61.25; H, 3.85; N, 13.39; found: C, 61.45; H,
3.80; N, 13.65%.
4-(2-Nitrophenyl)-2-oxo-1,2,5,6,7,8-hexahydroquinoline-3-
carbonitrile (2b): Crystallised from ethanol. Yield 55%; m.p. 276–
278°C; IR νmax/cm–1: 3300 (NH),3050 (CH aromatic), 2950, 2850
1
(CH aliphatic), 2200 (C≡N), 1640(C=O), 1520, 1340 (NO2); H NMR
(DMSO-d6) δ ppm: 1.51–1.66 (m, 4H, 2CH2), 1.81–1.99 (m, 2H, CH2),
2.63–2.69 (m, 2H, CH2), 7.52 (d, 1H, J=7.5 Hz, H6 of ArH), 7.84 (t, 1H,
J=7.5 Hz, H5 of ArH), 7.96 (t, 1H, J=7.8 Hz, H4 of ArH), 8.31 (d, 1H,
J=7.8 Hz, H3 of ArH), 12.51 (s, 1H, NH, exch. D2O). Anal. calcd for
C16H13N3O3 (295.28): C, 65.07; H, 4.43; N, 14.23; found: C, 64.90; H,
4.38; N, 14.45%.
4-(2-Nitrophenyl)-2-oxo-2,5,6,7,8,9-hexahydro-1H-cyclohepta[b]
pyridine-3-carbonitrile (2c): Crystallised from ethanol. Yield 68%;
m.p. 273–275°C; IR νmax/cm–1: 3300 (NH), 3050 (CH aromatic), 2920,
2820 (CH aliphatic), 2210 (C≡N), 1640 (C=O), 1540, 1350 (NO2);
1H NMR (DMSO-d6) δ ppm: 1.26–1.46 (m, 4H, 2CH2), 1.62–1.84 (m,
2H, CH2), 2.08–2.26 (m, 4H, 2CH2), 2.82–2.98 (m, 2H, CH2), 7.54–
8.32 (m, 4H, ArH), 12.74 (br s, 1H, NH, exch. D2O). Anal. calcd for
C17H15N3O3 (309.30): C, 66.00; H, 4.88; N, 13.58; found: C, 66.14; H,
4.92; N, 13.74%.
2-Chloro-4-(2-nitrophenyl)-5,6,7,8-tetrahydroquinoline-3-
carbonitrile (3b): Yield 55%; m.p. 154–156°C; IR νmax/cm–1: 3050(CH
1
aromatic), 2920 (CH aliphatic), 2200 (C≡N), 1520, 1340 (NO2); H
NMR (DMSO-d6) δ ppm: 1.64–1.66 (m, 2H, CH2), 1.78–1.80 (m, 2H,
CH2), 2.10–2.35 (m, 2H, CH2), 2.96–2.98 (m, 2H, CH2), 7.63 (d, 1H,
J=7.5 Hz, H6 of ArH), 7.88 (t, 1H, J=7.5 Hz, H5 of ArH), 7.98 (t, 1H,
J=7.5 Hz, H4 of ArH), 8.37 (d, 1H, J=8.1 Hz, H3 of ArH); MS m/z(%):
316.00 (M+3,12.45), 315(M+2, 5.95), 314(M+1, 25.71) 313(M+,18.67),
(C5H3,100). Anal. calcd for C16H12ClN3O2 (313.73): C, 61.25; H, 3.85; N,
13.39; found: C, 60.88; H, 3.80; N, 13.42%.
JCR1302284_FINAL.indd 150
04/03/2014 09:53:34

JOURNAL OF CHEMICAL RESEARCH 2014 151
2-Chloro-4-(2-nitrophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]
Synthesis of 2-amino-4,6-diarylpyridine-3-carbonitriles 5a,b;
general procedure
pyridine-3-carbonitrile (3c): Yield 60%; m.p. 165–166 °C; IR νmax
/
cm–1: 3060 (CH aromatic), 2920 (CH aliphatic), 2200 (C≡N), 1520,
A mixture of substituted acetophenone either Ie or 1f (0.01 mol),
appropriate aromatic aldehydes (0.01 mol), malononitrile (5.3 g,
0.08 mol) and ammonium acetate (6.16 g, 0.08 mol) in butan-1-ol
(10 mL) was heated under reflux for 8 h then cooled. The obtained
precipitate was filtered, washed with ethanol, dried and crystallised
from suitable solvent to afford 5a,b.
