Synthesis and anti-avian influenza virus (H5N1) evaluation of some novel nicotinonitriles and their N-acylic nucleosides

Article abstract of DOI:10.1002/jhet.966

Pyridine and fused pyridines derivatives are interesting compounds with diverse chemical properties and pharmacological activities. Herein, the synthesis and antiviral evaluation of some new pyridines are described.

Full text of DOI:10.1002/jhet.966

1130
Synthesis and Anti‐Avian Influenza Virus (H5N1) Evaluation of
Vol 49
Some Novel Nicotinonitriles and Their N‐Acylic Nucleosides
Aymn E. Rashad,a,b Ahmed H. Shamroukh, Maher A. El‐Hashash,d
a,c
*
Ahmed F. El‐Farargy,e Nabil M. Yousif,a Mowafia A. Salama,a
Ahmed Mostafa,f and Mahmoud El‐Shahata
aPhotochemistry Department, National Research Center, Dokki, Cairo, Egypt
bChemistry Department, Faculty of Science and Human Studies, Horimlaa, Shaqra Univerisity, KSA
cChemistry Department, Faculty of Science, Hail Univerisity, KSA
dChemistry Department, Faculty of Science, Ain Shams Univerisity, Cairo, Egypt
eChemistry Department Faculty of Science, Zagazig Univerisity, Zagazig, Egypt
fVirology Laboratory, Water Pollution Department, National Research Center, Dokki, Cairo, Egypt
*
E-mail: aymnelzeny@yahoo.com
Received February 23, 2011
DOI 10.1002/jhet.966
Published online 26 October 2012 in Wiley Online Library (wileyonlinelibrary.com).
Pyridine and fused pyridines derivatives are interesting compounds with diverse chemical properties and
pharmacological activities. Herein, the synthesis and antiviral evaluation of some new pyridines are
described.
J. Heterocyclic Chem., 49, 1130 (2012).
INTRODUCTION
acetate [22]. However, this procedure is time consuming
and gives low yield. So, we report here an easy one‐step
synthesis which gives a higher yield of 2 by a four‐
component modified Hantzch reaction catalyzed by glacial acetic
acid by heating a mixture of 4‐chloroacetophenone, 4‐benzalde-
hyde, ethyl cyanoacetate, ammonium acetate, and glacial acetic
acid (Scheme 1). The elemental analysis and spectroscopic data
are consistent with the assigned structure. IR spectrum of com-
pound 2 indicated absorption bands due to NH, CN, and C═O
Pyridines and their analogs have been well recognized and
documented in the literature as chemotherapeutic agents [1].
They are well known for their diverse therapeutic properties
and exhibited antibacterial [2,3], anticancer [4,5], antiulcer
[6], diuretics [7], anticonvulsant [8], antihypertensive [9],
antitumor [10], antifungal [11,12], anti‐AIDS [13], and
antiviral [14] properties. On the other hand, the viral resis-
tance to the used drugs is a major health problem and creates
a pressing need for novel antiviral agents. Although, signifi-
cant research is continuing in the area of antiviral drug
discovery and there is still an urgent need to discover and
develop new antiviral agents due to the increasing viral
resistance to some commonly used drugs [15,16]. The
manifold of diverse pharmacological activities shown by
pyridine and its analogs and our interest in this area [17–20]
led us to synthesize some new pyridin‐2(1H)‐ones/thione,
tetrazolo‐, triazolopyridines, and their N‐acyclic nucleoside
derivatives hoping to exhibit promising antiviral applications.
1
groups and its H NMR spectrum showed signals at δ 12.93
for NH, D2O exchangeable. Also, its 13C NMR spectrum
revealed CN and C═O at δ 116.14 ppm and 161.97 ppm, re-
spectively. Mass spectrum showed the molecular ion peak M+
at m/z 340 as the base peak as well as the presence of isotopic
pattern of two chlorine atoms.
Compound 2 was utilized as a key starting material in the
synthesis of many novel heterocyclic compounds (Scheme 2).
Thus, compound 2 was treated with phosphorus penta sulfide
in xylene to afford the corresponding pyridinethione derivative
3. The spectral data of compound 3 was in agreement with the
proposed structure; in particular, the 13C NMR spectrum which
revealed C═S at δ 175.03 ppm (c.f.experimental).
