Synthesis and cytotoxic evaluation of pyran, dihydropyridine and thiophene derivatives of 3-acetylcoumarin

Article abstract of DOI:10.1248/cpb.c15-00115

A series of coumarin analogues bearing 4H-pyran rings 2a-d, 11a-d and 1,4-dihydropyridine rings 3a-d, 12a-d at position 3 were synthesized starting from either 3-acetyl coumarin (1) or the coumarin acetohydrazide derivative 4. Condensation of 3-acetylcoum

Full text of DOI:10.1248/cpb.c15-00115

678
Chem. Pharm. Bull. 63, 678–687 (2015)
Vol. 63, No. 9
Regular Article
Synthesis and Cytotoxic Evaluation of Pyran, Dihydropyridine and
Thiophene Derivatives of 3-Acetylcoumarin
,b
Rafat Milad Mohareba and Nadia Youssef Megally Abdo*
a Chemistry Department, Faculty of Science, Cairo University; Giza, Cairo 12613, Egypt: and b Chemistry
Department, Faculty of Education, Alexandria University; Alexandria 21526, Egypt.
Received February 4, 2015; accepted May 21, 2015
A series of coumarin analogues bearing 4H-pyran rings 2a–d, 11a–d and 1,4-dihydropyridine rings
3a–d, 12a–d at position 3 were synthesized starting from either 3-acetyl coumarin (1) or the coumarin
acetohydrazide derivative 4. Condensation of 3-acetylcoumarin (1) with 2-cyanoacetohydrazide afforded
2-cyano-N′-{1-[2-oxo-2H-chromen-3-yl]ethylidene}acetohydrazide (4). Reaction of compound 4 with elemental
sulfur and either malononitrile or ethyl cyanoacetate afforded the thiophene derivatives 8 and 9, respectively.
The structures of the newly synthesized compounds were confirmed on the basis of their spectral data and
elemental analyses. All synthesized compounds were screened for their in vitro anticancer activity against
six human cancer cell lines and normal fibroblasts. Several compounds showed potent inhibition with an
IC50 value of ˂870nM. Compound 3d exhibited equivalent cytotoxic effect as the standard CHS 828 against
a breast cancer cell line (IC50 value=18 nM). Normal fibroblast cells (WI38) were affected to a much lesser
extent (IC50 value >10000nM).
Key words coumarin; 4H-pyran; dihydropyridine; thiophene; 2-cyanoacetohydrazide; anticancer activity
Coumarins (2H-chromen-2-one) have been established as inhibitory activity against Mycobacterium tuberculosis.13,14)
well known naturally occurring heterocyclic compounds that
Recently coumarin derivatives have been reported to pos-
can be either isolated from various plants including edible sess the potent anticancer effect through different mecha-
vegetables and fruits1,2) or can be carried out in the labora- nisms. The tricyclic coumarin sulfamate (STX64) (IC50=8nM),
tory.3)
a nonsteroid-based irreversible aromatase-steroid sulfatase
Among the oxygen heterocycles, coumarin derivatives are (STS) inhibitor provides remarkable activity for the cure of
an important class of natural, synthetic compounds and phar- prostate cancer, and most encouragingly, its clinical trials
macologically active substances displaying a broad range of have been accomplished in 2011.15–17) For instance, 3,8-di-
biological activities including anticancer,4) anti-human immu- bromo-7-hydroxy-4-methyl coumarin (DBC) (IC50=100 nM) is
nodeficiency virus (HIV),5) antituberculosis,6) anti-influenza,7) treated as a CK2 inhibitor to suppress neoplastic growth.18)
anti-Alzheimer8) and anti-inflammatory.9) They have also been Novobiocin, a known DNA gyrase inhibitor, binds to a
shown to be novel lipid lowering agents that possess moderate nucleotide-binding site located on the Hsp90-C terminus
triglyceride lowering activity.10) Certain coumarin derivatives and induces degradation of Hsp90-dependent client proteins
have been shown to function as HIV integrase inhibitors and at ca. 700µM in breast cancer cells.19,20) Some biologically
evaluated in the treatment of HIV infection,11) whereas others active anticancer agents, such as Geipavarin,21) Auraptene,
evaluated as anti-invasive compounds due to their inhibitory Collinin22) and Scopoletin23) having substituted coumarin
activity against some serine proteases and matrix metallopro- moiety are presented in Fig. 1. Moreover, 7-hydroxycoumarin
teases (MMPs).12) 7-O-Alkoxy-4-methylumbelliferone deriva- (Fig. 1) was shown to inhibit the release of cyclin D1, which
tives with longer chains, especially nonyl and decyl have good is over expressed in many types of cancers.24) In addition,
Fig. 1. Structure of Some Potent Anticancer Compounds
*To whom correspondence should be addressed. e-mail: nadiamegally@yahoo.com
© 2015 The Pharmaceutical Society of Japan

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
679
1
3-acetylcoumarin received considerable attention as a target Thus, the H-NMR spectrum of 2c (as an example) showed
molecule for the synthesis of pyridine, thiazole and other het- the presence of two singlets at δ 2.49 (D2O exchangeable) and
erocyclic derivatives.25–28)
6.97ppm due to the presence of NH2 group and coumarin H-4
In this work, we are demonstrating the reaction of 3-acetyl beside another two singlets at δ 6.94 and 8.39ppm correspond-
coumarin with 2-cyanoacetohydrazide to give the 2-cyano- ing to the presence of pyran H-4 and H-5, respectively. The
N′-(1-(2-oxo-2H-chromen-3-yl)ethylidene)acetohydrazide (4). 13C-NMR spectrum revealed the presence of signals at δ 88.1
Reaction of 4 with elemental sulfur and either malononitrile (pyran C-4), 116.0 (CN) and 162.1 (CO) (C=O). On the other
or ethyl cyanoacetate in ethanol using triethylamine as a hand, carrying the same reaction but using a catalytic amount
catalyst produced the (2-oxo-2H-chromen-3-yl)ethylidene)- of ammonium acetate gave the 1,4-dihydropyridine derivatives
hydrazinyl)thiophene derivatives 8 and 9, respectively. More- 3a–d, respectively (Chart 1). The structures of the latter prod-
1
over, the (2-oxo-2H-chromen-3-yl)-4H-pyran derivatives 2a–d, ucts were confirmed on the basis of their respective H-NMR
1
11a–d and the (2-oxo-2H-chromen-3-yl)-1,4-dihydropyridine and 13C-NMR spectra. Thus, the H-NMR spectrum of 3a (as
derivatives 3a–d, 12a–d have been prepared by condensation an example) displayed the presence of two signals (D2O ex-
of either 3-acetyl coumarin (1) or coumarin acetohydrazide changeable) at δ 2.89 and 9.04ppm due to the presence of NH2
derivative 4 with substituted aromatic aldehyde and malononi- and pyridine-NH beside the presence of another three signals
trile in presence of either triethylamine or ammonium acetate at δ 6.97 (pyridine H-4), 7.14 (coumarin H-4), 8.67 (pyridine
as catalyst, respectively.