2-Amino-6-(4-chlorophenyl)-4-(3-nitrophenyl)nicotinonitrile (5a):
Crystallised from butan-1-ol, yield 73%; m.p.>300 °C; IR νmax/cm–1:
3300–3100 (NH2), 3020(CH aromatic), 2200 (CN), 1550, 1350 (NO2);
1H NMR (DMSO-d6) δ ppm: 6.61 (s, 2H, NH2, exch. D2O), 7.11–8.86
(m, 9H, 8ArH and H5). Anal. calcd for C18H11ClN4O2 (350.76): C, 61.64;
H, 3.16; N, 15.97; found: C, 61.47; H, 3.13; N, 15.64%.
2-Amino-6-(3-nitrophenyl)-4-(thiophen-2-yl)nicotinonitrile (5b):
Crystallised from ethanol. Yield 80%; m.p.>300 °C; IR νmax/cm–1:
3362–3140 (NH2), 3080(CH aromatic), 2203 (CN), 1537, 1330 (NO2);
1H NMR (DMSO-d6) δ ppm: 6.86 (s, 2H, NH2, exch. D2O), 7.11–7.86
(m, 8H, 7ArH and H5). Anal. calcd for C16H10N4O2S (322.34): C, 59.62;
H, 3.13; N, 17.38; found: C, 59.52; H, 3.12; N, 17.35%.
1
1340 (NO2); H NMR (DMSO–d6) δ ppm: 1.33–1.46 (m, 2H, CH2),
1.66–1.76 (m, 4H, 2CH2), 2.40–2.43 (m, 2H, CH2), 3.10–3.12 (m, 2H,
CH2), 7.60 (d, 1H, J=7.5 Hz, H6 of ArH), 7.87 (t, 1H, J=7.5 Hz, H5 of
ArH), 7.96 (t, 1H, J=7.8 Hz, H4 of ArH), 8.36 (d, 1H, J=7.8 Hz, H3
of ArH). Anal. calcd for C17H14ClN3O2 (327.75): C, 62.29; H, 4.30; N,
12.82; found: C, 62.47; H, 4.28; N, 13.01%.
2-Chloro-4-(2-nitrophenyl)-5,6,7,8,9,10-hexahydrocycloocta[b]
pyridine-3-carbonitrile (3d): Yield 54%; m.p. 157–158 °C; IR νmax/cm–1:
3064 (CH aromatic), 2937, 2926, 2856 (CH aliphatic), 2210 (C≡N), 1520,
1
1320 (NO2); H NMR (DMSO–d6) δ ppm: 1.18–1.51 (m, 6H, 3CH2),
1.71–1.82 (m, 2H, CH2), 2.12–2.14 (m, 2H, CH2), 3.05–3.12 (m, 2H, CH2),
7.71–8.43 (m, 4H, ArH). Anal. calcd for C18H16ClN3O2 (341.78): C, 63.25;
H, 4.71; N, 12.29; found: C, 63.41; H, 4.76; N, 12.42%.
Synthesis of 2,4-diamino-5,6,7-trisubstituted pyrido[2,3-d]pyrimidines
(4a–d); general procedure
Guanidine hydrochloride (1.84 g, 0.022 mol) was added to a cold
solution of sodium (0.506 g, 0.022 mol) in absolute ethanol (50 mL).
The separated sodium chloride was filtered off and washed with
ethanol, the filtrate was evaporated under reduced pressure. The
formed oily mass was diluted with pyridine (5 mL) and finally the
corresponding 2-chloro-4,5,6-trisubstituted pyridine-3-carbonitrile
3a–d (0.0033 mol) was added. The reaction mixture was heated under
reflux for 5 h, cooled and finally poured onto water (25 mL). The
formed precipitate was filtered, dried and crystallised from DMF/H2O.
5-(3-Nitrophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline-2,4-
diamine (4a): Yield 72%; m.p.>300 °C; IR νmax/cm–1: 3283, 3180 (NH2),
3050(CH aromatic), 2950, 2850 (CH aliphatic), 1580, 1380 (NO2);
1H NMR (DMSO-d6) δ ppm: 1.22–1.25 (m, 2H, CH2), 1.82–1.90(m,
2H, CH2), 2.73–2.91(m, 4H, 2CH2), 6.58 (br s, 2H, NH2, exch. D2O),
7.19–8.37 (m, 4H, ArH), 11.89 (s, 2H, NH2, exch. D2O). Anal. calcd for
C17H16N6O2 (336.35): C, 60.71; H, 4.79; N, 24.99; found: C, 60.73; H,
4.79; N, 25.00%.