When compound 2 was reacted with ethyl bromoacetate, in
the presence of anhydrous potassium carbonate, it produced the
ethyl ester derivative 4 (Scheme 2). The IR spectrum of the latter
compound showed absorption bands at 2220 and 1742 cm−1 for
CN and C═O ester, respectively. Its 1H NMR spectrum showed
RESULTS AND DISCUSSION
In this work, enones are excellent starting materials
for the synthesis of 4,6‐bis(4‐chlorophenyl)‐2‐oxo‐1,2‐
dihydropyridine‐3‐carbinitrile (2) via the reaction of enone 1
[21] with ethyl cyanoacetate in the presence of ammonium
© 2012 HeteroCorporation

September 2012
Synthesis and Anti‐Avian Influenza Virus (H5N1) Evaluation of Some Novel
Nicotinonitriles and Their N‐Acylic Nucleosides
1131
Scheme 1
Scheme 2
signals at δ 1.16 (t, 3H, CH3CH2), 4.15 (q, 2H, CH3CH2), and
5.16 (s, 2H, OCH2). Moreover, the absence of C═O signal in
IR and 13C NMR spectra of compound 4 revealed that the site
of attack was on the O‐ and not N‐atom [18].
When compound 4 was treated with hydrazine hydrate, in
ethanol, the hydrazine derivative 6 was obtained and not the
1
hydrazide derivative 5. The IR and H NMR spectra for
compound 6 showed absorption bands for NH2+ NH, CN,
and the absence of C═O signals (c.f.experimental).
Due to the synthesis of acyclovir as one of the most
potent antiviral drugs by Schaffer et al. [23], many attempts
have been made by nucleoside chemists to prepare a number
of related compounds with various side chains and glycons
[23,24]. Thus, when the sodium salt of compound 2
(generated in situ) was treated with 2‐chloroethyl methyl ether,
chloroacetaldehyde dimethyl acetal, 2‐chloroethanol, or 2‐(2‐
chloroethoxy)ethanol, it afforded the corresponding N‐acyclic
nucleosides derivatives 7–10, respectively (Scheme 2).
The structure of the aforementioned acyclic nucleosides
was confirmed with spectral data and the NMR spectra
revealed methoxyethyl, dimethoxyethyl, hydroxyethyl,
and hydroxylethoxyethyl signals. In addition, the IR and
13C NMR spectra revealed that the site of attack was on
the N‐ and not O‐atom. This is due to the fact that the
nitrogen atom behaves as a nucleophile which attacks an
electrophilic carbon of an alkyl halide [17].
[1,2,4]triazolo[4,3‐a]pyridine‐8‐carbonitriles 13 and 14, re-
spectively (Scheme 3). The structure of the new products was
established according to their elemental and spectroscopic
data. The IR spectrum of 13 and 14 revealed the absence of
NH2, NH groups, and its 1H NMR spectrum showed signals
for CH3 and triazol‐H3.
Nitrozation of compound 6 gave the corresponding
5,7‐bis(4‐chlorophenyl)‐tetrazolo[1,5‐a]pyridine‐8‐carbonitrile
(11). The IR and 1H NMR spectra of 11 revealed the absence
of NH2 and NH groups and its mass spectrum showed
the molecular ion peak M+ at m/z 365 as the base peak (c.f.
experimental).
Condensation of compound 6 with acetyl acetone, gave
4,6‐bis (4‐chlorophenyl)‐2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)‐
nicotinonitrile (15). The IR spectrum of 15 showed bands
1
at 2210 cm−1 of CN and its H NMR spectrum showed
Synthesis of 2‐amino‐4,6‐bis(4‐chlorophenyl)nicotinonitrile
12 was achieved when compound 11 was heated with zinc dust
in glacial acetic acid (c.f. experimental). The IR spectrum of 12
showed bands for NH2 and CN groups and its 1H NMR spec-
trum showed signals at 7.08 (s, 2H, NH2, D2O exchangeable).
On the other hand, when compound 6 was refluxed with
acetic anhydride or trimethyl orthoformate, it afforded
signals at δ 2.3, 2.5 for two CH3 and at 6.1 (s, 1H, pyrazole‐H).
Also, its mass spectrum showed the molecular ion peak M+
at m/z 418 as the base peak.
