Multicomponent reactions of acetoacetanilide derivatives with aromatic aldehydes and cyanomethylene reagents to produce 4H-pyran and 1,4-dihydropyridine derivatives with antitumor activities

Article abstract of DOI:10.1248/cpb.c15-00685

The multi-component reaction of either acetoacetanilide derivative 1a or b with any of the aldehyde derivatives 2a-d and malononitrile 3 in the presence of triethylamine as a catalyst gave the 4H-pyran derivatives 4a-g, respectively. Carrying the same rea

Full text of DOI:10.1248/cpb.c15-00685

Vol. 63, No. 12
Chem. Pharm. Bull. 63, 1055–1064 (2015)
1055
Regular Article
Multicomponent Reactions of Acetoacetanilide Derivatives with Aromatic
Aldehydes and Cyanomethylene Reagents to Produce 4H-Pyran and
1,4-Dihydropyridine Derivatives with Antitumor Activities
,a
Rasha Abdallah Azzam* and Rafat Milad Moharebb
a Department of Chemistry, Faculty of Science, Helwan University; Cairo 11795, Egypt: and b Department of
Chemistry, Faculty of Science, Cairo University; Giza 12613, Egypt.
Received September 6, 2015; accepted September 27, 2015
The multi-component reaction of either acetoacetanilide derivative 1a or b with any of the aldehyde
derivatives 2a–d and malononitrile 3 in the presence of triethylamine as a catalyst gave the 4H-pyran deriva-
tives 4a–g, respectively. Carrying the same reaction but using a catalytic amount of ammonium acetate gave
the 1,4-dihydropyridine derivatives 5a–f, respectively. The use of ethyl cyanoacetate instead of malononitrile
in the presence of a catalytic amount of triethylamine gave the 4H-pyran derivatives 7a–d, respectively.
Compound 4e was used to synthesize 1,4-dihydropyridine 9a–c and arylhydraone 11a–e derivatives were syn-
thesized from 4a and e. The anti-tumor evaluations of the newly synthesized products were tested against six
human cancer and normal cell lines. The results showed that compounds 4a, b, f, 5d, f, 9 and 11a–d had op-
timal cytotoxic effect against cancer cell lines with IC50<550nM. The toxicity of the most active compounds
was further measured against shrimp larvae.
Key words 4H-pyran; 1,4-dihydropyridine; cyanomethylene; cytotoxicity
Multicomponent reactions (MCRs) are one-pot reactions of 4H-pyran derivatives through the reaction of acetoacetani-
...
employing more than two starting materials, e.g. 3, 4, , 7, lide derivatives with cyanomethylene derivatives and aromatic
where most of the atoms of the starting materials are incor- aldehydes. All the synthesized compounds were characterized
1
porated in the final product.1) Several descriptive tags are using IR (infrared), H-NMR, and 13C-NMR (nuclear mag-
regularly attached to MCRs they are atom economic, e.g. the netic resonance) spectrometry and were subjected to screening
majority if not all of the atoms of the starting materials are towards cancer cell lines.
incorporated in the product; they are efficient, e.g. they ef-
ficiently yield the product since the product is formed in one-
Results and Discussion
step instead of multiple sequential steps; they are convergent,
Chemistry The reaction of either acetoacetanilide 1a or
e.g. several starting materials combine in one reaction to form b with either benzaldehyde (2a), 4-chlorobenzaldehyde (2b),
the product; they exhibit a very high bond-forming-index 3-nitrobenzaldehyde (2c) or furfural (2d) and malononitrile
(BFI), e.g. several non-hydrogen atom bonds are formed in (3) in absolute ethanol containing a catalytic amount of tri-
one synthetic transformation.2,3) Therefore MCRs are often a ethylamine gave the 4H-pyran derivatives 4a–g, respectively
useful alternative to sequential multistep synthesis.
(Chart 1). The structures of 4a–g were established on the basis
1
In view of the increasing interest in the preparation of a of analytical and spectral data. Thus, the H-NMR spectrum
large variety of heterocyclic compound libraries, the devel- of 4a (as an example) showed a singlet at δ 1.91 for the methyl
opment of new synthetically valuable MCRs with several group, a singlet at δ 4.10ppm (D2O exchangeable) indicat-
diversity points remains a challenge for both academic and ing the NH2 group, a singlet at δ 5.91ppm equivalent to the
industrial institutions.4) 4H-Pyran and its derivatives are an pyran H-4, a multiplet at δ 7.10–7.52ppm equivalent to the two
important class of heterocyclic compounds possessing broad phenyl groups and a singlet at δ 7.80ppm for the NH group.
biological activities, such as anti-inflammatory,5) analgesic,6) Moreover, the 13C-NMR spectrum revealed δ 29.8 (CH3), 64.2
antioxidant,7) antitubercular,8) antidepressant,9) sedative,10) (4H-pyranC-4), 116.9 (CN), 119.0, 120.5, 120.9, 121.3, 121.7,
antiamoebic11) and oral analgesic.12) From the aforementioned 122.6, 124.0, 125.3, 130.2, 133.3, 136.9, 140.0 (2C6H5, 4H-
reports, it seemed that the development of an efficient, rapid, pyranC), 164.3 (CO).
and clean synthetic route towards focused libraries of such
The formation of the 4H-pyran derivatives 4a–g can be ex-
compounds is of great importance to both medicinal and syn- plained by the possible mechanism presented in Chart 2. The
thetic chemists. The combination of multicomponent reactions reaction can occur via the initial formation of the intermediate
(MCR2),13–15) which combines different types of MCRs in acrylonitrile derivative A followed by its nucleophilic attack
one pot process inheriting the high selectivity, efficiency, and of the anion of the acetoacetanilide derivatives to produce
atom economy of MCR, has gained more and more attention, the intermediate B. The final products D were formed via the
since Dömling and Ugi reported the first MCR2 of a modified initial cyclization and subsequent tautomerization of the cyclic
four-component reaction (4CR) and the Ugi 4CR.16) However, intermediate C.22)
most of the reported MCR2 cases are limited in isonitrile
To survey the scope of this reaction for the synthesis of 4H-
based multicomponent reactions.17–21) Hence in this work, we pyran and 1,4-dihydropyridine derivatives, different catalyst
report a one-pot, three-component reaction for the synthesis was used. Thus, the multicomponent reaction of acetoacetani-
*To whom correspondence should be addressed. e-mail: rashaazzam8@gmail.com
© 2015 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
lide 1a or b with either 2a, b, c or d and 3 in absolute ethanol noacetate (6) in absolute ethanol containing a catalytic amount
containing a catalytic amount of ammoniumacetate gave the of triethylamine gave the 4H-pyran derivatives 7a–d (Chart 4).
1,4-dihydropyridine derivatives 5a–g, respectively (Chart 3).