5-(2-Nitrophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline-
2,4-diamine (4b): Yield 68%; m.p.>300 °C; IR νmax/cm–1: 3400–3200
(NH2), 3100 (CH aromatic), 2920, 2850 (CH aliphatic), 1580, 1330
(NO2); 1H NMR (DMSO-d6) δ ppm: 1.17–1.23 (m, 2H, CH2), 1.71–1.98
(m, 2H, CH2), 2.74–2.89(m, 4H, 2CH2), 7.15 (s, 2H, NH2, exch. D2O),
7.48–7.96 (m, 4H, ArH), 12.02 (s, 2H, NH2, exch. D2O); 13C NMR
(DMSO) δ ppm: 22.98, 24.69, 46.19, 52.15.23, 93.91, 110.03, 115.54,
116.28, 130.82, 134.69, 136.13, 138.22, 141.21, 148.49, 155.58, 158.68,
162.23; Anal. calcd for C17H16N6O2 (336.35): C, 60.71; H, 4.79; N, 24.99;
found: C, 60.76; H, 4.69; N, 25.20%.
Synthesis of 4-amino-5,7-disubstituted pyrido[2,3-d]pyrimidines
(6a,b); general procedure
A mixture of the appropriate 2-aminopyridine-3-carbonitriles 5a,b
(0.01 mol) and excess formamide (15 mL) was refluxed in an oil-bath
for 15 h at 210 °C. The mixture was cooled. The separated solid was
filtered, dried and crystallised from ethanol.
7-(4-Chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]pyrimidin-4-
amine (6a): Yield 58%; m.p. 255–257 °C; IR νmax/cm–1: 3293, 3168
(NH2), 3050 (CH aromatic), 1559, 1337 (NO2); 1H NMR (DMSO–d6) δ
ppm: 7.39–8.91 (m, 8H, ArH), 8.02 (s, 1H, H2), 8.53 (s, 1H, H6), 10.44 (s,
2H, NH2,exch.D2O); 13C NMR (DMSO) δ ppm: 106.88, 121.19, 122.08,
123.99, 129.26, 129.77, 130.62, 130.89, 131.08, 133.24, 135.06, 135.85,
136.56, 139.90, 147.94, 148.60, 155.01, 156.05, 159.66; MS m/z (%):
379.22 (M+2, 6.52), 377.70 (M+, 22.23). Anal. calcd for C19H12ClN5O2
(377.78): C, 60.41; H, 3.20; N, 18.54; found: C, 60.98; H, 3.45; N, 18.88%.
7-(3-Nitrophenyl)-5-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4-amine
(6b): Yield 83%; m.p.>300 °C; IR νmax/cm–1: 3327–3193 (NH2), 3050
(CH aromatic), 1561, 1338 (NO2); 1H NMR (DMSO-d6) δ ppm: 6.85–
8.95 (m, 7H, ArH and thienyl H), 7.99 (s, 1H, H2), 8.35 (s,1H, H6),
10.01 (s, 2H, NH2, exch.D2O). Anal. calcd for C17H11N5O2S (349.37): C,
58.44; H, 3.17; N, 20.05; found: C, 58.89; H, 3.09; N, 20.43%.
Synthesis of 4-amino-5,7-disubstituted pyrido[2,3-d]pyrimidin-2(1H)-
ones (7a,b); general procedure
A mixture of the appropriate 2-aminopyridine-3-carbonitrile (5a,b)
(0.01 mol) and urea (1.2 g, 0.02 mol) was heated in an oil bath at 120–
130 °C for 2 h. The temperature was then raised to 180 °C for another
2 h. The residue was washed with water then ethanol and finally
crystallised from appropriate solvent.
5-(2-Nitrophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[b]
pyrido[2,3-d]pyrimidine-2,4-diamine (4c): Yield 84%; m.p. 255–
258 °C; IR νmax/cm–1: 3480, 3310 (NH2), 3090 (CH aromatic), 2920 (CH
4-Amino-7-(4-chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]
pyrimidin-2(1H)-one (7a): Crystallised from butan-1-ol. Yield 55%;
m.p. 275–277 °C; IR νmax/cm–1: 3330–3200 (NH, NH2), 3050 (CH
aromatic), 1700 (C=O),1520, 1340 (NO2); 1H NMR (DMSO-d6) δ ppm:
6.93 (s, 1H, H6), 7.01 (s, H, NH, exch.D2O), 7.58–8.13 (m, 3H, H4, H5
and H6 of 3-NO2C6H4), 8.85 (d, 2H, J=8 Hz, H3, H5 of 4-ClC6H4), 8.36
(d, 2H, J=7.8 Hz, H2, H6 of 4-ClC6H4), 8.47 (s, 1H, H2 of 3-NO2C6H4),
11.18 (s, 2H, NH2, exch.D2O); 13C NMR (DMSO) δ ppm: 94.87, 95.32,
116.19, 119.08, 123.99, 124.71, 129.26, 130.87, 131.08, 135.06, 135.85,
136.56, 139.25, 147.96, 148.32, 149.37, 150.46, 154.54; MS m/z (%):
393.15 (M+, 41.18), 104.20 (100). Anal. calcd for C19H12ClN5O3 (393.78):
C, 57.95; H, 3.07; N, 17.78; found: C, 57.69; H, 3.30; N, 17.20%.