Moreover, refluxing of compound 6 in dry benzene afforded
4,6‐bis(4‐chlorophenyl)‐1H‐pyrazolo[3,4‐b]pyridine‐3‐amine
(16); which was also obtained by heating compound 12
with hydroxyl amine in the presence of sodium acetate in
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

1132
A. E. Rashad, A. H. Shamroukh, M. A. El-Hashash, A. F. El-Farargy, N. M. Yousif,
M. A. Salama, A. Mostafa, and M. El-Shahat
Vol 49
1
a high yield [25]. The IR and H NMR spectra of com-
pound 16 showed bands characterized for NH and NH2.
On the other hand, the 4,6‐bis(4‐chlorophenyl)‐2‐(2,3,4,5,6‐
pentahydroxyhexylidene) hydrazinyl)‐nicotinonitriles (17)
was prepared by heating compound 6 with D‐glucose
(Scheme 3). The formed product revealed absorption
frequencies due to OH, NH, CN, and C═N in IR spectrum
and its 1H NMR spectrum showed the presence of the sugar
protons, NH, and azomethine (CH═N) [26]. Acetylation of
the hydrazone derivative 17 by heating in acetic anhydride
gave its corresponding O‐acetylated sugar hydrazone
derivative 18. The IR spectrum of the latter compound
revealed the absence of hydroxyl groups and showed
absorption bands due to NH and C═O groups, whereas its
1H NMR spectrum showed the presence of OAc and NH
signals (c.f.experimental).
Antiviral bioassays. Antiviral bioassays were carried out to
test compounds 2–4, 6–18. The concentrations of the tested
compounds which exhibited 50% cytotoxicity (LD50) and
the 50% effective antiviral concentration (EC50) were
determined in addition to the cytotoxicity (TC) and the
therapeutic index (TI) (Table 1). It was obvious that, at
different concentrations (5, 10, 20, and 40 μg), compounds
2, 3, 6, 10, 12, and 16 showed higher therapeutic indices
than the other tested compounds and the results were
compared with the anti‐influenza drug zanamivir.
CONCLUSIONS
Structural activity correlations of the obtained results
indicated that, replacement of the C═O group in com-
pound 2 with thio (compound 3), alkoxy (compound 4), hydra-
zino group (compound 6), or substituted with acyclic sugar
chain (compounds 7–10) decreased the antiviral activity. While,
the presence of pyrazolyl ring (compound 15) and side chain of
acyclic sugar hydrazone (compounds 17 and 18) increased the
H5N1 activity but not as much as that of compound 2 (Fig. 1).
EXPERIMENTAL
All melting points are uncorrected and measured using Electro‐
Thermal IA 9100 apparatus (Shimadzu, Japan). Infrared spectra
were recorded as potassium bromide pellets on a Perkin‐Elmer
1650 spectrophotometer, National Research Center, Cairo, Egypt.
1H NMR and 13C NMR spectra were determined on a Jeol‐Ex‐
500 NMR spectrometer and chemical shifts were expressed as part
per million; (δ values, ppm) against TMS as internal reference,
National Research Center, Cairo, Egypt. Mass spectra were
recorded on EI + Q1 MSLMR UPLR, National Research Center,
Cairo, Egypt. Microanalyses were operated using Mario Elmentar
apparatus, Organic Microanalysis Unit, National Research Center,
Cairo, Egypt. Column Chromatography was performed on
(Merck) Silica gel 60 (particle size 0.06–0.20 mm).
4,6‐Bis(4‐chlorophenyl)‐2‐oxo‐1,2‐dihydropyridine‐3‐
carbonitrile (2). Method A. A mixture of compound 1 [21] (0.01
mole), ethyl cyanoacetate (0.01 mole), and ammonium acetate
(0.08 mole) in ethanol (40 mL) was refluxed for 10 h. After
cooling, the precipitate was filtered off, dried, and recrystallized
from glacial acetic acid to give 2.
Scheme 3
Method B. A mixture of 4‐chloroacetophenone (0.01 mole),
4‐chlorobenzaldehyde (0.01 mole), ethyl cyanoacetate (0.01
mole), and ammonium acetate (0.08 mole) in glacial acetic acid
(40 mL) was refluxed for 3 h. After cooling, the precipitate was
Table 1
Antiviral activity against H5N1 virus of the prepared compounds by
determination of both EC50 and LD50.