To explore the scope and limitations of the products of the
The IR spectrum of 5a exhibits absorption band at 3339 and multicomponent reactions we have examined the reactivity
3246 (NH, NH2), 3061 (CH-aromatic), 2921 (CH3), 2207 (CN) of one of the 4H-pyran derivatives 4a–g towards chemical
1
and 1700 (C=O) cm−1. The H-NMR of compound 4a revealed reagents. For such purpose, the high yield of compound 4e
the presence of the 4H-pyran H-4 at δ 5.91ppm. The presence encouraged us to do further reactions. Thus, the reaction of
of the pyridine H-4 singlet at δ 4.99ppm, the aromatic protons compound 4e with either of aniline (8a), 4-chloroaniline (8b)
in the range δ 7.24–7.61ppm and a singlet at δ 8.69ppm (D2O or p-toluidine (8c) gave the N-arylpyridine derivatives 9a–c,
exchangeable) assigning for the NH group are consistent with respectively (Chart 5). Analytical and spectral data of the lat-
the proposed structure of compound 5a.
ter products were consistent with their respective structures.
With the optimized conditions established above, we de- The methyl group present in compounds 4a and e showed in-
cided to probe the generality of this multicomponent reaction teresting reactivity towards electrophilic reagents. Thus either
using different cyanomethylene reagent. Thus, the reaction of compound 4a or e reacted with the either benzenediazonium
acetoacetanilide 1a or b with either 2a, b or d and ethyl cya- chloride (10a), 4-chlorobenzenediazonium chloride (10b) or
4-methylbenzenediazonium chloride (10c) to afford the corre-
sponding arylhydrazone derivatives 11a–e (Chart 5).
The newly synthesized products were screened towards six
cancer cell lines where some of them showed high cytotoxicity.
In Vitro Cytotoxicity The heterocyclic compounds,
prepared in this study, were evaluated according to standard
protocols for their in vitro cytotoxicity23–25) against six human
cancer cell lines including cells derived from human gastric
cancer (NUGC), human colon cancer (DLD1), human liver
cancer (HA22T and HEPG2), human breast cancer (MCF)
and nasopharyngeal carcinoma (HONE1) as well as a normal
fibroblast cells (WI38). The reference compound used is the
CHS-828 (Fig. 1) which is a pyridylcyano guanidine anti-
tumor agent.26) All of the IC50 values (the sample concentra-
tion that produces 50% reduction in cell growth) in nanomolar
(nM) are listed in Table 1. Some heterocyclic compounds was
observed with significant cytotoxicity against most of the can-
cer cell lines tested (IC50=10–1000 nM). All the synthesized
compounds were tested for their cytotoxicity against normal
fibroblast cells. The results obtained showed that normal
fibroblasts cells (WI38) were affected to a much lesser extent
(IC50>10000nM).
Structure–Activity Relationship
From Table 1, it is convenient to notice that compounds 4b
and f have higher cytotoxicity than 4a and c–e and g. Such
high cytotoxicity of both compounds is attributed to the pres-
ence of Cl atom at position 4 of one of aryl moieties. However,
compound 4c showed high potency against breast cancer MCF
which is equivalent to the cytotoxic effect of the standard
Chart 1. Synthesis of 4-Aryl-4H-pyran-3-carboxylic Acid Arylamide
CHS 828 (IC50=18 nM). Considering the 1,4-dihydropyridine
Chart 2. Mechanism of Formation of the 4H-Pyran Derivatives

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1057
Chart 3. Synthesis of 4-Aryl-1,4-dihydropyridine-3-carboxylic Acid Arylamide
derivatives 5a–f, each one of these derivative revealed selec-
tive activity against certain cancer cell lines. Compound 5a
showed selective higher activity against liver cancer HEPG2
(IC50=22nM) than 5b–f. The introduction of 3-nito group
in 5d exhibited remarkable increase in the activity against
colon cancer DLD1 than 5a–c and f. Moreover compound
5f exhibited high cytotoxicity effect (IC50=38nM) against
gastric cancer (NUGC) compared to the standard CHS 828
(IC50=25nM). The remarkable activity of 5f is due to the pres-
ence of the furan moiety. Comparing compound 4f with 5d,
it is obvious that 4H-pyran ring present in 4f showed lower
growth inhibitory effect than 1,4-dihydropyridine ring in 5d.
Comparing the activities of compounds 4a, d and f with 7a–c,
it is observed that compounds 4a, d and f showed higher cyto-
toxicity compared to compounds 7a–c. Higher cytotoxicity of
4a, d and f is due to the presence of CN group attached to the
4H-pyran ring while in case of compounds 7a–c, the COOEt
group lowered their cytotoxicity. The high activity of 9b com-
pared to 9a and c as well as 5c is due to the presence of three
aryl moieties and Cl atom on one of aryl groups. Considering
the 4H-pyran derivatives 11a–e, compounds 11a–d showed
high growth inhibitory effects. This may be attributed to the
presence of arylhydrazonomethyl group attached to 4H-pyran
ring. Moreover, 11d substituted with 4-chloro group showed
the highest cytotoxicity among the five compounds with re-
markable activity against the five human cancer cell lines
namely: NUGC, DLD1, HA22T, HEPG2 and HONE1 with
Chart 4. Synthesis of 4-Aryl-4H-pyran-3-carboxylic Acid Ethyl Ester

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
Chart 5. Synthesis of 1,4-Diaryl-1,4-dihydropyridine 9a–c and 2-(Arylhydrazonomethyl)-4H-pyran 11a–e
tween A. salina toxicity test and the rodent data. Likewise, the
predictive screening potential of the aquatic invertebrate tests
for acute oral toxicity in man, including A. salina toxicity test,
was slightly better than the rat test for test compounds.31)
In order to prevent the toxicity results from possible false
effects originated from solubility of compounds and dimethyl
sulfoxides (DMSO’s) possible toxicity effect, compounds were
prepared by dissolving in DMSO in the suggested DMSO
volume ranges. It is clear from Table 2 that compounds 4f and
11a–d showed non toxicity against the tested organisms. On
the other hand, compounds 5f and 9b are very toxic, in addi-
tion, compounds 4a, b and 5d are harmful.
Fig. 1. Chemical Structure of CHS 828
IC50’s 32, 50, 27, 221 and 228nM, respectively.
Toxicity Bioactive compounds are often toxic to shrimp
larvae. Thus, in order to monitor these chemicals’ in vivo
lethality to shrimp larvae (Artemia salina), Brine-Shrimp
Conclusion
In summary, we have developed a convenient synthetic
Lethality Assay27) was used. Results were analysed with approach for novel 4H-pyran and 1,4-dihydropyridine deriva-
LC50 program to determine LC50 values and 95% confidence tives. The region selective attack by different reagents on the
intervals.28) Results are given in Table 2 for the compounds active center moiety in acetoacetanilide derivatives led to the
which exhibited optimal cytotoxic effect against cancer cell diversity of the produced systems. Most of the newly synthe-
lines which are the compounds 4a, b, f, 5d, f, 9 and 11a–d. sized compounds were found to be promising anti-prolifera-
The shrimp lethality assay is considered as a useful tool for tive agents. Results showed that compounds 4a, f, 5d, f, 9 and
preliminary assessment of toxicity, and it has been used for 11a–d are the most active compounds towards the tumor cell
the detection of fungal toxins, plant extract toxicity, heavy lines. In addition, compounds 4f and 11a–d showed non toxic-
metals, cyanobacteria toxins, pesticides, and cytotoxicity test- ity against shrimp larvae.