4-Amino-7-(3-nitrophenyl)-5-(thiophen-2-yl)pyrido[2,3-d]
pyrimidin-2(1H)-one (7b): Crystallised from ethanol. Yield 80%;
m.p.>300 °C; IR νmax/cm–1: 3419, 3350, 3197 (NH, NH2), 3100 (CH
aromatic), 1676 (C=O),1540, 1368 (NO2); 1H NMR (DMSO-d6) δ ppm:
6.88 (s, 1H, NH, exch. D2O), 7.13–7.92 (m, 8H, 7ArH+H6), 11.22 (s,
2H, NH2, exch.D2O); MS m/z (%): 365.24 (M+, 58.59), 359 (100). Anal.
calcd for C17H11N5O3S (365.37): C, 55.88; H, 3.03; N, 19.17; found: C,
55.83; H, 3.40; N, 19.30%.
1
aliphatic) 1580, 1340 (NO2); H NMR (DMSO–d6) δ ppm: 1.76–1.91
(m, 6H, 3CH2), 3.01–3.21 (m, 4H, 2CH2), 6.46 (s, 2H, NH2, exch. D2O),
7.25–8.35 (m, 4H, ArH), 12.02 (s, 2H, NH2, exch. D2O); 13C NMR
(DMSO) δ ppm: 26.09, 29.34, 29.77, 31.23, 38.15, 112.12, 114.41, 118.42,
119.0, 127.89, 132.36, 138.33, 149.74, 155.70, 158.58, 158.88, 167.2,
169.87. Anal. calcd for C18H18N6O2 (350.37): C, 61.70; H, 5.18; N, 23.99;
found: C, 61.80; H, 5.00; N, 24.06%.
5-(2-Nitrophenyl)-5,6,7,8,9,10-hexahydrocycloocta[b]pyrido[2,3-d]
pyrimidine-2,4-diamine (4d):Yield 75%; m.p.>300 °C; IR νmax/cm–1:
3400–3200 (NH2), 3050 (CH aromatic), 2920, 2820 (CH aliphatic),
1
1520, 1340 (NO2); H NMR (DMSO–d6) δ ppm: 1.36–1.50 (m, 4H,
2CH2), 1.76–1.80 (m, 2H, CH2), 1.85–1.92 (m, 2H, CH2), 2.86–2.88 (m,
2H, CH2), 3.06–3.12 (m, 2H, CH2), 6.41 (s, 2H, NH2, exch. D2O), 7.23
(d, 1H, J=6.9 Hz, H6 of ArH), 7.47 (t, 1H, J=8.7 Hz, H4 of ArH), 7.52
(t, 1H, J=6.9 Hz, H5 of ArH), 8.33 (d, 1H, J=8.7 Hz, H3 of ArH), 12.01
(s, 2H, NH2, exch. D2O); 13C NMR (DMSO) δ ppm: 26.41, 26.61, 30.12,
30.57, 35.50, 42.55, 96.91, 112.17, 125.72, 128.49, 131.59, 131.89, 135.32,
146.8, 149.9, 156.28, 156.19, 161.69, 165.37. Anal. calcd for C19H20N6O2
(364.40): C, 62.62; H, 5.53; N, 23.06; found: C, 62.47; H, 5.51; N, 23.01%.
JCR1302284_FINAL.indd 151
04/03/2014 09:53:34

152 JOURNAL OF CHEMICAL RESEARCH 2014
Synthesis of 4-amino-5,7-disubstituted pyrido[2,3-d]pyrimidine-
2(1H)-thiones (8a,b); general procedure
7-(3-Nitrophenyl)-5-(thiophen-2-yl)pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-dithione (10b): Crystallise from ethanol, Yield 58%; m.p.
258–260 °C; IR νmax/cm–1: 3377, 3277 (NH), 3095 (CH aromatic), 1521,
1340 (NO2), 1085 (C=S); 1H NMR (DMSO-d6) δ ppm: 6.89 (s, 1H, N1H
exch. D2O), 7.06 (s, 1H, N3H, exch D2O), 7.17–8.6 (m, 8H, 7ArH+H6);
MS m/z (%): 398.30 (M+, 100). Anal. calcd for C17H10N4O2S3 (398.48): C,
51.24; H, 2.53; N, 14.06; found: C, 51.60; H, 2.92; N, 14.28%.