TC50
(μg/mL)
Avian influenza virus
(H5N1) IC50 (μg/mL)
Therapeutic
index (TI)
Compound
2
3
4
6
7
8
9
10
11
12
13
14
15
16
17
18
215.25
168.9
138.18
133.38
151.15
176.12
13.30
111.1
133.3
111.19
133
169.19
174.59
155.19
158
141.3
76
152.66
178.26
153.54
99.54
160.8
238
134.44
113.4
156.94
84.24
137.16
176.24
185.74
156.76
164.62
153.6
5
1.41
0.948
0.9
1.34
0.94
0.74
0.099
0.98
0.85
1.32
0.97
0.96
0.94
0.99
0.96
0.92
15.2
Zanamivir
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Synthesis and Anti‐Avian Influenza Virus (H5N1) Evaluation of Some Novel
Nicotinonitriles and Their N‐Acylic Nucleosides
1133
9H, 8Ar‐H + pyridine‐H5), 8.49 (s, 1H, NH, D2O exchangeable).
Ms: m/z (358, 4M+, 3%), (356, 2M+, 81%), (354, M+, 100%).
Anal. Calcd. for C18H12Cl2N4 (355.23): C, 60.86; H, 3.41; Cl,
19.96; N, 15.77; found C, 60.81; H, 3.36; Cl, 19.87; N, 15.72.
General procedure for synthesis of 7–10. To a solution of
compound 2 (0.01 mole) in dry DMF (50 mL), 50% oil‐immersed
sodium hydride (0.20 g) was added. Thereafter, the reaction mixture
was stirred at room temperature for 1 h. Then, 2‐chloroethyl methyl
ether, chloroacetaldehyde dimethylacetal, 2‐chloroethanol or 2‐(2‐
chloroethoxy)ethanol (0.02 mol) was added and the reaction
mixtures were stirred at 70°C for 3 h, 5 h, 4 h, 3 h, and 2 h,
respectively. After evaporation under reduced pressure, the residues
were purified on silica gel column using chloroform: methanol (9:1)
as an eluent to give compounds 7, 8, 9, and 10, respectively.
4,6‐Bis(4‐chlorophenyl)‐1‐(2‐methoxyethyl)‐2‐oxo‐1,2‐
dihydropyridine‐3‐carbonitrile (7). Yield 68%, mp 153–154°C. IR
(KBr) ν, cm−1: 2218 (CN), 1673 (C═O). 1H NMR spectrum
(CDCl3, δ ppm): 3.48 (s, 3H, OCH3), 3.85 (t, J = 4.6 Hz, 2H, NCH2),
4.72 (t, J = 3.8 Hz, 2H, CH2O), 7.39 (s, 1H, pyridine‐H5), 7.44–8.00
(m, 8H, Ar‐H). 13C NMR spectrum (CDCl3, δ ppm): 30.43 (NCH2),
59.49 (OCH3), 70.50 (CH2O), 115.11 (CN), 128.68–136.95 (16 Ar‐C),
164.60 (C═O). Anal. Calcd. for C21H16Cl2N2O2 (399.28): C, 63.17;
H, 4.04; Cl, 17.76; N, 7.02; found C, 63.09; H, 3.95; Cl, 17.70; N, 6.95.
4,6‐Bis(4‐chlorophenyl)‐1‐(2,2‐dimethoxyethyl)2‐oxo‐1,2‐
dihydropyridine‐3‐carbonitrile (8). Yield 53%, mp 171–172°C. IR
(KBr) ν, cm−1: 2220 (CN), 1671 (C═O). 1H NMR spectrum (CDCl3,
δ ppm): 3.51(s, 6H, 2OCH3), 4.60 (d, J = 4.55 Hz, 2H, NCH2), 4.84
(t, J = 7.3 Hz, 1H, CH), 7.41 (s, 1H, pyridine‐H5), 7.45–8.00 (m, 8H,
Ar‐H). Anal. Calcd. for C22H18Cl2N2O3 (429.31): C, 61.55; H, 4.23;
Cl, 16.52; N, 6.53; found C, 61.49; H, 4.17; Cl, 16.46; N, 6.45.
Figure 1. Antiviral activity of compounds 2–4, 6–18 at different concentrations.
filtered off, dried, and recrystallized from glacial acetic acid to
give 2. Yield (54% from method A and 91% from method B),
mp 305–306°C. IR (KBr) ν, cm−1: 3279 (NH), 2215 (CN), and
1
1629 (C═O). H NMR spectrum (DMSO‐d6, δ ppm): 6.88 (s, 1H,
pyridine‐H5), 7.57–7.90 (m, 8H, Ar‐H), 12.87 (s, 1H, NH, D2O
exchangeable). 13C NMR spectrum (DMSO‐d6, δ ppm): 116.14
(CN), 128.77–150.60 (16 Ar‐C), 161.97 (C═O). Ms: m/z (344,
4M+, 9%), (342, 2M+, 51%), (340, M+, 100%). Anal. Calcd. for
C18H10Cl2N2O (341.20): C, 63.36; H, 2.95; Cl, 20.78; N, 8.21;
found C, 63.27; H, 2.88; Cl, 20.71; N, 8.16.