H-5), respectively. In addition, the 13C-NMR spectrum re-
The design and development of new bioactive agents based vealed the presence of signals δ 84.8 (pyridine C-4), 116.4
on the molecular hybridization strategy, involving the integra- (CN) and 164.8 (C=O).
tion of two or more pharmacophoric units having different
It is well known that the hydrazide-hydrazones play an
mechanisms of action in the same molecule, is a rationally important role for the antitumor activity.35–37) With the aim
attractive approach.29,30) These combined pharmacophores of obtaining new hydrazide-hydrazones with such wide spec-
probably offer some advantages such as in overcoming drug trum of pharmaceutical applications,38–48) we report here the
resistance31) as well as improving their biological potency.32) synthesis of a series of hydrazide-hydrazones via the reaction
Therefore, in the present study it was planned to synthesize of 3-acetylcoumarine (1) with 2-cyanoacetohydrazide fol-
hybrid compounds that comprise 3-acetylcoumarin and the lowed by heterocyclizations of the reaction product. Moreover,
aforementioned heterocyclic ring systems in order to identify the cytotoxic evaluations of the synthesized products were
new candidates that may be of value in designing new, potent, measured. Thus, the reaction of compound 1 with 2-cyano-
selective and less toxic anticancer agents. All the synthesized acetohydrazide in 1,4-dioxane under the reflux conditions
compounds were evaluated for their in vitro cytotoxicity gave the hydrazide-hydrazone derivative 4. The structure of
1
against six human cancer cell lines and normal fibroblast cells. compound 4 was confirmed on the basis of its H-NMR and
1
13C-NMR spectra. The H-NMR spectrum revealed the pres-
ence of two singlets at δ 2.15 and 5.08ppm for the CH3 and
Results and Discussion
Chemistry The multicomponent reaction of 3-acetylcou- CH2 groups beside the presence of two signals at δ 6.63ppm
marin with either of benzaldehyde, 4-methoxybenzaldehyde, and δ 8.92ppm equivalent to the coumarin H-4 and NH (D2O
4-chlorobenzaldehyde or furfural in ethanol containing a exchangeable) of the acetohydrazide moiety. Moreover, the
catalytic amount of triethylamine gave the 4H-pyran deriva- 13C-NMR spectrum showed the presence of signals at δ 28.3
tives 2a–d,33,34) respectively. The analytical and spectral data (CH3), 64.2 (CH2), 117.3 (CN), 160.1, 164.3 (2CO) and 172.1
of 2a–d were consistent with their respective structures. (C=N), respectively. Compound 4 was a good candidate in
Chart 1. Synthesis of Compounds 2a–d and 3a–d

680
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
Chart 2. Synthesis of Compounds 5–9, and 10a, b
synthesizing heterocyclic compounds and their fused deriva- of two signals (D2O exchangeable) at δ 3.83 and 8.95ppm cor-
tives with potential antitumor activities. Thus, compound 4 responding to NH2 and NH groups beside another two signals
reacted with benzenediazonium chloride at 0–5°C to af- at δ. 3.05 and 6.83ppm corresponding to CH3 and coumarin
ford the phenylhydrazone derivative 5. On the other hand, H-4, respectively. Moreover, the 13C-NMR spectrum revealed
it reacted with benzaldehyde in the presence of a catalytic the presence of signals at δ 28.8 (CH3), 116.6, 117.3 (2CN),
amount of piperidine to give the benzylidene derivative 6. In 163.8 (C=O) and 170.3 (C=N).
addition, compound 4 reacted with salicylaldehyde to afford
The reaction of compound 4 with either acetylacetone or
the 2-iminobenzo[b]pyran derivative 7. The analytical and ethyl acetoacetate gave the 1,2-dihydropyridine34) derivatives
spectral data of compounds 5–7 are in agreement with their 10a, b, respectively (Chart 2). The H-NMR and 13C-NMR
1
respective structures (see Experimental).
spectra were used to confirm the structures of 10a, b. Thus
Next, we moved towards studying the reactivity of com- the H-NMR spectrum of 10a (for example) showed the pres-
pound 4 towards thiophene synthesis through the well-known ence of three singlets at δ 2.09, 3.01, 3.48ppm due to the pres-
1
Gewald’s thiophene synthesis.49,50)
ence of three CH3 groups beside two singlets at δ 6.99 and
Thus, the reaction of compound 4 with either malononitrile 9.01ppm corresponding to the presence of coumarin H-4 and
or ethyl cyanoacetate gave the thiophene derivatives 8 and pyridine H-5, respectively. Moreover, the 13C-NMR spectrum
9,34) respectively. The structures of 8 and 9 were confirmed revealed the presence of signals at δ 30.0, 33.9, 42.1ppm for
on the basis of their 1H-NMR and 13C-NHR spectra. The three (CH3) groups beside the presence of signals at δ 116.2
1H-NMR spectrum of 8 (as an example) showed the presence (CN), 160.1, 164.1 (2C=O), 168.3 (C=N).

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
681
Chart 3. Synthesis of Compounds 11a–d and 12a–d
>10000nM). Among the 4H-pyran derivatives 2a–d, com-
pounds 2a and c are the most potent. The latter compound
showed high potency towards the six cancer cell lines, while
compound 2a was potent only against four cancer cell lines
namely: NUGC, DLD1, HA22T and MCF with IC50’s 48, 59,
122 and 480nM, respectively. The high potency of 2c is at-
tributed to the presence of the 4-chloro group. Considering
the 1,4-dihydropyridine derivatives 3a–d, each one of these
Fig. 2. Chemical Structure of CHS 828
Finally, the multicomponent reactions of compound derivative revealed selective activity against certain cancer
4
with either of benzaldehyde, 4-methoxybenzaldehyde, cell lines. Compound 3a showed selective higher activity
4-chlorobenzaldehyde or furfural in ethanol containing a against liver cancer HEPG2 (IC50=22nM) than 3b, c and d.
catalytic amount of triethylamine gave the 4H-pyran deriva- The introduction of 4-methoxy group in 3b exhibited remark-
tives 11a–d, respectively. On the other hand carrying the same able increase in the activity against NUGC and HA22T than
reaction but using ammonium acetate instead of triethylamine 3a, c and d. Moreover, the presence of furan moiety in 3d is
afforded the 1,4-dihydropyridine derivatives 12a–d, respec- responsible for its high potency against breast cancer MCF, it
tively (Chart 3). The structures of 11a–d and 12a–d were showed equivalent cytotoxic effect to the standard CHS 828
based on analytical and spectral data (see Experimental).
In Vitro Cytotoxicity
Effect on the Growth of Human Cancer Cell Lines
(IC50=18 nM).
Comparing the cytotoxicity of the hydrazide-hydrazone 4
with its condensation products 5 and 6, all of them showed
The heterocyclic compounds, prepared in this study, were low cytotoxicity. On the other hand the thiophene derivatives
evaluated according to standard protocols for their in vitro 8 and 9 showed optimal cytotoxic activity against the six can-
cytotoxicity against six human cancer cell lines including cells cer cell lines. Moreover compound 8 exhibited two fold higher
derived from human gastric cancer (NUGC), human colon activity (IC50=48nM) against NUGC compared to the standard
cancer (DLD1), human liver cancer (HA22T and HEPG2), CHS 828 (IC50=25nM). The remarkable activity of 8 and 9 is
nasopharyngeal carcinoma (HONE1), human breast cancer due to the presence of the thiophene ring.29)
(MCF) and normal fibroblast cells (WI38). For comparison
Considering the 1,2-dihydropyridine derivatives 10a, b, it
purposes, CHS 828, a pyridyl cyanoguanidine, was used as is clear that the cytotoxicity of 10b is higher than that of 10a.
standard antitumor drug51) (Fig. 2). All of the IC50 values (the Compound 10b showed more potency towards the three can-
sample concentration that produces 50% reduction in cell cer cell lines namely: NUGC, HEPG2 and MCF with IC50’s
growth) in nanomolar (nM) are listed in Table 1. Several com- 239, 125 and 36nM, respectively. Such high cytotoxicity of
pounds showed potent inhibition with an IC50 Values <870nM 10b is attributed to the presence of the electronegative OH
and the results are represented graphically in (Figs. 3, 4). All group.
the synthesized compounds were tested for their cytotoxicity
Considering the 4H-pyran derivatives 11a–d, compound
against normal fibroblast cells. The results obtained showed 11c substituted with 4-chloro group showed the highest cyto-
that normal fibroblast cells (WI38) were affected to a much toxicity among the four compounds with remarkable activity
lesser extent (IC50>10000nM).
Structure–Activity Relationship
against the six human cancer cell lines. Thus it is obvious that
while some of the compounds were not the most potent, their
In this study, when correlating the structures of the syn- specific activity against particular cell lines makes them of
thesized compounds with their anticancer activity, it has interest for further development as anticancer drugs.
been observed that several compounds showed significant
cytotoxic effect with IC50 values <870nM. Normal fibroblast
cells (WI38) were affected to a much lesser extent (IC50
Conclusion
The present study reports the successful synthesis, charac-

682
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
b)
Table 1. Cytotoxicity of Compounds 2a–d; 3a–d; 4; 5; 6; 7; 8; 9; 10a, b; 11a–d, and 12a–d against a Variety of Cancer Cell Linesa) [IC50 (nM)]
Cytotoxicity (IC50 in nM)
Compound No.