A
mixture of thiourea (1.52 g, 0.02 mol) and the appropriate
2-aminopyridine-3-carbonitrile 5a,b (0.01 mol) was heated in an oil-
bath at 120–130 °C for 2 h then the temperature was raised to 180°C
for additional 2 h. The residue was washed with water then ethanol and
finally crystallised from appropriate solvent.
4-Amino-7-(4-chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]
pyrimidine-2(1H)-thione (8a): Crystallised from n-butanol. Yield
62%; m.p. 262–264 °C; IR νmax/cm–1: 3450–3300 (NH, NH2), 3050 (CH
aromatic), 1090 (C=S), 1560, 1330 (NO2); 1H NMR (DMSO–d6) δ ppm:
6.97 (s, 1H, H6), 7.04 (s, 1H, NH2,exch. D2O), 7.62–7.90 (m, 3H, H4, H5
and H6 of 3-NO2C6H4), 7.88 (d, 2H, J=7.6 Hz, H3, H5 of 4-ClC6H4),
8.15 (d, 2H, J=7.6 Hz, H2, H6 of 4-ClC6H4), 8.51(s, 2H, NH2,exch.
D2O); MS m/z (%): 412 (M+2, 0.6), 410 (M+, 1.9), 68 (100). Anal. calcd
for C19H12ClN5O2S (409.85): C, 55.68; H, 2.95; N, 17.09; found: C, 55.85;
H, 2.68; N, 17.10%.
4-Amino-7-(3-nitrophenyl)-5-(thiophen-2-yl)pyrido[2,3-d]
pyrimidine-2(1H)-thione (8b): Crystallised from ethanol. Yield 55%;
m.p. 178–180 °C; IR νmax/cm–1: 3377, 3277, 3176 (NH, NH2), 3096 (CH
aromatic), 1520, 1340 (NO2), 1087 (C=S); 1H NMR (DMSO-d6) δ ppm:
6.93 (s, 1H, NH, exch.D2O), 7.42–8.07 (m, 8H, 7ArH+H6), 11, 32 (s,
2H, NH2, exch.D2O); MS m/z (%): 381.762 (M+, 100). Anal. calcd for
C17H11N5O2S2 (381.42): C, 53.53; H, 2.91; N, 18.36; found: C, 53.44; H,
3.26; N, 18.08%.
Synthesis of 4-amino-3-phenyl-5,7-disubstitutedpyrido[2,3-d]
pyrimidine-2(3H)-thiones (11a,b); general procedure
A few drops of triethylamine was added to a mixture of the appropriate
2-aminopyridine-3-carbonitrile 5a,b (0.005 mol) and phenyl
isothiocyanate (0.67 g, 0.005 mol) in DMF (30 mL). The mixture was
heated under reflux for 10 h, then left to cool and finally poured onto
cold water (25 mL). The formed solid was filtered, washed with ethanol
and crystallised from butan-1-ol.
4 - A m i n o -7- (4 - c h l o r o p h e n y l ) - 5 - (3 - n i t r o p h e n y l ) - 3 -
phenylpyrido[2,3-d]pyrimidine-2(3H)-thione (11a): Yield 62%; m.p.
262–263 °C; IR νmax/cm–1: 3377, 3277 (NH2), 3095 (CH aromatic), 1521,
1340 (NO2), 1083 (C=S); 1H NMR (DMSO-d6) δ ppm: 7.02 (s, 2H, NH2,
exch.D2O), 7.02–8.52 (m, 14H, 13ArH+H6); MS m/z (%): 487.2(M+2,
1.2), 485.1(M+, 3.7), 85.9 (100) Anal. calcd for C25H16ClN5O2S (485.94):
C, 61.79; H, 3.32; N, 14.41; found: C, 61.75; H, 3.24; N, 14.45%.
4-Amino-7-(3-nitrophenyl)-3-phenyl-5-(thiophen-2-yl)pyrido[2,3-d]
pyrimidine-2(3H)-thione (11b): Yield 58%; m.p. 188–190 °C; IR νmax
/
cm–1: 3350, 3250 (NH2), 3050 (CH aromatic),1520, 1370 (NO2) 1040
1
Synthesis of 2,4-diamino-5,7-disubstituted pyrido[2,3-d]pyrimidines
(9a,b); general procedure
(C=S); H NMR (DMSO-d6) δ ppm: 6.98 (s, 2H, NH2, exch.D2O),
7.2–7.84 (m, 12H, ArH), 7.99 (s, 1H, H6). Anal. calcd for C23H15N5O2S2
(457.51): C, 60.37; H, 3.30; N, 15.30; found: C, 60.69; H, 3.05; N,
15.00%.