ing of dental materials,29) natural and synthetic organic com-
pounds.30) It has also been shown that A. salina toxicity test
Experimental
results have a correlation with rodent and human acute oral
Chemistry All melting points were uncorrected on a Stu-
toxicity data. Generally, a good correlation was obtained be- art apparatus melting point apparatus. IR spectra (KBr, cm−1)

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1059
b)
Table 1. Cytotoxicity of the Newly Synthesized Products against a Variety of Cancer Cell Linesa) [IC50 (nM)]
Cytotoxicity (IC50 in nM)
Compound
NUGC
DLD1
HA22T
HEPG2
HONE1
MCF
WI38
4a
4b
389
228
1220
569
1480
213
125
1112
2177
1232
1890
239
1620
2052
2250
1824
2170
128
36
2011
18
na
632
262
549
na
4c
2265
1126
2377
880
2139
2168
1389
760
2257
1312
2076
1089
2377
2530
1650
1288
122
4d
2330
779
4e
4f
320
na
4g
1128
1288
1156
2211
48
1892
2187
1280
1070
59
1328
22
1290
2135
699
2673
1729
821
360
650
910
489
na
5a
5b
1226
1302
2334
2120
3744
2216
1284
350
5c
2179
3289
670
1229
480
5d
5e
1622
38
396
274
1180
1264
1286
2073
1140
2389
2690
1142
2457
128
490
860
na
5f
163
120
441
7a
1232
1280
2101
1562
1760
84
1166
2419
2458
770
2225
2160
2258
528
326
7b
2130
2180
139
872
428
na
7c
7d
238
9a
1429
167
1267
219
2309
2023
2560
128
1876
1210
1544
248
na
9b
na
9c
2210
48
2433
55
1650
128
520
838
829
na
11a
11b
11c
11d
11e
CHS 828
135
158
278
279
206
668
122
3210
50
59
1245
221
1140
228
1130
2055
326
32
27
780
650
na
3138
25
2366
2315
2228
2067
2130
1245
1584
15
18
a) NUGC, gastric cancer; DLD1, colon cancer; HA22T, liver cancer; HEPG2, liver cancer; HONE1, nasopharyngeal carcinoma; HR, gastric cancer; MCF, breast cancer;
WI38, normal fibroblast cells. b) The sample concentration produces a 50% reduction in cell growth.
were determined on a Shimadzu IR 435 spectrophotometer D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 29.8
1H- and 13C-NMR spectra were recorded on a Mercury-300BB (CH3), 64.2 (4H-pyranC-4), 116.9 (CN), 119.0, 120.5, 120.9,
(300MHz) (Cairo University) in DMSO-d6 as solvent using 121.3, 121.7, 122.6, 124.0, 125.3, 130.2, 133.3, 136.9, 140.0
TMS [Si(CH3)4] as internal standard and chemical shifts are (2C6H5, 4H-pyranC), 164.3 (CO). Anal. Calcd for C20H17N3O2
expressed as δ ppm. Elemental analyses were obtained from (331.37): C, 72.49; H, 5.17; N, 12.68. Found: C, 72.19; H, 4.09;
the Microanalytical Data Center at Cairo University, Egypt N, 11.39.
and were performed on Vario El III Elemental CHNS ana-
lyzer.
General Procedure for the Synthesis of 4-Aryl-4H-
6-Amino-4-(4-chlorophenyl)-5-cyano-2-methyl-4H-pyran-3-
carboxylic Acid Phenylamide (4b)
White crystals, yield 65%; mp 214°C; IR (KBr) cm−1: 3453,
pyran-3-carboxylic Acid Arylamide (4a–g) To a solution 3325 (NH, NH2), 3208 (CH-aromatic), 2911 (CH3), 2180 (CN),
1
of either acetoacetanilide (1.77g, 1.0mmol), or 2-methylace- 1707 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.92 (s, 3H,
toacetanilide (1.91g, 1.0mmol), in 1,4-dioxane (40mL), either CH3), 3.94 (s, 2H, D2O exchangeable, NH2), 5.80 (s, 1H, CH,
of benzaldehyde (1g, 1.0mmol), 4-chlorobenzaldehyde (1.4g, 4H-pyran), 7.18–7.71 (m, 9H, C6H5, C6H4), 7.82 (s, 1H, D2O ex-
1.0mmol), 3-nitrobenzaldehyde (1.5g, 1.0mmol), or furfural changeable, NH). 13C-NMR (75MHz, DMSO-d6): 30.0 (CH3),
(1g, 1.0mmol) in absolute ethanol (15mL) was added. Malo- 64.5 (4H-pyranC-4), 117.2 (CN), 119.3, 120.8, 122.3, 123.8,
nonitrile (0.66g, 1.0mmol) was added to the previous mixture 124.7, 124.9, 125.8, 127.3, 130.8, 131.8, 134.7, 140.3 (C6H5,
followed by few drops of Et3N. The whole reaction mixture C6H4, 4H-pyranC), 163.8 (CO). Anal. Calcd for C20H16ClN3O2
was heated under reflux for 3h then was allowed to cool and (365.81): C, 65.67; H, 4.41; N, 11.49. Found: C, 66.72; H, 5.02;
poured into ice cold water with stirring. The separated solid N, 11.83.
was filtered and washed with water. The crude product was
purified by recrystallization from ethanol.
6-Amino-5-cyano-2-methyl-4-(3-nitrophenyl)-4H-pyran-3-
carboxylic Acid Phenylamide (4c)
6-Amino-5-cyano-2-methyl-4-phenyl-4H-pyran-3-carboxylic
Acid Phenylamide (4a)
Yellowish brown crystals, yield 85%; mp 205°C; IR (KBr)
cm−1: 3429, 3321 (NH, NH2), 3076 (CH-aromatic), 2924 (CH3),
Pale yellow crystals, yield 70%; mp 202°C; IR (KBr) cm−1: 2189 (CN), 1702 (C=O). H-NMR (300MHz, DMSO-d6) δ:
3437, 3340 (NH, NH2), 3214 (CH-aromatic), 2936 (CH3), 2210 2.03 (s, 3H, CH3), 4.14 (s, 2H, D2O exchangeable, NH2), 5.94
(CN), 1680(C=O). 1H-NMR (300MHz, DMSO-d6) δ: 1.91 (s, 1H, CH, 4H-pyran), 7.12–8.21 (m, 9H, C6H5, C6H4), 8.25
(s, 3H, CH3), 4.10 (s, 2H, D2O exchangeable, NH2), 5.91 (s, (s, 1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-
1H, CH, 4H-pyran), 7.10–7.52 (m, 10H, 2C6H5), 7.80 (s, 1H, d6): 29.7 (CH3), 64.2 (4H-pyranC-4), 116.7 (CN), 119.6, 120.7,
1

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Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
Table 2. Toxicity of Newly Synthesized Compounds Which Exhibited Optimal Cytotoxic Effect against Cancer Cell Lines
Compound
Conc. (µg/mL)
Mortalitya)
Toxicity
Harmful
LC50
Upper 95% lim
110.23
Lower 95% lim
88.25
4a
10
100
1000
10
0
5
403.26
10
1
4b
4f
Harmful
Non toxic
Harmful
82.28
979.19
20.7
—
—
—
—
100
1000
10
3
10
0
100
1000
10
0
2
5d
0
216.29
—
162.51
—
100
1000
10
6
8
5f
2
Very toxic
Very toxic
Non toxic
Non toxic
Non toxic
Non toxic
12.8
100
1000
10
8
10
1
9b
16.22
981.41
999.09
991.05
892.41
—
—
100
1000
10
6
10
0
11a
11b
11c
11d
—
—
100
1000
10
0
2
0
—
—
100
1000
10
0
5
0
—
—
100
1000
10
0
3
0
—
—
100
1000
1
4
a) Ten organisms (A. salina) tested for each concentration.