NUGC
DLDI
HA22T
HEPG2
HONE1
MCF
WI38
2a
2b
48
1128
862
59
1892
207
122
2377
380
2334
1328
282
3289
1290
206
480
2673
264
na
360
660
428
650
na
2c
2d
2101
1288
122
2458
2187
3210
2266
2139
1166
2419
2366
2433
55
2258
2530
59
350
2180
2135
1140
2612
2250
326
1140
1729
1130
430
3a
22
3b
1245
2328
2177
2216
1284
2130
2560
128
3c
1289
2265
1232
1280
3138
2210
48
351
na
3d
2257
2225
2160
2228
1650
128
18
262
na
4
1286
2073
326
5
2130
1584
1544
248
872
650
520
838
829
549
na
6
7
2457
128
8
9
135
158
278
279
206
668
10a
10b
11a
11b
11c
11d
12a
12b
12c
12d
CHS 828
1126
389
2168
1220
2055
2150
80
1312
1480
2173
1165
31
1232
125
1824
1620
2149
4273
168
2330
36
2120
3242
470
1359
4321
132
2580
2533
447
883
na
276
370
1179
na
1040
1278
1488
2210
1175
25
2763
1830
1259
2186
2340
2315
2469
2067
1224
1160
1169
2067
3146
2634
3120
2178
1273
1245
1342
1970
1680
2562
2181
15
2293
2263
2328
1179
2834
18
na
na
na
a) NUGC, gastric cancer; DLDI, colon cancer; HA22T, liver cancer; HEPG2, liver cancer; HONEI, nasopharyngeal carcinoma; MCF, breast cancer; WI38, normal fibroblast cells.
b) The sample concentration produces a 50% reduction in cell growth.
Fig. 3. Cytotoxicity of Compounds 2a, c, d, 3a–d and CHS 828 against NUGC, Gastric Cancer; DLDI, Colon Cancer; HA22T, Liver Cancer;
HEPG2, Liver Cancer; HONEI, Nasopharyngeal Carcinoma; MCF, Breast Cancer
terization and anticancer activity of new series of 4H-pyran,
Experimental
dihydropyridine and thiophene derivatives starting from either
Chemistry All melting points were determined on a Stu-
3-acetyl coumarin (1) or coumarin acetohydrazide derivative art apparatus and the values given are uncorrected. IR spectra
4. Several compounds showed potent inhibition with an IC50 (KBr, cm−1) were determined on a Shimadzu IR 435 spec-
˂870 n M. Among these derivatives compound 3d exhibited trophotometer (Faculty of Science, Cairo University, Egypt).
equivalent cytotoxic effect to the standard CHS 828 against 1H-NMR spectra were recorded on Varian Gemini 300MHz
breast cancer cell line (IC50=18 nM). Normal fibroblast cells (Microanalysis Center, Cairo University, Egypt) using tetra-
(WI38) were affected to a much lesser extent (IC50>10000nM). methylsilane (TMS) as internal standard. Chemical shift
The obtained results suggest that these compounds may serve values are recorded in ppm on δ scale. The electron impact
as lead chemical entities for further modification in the search (EI) mass spectra were recorded on a Hewlett Packard 5988
of new classes of potential anticancer agents.
spectrometer (Microanalysis Center, Cairo University, Egypt).

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
683
Fig. 4. Cytotoxicity of Compounds 4, 6, 8, 9, 10b, 11c, and CHS 828 against NUGC, Gastric Cancer; DLDI, Colon Cancer; HA22T, Liver Cancer;
HEPG2, Liver Cancer; HONEI, Nasopharyngeal Carcinoma; MCF, Breast Cancer
Elemental analyses were carried out at the Microanalysis 1H-NMR (DMSO-d6) δ: 2.49 (s, 2H, NH2, D2O exchange-
Center, Cairo University, Egypt; found values were within able), 6.94 (s, 1H, pyran H-4), 6.97 (s, 1H, coumarin H-4),
0.35% of the theoretical ones. Progress of the reactions was 7.41–7.81 (m, 8H aromatic), 8.39 (s, 1H, pyran H-5); 13C-NMR
monitored using thin layer chromatography (TLC) sheets re- (DMSO-d6) δ: 88.1, 116.0, 120.5, 120.6, 122.8, 123.7, 124.6,
coated with UV fluorescent silica gel Merck 60F 254 and were 125.4, 126.1, 127.3, 128.5, 129.2, 129.5, 133.1, 134.5, 139.6,
visualized using UV lamp.
140.3, 142.9, 162.1; MS (EI): m/z (%) 376 (M+). Anal. Calcd
General Procedure for the Synthesis of Compounds 2a–d for C21H13ClN2O3: C, 66.94; H, 3.48; N, 7.43. Found: C, 66.83;
A mixture of 3-acetyl-2H-chromen-2-one (1) (1.88g, 0.01mol), H, 3.35; N, 7.08.
the appropriate arylaldehyde (0.1mol) and malononitrile
2-Amino-4-(furan-2-yl)-6-(2-oxo-2H-chromen-3-yl)-4H-
(0.66g, 0.1mol) were heated under reflux in ethanol (40mL) pyran-3-carbonitrile (2d)
containing triethylamine (1.0mL) for 3h. The reaction mixture
Yield: 66%; mp: 118–120°C; IR (KBr, cm−1): 3427, 3389
was left to cool, poured onto ice water and neutralized by (NH2), 3077 (CH, aromatic), 2214 (CN), 1722 (C=O); 1H-NMR
hydrochloric acid. The solid product was precipitated, filtered, (DMSO-d6) δ: 2.88 (s, 2H, NH2, D2O exchangeable), 6.69 (s,
washed with water, and crystallized from ethanol.
1H, pyran H-4), 6.81 (s, 1H, coumarin H-4), 7.05–7.95 (m,
2-Amino-6-(2-oxo-2H-chromen-3-yl)-4-phenyl-4H-pyran-3- 7H aromatic), 8.65 (s, 1H, pyran H-5); 13C-NMR (DMSO-d6)
carbonitrile (2a)
δ: 88.3, 116.8, 120.9, 121.2, 122.8, 123.9, 124.6, 125.4, 126.5,
Yield: 62%; mp: 98–100°C; IR (KBr, cm−1): 3436, 3328 127.9, 129.2, 129.5, 133.1, 134.9, 139.6, 140.8, 142.6, 149.9,
(NH2), 3066 (CH, aromatic), 2198 (CN), 1724 (C=O); 162.1. MS (EI): m/z (%) 332 (M+). Anal. Calcd for C19H12N2O4:
1H-NMR (dimethyl sulfoxide (DMSO)-d6) δ: 2.88 (s, 2H, C, 68.67; H, 3.64; N, 8.43. Found: C, 68.49; H, 3.31; N, 8.72.
NH2, D2O exchangeable), 6.76 (s, 1H, pyran H-4), 6.89 (s, 1H,
General Procedure for the Synthesis of Compounds 3a–d
coumarin H-4), 7.38–7.96 (m, 9H aromatic), 8.65 (s, 1H, pyran A mixture of 3-acetyl-2H-chromen-2-one (1) (1.88g, 0.01mol),
H-5); 13C-NMR (DMSO-d6) δ: 86.3, 116.6, 120.2, 122.1, 122.9, the appropriate arylaldehyde (0.1mol) and malononitrile
123.9, 124.7, 125.1, 127.5, 129.0, 130.7, 132.6, 133.4, 136.8, (0.66g, 0.1mol) were heated under reflux in ethanol (40mL)
138.9, 139.3, 140.1, 142.0, 164.6; MS electron impact (EI): m/z containing ammonium acetate (0.5g) for 2–4h. The reaction
(%) 342 (M+). Anal. Calcd for C21H14N2O3: C, 73.68; H, 4.12; mixture was left to cool, poured onto ice water and neutral-
N, 8.18. Found: C, 73.87; H, 4.18; N, 7.93.