Guanidine hydrochloride (2.1 g, 0.025 mol) was added to a solution
of sodium (2.6 g, 0.113 mol) in methanol (95 mL), the precipitated
sodium chloride was filtered off. The o-amino cyano derivative 5a,b
(0.015 mol) was added to the filtrate. The reaction mixture was heated
under reflux for 6 h. The mixture was cooled and the separated solid
was filtered, washed well with ethanol and crystallised from butan-1-
ol.
7-(4-Chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]pyrimidine-
2,4-diamine (9a): Yield 65%; m.p.>300 °C; IR νmax/cm–1: 3410–
3200 (NH2), 3050 (CH aromatic), 1540, 1350 (NO2); 1H NMR
(DMSO-d6) δ ppm: 6.95 (s, 2H, NH2, exch.D2O), 7.34–8.51 (m, 11H,
8ArH+H6 + NH2, exch. D2O); MS m/z (%): 395 (M+3, 45.16), 393
(M+1, 67.74), 258 (M–Cl+1, 61.29), 213.65 (100). Anal. calcd for
C19H13ClN6O2 (392.80): C, 58.10; H, 3.34; N, 21.40; found: C, 58.10; H,
3.39; N, 21.73%.
4-Amino-7-(4-chlorophenyl)-5-(3-nitrophenyl)-2-phenyl-
1,8-naphthyridine-3-carbonitrile (12):
A
mixture of the
benzylidenemalononitriles (0.15 g; 0.001 mol), and corresponding
2-aminopyridine-3-carbonitrile derivatives 5a (0.35 g; 0.001 mol) and
piperidine (1 mL) was dissolved in dioxane (10 mL). The mixture was
heated under reflux for 3 h. The solvent was removed under reduced
pressure. The formed solid was filtered, washed and crystallised
from DMF/H2O. Yield 74%; m.p. 273–274 °C; IR νmax/cm–1: 3300
1
(NH2), 3050 (CH aromatic), 2200 (CN), 1520, 1340 (NO2); H NMR
(DMSO-d6) δ ppm: 6.94 (s, 1H, H‑6 of naphthridine), 6.96 (s, 2H, NH2,
exch.D2O), 7.59–8.47 (m, 13H, ArH).; MS m/z (%): 479.75 (M+3, 9.3),
478 (M+1, 9.3), 374 (M–C6H4CN, 100). Anal. calcd for C27H16ClN5O2
(477.88): C, 67.85; H, 3.37; N, 14.65; found: C, 68.05; H, 3.70; N, 14.74%.
4-Amino-7-(4-chlorophenyl)-5-(3-nitrophenyl)-2-oxo-1,2-dihydro-
1,8-naphthyridine-3-carbonitrile (13): A mixture of 2-amino-6-(4-
chlorophenyl)-4-(3-nitrophenyl)nicotinonitrile (5a) (3.50 g, 0.01 mol),
ethyl cyanoacetate (1.13 g, 0.01 mol) and triethylamine (2 mL) in
ethanol 95% (30 mL) was heated under reflux for 3 h. The formed
solid was filtered, washed with ethanol and crystallised from ethanol.
Yield 58%; m.p. 270–272 °C; IR νmax/cm–1: 3480, 3360, 3200 (NH2,
7-(3-Nitrophenyl)-5-(thiophen-2-yl)pyrido[2,3-d]pyrimidine-
2,4-diamine (9b): Yield 68%; m.p.>300 °C; IR νmax/cm–1: 3463,
1
3349, 3245 (NH2), 3019 (CH aromatic), 1566, 1380 (NO2); H NMR
(DMSO-d6) δ ppm: 5.02 (s, 2H, NH2, exch.D2O), 7.23–8.28 (m, 10H,
7ArH+H6 + NH2, exch.D2O); 13C NMR (DMSO) δppm: 94.01, 112.12,
114.17, 114.70, 117.0, 119.0, 120.28, 129.44, 130.07, 133.71, 135.26,
154.02, 154.13, 159.39, 159.70, 160.43. Anal. calcd for C17H12N6O2S
(364.37): C, 56.03; H, 3.32; N, 23.06; found: C, 56.30; H, 3.29; N,
22.97%.