121.9, 122.4, 123.9, 128.2, 129.3, 129.9, 131.2, 132.3, 133.8, 116.9 (CN), 118.4, 119.5, 120.8, 123.2, 123.6, 126.4, 127.8,
139.7, 141.2, 142.4 (C6H5, C6H4, 4H-pyranC), 164.0 (CO). Anal. 130.6, 131.2, 132.3, 137.4, 140.9, 141.2, 144.4 (C6H5, C6H4, 4H-
Calcd for C20H16N4O4 (376.37): C, 63.82; H, 4.28; N, 14.89. pyranC), 164.5 (CO). Anal. Calcd for C21H19N3O2 (345.39): C,
Found: C, 63.21; H, 4.09; N, 14.92.
73.03; H, 5.54; N, 12.17. Found: C, 72.66; H, 5.23; N, 11.05.
6-Amino-4-(4-chlorophenyl)-5-cyano-2-methyl-4H-pyran-3-
carboxylic Acid o-Tolylamide (4f)
6-Amino-5-cyano-4-furan-2-yl-2-methyl-4H-pyran-3-
carboxylic Acid Phenylamide (4d)
Red crystals, yield 81%; mp 246°C; IR (KBr) cm−1: 3434,
White crystals, yield 75%; mp 214°C; IR (KBr) cm−1: 3437,
3284 (NH, NH2), 3141 (CH-aromatic), 2926 (CH3), 2177 (CN), 3343 (NH, NH2), 3061 (CH-aromatic), 2925 (CH3), 2179 (CN),
1
1
1723 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91 (s, 3H, 1690 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91 (s, 3H,
CH3), 4.12 (s, 2H, D2O exchangeable, NH2), 5.92 (s, 1H, CH, CH3), 2.27 (s, 3H, CH3), 4.14 (s, 2H, D2O exchangeable, NH2),
4H-pyran), 7.16–7.62 (m, 8H, C6H5, furan CH), 7.80 (s, 1H, 5.92 (s, 1H, CH, 4H-pyran), 6.94–7.81 (m, 8H, 2C6H4), 8.22
D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): (s, 1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-
30.2 (CH3), 64.8 (4H-pyranC-4), 117.0 (CN), 120.3, 121.5, d6): 29.4, 37.9 (2CH3), 64.6 (4H-pyranC-4), 117.0 (CN), 120.2,
124.8, 125.2, 129.0, 130.5, 133.5, 134.6, 137.7, 138.2, 146.6, 121.2, 124.5, 125.2, 125.9, 127.2, 129.1, 130.2, 131.7, 132.8,
152.8 (C6H5, furan, 4H-pyranC), 163.8 (CO). Anal. Calcd for 137.6, 140.5, 41.3, 143.8 (2C6H4, 4H-pyranC), 164.8 (CO).
C18H15N3O3 (321.33): C, 67.28; H, 4.71; N, 13.08. Found: C, Anal. Calcd for C21H18ClN3O2 (379.84): C, 66.40; H, 4.78; N,
67.66; H, 3.99; N, 12.88.
11.06. Found: C, 66.72; H, 5.39; N, 11.33.
6-Amino-5-cyano-2-methyl-4-phenyl-4H-pyran-3-
carboxylic Acid o-Tolylamide (4e)
6-Amino-5-cyano-2-methyl-4-(3-nitrophenyl)-4H-pyran-3-
carboxylic Acid o-Tolylamide (4g)
White crystals, yield 85%; mp 198°C; IR (KBr) cm−1:
Pale yellow crystals, yield 70%; mp 213°C; IR (KBr) cm−1:
3438, 3344 (NH, NH2), 3068 (CH-aromatic), 2954 (CH3), 2182 3437, 3346 (NH, NH2), 3070 (CH-aromatic), 2941 (CH3), 2182
1
1
(CN), 1709 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.89 (CN), 1706 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91 (s,
(s, 3H, CH3), 2.17 (s, 3H, CH3), 4.17 (s, 2H, D2O exchange- 3H, CH3), 2.17 (s, 3H, CH3), 4.30 (s, 2H, D2O exchangeable,
able, NH2), 5.94 (s, 1H, CH, 4H-pyran), 6.94–7.47 (m, 9H, NH2), 6.00 (s, 1H, CH, 4H-pyran), 6.98–8.23 (m, 8H, 2C6H4),
C6H5, C6H4), 8.00 (s, 1H, D2O exchangeable, NH). 13C-NMR 8.30 (s, 1H, D2O exchangeable, NH). 13C-NMR (75MHz,
(75MHz, DMSO-d6): 30.3, 38.3 (2CH3), 64.8 (4H-pyranC-4), DMSO-d6): 28.4, 38.6 (2CH3), 64.5 (4H-pyranC-4), 116.7

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
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(CN), 118.8, 119.8, 121.7, 122.9, 123.7, 125.9, 126.3, 130.9, (CN), 1680 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91
131.2, 131.8, 132.3, 138.7, 139.2, 140.7, 142.3, 144.5 (2C6H4, (s, 3H, CH3), 2.21 (s, 3H, CH3), 3.31 (s, 2H, D2O exchange-
4H-pyranC), 164.2 (CO). Anal. Calcd for C21H18N4O4 (390.39): able, NH2), 4.99 (s, 1H, CH, 4H-pyridine), 6.94–7.63 (m, 8H,
C, 64.61; H, 4.65; N, 14.35. Found: C, 63.92; H, 4.75; N, 13.92. 2C6H4), 8.22 (s, 1H, D2O exchangeable, NH), 9.70 (s, 1H,
General Procedure for the Synthesis of 4-Aryl-1,4- D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6):
dihydro-pyridine-3-carboxylic Acid Arylamide (5a–f) To 28.4 (CH3), 64.6 (1,4-dihydropyridineC-4), 116.4 (CN), 119.6,
a solution of either acetoacetanilide (1.77g, 1.0mmol) or 120.8, 121.9, 122.8, 124.6, 125.8, 126.3, 131.5, 132.6, 132.9,
2-methylacetoacetanilide (1.91g, 1.0mmol) in absolute etha- 136.8, 138.2, 141.9, 142.5 (2C6H4, 1,4-dihydropyridineC),
nol (15mL), either of benzaldehyde (1g, 1.0mmol), 4-chlo- 164.6 (CO). Anal. Calcd for C21H19ClN4O (378.85): C, 66.58;
robenzaldehyde (1.4g, 1.0mmol), 3-nitrobenzaldehyde (1.5g, H, 5.05; N, 14.79. Found: C, 67.38; H, 6.01; N, 13.72.