4,6‐Bis(4‐chlorophenyl)‐2‐thioxo‐1,2‐dihydropyridine‐3‐
carbonitrile (3). To a solution of compound 2 (0.01 mole) in dry
xylene (50 mL), phosphorus pent sulfide (0.04 mol) was added and
the reaction mixture was heated under reflux for 2 h. Then the excess
solvent was evaporated under reduced pressure and the residue was
recrystallized from dioxane to give 3. Yield 37%, mp 237–238°C. IR
4,6‐Bis(4‐chlorophenyl)‐1‐(2‐hydroxyethyl)‐2‐oxo‐1,2‐
dihydropyridine‐3‐carbonitrile (9). Yield 59%, mp 183–184°C. IR
(KBr) ν, cm−1: 3400 (OH), 2211 (CN), 1678 (C═O). 1H NMR
spectrum (DMSO‐d6, δ ppm): 3.79 (t, J = 4.50 Hz, 2H, NCH2),
4.58 (t, J = 4.55 Hz, 2H, CH2O), 5.43 (bs, 1H, OH, D2O
exchangeable), 7.50–8.22 (m, 9H, 8Ar‐H+ pyridine‐H5). Anal.
Calcd. for C20H14Cl2N2O2 (385.25): C, 62.35; H, 3.66; Cl, 18.41;
N, 7.27; found C, 62.27; H, 3.58; Cl, 18.32; N, 7.18.
1
(KBr) ν, cm−1: 3223 (NH), 2216 (CN), 1272 (C═S). The H NMR
spectrum (DMSO‐d6, δ ppm): 7.42–8.33 (m, 10H, 8Ar‐H + pyridine‐
H5 + NH, D2O exchangeable). 13C NMR spectrum (DMSO‐d6, δ
ppm): 120.70 (CN), 128.34–159.22 (16 Ar‐C), 175.03 (C═S). Ms:
m/z (360, 4M+, 7%), (358, 2M+, 42%), (356, M+, 85%). Anal. Calcd.
for C18H10Cl2N2S (357.26): C, 60.52; H, 2.82; Cl, 19.85; N, 7.84; S,
8.97; found C, 60.44; H, 2.77; Cl, 19.79; N, 7.80; S, 8.88.
4,6‐Bis(4‐chlorophenyl)‐1‐(2‐(2‐hydroxyethoxy)ethyl)‐2‐oxo‐
1,2‐dihydropyridine‐3‐carbonitrile (10). Yield 54%, mp 191–192°C.
IR (KBr) ν, cm−1: 3340 (OH), 2216 (CN), 1670 (C═O). 1H NMR
spectrum (CDCl3, δ ppm): 2.10 (t, J = 4.7 Hz, 2H, NCH2), 2.91 (t,
J = 4.49 Hz, 2H, CH2O), 3.6 (m, 4H, OCH2CH2OH), 5.0 (bs, OH,
D2O exchangeable), 7.50–8.22 (m, 9H, 8Ar‐H+ pyridine‐H5). Anal.
Calcd. for C22H18Cl2N2O3 (429.31): C, 61.55; H, 4.23; Cl, 16.52;
N, 6.53; found C, 61.47; H, 4.19; Cl, 16.42; N, 6.45.
5,7‐Bis(4‐chlorophenyl)‐tetrazolo[1,5‐a]pyridine‐8‐carbonitrile
(11). To an ice‐cold solution of compound 6 (0.01 mole) in glacial
acetic acid (10 mL), a solution of sodium nitrite [prepared by
dissolving sodium nitrite (0.01 mole) in water (3 mL)] was
added drowsily in an ice bath. The reaction mixture was
allowed to stand overnight at room temperature and then was
poured into water. The formed solid was filtered off, washed
with water, dried, and recrystallized from ethanol to give 11.