2-Amino-4-(4-methoxyphenyl)-6-(2-oxo-2H-chromen-3- filtered, washed with water, and crystallized from ethanol.
yl)-4H-pyran-3-carbonitrile (2b)
2-Amino-6-(2-oxo-2H-chromen-3-yl)-4-phenyl-1,4-
Yield: 65%; mp: >300°C; IR (KBr, cm−1): 3440, 3342 dihydropyridine-3-carbonitrile (3a)
ized by hydrochloric acid. The solid product was precipitated,
(NH2), 3089 (CH, aromatic), 2212 (CN), 1722 (C=O);
Yield: 70%; mp: 178–180°C; IR (KBr, cm−1): 3423–3254
1H-NMR (DMSO-d6) δ: 2.57 (s, 2H, NH2, D2O exchangeable), (NH2, NH), 3089 (CH, aromatic), 2204 (CN), 1719 (C=O);
3.81 (s, 3H, OCH3), 6.79 (s, 1H, pyran H-4), 7.01 (s, 1H, cou- 1H-NMR (DMSO-d6) δ: 2.89 (s, 2H, NH2, D2O exchange-
marin H-4), 7.39–8.57 (m, 8H aromatic), 8.90 (s, 1H, pyran able), 6.97 (s, 1H, pyridine H-4), 7.14 (s, 1H, coumarin H-4),
H-5); 13C-NMR (DMSO-d6) δ: 33.2, 86.2, 116.8, 120.8, 121.3, 7.42–7.81 (m, 9H aromatic), 8.67 (s, 1H, pyridine H-5), 9.04 (s,
122.8, 123.2, 124.2, 125.2, 126.3, 128.2, 129.4, 130.2, 133.1, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 84.8,
134.6, 138.6, 139.1, 140.1, 142.3, 163.8; MS (EI): m/z (%) 372 116.4, 119.6, 120.7, 121.8, 122.3, 123.2, 124.5, 125.4, 126.3,
(M+). Anal. Calcd for C22H16N2O4: C, 70.96; H, 4.33; N, 7.52. 128.3, 129.8, 132.6, 134.6, 136.8, 138.2, 139.1, 140.2, 164.8;
Found: C, 70.63; H, 4.65; N, 7.83.
2-Amino-4-(4-chlorophenyl)-6-(2-oxo-2H-chromen-3- 73.89; H, 4.43; N, 12.31. Found: C, 73.58; H, 4.41; N, 11.98.
yl)-4H-pyran-3-carbonitrile (2c) 2-Amino-4-(4-methoxyphenyl)-6-(2-oxo-2H-chromen-3-
Yield: 61%; mp: >300°C; IR (KBr, cm−1): 3430, 3410 yl)-1,4-dihydropyridine-3-carbonitrile (3b)
(NH2), 3087 (CH, aromatic), 2220 (CN), 1719 (C=O);
Yield: 74%; mp: 169–171°C; IR (KBr, cm−1): 3428–3263
MS (EI): m/z (%) 341 (M+). Anal. Calcd for C21H15N3O2: C,

684
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
(NH2, NH), 3067 (CH, aromatic), 2191 (CN), 1722 (C=O); collected by filtration and crystalized from ethanol.
1H-NMR (DMSO-d6) δ: 2.95 (s, 2H, NH2, D2O exchange- Yield: 75%; mp: 278–280°C; IR (KBr, cm−1): 3444–3104
able), 3.94 (s, 3H, OCH3), 6.83 (s, 1H, pyridine H-4), 6.95 (s, (2NH), 3044 (CH, aromatic), 2230 (CN), 1730, 1680 (2C=O);
1H, coumarin H-4), 7.05–7.48 (m, 8H aromatic), 8.89 (s, 1H, 1H-NMR (DMSO-d6) δ: 2.49 (s, 3H, CH3), 7.04 (s, 1H, couma-
pyridine H-5), 9.97 (s, 1H, NH, D2O exchangeable); 13C-NMR rin H-4), 7.44–7.83 (m, 9H aromatic), 8.95 (s, 2H, 2 NH, D2O
(DMSO-d6) δ: 32.8, 86.1, 116.4, 120.3, 120.9, 121.6, 123.1, exchangeable); 13C-NMR (DMSO-d6) δ: 22.8, 116.7, 120.8,
123.9, 124.3, 125.8, 127.4, 129.4, 130.3, 132.5, 133.9, 137.3, 121.3, 123.7, 124.6, 125.4, 126.5, 127.3, 129.2, 130.1, 132.8,
139.5, 140.2, 141.9, 165.0; MS (EI): m/z (%) 371 (M+). Anal. 133.2, 142.9, 162.2, 164.1, 169.8, 170.3; MS (EI): m/z (%) 373
Calcd for C22H17N3O3: C, 71.15; H, 4.61; N, 11.31. Found: C, (M+). Anal. Calcd for C20H15N5O3: C, 64.34; H, 4.05; N, 18.76.
71.43; H, 4.39; N, 11.52.
Found: C, 64.08; H, 4.22; N, 18.59.
2-Amino-4-(4-chlorophenyl)-6-(2-oxo-2H-chromen-3-
yl)-1,4-dihydropyridine-3-carbonitrile (3c)
General Procedure for the Synthesis of Compounds 6
and 7 The mixture of hydrazide-hydrazone derivative (4)
Yield: 75%; mp: 188–190°C; IR (KBr, cm−1): 3455–3240 (2.69g, 0.01mol) with either of benzaldehyde or salicylal-
(NH2, NH), 3078 (CH, aromatic), 2213 (CN), 1735 (C=O); dehyde (0.01mol) in absolute ethanol containing piperidine
1H-NMR (DMSO-d6) δ: 3.09 (s, 2H, NH2, D2O exchange- (1mL) was heated under reflux for 1h. The reaction mixture
able), 6.80 (s, 1H, pyridine H-4), 7.21 (s, 1H, coumarin H-4), was left to cool, poured onto ice water and neutralized by
7.41–8.59 (m, 8H aromatic), 8.96 (s, 1H, pyridine H-5), 11.20 hydrochloric acid. The solid product was precipitated, filtered,
(s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 88.2, washed with water, and crystallized from ethanol.
116.1, 120.6, 121.0, 121.3, 122.1, 123.1, 124.2, 125.3, 126.0,
2-Cyano-N′-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-3-
127.9, 129.5, 129.8, 133.0, 136.1, 138.5, 140.0, 145.3, 163.1; phenylacrylohydrazide (6)
MS (EI): m/z (%) 375 (M+). Anal. Calcd for C21H14ClN3O2: C,
Yield: 62%; mp: 230°C; IR (KBr, cm−1): 3413–3328 (NH),
1
67.12; H, 3.75; N, 11.18. Found: C, 67.43; H, 3.47; N, 11.51.
3013 (CH, aromatic), 2217 (CN), 1704, 1687 (2C=O); H-NMR
2-Amino-4-(furan-2-yl)-6-(2-oxo-2H-chromen-3-yl)-1,4- (DMSO-d6) δ: 2.14 (s, 3H, CH3), 6.68 (s, 1H, coumarin H-4),
dihydropyridine-3-carbonitrile (3d)
7.12 (s, 1H, C=CH) 7.44–7.92 (m, 9H aromatic), 10.09 (s, 1H,
Yield: 75%; mp: 105–107°C; IR (KBr, cm−1): 3424–3267 NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 22.8, 116.8,
(NH2, NH), 3079 (CH, aromatic), 2215 (CN), 1718 (C=O); 120.8, 121.4, 122.8, 123.7, 124.6, 125.8, 126.4, 127.2, 128.0,
1H-NMR (DMSO-d6) δ: 2.90 (s, 2H, NH2, D2O exchange- 129.5, 132.1, 134.5, 140.3, 164.8, 166.2, 169.5, 170.1; MS (EI):
able), 6.61 (s, 1H, pyridine H-4), 6.78 (s, 1H, coumarin H-4), m/z (%) 357 (M+). Anal. Calcd for C21H15N3O3: C, 70.58; H,
6.80–7.96 (m, 7H aromatic), 8.29 (s, 1H, pyridine H-5), 9.08 4.23; N, 11.76. Found: C, 70.69; H, 4.39; N, 12.05.