1.0mmol) or furfural (1g, 1.0mmol) was added. Ammonium
6-Amino-5-cyano-2-methyl-4-(3-nitrophenyl)-1,4-dihydro-
acetate (0.77g, 1.0mmol) and malononitrile (0.66g, 1.0mmol) pyridine-3-carboxylic Acid o-Tolylamide (5e)
were added and the mixture heated under reflux for 2h. The
Yellow crystals, yield 65%; mp 229°C; IR (KBr) cm−1:
reaction mixture was allowed to cool and poured into ice 3438, 3362 (NH, NH2), 3052 (CH-aromatic), 2997 (CH3), 2202
1
cold water with stirring. The separated solid was filtered and (CN), 1705 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91
washed with water. The crude product was purified by recrys- (s, 3H, CH3), 2.18 (s, 3H, CH3), 3.31 (s, 2H, D2O exchange-
tallization from ethanol.
able, NH2), 5.00 (s, 1H, CH, 4H-pyridine), 6.98–8.20 (m,
6-Amino-5-cyano-2-methyl-4-phenyl-1,4-dihydro- 8H, 2C6H4), 8.30 (s, 1H, D2O exchangeable, NH), 9.30 (s,
pyridine-3-carboxylic Acid Phenylamide (5a)
1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6):
Yellow crystals, yield 65%; mp 302°C; IR (KBr) cm−1: 28.4 (CH3), 64.6 (1,4-dihydropyridineC-4), 116.5 (CN), 119.3,
3339, 3246 (NH, NH2), 3061 (CH-aromatic), 2921 (CH3), 2207 120.8, 122.6, 122.8, 123.0, 124.9, 126.5, 134.9, 135.4, 136.8,
1
(CN), 1700 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.91 (s, 137.5, 137.9, 138.7, 141.8, 142.7, 143.8, (2C6H4, 1,4-dihydro-
3H, CH3), 3.54 (s, 2H, D2O exchangeable, NH2), 4.99 (s, 1H, pyridineC), 164.7 (CO). Anal. Calcd for C21H19N5O3 (389.41):
CH, 4H-pyridine), 7.24–7.61 (m, 10H, 2C6H5), 8.69 (s, 1H, C, 64.77; H, 4.92; N, 17.98. Found: C, 64.07; H, 3.86; N, 16.81.
D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 28.6
6-Amino-5-cyano-4-furan-2-yl-2-methyl-1,4-dihydro-
(CH3), 64.7 (1,4-dihydropyridineC-4), 116.5 (CN), 118.5, 120.3, pyridine-3-carboxylic Acid o-Tolylamide (5f)
120.9, 123.4, 124.9, 125.2, 126.8, 130.4, 131.7, 132.3, 139.4,
Reddish brown crystals, yield 77%; mp 272°C; IR (KBr)
141.9 (2C6H5, 1,4-dihydropyridineC), 164.8 (CO). Anal. Calcd cm−1: 3438, 3350 (NH, NH2), 3258 (CH-aromatic), 2923
1
for C20H18N4O (330.38): C, 72.71; H, 5.49; N, 16.96. Found: C, (CH3), 2216 (CN), 1710 (C=O). H-NMR (300MHz, DMSO-
70.09; H, 5.28; N, 16.96.
d6) δ: 1.91 (s, 3H, CH3), 2.22 (s, 3H, CH3), 3.31 (s, 2H, D2O
6-Amino-4-(4-chlorophenyl)-5-cyano-2-methyl-1,4-dihydro- exchangeable, NH2), 4.01 (s, 1H, CH, 4H-pyridine), 6.70–7.38
pyridine-3-carboxylic Acid Phenylamide (5b)
(m, 7H, C6H4, furan CH), 7.98 (s, 1H, D2O exchangeable,
Yellow crystals, yield 68%; mp 343°C; IR (KBr) cm−1: NH), 9.63 (s, 1H, D2O exchangeable, NH). 13C-NMR (75MHz,
3325, 3287 (NH, NH2), 3062 (CH-aromatic), 2920 (CH3), 2210 DMSO-d6): 28.5 (CH3), 63.9 (1,4-dihydropyridineC-4),
1
(CN), 1688 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.90 (s, 116.4 (CN), 119.4, 120.8, 122.6, 123.9, 124.6, 124.8, 125.2,
3H, CH3), 3.54 (s, 2H, D2O exchangeable, NH2), 5.00 (s, 1H, 132.3, 133.1, 134.8, 136.9, 138.9, 140.9, 141.2, 143.7 (C6H4,
CH, 4H-pyridine), 6.94–7.47 (m, 9H, C6H5, C6H4), 8.63 (s, furan, 1,4-dihydropyridineC), 164.4 (CO). Anal. Calcd for
1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): C19H18N4O2 (334.37): C, 68.25; H, 5.43; N, 16.76. Found: C,
28.9 (CH3), 64.3 (1,4-dihydropyridineC-4), 116.8 (CN), 118.8, 67.94; H, 5.04; N, 15.82.
120.6, 122.3, 123.6, 125.0, 125.2, 126.8, 130.8, 132.1, 132.3,
General Procedure for the Synthesis of 4-Aryl-5-aryl-
138.7, 142.2 (C6H5, C6H4, 1,4-dihydropyridineC), 164.4 (CO). carbamoyl-4H-pyran-3-carboxylic Acid Ethyl Ester (7a–d)
Anal. Calcd for C20H17ClN4O (364.83): C, 65.84; H, 4.70; N, To a solution of either acetoacetanilide (1.77g, 1.0mmol) or
15.36. Found: C, 65.21; H, 5.93; N, 14.11. 2-methylacetoacetanilide (1.91g, 1.0mmol) and either benzal-
6-Amino-5-cyano-2-methyl-4-phenyl-1,4-dihydro- dehyde (1g, 1.0mmol), 4-chlorobenzaldehyde (1.4g, 1.0mmol)
pyridine-3-carboxylic Acid o-Tolylamide (5c)
or furfural (1g, 1.0mmol) in absolute ethanol (15mL) contain-
Yellow crystals, yield 72%; mp 344°C; IR (KBr) cm−1: ing Et3N (0.50mL), ethyl cyanoacetate (1.2g, 1.0mmol) was
3455, 3329 (NH, NH2), 3063 (CH-aromatic), 2978 (CH3), 2200 added. The whole reaction mixture was heated under reflux
1
(CN), 1681 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.92 for 2h then allowed to cool and poured into ice cold water
(s, 3H, CH3), 2.21 (s, 3H, CH3), 3.31 (s, 2H, D2O exchange- with stirring. The separated solid was filtered and washed
able, NH2), 4.99 (s, 1H, CH, 4H-pyridine), 6.90–7.49 (m, 9H, with water. The crude product was purified by recrystalliza-
C6H5, C6H4), 7.59 (s, 1H, D2O exchangeable, NH), 8.39 (s, tion from ethanol.