Yield 60%, mp 157–158°C. IR (KBr) ν, cm−1: 2213 (CN). 1H NMR
spectrum (DMSO‐d6, δ ppm): 7.64–8.41 (m, 9H, 8Ar‐H+pyridine‐
H5). Ms: m/z (369, 4M+, 10%), (367, 2M+, 61%), (365, M+, 100%).
Anal. Calcd. for C18H9Cl2N5 (366.21): C, 59.04; H, 2.48; Cl, 19.36;
N, 19.12; found C, 59.00; H, 2.39; Cl, 19.28; N, 19.05.
Ethyl 2‐(4,6‐bis(chlorophenyl)‐3‐cyanopyridin‐2‐yloxy)acetate
(4). A mixture of compound 2 (0.01 mole), ethyl bromoacetate
(0.01 mole), and anhydrous potassium carbonate (0.04 mole) in
dry acetone (30 mL) was refluxed for 20 h. The excess solvent
was evaporated under reduced pressure and the solid obtained was
recrystallized from ethanol to give 4. Yield 83%, mp 187–188°C.
1
IR (KBr) ν, cm−1: 2220 (CN), 1742 (C═O). The H NMR spectrum
(DMSO‐d6, δ ppm): 1.16 (t, J = 8 Hz, 3H, CH3CH2O), 4.20 (q, J = 8
Hz, 2H, CH3CH2O), 5.16 (s, 2H, CH2), 7.58–8.20 (m, 9H, 8Ar‐H +
pyridine‐H5). 13C NMR spectrum (DMSO‐d6, δ ppm): 14.99 (CH3),
61.23 (CH2), 64.26 (OCH2CO), 115.00 (CN), 129.38–136.34 (16 Ar‐
C), 168.62 (C═O). Ms: m/z (430, 4M+, 11%), (428, 2M+, 45%), (426,
M+, 100%). Anal. Calcd. for C22H16Cl2N2O3 (427.29): C, 61.84; H,
3.77; Cl, 16.59; N, 6.56; found C, 61.79; H, 3.70; Cl, 16.53; N, 6.46.
4,6‐Bis(4‐chlorophenyl)‐2‐hydrazinylnicotinonitrile (6). A
solution of compound 4 (0.01 mole) in ethanol (50 mL) and
hydrazine hydrate (0.02 mole, 99%) was refluxed for 6 h. After
cooling, the separated solid was collected and recrystallized from
ethanol to give 6. Yield 77%, mp178–179°C. IR (KBr) ν, cm−1:
2‐Amino‐4,6‐bis(4‐chlorophenyl)‐nicotinonitrile (12). A mixture
of compound 11 (0.01 mole), zinc dust (0.01 mole), and glacial
acetic acid (10 mL) was stirred at room temperature for 2 h and
1
3398–3322 (NH2, NH), 2203 (CN). H NMR spectrum (DMSO‐
d6, δ ppm): 4.61 (bs, 2H, NH2, D2O exchangeable), 7.37–8.33 (m,
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A. E. Rashad, A. H. Shamroukh, M. A. El-Hashash, A. F. El-Farargy, N. M. Yousif,
M. A. Salama, A. Mostafa, and M. El-Shahat
Vol 49
then was heated on water bath at 80°C for 8 h. The reaction
mixture was poured into water, extracted with chloroform, and
the extract was evaporated under reduced pressure. The formed
solid was recrystallized from ethanol to give 12. Yield 40%,
Calcd. for C18H12Cl2N4 (355.23): C, 60.86; H, 3.41; Cl, 19.96;
N, 15.77; found C, 60.80; H, 3.33; Cl, 19.87; N, 15.70.
4,6‐Bis(4‐chlorophenyl)‐2‐(2,3,4,5,6‐pentahydroxyhexylidene)
hydrazinyl)‐ nicotinonitrile (17). A mixture of compound 6 (0.01
mole), D‐glucose (0.01 mole) in ethanol (30 mL) and a catalytic
amount of glacial acetic acid (three drops) was heated at 80°C
for 2 h. The formed precipitate was filtered off, washed with
water several times, and dried to give 17. Yield 47%, mp 147–149°C.
1
mp 193–194°C. IR (KBr) ν, cm−1: 3392 (NH2), 2205 (CN). H
NMR spectrum (DMSO‐d6, δ ppm): 7.08 (bs, 2H, NH2, D2O
exchangeable), 7.28 (s, 1H, pyridine‐H5) 7.52–814 (m, 8H, Ar‐H).
Ms: m/z (343, 4M+, 11%), (341, 2M+, 70%), (339, M+, 100%).