1
NH), 3050 (CH aromatic), 1640 (C=O), 1520, 1350 (NO2); H NMR
(DMSO-d6) δ ppm: 7.99 (s, 2H, NH2, exch.D2O), 6.99–8.4 (m, 9H,
8ArH+H6), 8.51(s, 1H, NH, exch.D2O); MS m/z (%): 419.75 (M+3,
16.67), 418.25 (M+1, 18.33), 55 (100). Anal. calcd for C21H12ClN5O3
(417.79): C, 60.36; H, 2.89; N, 16.76; found: C, 60.69; H, 3.19; N,
16.36%.
Synthesis of 5,7-disubstituted pyrido[2,3-d]pyrimidine-2,4(1H,3H)-
dithiones (10a,b); general procedure
Carbon disulfide (3.04 g, 0.04 mol) was added to a solution of
2-aminopyridine-3-carbonitriles 5a,b (0.01 mol) in pyridine (15 mL),
the mixture was heated under reflux for 15 h. After cooling excess
pyridine was removed by distillation, the precipitated solid was
filtered, washed with ethanol and finally crystallised from appropriate
solvent.
7-(4-Chlorophenyl)-5-(3-nitrophenyl)pyrido[2,3-d]pyrimidine-
2,4(1H,3H)-dithione (10a): Crystallise from DMF/H2O. Yield 62%;
m.p. 266–268 °C; IR νmax/cm–1: 3360, 3200 (NH), 3035 (CH aromatic),
1527, 1346 (NO2), 1100 (C=S); 1H NMR (DMSO–d6) δ ppm: 6.95–8.5
(m, 9H, 8ArH+H6), 7.03 (s, 1H, N1H, exch.D2O), 8.5 (s, 1H, N3H,
exch. D2O); MS m/z (%): 428 (M+2, 10.4), 426 (M+,3.7), 374 (100).
Anal. calcd for C19H11ClN4O2S2 (426.90): C, 53.46; H, 2.60; N, 13.12;
found: C, 53.35; H, 2.29; N, 13.28%.
Cytotoxic activity studies
Anticancer activity studies were done at Cairo University, National
Cancer Institute, Cancer Biology Department, Pharmacology Unit.
Measurement of potential cytotoxicity: The cytotoxic activity of the
newly synthesised compounds was measured in vitro on human breast
adenocarcinoma cell line (MCF-7) using Sulforhodamine-B stain
(SRB) assay applying the method of Skehan et al. 26 Cells were plated
in 96-multiwell plate (104 cells/well) for 24 h before treatment with the
test compounds to allow attachment of the cells to the wall of the plate.
Test compounds were dissolved in DMSO and diluted with saline to the
appropriate volume. Different concentrations of the test compounds (0,
1, 2.5, 5 and 10 mg mL–1) were added to the cell monolayer. Triplicate
JCR1302284_FINAL.indd 152
04/03/2014 09:53:34

JOURNAL OF CHEMICAL RESEARCH 2014 153
7
J.M. Hamby, J.C. Connolly, M.C. Schroeder, T. Winters, H.D.H. Showalter,
R.L. Panek, T.C. Major, B. Olsewski, M.J. Ryan, T. Dahring, G.H. Lu, J.
Keiser, A. Amar, C. Shen, A.J. Kraker, V. Slintak, J.M. Nelson, D.W. Fry,
L. Bradford, H. Hallak and A.M. Doherty, J. Med. Chem., 1997, 40, 2296.
A.J. Kraker, B.G. Hartl, A.M. Amar, M.R. Barvaian, H.D.H. Showalter
and C.W. Moore, Biochem. Pharm., 2000, 60, 885.
J.C. Connolly, J.M. Hamby, M.C. Schroeder, M. Barvaian, G.H. Lu, R.L.
Panek, A. Amar, C. Shen, A.J. Kraker, D.W. Fry, W.D. Klohs and A.M.
Doherty, Bioorg. Med. Chem., Lett., 1997, 7, 2415.
wells were prepared for each individual dose. Monolayer cells were
incubated with the test compound for 48 h at 37 °C in atmosphere of 5%
CO2. After 48 h, cells were fixed with trichloroacetic acid, washed with
water and stained for 30 min with 0.4% (wt/vol). Sulforhodamine-B
stain dissolved with 1% acetic acid. Excess stain was removed by four
washes with 1% acetic acid and attached stain was recovered with Tris
EDTA buffer. Colour intensity was measured in an ELISA reader. The
relation between surviving fraction and compound concentration was
plotted and IC50 [the concentration required for 50% inhibition of cell
viability] was calculated for each compound and results are given in
Table 1.