1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6):
2-Amino-6-methyl-4-phenyl-5-phenylcarbamoyl-4H-
28.6 (CH3), 64.8 (1,4-dihydropyridineC-4), 116.6 (CN), 119.6, pyran-3-carboxylic Acid Ethyl Ester (7a)
120.8, 122.6, 122.9, 124.6, 125.8, 128.3, 129.4, 132.7, 135.2,
White crystals, yield 60%; mp 191°C; IR (KBr) cm−1:
137.4, 138.4, 142.8, 143.9 (C6H5, C6H4, 1,4-dihydropyridineC), 3332, 3236 (NH, NH2), 3058 (CH-aromatic), 2984 (CH3),
164.2 (CO). Anal. Calcd for C21H20N4O (344.41): C, 73.23; H, 1763, 1673 (C=O). 1H-NMR (300MHz, DMSO-d6) δ: 1.31
5.85; N, 16.27. Found: C, 73.83; H, 4.99; N, 14.93.
(t, J=7.2, 3H, CH2CH3), 2.20 (s, 3H, CH3), 3.54 (s, 2H, D2O
6-Amino-4-(4-chlorophenyl)-5-cyano-2-methyl-1,4-dihydro- exchangeable, NH2), 4.32 (q, J=7.2Hz, 2H, CH2CH3), 4.80
pyridine-3-carboxylic Acid o-Tolylamide (5d)
(s, 1H, CH, 4H-pyran), 7.02–8.06 (m, 10H, 2C6H5), 8.40 (s,
Orange crystals, yield 75%; mp 378°C; IR (KBr) cm−1: 1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6):
3446, 3374 (NH, NH2), 3062 (CH-aromatic), 2977 (CH3), 2200 16.2 (OCH2CH3), 28.8 (CH3), 56.3 (OCH2CH3), 64.4 (4H-py-

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ranC-4), 119.8, 121.6, 122.6, 124.0, 124.8, 125.3, 127.4, 131.8, (s, 3H, CH3), 2.20 (s, 3H, CH3), 4.27 (s, 2H, D2O exchange-
133.8, 133.9, 134.7, 139.8 (2C6H5, 4H-pyranC), 164.8, 166.3 able, NH2), 5.80 (s, 1H, CH, 4H-pyridine), 6.94–7.46 (m, 15H,
(2CO). Anal. Calcd for C22H22N2O4 (378.42): C, 69.83; H, 5.86; 2C6H5, C6H4 and NH). 13C-NMR (75MHz, DMSO-d6): 28.1
N, 7.40. Found: C, 71.69; H, 5.39; N, 8.52.
(CH3), 32.8 (CH3), 64.5 (1,4-dihydropyridineC-4), 116.5 (CN),
2-Amino-4-furan-2-yl-6-methyl-5-phenylcarbamoyl-4H- 120.9, 121.5, 122.5, 123.2, 125.8, 126.9, 127.4, 131.8, 132.6,
pyran-3-carboxylic Acid Ethyl Ester (7b)
135.3, 136.0, 136.2, 137.0, 138.0, 138.4, 139.2, 142.6 (2C6H5,
Red crystals, yield 62%; mp 220°C; IR (KBr) cm−1: 3426, C6H4, 1,4-dihydropyridineC), 166.6 (CO). Anal. Calcd for
3282 (NH, NH2), 3064 (CH-aromatic), 2971 (CH3), 1725, 1661 C27H24N4O (420.51): C, 77.12; H, 5.75; N, 13.32. Found: C,
1
(C=O). H-NMR (300MHz, DMSO-d6) δ: 1.31 (t, J=7.2Hz, 76.44; H, 5.41; N, 13.52.
3H, CH2CH3), 1.99 (s, 3H, CH3), 3.89 (s, 2H, D2O exchange-
6-Amino-1-(4-chlorophenyl)-5-cyano-2-methyl-4-phenyl-1,4-
able, NH2), 4.27 (q, J=7.2Hz, 2H, CH2CH3), 5.10 (s, 1H, CH, dihydro-pyridine-3-carboxylic Acid o-Tolylamide (9b)
4H-pyran), 7.01–7.54 (m, 8H, C6H5, furan CH), 8.40 (s, 1H,
Yellow crystals, yield 58%; mp 183°C; IR (KBr) cm−1:
D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 3452, 3343 (NH, NH2), 3058 (CH-aromatic), 2946 (CH3), 2179
1
16.2 (OCH2CH3), 28.6 (CH3), 56.4 (OCH2CH3), 64.6 (4H-py- (CN), 1714 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.87
ranC-4), 120.7, 121.5, 122.3, 125.2, 126.8, 128.6, 129.3, 129.5, (s, 3H, CH3), 2.17 (s, 3H, CH3), 4.35 (s, 2H, D2O exchange-
138.9, 147.2, 148.8, 154.4 (C6H5, furan, 4H-pyranC), 164.9, able, NH2), 5.79 (s, 1H, CH, 4H-pyridine), 6.94–7.43 (m, 13H,
167.7 (2CO). Anal. Calcd for C20H20N2O5 (368.38): C, 65.21; H, 2C6H4, C6H5), 7.92 (s, 1H, D2O exchangeable, NH). 13C-NMR
5.47; N, 7.60. Found: C, 67.31; H, 5.09; N, 7.19. (75MHz, DMSO-d6): 28.6 (CH3), 32.4 (CH3), 64.9 (1,4-dihy-
2-Amino-4-(4-chlorophenyl)-6-methyl-5-o-tolylcarbamoyl- dropyridineC-4), 116.9 (CN), 121.2, 121.5, 122.8, 124.6, 124.9,
4H-pyran-3-carboxylic Acid Ethyl Ester (7c)
127.2, 127.4, 130.3, 132.8, 134.1, 135.2, 136.4, 137.0, 138.2,
White crystals, yield 58%; mp 254°C; IR (KBr) cm−1: 3470, 38.7, 139.5, 140.9, 141.5 (C6H5, 2C6H4, 1,4-dihydropyridineC),
3292 (NH, NH2), 3063 (CH-aromatic), 2977 (CH3), 1723, 1658 164.8, 166.6 (2CO). Anal. Calcd for C27H23ClN4O (454.95): C,
1
(C=O). H-NMR (300MHz, DMSO-d6) δ: 1.12 (t, J=7.2Hz, 71.28; H, 5.10; N, 12.31. Found: C, 71.73; H, 4.90; N, 12.04.