(s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 86.8,
116.8, 120.6, 120.9, 122.3, 123.9, 125.7, 127.4, 129.8, 130.5, chromene-3-carbohydrazide (7)
132.8, 134.3, 137.3, 137.8, 139.5, 140.2, 142.3, 144.9, 163.9; MS
Yield: 60%; mp: 291°C; IR (KBr, cm−1): 3434–3328 (2 NH),
(EI): m/z (%) 331 (M+). Anal. Calcd for C19H13N3O3: C, 68.88; 3023 (CH, aromatic), 1714, 1681 (2C=O); H-NMR (DMSO-
H, 3.95; N, 12.86. Found: C, 68.59; H, 3.61; N, 12.64. d6) δ: 2.92 (s, 3H, CH3), 6.78 (s, 1H, coumarin H-4), 6.81 (s,
2-Imino-N′-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-2H-
1
Synthesis of 2-Cyano-N′-(1-(2-oxo-2H-chromen-3-yl)- 1H, coumarin H-4), 6.83–8.76 (m, 8H aromatic), 9.98 (s, 1H,
ethylidene)acetohydrazide (4) A mixture of 3-acetyl-2H- NH, D2O exchangeable), 11.19 (s, 1H, NH, D2O exchange-
chromen-2-one (1) (1.88g, 0.01mol) and 2-cyanoacetohydra- able); 13C-NMR (DMSO-d6) δ: 32.2, 120.6, 121.0, 121.4, 122.1,
zide (0.99g, 0.01mol) in 1,4-dioxane was heated under reflux 122.5, 123.4, 124.2, 125.0, 126.2, 127.7, 128.3, 129.4, 130.6,
for 2h. The reaction mixture was left to cool, poured onto ice 131.9, 132.1, 140.1, 160.3, 164.5, 166.1, 168.9; MS (EI): m/z (%)
water and neutralized by hydrochloric acid. The solid product 373 (M+). Anal. Calcd for C21H15N3O4: C, 67.56; H, 4.05; N,
was precipitated, filtered, washed with water, and crystallized 11.25. Found: C, 67.29; H, 4.37; N, 11.58.
from ethanol.
General Procedure for the Synthesis of Compounds
Yield: 65%; mp: 268–270°C; IR (KBr, cm−1): 3348–3259 8 and 9 A mixture of hydrazide-hydrazone derivative (4)
(NH), 3063 (CH, aromatic), 2236 (CN), 1734, 1685 (2C=O); (2.69g, 0.01mol), sulphur metal (0.32g, 0.01mol), and either
1H-NMR (DMSO-d6) δ: 2.15 (s, 3H, CH3), 5.08 (s, 2H, CH2), malononitrile or ethyl cyanoacetate (0.01mol) in absolute etha-
6.63 (s, 1H, coumarin H-4), 6.88–7.83 (m, 4H aromatic), 8.92 nol containing triethylamine (1mL) was heated under reflux
(s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 28.3, for 1h. The reaction mixture was left to cool, poured onto ice
64.2, 117.3, 120.4, 120.9, 124.3, 126.2, 128.5, 130.3, 133.2, water and neutralized by hydrochloric acid. The solid product
142.3, 160.1, 164.3, 172.1; MS (EI): m/z (%) 269 (M+). Anal. was precipitated, filtered, washed with water, and crystallized
Calcd for C14H11N3O3: C, 62.45; H, 4.12; N, 15.61. Found: C, from ethanol.
62.23; H, 4.29; N, 15.69.
Synthesis of 2-Oxo-2-(2-(1-(2-oxo-2H-chromen-3-yl)eth- hydrazinyl)thiophene-2,4-dicarbonitrile (8)
ylidene)hydrazinyl)-N′-phenylacetohydrazonoyl Cyanide
Yield: 71%; mp: 173–175°C; IR (KBr, cm−1): 3422–3278
5-Amino-3-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-
(5) To a cold solution of the hydrazide-hydrazone deriva- (NH2, NH), 3091 (CH, aromatic), 2205 (CN), 1706 (C=O);
tive 4 (2.69g, 0.01mol) in ethanol (30mL) containing sodium 1H-NMR (DMSO-d6) δ: 3.05 (s, 3H, CH3), 3.83 (s, 2H, NH2,
acetate (2.5g), a cold solution benzenediazonium chloride D2O exchangeable), 6.83 (s, 1H, coumarin H-4), 6.97–7.93
(0.01mol) [prepared by the addition of sodium nitrite solution (m, 4H aromatic), 8.95 (s, 1H, NH, D2O exchangeable);
(0.7g, 0.01mol) to a cold solution of aniline (0.01mol) in con- 13C-NMR (DMSO-d6) δ: 28.8, 116.6, 117.3, 120.3, 120.7, 121.6,
centrated hydrochloric acid (3mL, 18N) with continuous stir- 122.8, 126.8, 128.4, 129.9, 132.8, 133.2, 140.2, 143.6, 144.0,
ring] was added while stirring. The reaction mixture was kept 163.8, 170.3; MS (EI): m/z (%) 349 (M+). Anal. Calcd for
at room temperature for 1h and the formed solid product, was C17H11N5O2S: C, 58.44; H, 3.17; N, 20.05; S, 9.18. Found: C,

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
685
58.79; H, 3.49; N, 20.39; S, 9.04.
117.4, 120.8, 121.3, 122.4, 123.7, 124.6, 125.8, 127.1, 128.4,
Ethyl 2-Amino-5-cyano-4-(2-(1-(2-oxo-2H-chromen-3-yl)- 130.3, 132.2, 136.9, 137.0, 138.2, 140.2, 142.8, 143.6, 164.3,
ethylidene)hydrazinyl)thiophene-3-carboxylate (9)
171.6; MS (EI): m/z (%) 423 (M+). Anal. Calcd for C24H17N5O3:
Yield: 68%; mp: 273–275°C; IR (KBr, cm−1): 3433–3252 C, 68.08; H, 4.05; N, 16.54. Found: C, 68.31; H, 4.40; N, 16.72.
(NH2, NH), 3025 (CH, aromatic), 2220 (CN), 1734, 1685 2-Amino-4-(4-methoxyphenyl)-6-(2-(1-(2-oxo-2H-
1
(2C=O); H-NMR (DMSO-d6) δ: 1.15 (t, 3H, J=7.2Hz, CH2- chromen-3-yl)ethylidene)hydrazinyl)-4H-pyran-3,5-
CH3), 3.08 (s, 3H, CH3), 3.77 (s, 2H, NH2, D2O exchangeable), dicarbonitrile (11b)
3.85 (q, 2H, J=7.2Hz, CH2-CH3), 6.96 (s, 1H, coumarin H-4),
Yield: 75%; mp: 138–140°C; IR (KBr, cm−1): 3441–3104
7.38–7.83 (m, 4H aromatic), 8.95 (s, 1H, NH, D2O exchange- (NH2, NH), 3043 (CH, aromatic), 2223 (CN), 1729 (C=O);
able); 13C-NMR (DMSO-d6) δ: 28.1, 30.0, 65.3, 116.9, 120.6, 1H-NMR (DMSO-d6) δ: 2.93 (s, 3H, CH3), 3.72 (s, 2H, NH2,
121.4, 122.4, 123.4, 124.2, 126.1, 127.7, 129.4, 130.6, 140.0, D2O exchangeable), 3.89 (s, 3H, OCH3), 5.02 (s, 1H, pyran
146.9, 147.9, 160.3, 163.4, 168.2; MS (EI): m/z (%) 396 (M+). H-4), 6.96 (s, 1H, coumarin H-4), 7.02–7.99 (m, 8H aromatic),
Anal. Calcd for C19H16N4O4S: C, 57.57; H, 4.07; N, 14.13; S, 8.95 (s, 1H, NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ:
8.09. Found: C, 57.36; H, 4.29; N, 13.97; S, 7.99.
30.0, 56.3, 88.0, 116.0, 117.2, 120.6, 121.3, 122.3, 123.1, 124.2,
General Procedure for the Synthesis of Compounds 10a 125.4, 126.5, 127.6, 129.4, 129.8, 133.0, 136.0, 138.6, 140.1,
and b The reaction of hydrazide-hydrazone derivative (4) 144.5, 148.4, 160.0, 168.1; MS (EI): m/z (%) 453 (M+). Anal.