8
9
10 S. Tu, J. Zhang, X. Zhu, J. Xu, Y. Zhang, Q. Wang, R. Jia, B. Jiang and J.
Zhang, Bioorg. Med. Chem., 2006, 16, 3578.
11 D.A. Ibrahim and N.S.M. Ismail, Eur. J. Med. Chem., 2011, 46, 5825.
12 A. Gangjee, A. Vasudevan, S.F. Queener and R.L. Kisliuk, J. Med. Chem.,
1995, 38, 1778.
13 M. Zink, H. Lanig and R. Troschütz, Eur. J. Med. Chem., 2004, 39, 1079.
14 C.W. Sigel, A.W. Macklin, J.L. Jr. Woolley, N.W. Johnson, M.A. Collier,
M.R. Blum, N.J. Clendeninn, J.M. Everitt, G. Grebe, A. Mackars, R.G.
Foss, D.S. Duch, S.W. Bowers and C.A. Nichol, NCI Monogr., 1987, 5, 111.
15 C. Sessa, J. d. Jong, M. D’Incalci, S. Hatty, O. Pagani and F. Cavalli, Clin.
Cancer Res., 1996, 2, 1123.
16 G. Roma, G. Grossi, M.D. Braccio, D. Piras, V. Ballabeni, M. Tognolini, S.
Bertoni and E. Barocelli, Eur. J. Med. Chem., 2008, 43, 1665.
17 G. Roma, M.D. Braccio, G. Grossi, F. Mattioli and M. Ghia., Eur. J. Med.
Chem, 2000, 35, 1021.
The authors thank all members of the department of Cancer
Biology, Pharmacology Unit, National Cancer Institute, Cairo,
Egypt, for carrying out the cytotoxicity testing.
Received 15 November 2013; accepted 9 January 2014
Paper 1302284 doi: 10.3184/174751914X13910886393992
Published online: 7 March 2014
18 M. Atanasova, S. Ilieva and B. Galabov, Eur. J. Med. Chem., 2007, 42,
1184.
19 K. Chen, S.C. Kuo, M.C. Hsieh, A. Mauger, C.M. Lin, E. Hamel and K.H.
Lee, J. Med. Chem., 1997, 40, 2266.
20 O.G. Schramm, N. Dediu, T. Oeser and T.J.J. Müller, J. Org. Chem., 2006,
71, 3494.
21 A.K. Elansary, A.A. Moneer, H.H. Kadry and E.M. Gedawy, Arch.Pharm.
Res., 2012, 35, 1909.
22 A.O. Fathalla, W.A. Zaghary, E.M.M. Kassem, Y.M. Zohny and M.S.
Mohamed, Bull. Fac. Pharm. Cairo Univ., 2002, 40, 185.
23 R. Sharma, R.D. Goyal and L. Prakash, Indian J. Chem., 1992, 31B, 719.
24 A. Essawy, M. Abdalla and A. Deeb, Pakistan J. Sci. Ind. Res., 1979, 22, 1.
25 S. Patai and Y. Israeli, J. Chem. Soc., 1960, 2020.
26 P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.R. Warren, H. Bokesch, S. Kenney and M.R. Boyd, J. Natl. Cancer Inst.,
1990, 82, 1107.
References
1
2
3
4
B.L. Narayana, A.R.R. Rao and P.S. Rao, Eur. J. Med. Chem., 2009, 44,
1369.
G.F. Hardtmann, B. Huegi, G. Koletar, S. Kroin, H. Ott, J.W. Perrine and
E.I. Takesue. J. Med. Chem., 1974, 17, 636.
L.R. Bennett, C.J. Blankley, R.W. Fleming, R.D. Smith and D.K. Tessman,
J. Med. Chem., 1981, 24, 382.
A. Monge, V. Martinez-Merino, C. Sanmartin, F.J. Fernandez, M.C.
Ochoa, C. Bellver, P. Artigas and E.F. Alvarez, Eur. J. Med. Chem., 1989,
24, 209.
5
J.M. Quintela, C. Peinador, L. Botana, M. Estevez and R. Riguera, Bioorg.
Med. Chem., 1997, 5, 1543.
6
F. Shia, J. Ding, S. Zhang, W.J. Hao, C. Cheng and S. Tua, Bioorg. Med.
Chem. Lett., 2011, 21, 1554.
JCR1302284_FINAL.indd 153
04/03/2014 09:53:34

Copyright of Journal of Chemical Research is the property of Science Reviews 2000 Ltd. and
its content may not be copied or emailed to multiple sites or posted to a listserv without the
copyright holder's express written permission. However, users may print, download, or email
articles for individual use.