3H, CH2CH3), 1.85 (s, 3H, CH3), 2.09 (s, 3H, CH3), 3.52 (s,
6-Amino-5-cyano-2-methyl-4-phenyl-1-p-tolyl-1,4-dihydro-
2H, D2O exchangeable, NH2), 4.05 (q, J=7.2Hz, 2H, CH2CH3), pyridine-3-carboxylic Acid o-Tolylamide (9c)
4.70 (s, 1H, CH, 4H-pyran), 6.99–7.48 (m, 8H, 2C6H4), 9.10 (s,
Pale yellow crystals, yield 63%; mp 168°C; IR (KBr) cm−1:
1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 3453, 3343 (NH, NH2), 3059 (CH-aromatic), 2947 (CH3), 2181
1
16.4 (OCH2CH3), 28.5 (CH3), 56.6 (OCH2CH3), 64.8 (4H-py- (CN), 1713 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.87
ranC-4), 120.7, 121.8, 122.5, 123.8, 124.1, 126.9, 128.6, 131.6, (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.34 (s, 3H, CH3), 4.33 (s,
132.6, 133.9, 134.9, 136.4, 138.4, 141.8 (2C6H4, 4H-pyranC), 2H, D2O exchangeable, NH2), 5.79 (s, 1H, CH, 4H-pyridine),
164.8, 166.3 (2CO). Anal. Calcd for C23H23ClN2O4 (426.89): C, 6.94–7.47 (m, 13H, 2C6H4, C6H5), 7.93 (s, 1H, D2O exchange-
64.71; H, 5.43; N, 6.56. Found: C, 66.40; H, 6.73; N, 6.05.
2-Amino-4-furan-2-yl-6-methyl-5-o-tolylcarbamoyl-4H- (CH3), 32.4 (CH3), 64.9 (1,4-dihydropyridineC-4), 116.9 (CN),
able, NH).13C-NMR (75MHz, DMSO-d6): 28.6 (CH3), 29.5
pyran-3-carboxylic Acid Ethyl Ester (7d)
121.2, 121.5, 122.8, 124.6, 124.9, 127.2, 127.4, 130.3, 132.8,
White crystals, yield 60%; mp 232°C; IR (KBr) cm−1: 134.1, 135.0, 135.8, 136.4, 137.1, 137.4, 138.0, 141.5, 142.8,
3416, 3274 (NH, NH2), 3052 (CH-aromatic), 2973 (CH3), (C6H5, 2C6H4, 1,4-dihydropyridineC), 166.6 (CO). Anal. Calcd
1
1723, 1661 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.38 for C28H26N4O (434.53): C, 77.39; H, 6.03; N, 12.89. Found: C,
(t, J=7.2Hz, 3H, CH2CH3), 2.00 (s, 3H, CH3), 2.08 (s, 3H, 77.59; H, 5.76; N, 12.53.
CH3), 3.89 (s, 2H, D2O exchangeable, NH2), 4.29 (q, J=7.2Hz,
General Procedure for the Synthesis of 2-(Aryl-
2H, CH2CH3), 5.10 (s, 1H, CH, 4H-pyran), 7.04–7.54 (m, hydrazonomethyl)-4H-pyran-3-carboxylic Acid Arylamide
7H, C6H4, furan CH), 7.69 (s, 1H, D2O exchangeable, NH). (11a–e) To a cold solution (0–5°C) of either 4a (3.31g,
13C-NMR (75MHz, DMSO-d6): 16.2 (OCH2CH3), 28.6 (CH3), 1.0mmol) or 4e (3.45g, 1.0mmol) in absolute ethanol (20mL)
56.4 (OCH2CH3), 64.6 (4H-pyranC-4), 120.7, 121.5, 122.3, containing sodium hydroxide (1.00g), an equimolar amount of
125.2, 126.8, 128.6, 129.3, 129.5, 138.9, 147.2, 149.3, 154.8 either diazotized aniline, diazotized p-chloroaniline or diazo-
(C6H4, furan, 4H-pyranC), 164.3, 165.6 (2CO). Anal. Calcd tized p-toluidene [prepared by the addition of sodium nitrite
for C21H22N2O5 (382.41): C, 65.96; H, 5.80; N, 7.33. Found: C, solution (0.70g, 1.0mmol in 5mL water) to a cold solution
65.84; H, 4.55; N, 7.31.
(0–5°C) of appropriate aromatic amine (1.0mmol) in concen-
General Procedure for the Synthesis of 1,4-Diaryl-1,4- trated hydrochloric acid (12mL) with continuous stirring] was
dihydro-pyridine-3-carboxylic Acid o-Tolylamide (9a–c) gradually added while stirring. The solid products formed
To a mixture of equimolar amounts of aniline (1g, 1.0mmol), upon cooling in an ice-bath were collected by filtration,
4-chloroaniline (1.3g, 1.0mmol), or p-toluidene (1.1g, 1.0mmol) washed with water and crystallized from 1,4-dioxane.
in N,N-dimethylformamide (DMF) (15mL), compound 4e
5-Amino-6-cyano-4-phenyl-2-(phenylhydrazonomethyl)-4H-
(3.45g, 1.0mmol) was added. The reaction mixture was heated pyran-3-carboxylic Acid Phenylamide (11a)
under reflux for 5h. The solid products formed upon pouring
Orange crystals, yield 78%; mp 180°C; IR (KBr) cm−1:
onto ice/water mixture and collected by filtration and crystal- 3439, 3342 (NH, NH2), 3033 (CH-aromatic), 2177 (CN), 1691
1
lized from ethanol.
(C=O). H-NMR (300MHz, DMSO-d6) δ: 4.10 (s, 2H, D2O
6-Amino-5-cyano-2-methyl-1,4-diphenyl-1,4-dihydro- exchangeable, NH2), 5.90 (s, 1H, CH, 4H-pyran), 6.94–7.46
pyridine-3-carboxylic Acid o-Tolylamide (9a)
(m, 16H, 3C6H5, CH=N), 10.58 (b, 1H, D2O exchange-
Yellow crystals, yield 60%; mp 184°C; IR (KBr) cm−1: able, NH), 12.36 (s, 1H, D2O exchangeable, NH). 13C-NMR
3451, 3343 (NH, NH2), 3058 (CH-aromatic), 2945 (CH3), 2179 (75MHz, DMSO-d6): 64.5 (4H-pyranC-4), 116.6 (CN), 120.8,
1
(CN), 1715 (C=O). H-NMR (300MHz, DMSO-d6) δ: 1.89 121.7, 123.6, 124.8, 125.3, 126.8, 127.8, 131.6, 133.4, 134.6,

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Chem. Pharm. Bull.
1063
135.3, 136.9, 137.9, 138.3, 139.6, 141.3 (3C6H5, 4H-pyranC),
164.8 (CO), 169.8 (C=N). Anal. Calcd for C26H21N5O2 (435.48):
C, 71.71; H, 4.86; N, 16.08. Found: C, 71.49; H, 5.03; N, 16.58.