(2.69g, 0.01mol) with either acetylacetone or ethyl acetoace- Calcd for C25H19N5O4: C, 66.22; H, 4.22; N, 15.44. Found: C,
tate (0.01mol) in absolute ethanol containing piperidine (1mL) 66.52; H, 3.99; N, 15.29.
was heated under reflux for 8h. The reaction mixture was left
2-Amino-4-(4-chlorophenyl)-6-(2-(1-(2-oxo-2H-chromen-3-
to cool, poured onto ice water and neutralized by hydrochloric yl)ethylidene)hydrazinyl)-4H-pyran-3,5-dicarbonitrile (11c)
acid. The solid product was precipitated, filtered, washed with
Yield: 77%; mp: 159–161°C; IR (KBr, cm−1): 3441–3282
(NH2, NH), 3054 (CH, aromatic), 2214 (CN), 1715 (C=O);
water, and crystallized from ethanol.
4,6-Dimethyl-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)- 1H-NMR (DMSO-d6) δ: 2.68 (s, 3H, CH3), 3.57 (s, 2H, NH2,
ethylidene)amino)-1,2-dihydropyridine-3-carbonitrile (10a)
D2O exchangeable), 5.09 (s, 1H, pyran H-4), 6.82 (s, 1H,
Yield: 59%; mp: 200–202°C; IR (KBr, cm−1): 3045 (CH, coumarin H-4), 7.15–7.84 (m, 8H aromatic), 8.94 (s, 1H,
1
aromatic), 2231 (CN), 1726, 1680 (2C=O); H-NMR (DMSO- NH, D2O exchangeable); 13C-NMR (DMSO-d6) δ: 28.8, 84.2,
d6) δ: 2.09 (s, 3H, CH3), 3.01 (s, 3H, CH3), 3.48 (s, 3H, CH3), 116.4, 117.1, 119.8, 120.3, 122.4, 123.9, 124.0, 125.8, 126.4,
6.99 (s, 1H, coumarin H-4), 7.34–7.69 (m, 4H aromatic), 9.01 128.6, 132.7, 136.3, 138.4, 139.8, 140.4, 143.9, 144.1, 148.3,
(s, 1H, pyridine H-5); 13C-NMR (DMSO-d6) δ: 30.0, 33.9, 42.1, 164.4, 171.2; MS (EI): m/z (%) 457 (M+). Anal. Calcd for
116.2, 120.4, 120.6, 121.1, 122.8, 124.3, 125.8, 126.9, 128.3, C24H16ClN5O3: C, 62.96; H, 3.52; N, 15.30. Found: C, 62.91; H,
129.4, 130.6, 140.0, 148.4, 160.1, 164.1, 168.3; MS (EI): m/z 3.29; N, 15.02.
(%) 333 (M+). Anal. Calcd for C19H15N3O3: C, 68.46; H, 4.54;
N, 12.61. Found: C, 68.73; H, 4.66; N, 12.72.
6-Hydroxy-4-methyl-2-oxo-1-((1-(2-oxo-2H-chromen-3-yl)-
ethylidene)amino)-1,2-dihydropyridine-3-carbonitrile (10b)
2-Amino-4-(furan-2-yl)-6-(2-(1-(2-oxo-2H-chromen-3-yl)-
ethylidene)hydrazinyl)-4H-pyran-3,5-dicarbonitrile (11d)
Yield: 72%; mp: 238–240°C; IR (KBr, cm−1): 3398–3274
(NH2, NH), 3044 (CH, aromatic), 2212 (CN), 1720 (C=O);
Yield: 61%; mp: 243–245°C; IR (KBr, cm−1): 3427 (OH), 1H-NMR (DMSO-d6) δ: 2.92 (s, 3H, CH3), 3.44 (s, 2H, NH2,
3034 (CH, aromatic), 2210 (CN), 1724, 1687 (2C=O); D2O exchangeable), 5.23 (s, 1H, pyran H-4), 6.75 (s, 1H, cou-
1H-NMR (DMSO-d6) δ: 2.42 (s, 3H, CH3), 3.61 (s, 3H, CH3), marin H-4), 6.92–7.80 (m, 7H aromatic), 8.97 (s, 1H, NH,
6.78 (s, 1H, coumarin H-4), 6.83–7.68 (m, 4H aromatic), 9.01 D2O exchangeable); 13C-NMR (DMSO-d6) δ: 28.6, 84.5, 116.3,
(s, 1H, pyridine H-5), 11.14 (s, 1H, OH, D2O exchangeable); 117.0, 119.8, 120.4, 122.3, 123.8, 124.2, 125.3, 126.0, 127.2,
13C-NMR (DMSO-d6) δ: 22.8, 30.0, 116.9, 120.5, 120.9, 122.8, 128.3, 129.4, 132.2, 136.1, 138.8, 140.2, 143.6, 143.8, 164.6,
123.9, 124.6, 125.4, 126.1, 127.9, 129.2, 130.2, 134.8, 136.6, 171.8; MS (EI): m/z (%) 413 (M+). Anal. Calcd for C22H15N5O4:
162.8, 164.0, 169.6; MS (EI): m/z (%) 335 (M+). Anal. Calcd C, 63.92; H, 3.66; N, 16.94. Found: C, 63.69; H, 4.01; N, 16.76.
for C18H13N3O4: C, 64.47; H, 3.91; N, 12.53. Found: C, 64.69;
General Procedure for the Synthesis of Compounds
H, 4.22; N, 12.67.
12a–d A mixture of hydazide-hydrazone derivative (4)
General Procedure for the Synthesis of Compounds 11a– (2.69g, 0.01mol), the appropriate arylaldehyde (0.1mol) and
A mixture of hydazide-hydrazone derivative (4) (2.69g, malononitrile (0.66g, 0.1mol) were heated under reflux in
d
0.01mol, the appropriate arylaldehyde (0.1mol) and malononi- 1,4-dioxane (30mL) containing ammonium acetate (0.5g) for
trile (0.66g, 0.1mol) were heated under reflux in 1,4-dioxane 5h. The reaction mixture was left to cool, poured onto ice
(30mL) containing triethylamine (1.0mL) for 3h. The reaction water and neutralized by hydrochloric acid. The solid product
mixture was left to cool, poured onto ice water and neutral- was precipitated, filtered, washed with water, and crystallized
ized by hydrochloric acid. The solid product was precipitated, from ethanol.
filtered, washed with water, and crystallized from ethanol. 2-Amino-6-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)-
2-Amino-6-(2-(1-(2-oxo-2H-chromen-3-yl)ethylidene)- hydrazinyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarbonitrile
hydrazinyl)-4-phenyl-4H-pyran-3,5-dicarbonitrile (11a)
(12a)
Yield: 70%; mp: 123–125°C; IR (KBr, cm−1): 3431–3254
Yield: 66%; mp: 189–191°C; IR (KBr, cm−1): 3447–3259
(NH2, NH), 3065 (CH, aromatic), 2212 (CN), 1723 (C=O); (NH2, 2NH), 3046 (CH, aromatic), 2230 (CN), 1737 (C=O);
1H-NMR (DMSO-d6) δ: 2.49 (s, 3H, CH3), 3.48 (s, 2H, NH2, 1H-NMR (DMSO-d6) δ: 2.09 (s, 3H, CH3), 3.08 (s, 2H, NH2,
D2O exchangeable), 5.12 (s, 1H, pyran H-4), 6.77 (s, 1H, cou- D2O exchangeable), 4.22 (s, 1H, pyridine H-4), 7.20 (s, 1H,
marin H-4), 6.79–7.83 (m, 9H aromatic), 8.95 (s, 1H, NH, coumarin H-4), 7.30–8.03 (m, 9H aromatic), 8.33 (s, 1H, NH,
D2O exchangeable); 13C-NMR (DMSO-d6) δ: 29.3, 86.9, 116.8, D2O exchangeable), 10.73 (s, 1H, NH, D2O exchangeable);

686
Chem. Pharm. Bull.