Anal. Calcd. for C18H11Cl2N3 (340.21): C, 63.55; H, 3.26; Cl,
20.84; N, 12.35; found C, 63.48; H, 3.20; Cl, 20.79; N, 12.26.
5,7‐Bis(4‐chlorophenyl)‐3‐methyl‐[1,2,4]triazolo[4,3‐a]
pyridine‐8‐carbonitrile (13). A mixture of compound 5 (0.01 mole)
and acetic anhydride (10 mL) was refluxed for 6 h. The reaction
mixture was cooled and poured into cold water. The separated solid
was filtered off, dried, and recrystallized from ethanol to give 13.
Yield 82%, mp 174–175°C. IR (KBr) ν, cm−1: 2210 (CN). 1H NMR
spectrum (CDCl3, δ ppm): 2.21 (s, 3H, CH3), 7.64–8.41 (m, 9H,
8Ar‐H + pyridine‐H5). Ms: m/z (382, 4M+, 3%) (380, 2M+, 34%),
(378, M+, 78%), (363, M+‐CH3, 100%). Anal. Calcd. for
C20H12Cl2N4 (379.25): C, 63.34; H, 3.19; Cl, 18.70; N, 14.77;
found C, 63.24; H, 3.11; Cl, 18.62; N, 14.68.
1
IR (KBr) ν, cm−1: 3350–3260 (OH + NH), 2211 (CN). The H
NMR spectrum (DMSO‐d6, δ ppm): 2.90–3.37 (protons of the
alditol congregated with the solvent absorption) [23], 3.38–3.40
(m, 2H, CH2OH), 4.33–4.86 (m, 5H, 5OH, D2O exchangeable),
6.15 (s,1H, NH, D2O exchangeable), 7.42–8.10 (m, 9H, 8Ar‐H
+pyridine‐H5), 8.30 (s, 1H, CH═N). 13C NMR spectrum (CDCl3, δ
ppm): 61.73–73.59 (5C‐alditol), 115.11 (CN), 128.68–136.95
(17Ar‐C+N═CH). Anal. Calcd. for C24H22Cl2N4O5 (517.37): C,
55.72; H, 4.29; Cl, 13.71; N, 10.83; found C, 55.65; H, 4.22; Cl,
13.61; N, 10.74.
6‐(2‐[4,6‐Bis(4‐chlorophenyl)‐3‐cyanopyridin‐2‐yl]hydrazono)
hexane‐1,2,3,4,5‐pentaylpentaacetate (18). Compound 17 (0.01
mole) in acetic anhydride (20 mL) was refluxed on water bath for
3 h. The reaction mixture was poured onto ice water with stirring
and the solid that precipitated was collected by filtration, washed
with water, dried, and purified using silica gel column using
petroleum ether:ethyl acetate (8:2) as an eluent to give 18. Yield
63%, mp 189–191°C. IR (KBr) ν, cm−1: 3230 (NH), 2220 (CN),
1730 (C═O). The 1H NMR spectrum (DMSO‐d6, δ ppm): 2.01–2.11
(5s, 15H, 5OAc), 3.02–3.05 (m, 2H, CH2OAc), 3.86–3.88
(m, 1H, CHOAc), 4.06–4.12 ((m, 2H, CH2OAc), 5.10–5.15
(m, 1H, CHOAc), 5.70–7.73 (m, 1H, CHOAc), 7.46–8.00 (m, 9H,
8Ar‐H+pyridine‐H5), 8.02 (s, 1H, CH═N), 11.05 (s, 1H, NH, D2O
exchangeable). Anal. Calcd. for C34H32Cl2N4O10 (727.56): C, 56.13;
H, 4.43; Cl, 9.75; N, 7.70; found C, 56.04; H, 4.35; Cl, 9.68; N, 7.60.
Antiviral bioassay. Virus and cells. Avian influenza A virus
(H5N1), isolated from Egypt in 2006, was used in this study to
evaluate antiviral activity of some synthetic compounds. Madin‐
Darby canine kidney (MDCK) cells used for virus propagation
were friendly obtained from St. Jude Children’s Research
Hospital. The MDCK cells were routinely passaged in Dulbecco’s
modified Eagle medium (DMEM) containing 10% fetal
bovine serum and 1% antibiotic‐antimycotic mixture (penicillin‐
streptomycin‐amphotericin B).