In Vitro Cytotoxic Assay
Chemicals
Fetal bovine serum (FBS) and L-glutamine, were purchased
5-Amino-2-[(4-chloro-phenyl)hydrazonomethyl]-6-cyano-4- from Gibco Invitrogen Co. (Scotland, U.K.). RPMI-1640
phenyl-4H-pyran-3-carboxylic Acid Phenylamide (11b)
medium was purchased from Cambrex (NJ, U.S.A.). DMSO,
Red crystals, yield 79%; mp 145°C; IR (KBr) cm−1: 3450, doxorubicin, penicillin, streptomycin and sulforhodamine B
3343 (NH, NH2), 3025 (CH-aromatic), 2181 (CN), 1677 (SRB) were purchased from Sigma Chemical Co. (Saint Louis,
1
(C=O). H-NMR (300MHz, DMSO-d6) δ: 4.66 (s, 2H, D2O MO, U.S.A.).
exchangeable, NH2), 5.80 (s, 1H, CH, 4H-pyran), 6.80 (s, 1H,
CH=N), 6.94–7.46 (m, 14H, 2C6H5, C6H4), 10.58 (s, 1H, D2O
Cell Cultures
The cell cultures were obtained from the European Collec-
exchangeable, NH), 12.37 (s, 1H, D2O exchangeable, NH). tion of cell Cultures (ECACC, Salisbury, U.K.) and human
13C-NMR (75MHz, DMSO-d6): 64.2 (4H-pyranC-4), 116.8 gastric cancer (NUGC and HR), human colon cancer (DLD1),
(CN), 119.3, 120.9, 122.7, 123.5, 125.9, 127.0, 129.2, 132.8, human liver cancer (HA22T and HEPG2), human breast can-
133.8, 135.0, 135.3, 136.8, 137.3, 139.2, 139.6, 140.6 (2C6H5, cer (MCF), nasopharyngeal carcinoma (HONE1) and normal
C6H4, 4H-pyranC), 164.6 (CO), 169.9 (C=N). Anal. Calcd for fibroblast cells (WI38) were kindly provided by the National
C26H20ClN5O2 (469.92): C, 66.45; H, 4.29; N, 14.90. Found: C, Cancer Institute (NCI, Cairo, Egypt). They grow as monolayer
65.84; H, 4.51; N, 14.73.
and routinely maintained in RPMI-1640 medium supplement-
5-Amino-6-cyano-4-phenyl-2-(phenylhydrazonomethyl)-4H- ed with 5% heat inactivated FBS, 2µM glutamine and antibiot-
pyran-3-carboxylic Acid o-Tolylamide (11c)
ics (penicillin 100U/mL, streptomycin 100g/mL), at 37°C in
Yellow crystals, yield 82%, mp 95°C; IR (KBr) cm−1: a humidified atmosphere containing 5% CO2. Exponentially
3446, 3340 (NH, NH2), 3026 (CH-aromatic), 2177 (CN), 1693 growing cells were obtained by plating 1.5×105 cells/mL for
1
(C=O). H-NMR (300MHz, DMSO-d6) δ: 2.18 (s, 3H, CH3), the seven human cancer cell lines including cells derived
4.27 (s, 2H, D2O exchangeable, NH2), 5.75 (s, 1H, CH, 4H- from 0.75×104 cells/mL followed by 24h of incubation. The
pyran), 6.77 (s, 1H, CH=N), 6.94–7.45 (m, 14H, 2C6H5, C6H4), effect of the vehicle solvent (DMSO) on the growth of these
10.50 (s, 1H, D2O exchangeable, NH), 12.30 (s, 1H, D2O cell lines was evaluated in all the experiments by exposing
exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 64.7 (4H- untreated control cells to the maximum concentration (0.5%)
pyranC-4), 116.5 (CN), 119.6, 121.4, 122.2, 123.8, 124.2, 127.4, of DMSO used in each assay.
129.8, 130.2, 133.9, 135.4, 136.2, 136.8, 137.0, 138.8, 139.1,
Toxicity All toxicity tests were 96-h static renewal tests
139.8, 140.9, 141.2, (2C6H5, C6H4, 4H-pyranC), 164.6 (CO), and water quality measurements (dissolved oxygen, pH, tem-
169.9 (C=N). Anal. Calcd for C27H23N5O2 (449.50): C, 72.14; perature, salinity) were taken in the control containers each
H, 5.16; N, 15.58. Found: C, 72.63; H, 5.29; N, 11.70.
day. Tests were run in a Revcos Environmental Chamber at
5-Amino-2-[(4-chlorophenyl)hydrazonomethyl]-6-cyano-4- 25°C, 20% salinity, and a 16-h light:8-h dark cycle. Amedia
phenyl-4H-pyran-3-carboxylic Acid o-Tolylamide (11d)
change was made every 24h. Larvae used for all tests were
Orange crystals, yield 85%; mp 110°C; IR (KBr) cm−1: 3611 one to two days old and exposed in 600-mL glass beakers
(NH), 3459, 3340 (NH2), 3027 (CH-aromatic), 2166 (CN), containing 400mL of media with 10 larvae/beaker and three
1
1686 (C=O). H-NMR (300MHz, DMSO-d6) δ: 2.02 (s, 3H, replicates/concentration. Larvae were fed newly hatched Ar-
CH3), 4.68 (s, 2H, D2O exchangeable, NH2), 5.87 (s, 1H, CH, temia after daily media change. For the individual pesticide
4H-pyran), 6.77 (s, 1H, CH=N), 7.25–7.44 (m, 13H, C6H5, toxicity tests, the following concentrations were used. Larval
2C6H4), 10.56 (s, 1H, D2O exchangeable, NH), 12.36 (s, 1H, nominal atrazine concentrations were 625, 1250, 2500, 5000,
D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6): 64.6 10000mg/L and control. Larval nominal imidacloprid con-
(4H-pyranC-4), 116.8 (CN), 120.8, 120.8, 124.7, 124.9, 126.2, centrations were 100, 200, 400, 600, 800mg/L and control.
128.4, 129.3, 132.6, 133.2, 134.2, 135.8, 137.0, 137.8, 137.9, For imidacloprid, adult shrimp toxicity tests were also run to
138.5, 139.2, 140.1, 142.8, (C6H5, 2C6H4, 4H-pyranC), 164.8 complete the grass shrimp toxicity profile. Adult shrimp (ac-
(CO), 169.4 (C=N). Anal. Calcd for C27H22ClN5O2 (483.95): C, climated for two weeks before testing) were exposed in 4-L
67.01; H, 4.58; N, 14.47. Found: C, 66.83; H, 4.38; N, 14.33.
wide mouth glass jars containing 2-L of media and 10 shrimp/
5-Amino-6-cyano-4-phenyl-2-(p-tolyl-hydrazonomethyl)-4H- jar with two replicates/concentration, and were run under
pyran-3-carboxylic Acid o-Tolylamide (11e)
conditions as described above for larvae. Adults were not fed
Yellow crystals, yield 78%; mp 145°C; IR (KBr) cm−1: 3452 during the test. Adult nominalimidacloprid concentrations
and 3333 (NH, NH2), 3056 (CH-aromatic), 2181 (CN) and 1675 were 226, 328, 475.6, 689.6, 1000mg/L and control.
1
(C=O). H-NMR (300MHz, DMSO-d6) δ: 2.03 (s, 3H, CH3),
2.16 (s, 3H, CH3), 4.64 (s, 2H, D2O exchangeable, NH2), 5.79
Conflict of Interest The authors declare no conflict of
(s, 1H, CH, 4H-pyran), 6.80 (s, 1H, CH=N), 7.11–7.44 (m, 13H, interest.
C6H5, 2C6H4), 10.36 (s, 1H, D2O exchangeable, NH), 12.42 (s,
1H, D2O exchangeable, NH). 13C-NMR (75MHz, DMSO-d6):
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