Vol. 63, No. 9 (2015)
13C-NMR (DMSO-d6) δ: 28.9, 88.4, 116.8, 117.2, 120.3, 120.9, sopharyngeal carcinoma (HONE1) and normal fibroblast cells
121.8, 122.9, 124.4, 125.9, 126.3, 127.4, 128.6, 129.7, 132.8, (WI38) were kindly provided by the National Cancer Institute
136.3, 138.3, 140.6, 142.3, 143.6, 164.8, 170.9; MS (EI): m/z (NCI, Cairo, Egypt). They were grown as monolayer and
(%) 422 (M+). Anal. Calcd for C24H18N6O2: C, 68.24; H, 4.29; routinely maintained in RPMI-1640 medium supplemented
N, 19.89. Found: C, 68.50; H, 3.99; N, 19.63.
with 5% heat inactivated FBS, 2mM glutamine and antibiot-
2-Amino-4-(4-methoxyphenyl)-6-(2-(1-(2-oxo-2H- ics (penicillin 100U/mL, streptomycin 100lg/mL), at 37°C in
chromen-3-yl)ethylidene)hydrazinyl)-1,4-dihydropyridine-3,5- a humidified atmosphere containing 5% CO2. Exponentially
dicarbonitrile (12b)
growing cells were obtained by plating 1.5×105 cells/mL for
Yield: 69%; mp: 159–161°C; IR (KBr, cm−1): 3438–3265 the six human cancer cell lines followed by 24h of incubation.
(NH2, 2NH), 3038 (CH, aromatic), 2222 (CN), 1731 (C=O); The effect of the vehicle solvent (DMSO) on the growth of
1H-NMR (DMSO-d6) δ: 2.35 (s, 3H, CH3), 2.59 (s, 2H, NH2, these cell lines was evaluated in all the experiments by ex-
D2O exchangeable), 2.71 (s, 3H, OCH3), 3.98 (s, 1H, pyridine posing untreated control cells to the maximum concentration
H-4), 6.97 (s, 1H, coumarin H-4), 7.08–7.99 (m, 8H aromatic), (0.5%) of DMSO used in each assay.
8.75 (s, 1H, NH, D2O exchangeable), 11.12 (s, 1H, NH, D2O
exchangeable); 13C-NMR (DMSO-d6) δ: 28.2, 54.0, 88.3,
116.3, 117.4, 120.4, 120.6, 121.1, 121.8, 122.8, 124.3, 125.4,
Conflict of Interest The authors declare no conflict of
126.2, 127.4, 128.3, 129.5, 133.4, 139.2, 140.5, 146.0, 147.7, interest.
163.9, 168.1; MS (EI): m/z (%) 452 (M+). Anal. Calcd for
C25H20N6O3: C, 66.36; H, 4.46; N, 18.57. Found: C, 66.08; H,
4.72; N, 18.49.
2-Amino- 4-(4-chlorophenyl)-6 -(2-(1-(2-oxo-2H-
chromen-3-yl)ethylidene)hydrazinyl)-1,4-dihydropyridine-3,5-
dicarbonitrile (12c)
References
1) Curini M., Cravotto G., Epifano F., Giannone G., Curr. Med.
Chem., 13, 199–222 (2006).
2) Sankappa Rai U., Isloor A. M., Shetty P., Vijesh A. M., Prabhu N.,
Isloor S., Thiageeswaran M., Fun H. K., Eur. J. Med. Chem., 45,
2695–2699 (2010).
3) Nikhil B., Shikha B., Anil P., Prakash N. B., Int. Res. J. Pharm., 3,
24–29 (2013).
4) Devji T., Reddy C., Woo C., Awale S., Kadota S., Carrico-Moniz
D., Bioorg. Med. Chem. Lett., 21, 5770–5773 (2011).
5) Xue H., Lu X., Zheng P., Liu L., Han C., Hu J., Liu Z., Ma T., Li Y.,
Wang L., Chen Z., Liu G., J. Med. Chem., 53, 1397–1401 (2010).
6) Manvar A., Bavishi A., Radadiya A., Patel J., Vora V., Dodia N.,
Rawal K., Shah A., Bioorg. Med. Chem. Lett., 21, 4728–4731 (2011).
7) Yeh J. Y., Coumar M. S., Horng J. T., Shiao H. Y., Kuo F. M., Lee
H. L., Chen I. C., Chang C. W., Tang W. F., Tseng S. N., Chen C. J.,
Shih S. R., Hsu J. T., Liao C. C., Chao Y. S., Hsieh H. P., J. Med.
Chem., 53, 1519–1533 (2010).
Yield: 70%; mp: 176–178°C; IR (KBr, cm−1): 3435–3276
(NH2, 2NH), 3097 (CH, aromatic), 2227 (CN), 1727 (C=O);
1H-NMR (DMSO-d6) δ: 2.09 (s, 3H, CH3), 2.90 (s, 2H, NH2,
D2O exchangeable), 4.02 (s, 1H, pyridine H-4), 6.92 (s, 1H,
coumarin H-4), 7.09–7.92 (m, 8H aromatic), 8.95 (s, 1H, NH,
D2O exchangeable), 10.72 (s, 1H, NH, D2O exchangeable);
13C-NMR (DMSO-d6) δ: 28.2, 87.9, 116.5, 117.3, 120.4, 120.9,
122.6, 123.8, 124.7, 125.6, 126.8, 127.4, 128.8, 129.3, 132.8,
136.9, 139.5, 140.9, 142.8, 143.6, 164.1, 172.2; MS (EI): m/z (%)
456 (M+). Anal. Calcd for C24H17ClN6O2: C, 63.09; H, 3.75; N,
18.39. Found: C, 63.39; H, 3.47; N, 18.69.
2-Amino-4-(furan-2-yl)-6-(2-(1-(2-oxo-2H-chromen-3-yl)-
ethylidene)hydrazinyl)-1,4-dihydropyridine-3,5-dicarbonitrile
(12d)
8) Piazzi L., Cavalli A., Colizzi F., Belluti F., Bartolini M., Mancini F.,
Recanatini M., Andrisano V., Rampa A., Bioorg. Med. Chem. Lett.,
18, 423–436 (2008).
Yield: 61%; mp: 193–195°C; IR (KBr, cm−1): 3439–3259
(NH2, 2NH), 3042 (CH, aromatic), 2226 (CN), 1728 (C=O);
1H-NMR (DMSO-d6) δ: 2.32 (s, 3H, CH3), 2.71 (s, 2H, NH2,
D2O exchangeable), 3.98 (s, 1H, pyridine H-4), 6.97 (s, 1H,
coumarin H-4), 7.14–8.00 (m, 7H aromatic), 8.65 (s, 1H, NH,
D2O exchangeable), 11.05 (s, 1H, NH, D2O exchangeable);
13C-NMR (DMSO-d6) δ: 26.8, 88.1, 116.2, 117.0, 121.3, 121.8,
122.9, 123.2, 125.1, 126.3, 128.8, 129.3, 132.8, 136.3, 138.9,
140.6, 142.3, 143.6, 144.2, 145.3, 164.3, 172.3; MS (EI): m/z
(%) 412 (M+). Anal. Calcd for C22H16N6O3: C, 64.07; H, 3.91;
N, 20.38. Found: C, 64.39; H, 3.78; N, 20.11.
9) Melagraki G., Afantitis A., Igglessi-Markopoulou O., Detsi A.,
Koufaki M., Kontogiorgis C., Hadjipavlou-Litina D. J., Eur. J. Med.
Chem., 44, 3020–3026 (2009).
10) Madhavan G. R., Balraju V., Mallesham B., Chakrabarti R., Lohray
V. B., Bioorg. Med. Chem. Lett., 13, 2547–2551 (2003).
11) Yu D., Suzuki M., Xie L., Morris-Natschke S. L., Lee K. L., Med.
Res. Rev., 23, 322–345 (2003).
12) Kempen I., Papapostolou D., Thierry N., Pochet L., Counerotte S.,
Masereel B., Foidart J. M., Reboud-Ravaux M., Noel A., Pirotte B.,
Br. J. Cancer, 88, 1111–1118 (2003).
13) Ananthan S., Faaleolea E. R., Goldman R. C., Hobrath J. V., Kwong
C. D., Laughon B. E., Maddry J. A., Mehta A., Rasmussen L.,
Reynolds R. C., Secrist J. A. III, Shindo N., Showe D. N., Sosa M.
I., Suling W. J., White E. L., Tuberculosis, 89, 334–353 (2009).
14) Reynolds R. C., Ananthan S., Faaleolea E., Hobrath J. V., Kwong
C. D., Maddox C., Rasmussen L., Sosa M. I., Thammasuvimol E.,
White E. L., Zhang W., Secrist J. A. III, Tuberculosis, 92, 72–83
(2012).
15) Woo L. W. L., Purohit A., Malini B., Reed M. J., Potter B. V. L.,
Chem. Biol., 7, 773–791 (2000).
16) Basanagouda M., Jambagi V. B., Barigidad N. N., Laxmeshwar
S. S., Devaru V., Narayanachar, Eur. J. Med. Chem., 74, 225–233
(2014).
In Vitro Cytotoxic Assay
Chemicals
Fetal bovine serum (FBS) and L-glutamine were purchased
from Gibco Invitrogen Co. (Scotland, U.K.). RPMI-1640 me-
dium was purchased from Cambrex (New Jersey, NJ, U.S.A.).
Dimethyl sulfoxide (DMSO), CHS 828, penicillin, streptomy-
cin and sulforhodamine B (SRB) were purchased from Sigma
Chemical Co. (St. Louis, MO, U.S.A.).
Cell Cultures
Cell cultures were obtained from the European Collection
of cell Cultures (ECACC, Salisbury, U.K.) and human gastric
cancer (NUGC), human colon cancer (DLD1), human liver
cancer (HA22T and HEPG2), human breast cancer (MCF), na-
17) Purohit A., Woo L. W. L., Potter B. V. L., Reed M. J., Cancer Res.,
60, 3394–3396 (2000).
18) Chilin A., Battistutta R., Bortolato A., Cozza G., Zanatta S., Poletto

Vol. 63, No. 9 (2015)
Chem. Pharm. Bull.
687
G., Mazzorana M., Zagotto G., Uriarte E., Guiotto A., Pinna L. A.,
Meggio F., Moro S., J. Med. Chem., 51, 752–759 (2008).
35) Nasr T., Bondock S., Youns M., Eur. J. Med. Chem., 76, 539–548
(2014).
19) Zhao H., Donnelly A. C., Kusuma B. R., Brandt G. E. L., Brown D.,
Rajewski R. A., Vielhauer G., Holzbeierlein J., Cohen M. S., Blagg
B. S. J., J. Med. Chem., 54, 3839–3853 (2011).
36) Yadagiri B., Holagunda U. D., Bantu R., Nagarapu L., Guguloth
V., Polepally S., Jain N., Bioorg. Med. Chem. Lett., 24, 5041–5044
(2014).
20) Zhao H. P., Yan B., Peterson L. B., Blagg B. S. J., ACS Med. Chem. 37) Mohareb R. M., Al-Omran F., Steroids, 77, 1551–1559 (2012).
Lett., 3, 327–331 (2012).
38) Rollas S., Gulerman N., Erdeniz H., II Farmaco, 57, 171–174 (2002).
39) Gürsoy A., Terzioglu N., Otuk G., Eur. J. Med. Chem., 32, 753–757
(1997).
40) Vicini P., Zani F., Cozzini P., Doytchinova I., Eur. J. Med. Chem.,
37, 553–564 (2002).
21) Kohno H., Suzuki R., Curini M., Epifano F., Maltese F., Gonzales
S. P., Tanaka T., Int. J. Cancer, 118, 2936–2942 (2006).
22) Liu W., Hua J., Zhou J., Zhang H., Zhu H., Cheng Y., Gust R.,
Bioorg. Med. Chem. Lett., 22, 5008–5012 (2012).
23) Sashidhara K. V., Avula S. R., Sharma K., Palnati G. R., Bathula S.
R., Eur. J. Med. Chem., 60, 120–127 (2013).
41) Mamolo M. G., Falagiani V., Zampieri D., Vio L., Banfi E., II
Farmaco, 56, 587–592 (2001).
24) Lacy A., O'Kennedy R., Curr. Pharm. Des., 10, 3797–3811 (2004).
25) Zhou J. F., Gong G. X., Zhu F. X., Zhi S. J., Chin. Chem. Lett., 20,
37–39 (2009).
26) Kurt B. Z., Gazioglu I., Sonmez F., Kucukislamoglu M., Bioorg.
Chem., 59, 80–90 (2015).
27) Hamama W. S., Berghot M. A., Baz E. A., Gouda M. A., Arch.
Pharm. Chem. Life Sci., 344, 710–718 (2011).
28) Liu X.-H., Liu H.-F., Chen J., Yang Y., Song B.-A., Bai L.-S., Liu
J.-X., Zhu H.-L., Qi X.-B., Bioorg. Med. Chem. Lett., 20, 5705–5708
(2010).
42) Rahman V. P., Mukhtar S., Ansari W. H., Lemiere G., Eur. J. Med.
Chem., 40, 173–184 (2005).
43) Dimmock J. R., Vashishtha S. C., Stables J. P., Eur. J. Med. Chem.,
35, 241–248 (2000).
44) Tapia R., De La Mora M. P., Massieu G. H., Biochem. Pharmacol.,
16, 1211–1218 (1967).
45) Sava G., Perissin L., Lassiani L., Zabucchi G., Chem. Biol. Inter-
act., 53, 37–43 (1985).
46) Xia Y., Fan C. D., Zhao B. X., Zhao J., Shin D. S., Miao J. Y., Eur.
J. Med. Chem., 43, 2347–2353 (2008).
29) Nepali K., Sharma S., Sharma M., Bedi P. M. S., Dhar K. L., Eur. J. 47) Melnyk P., Leroux V., Sergheraert C., Grellier P., Bioorg. Med.
Med. Chem., 77, 422–487 (2014).
Chem. Lett., 16, 31–35 (2006).
30) Muregi F. W., Ishih A., Drug Dev. Res., 71, 20–32 (2010).
31) Hubschwerlen C., Specklin J. L., Sigwalt C., Schroeder S., Locher
H. H., Bioorg. Med. Chem., 11, 2313–2319 (2003).
32) Pingaew R., Prachayasittikul S., Ruchirawat S., Prachayasittikul V.,
Med. Chem. Res., 23, 1768–1780 (2014).
33) Mohareb R. M., Abbas N. S., Abdelaziz M. A., Steroids, 86, 45–55
(2014).
34) Mohareb R. M., Zaki M. Y., Abbas N. S., Steroids, 98, 80–91
(2015).
48) Ajani O. O., Obafemi C. A., Nwinyi O. C., Akinpelu D. A., Bioorg.
Med. Chem., 18, 214–221 (2010).
49) Puterová Z., Andicsová A., Végh D., Tetrahedron, 64, 11262–11269
(2008).
50) Srikrishna D., Dubey P. K., Tetrahedron Lett., 55, 6561–6566
(2014).
51) Hjarnaa P. J, Jonsson E., Latini S., Dhar S., Larsson R., Bramm E.,
Skov T., Binderup L., Cancer Res., 59, 5751–5757 (1999).