MTT assay (cytotoxicity assay). The stock samples were diluted
with Dulbecco’s Modified Eagle’s Medium (DMEM) to desired
concentrations. Stock solutions of the test compounds were
prepared in DMSO at a concentration of 10% in ddH2O. The
cytotoxic activity of the extracts were tested in MDCK cell line
by using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium
bromide (MTT) method (Mossman, 1983) [27] with minor
modification. Briefly, the cells were seeded in 96‐well plates
(100 μL/well at a density of 3 × 105 cells/mL) and treated
with various concentrations of the sample solutions. At 24 h,
cells were washed with sterile phosphate buffer (PBS) three
times and the supernatant was discarded. MTT solution
(20 μL of 5 mg/mL) was added to each well and incubated
at 37°C for 4 h.
5,7‐Bis(4‐chlorophenyl)‐[1,2,4]triazolo[4,3‐a]pyridine‐8‐
carbonitrile (14). A mixture of compound 6 (0.01 mole) and
trimethyl orthoformate (20 mL) was refluxed for 15 h. The
reaction mixture was filtered off on hot and the separated
solid was recrystallized from ethanol to give 14. Yield 79%, mp
279–280°C. IR (KBr) ν, cm−1: 2211(CN). 1H NMR spectrum
(DMSO‐d6, δ ppm): 7.77–8.41 (m, 10H, 8Ar‐H + pyridine‐H5+
triazol‐H3). Ms: m/z (368, 4M+, 4%), (366, 2M+, 53%), (364,
M+, 100%). Anal. Calcd. for C19H10Cl2N4 (365.22): C, 62.49; H, 2.76;
Cl, 19.41; N, 15.34; found C, 62.39; H, 2.71; Cl, 19.35; N, 15.26.
4,6‐Bis(4‐chlorophenyl)‐2‐(3,5‐dimethyl‐1H‐pyrazol‐1‐yl)
nicotinonitrile (15). A mixture of compound 6 (0.01 mole) and
acetyl acetone (0.02 mole) in ethanol (20 mL) was refluxed for 8 h.
After cooling, the separated solid was filtered off, dried, and
recrystallized from ethanol to give 15. Yield 79%, mp 192–193°C.
1
IR (KBr) ν, cm−1: 2210 (CN). H NMR spectrum (CDCl3, δ ppm):
2.35 (s, 3H, CH3), 2.64 (s, 3H, CH3), 6.11 (s, 1H, pyrazole) 7.26–8.08 (m,
9H, 8Ar‐H + pyridine‐H5). Ms: m/z (422, 4M+, 14%), (420, 2M+, 67%),
(418, M+, 100%). Anal. Calcd. for C23H16Cl2N4 (419.32): C, 65.88; H,
3.85; Cl,16.91; N, 13.36; found C, 65.79; H, 3.75; Cl, 16.82; N, 13.30.
4,6‐Bis(4‐chlorophenyl)‐1H‐pyrazolo[3,4‐b]pyridin‐3‐
amine (16). Method A. Compound 6 (0.01 mole) in dry benzene
(20 mL) was refluxed for 5 h. After cooling, the separated solid
was filtered off, dried, and recrystallized from dioxane to give 16.
Method B. A mixture of compound 12 (0.01 mole), hydroxylamine
hydrochloride (0.02 mole), sodium acetate (0.01 mole), and
glacial acetic acid (15 mL) was refluxed for 7 h; left overnight
at room temperature, and then poured into cold water. The solid
that precipitated was filtered off, dried, and recrystallized
from dioxane to give 16. Yield (39% from method A, 73 %
from method B), mp 257–258°C. IR (KBr) ν, cm−1: 3340–3320
(NH+NH2). 1H NMR spectrum (DMSO‐d6, δ ppm): 4.61 (bs,
2H, NH2, D2O exchangeable), 7.77–9.21 (m, 9H, 8Ar‐H+
pyridine‐H5), 9.32 (s, 1H, NH, D2O exchangeable). Ms: m/z
(358, 4M+, 12%), (356, 2M+, 61%), (354, M+, 100%). Anal.
ðAbsorbance of cell without treatment ꢀ Absorbance of cell with treatmentÞ ꢁ 100
% Cytotoxicity ¼
:
Absorbance of cell without treatment
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2012
Synthesis and Anti‐Avian Influenza Virus (H5N1) Evaluation of Some Novel
Nicotinonitriles and Their N‐Acylic Nucleosides
1135
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet