Synthesis, antitumor and antimicrobial activities of 4-(4-chlorophenyl)-3- cyano-2-(β-O-glycosyloxy)-6-(thien-2-yl)-nicotinonitrile

Article abstract of DOI:10.1016/j.ejmech.2011.04.019

4-(4-Chlorophenyl)-3-cyano-6-(thien-2-yl)-1H-pyridin-2-one (2) was obtained by reaction of 2-acetyl thiophene with 4-chlorobenzaldehyde and ethyl cyanoacetate in presence of ammonium acetate or by the reaction of α,β-unsaturated compound 1 with ethyl cyan

Full text of DOI:10.1016/j.ejmech.2011.04.019

European Journal of Medicinal Chemistry 46 (2011) 2948e2954
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, antitumor and antimicrobial activities of 4-(4-chlorophenyl)-3-
cyano-2-( -O-glycosyloxy)-6-(thien-2-yl)-nicotinonitrile
b
,
a
b
Hassan A. El-Sayed a, Ahmed H. Moustafa a , Abd El-Fattah Z. Haikal , Rajab Abu-El-Halawa ,
*
El Sayed H. El Ashry c
a Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt
b Department of Chemistry, Al al-Bayt University, Mafraq, Jordan
c Department of Chemistry, Faculty of Science, Alexandria University, Alexandria, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
4-(4-Chlorophenyl)-3-cyano-6-(thien-2-yl)-1H-pyridin-2-one (2) was obtained by reaction of 2-acetyl
thiophene with 4-chlorobenzaldehyde and ethyl cyanoacetate in presence of ammonium acetate or by
Received 21 July 2010
Received in revised form
5 April 2011
Accepted 5 April 2011
Available online 15 April 2011
the reaction of
a
,
b
-unsaturated compound 1 with ethyl cyanoacetate in the presence of ammonium
-gluco/galactopyranosyloxy)-6-(thien-2-yl)
acetate. 4-(4-Chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b-D
nicotinonitrile (5a and 5b), riboside 11, xyloside 12 and lactoside 16 were prepared by the reaction of 2
with glycosyl/galactosyl/xylosyl/lactosyl bromide and peracetylated xylose/ribose under the conven-
tional and microwave irradiation methods. The reaction has regioselectively gave the O-glycosides and
not the N-glycosides. The glycosides 5a,b, riboside 11, xyloside 12 and lactoside 16 were deacetylated in
the presence of Et3N/MeOH and few drops of water to give 7a,b, 13, 14 and 17. The structure of the new
synthesized compounds was confirmed by using IR, 1H, 13C NMR spectra and microanalysis. Selected
members of these compounds were screened for antitumor and antibacterial activity.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
3-Cyanopyridin-2(1H)-one
Nicotinonitrile 2-O-glycosides/ribosides
2-O-Lactoside
Microwave
Antitumor
Antimicrobial activity
1. Introduction
Several glycosides have exhibited good biological inhibitions
[22e29], inducers and ligands [30], in addition of having excellent
Pyridine derivatives have shown a broad spectrum of biological
activities [1e3]. Pyridine derivatives and their nucleoside analogs
showed strong cytotoxicity against several human cancer cell [4,5],
potent and selective farnesyltransferase inhibitions [6] and inhib-
itors of HCV NS5B polymerase inhibitions [7], as well as antimi-
crobial [8], and antimycobacterial activities [9]. The
pharmacological and physiological activity of 3-cyanopyridin-
2(1H)-ones has attracted much attention in recent years. Thus the
non-glycosidic cardiotonic agent milrinone (I) (Fig. 1) [10,11] and
other pyridine derivatives proved to be active against herpes and
the human immunodeficiency virus [12e15]. The 3-cyanopyridin-
2-(1H)-one nucleus is also the structural basis of the alkaloid
ricinine (II) (Fig. 1) [16]. Moreover, the thiophene nucleus has
constituted the active part of several biologically active compounds
[17e21].
chemoselectivity in glycosylation processes as donors and accep-
tors [31]. Having the above aspects in mind and a continuation of
our work on the synthesis of glycosides and nucleosides with
having biological activity [32e34], glycosylative have planned to
target a group of pyridine compounds functionalized with cyano
group, thiophene and p-chlorophenyl rings. The presence of
glycosyl residue is expected to enhance the biological activity. The
antitumor and antimicrobial activities of the namely synthesized
compounds have been established.
H3C
N
O
CN
O
CN
O
CH3
N
H
CH3
N
CH3
milrinone
(I)
ricinine
(II)
* Corresponding author. Tel.: þ20 167517333; fax: þ20 552366555.
E-mail addresses: ah_hu_mostafa@yahoo.com (A.H. Moustafa), eelashry60@
hotmail.com (E.S.H. El Ashry).
Fig. 1.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.019

H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
2949
2. Chemistry
configuration. Their IR spectra showed the absence of the amide
carbonyl band which indicated the formation of the O-glycoside
and not the N-glycoside. 13C NMR spectrum of 11 showed signal at
4-(4-Chlorophenyl)-3-cyano-6-(thien-2-yl)-pyridin-2(1H)-one
(2) selected as a starting material for this study. It was obtained
from the condensation of the 2-acetyl thiophene and 4-chlor-
obenzaldehyde in presence of sodium hydroxide to give 1 whose
heterocyclization with ethyl cyanoacetate in presence of ammo-
nium acetate gave 2 (Scheme 1) [35]. Alternatively, 2 was obtained
in a better yield by an one pot synthesis of the same reactants [35].
d
99.9 ppm consistent with the anomeric carbon.
Deacetylation of compounds 5a, 5b, 11 and 12 (Schemes 2 and
3), in the presence of methanol/Et3N and few drops of water, led
to the formation of the free glycosides 7a, 7b, 13 and 14. The 1H
NMR data of these latter compounds revealed the absence of the
acetyl groups at
d 1.96e2.25 ppm and the appearance of the D2O
exchangeable OH protons at
d 3.99e5.44 ppm. Their IR spectra
indicated the presence of broad band at 3423 cmꢀ1 for OH
groups.
Ar
CN
Furthermore reaction of pyridin-2-(1H)-one 2 with 2,3,6-tri-
O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)-a-D-
O
ArCHO
CN-CH2-CO2Et
+
N
H
O
CH3
CH3CO2NH4
glucopyranosyl bromide (15) in the presence of potassium
carbonate gave the respective O-lactoside 16, which was
deacetylated in the presence of methanol/Et3N and few drops of
water to give the corresponding deprotected lactoside 17
(Scheme 4).
S
S
NaOH
ArCHO
The 1H NMR spectrum of 16 showed signals at
for the seven acetoxy groups and a doublet at
teristic for the anomeric proton with coupling constant J1 a,
d 1.90e2.10 ppm
6.35 ppm charac-
Ar
d
CN
OH
O
0
NCCH2CO2Et
CH3CO2NH4
Ar
0
¼ 8.7 Hz, which confirmed the
b-configuration. Its IR spectrum
2 a
N
S
showed the absence of the amide carbonyl group which character-
istic the formation of the O-glycoside and not N-glycoside. 1H NMR
spectrum of compound 17 revealed the absence of the acetyl groups
S
2
1
Ar = p.Cl-C6H4
and the appearance of OH protons at d
4.40e5.29 ppm. Its 13C NMR
spectrum indicated the absence the acetoxy groups and the pres-
Scheme 1. Synthesis of pyridin-2(1H)-one.
ence of the two anomeric carbons (C-10b) and (C-10a) at
99.3 ppm, respectively.
d 97.3 and
The IR spectrum of 2 showed bands at 1687, 2216 and 3212 cmꢀ1
indicating the presence of C]O, C^N and NH groups respectively.
Glycosylation of pyridin-2(1H)-one 2 with 1.1 M equivalent of
3. Pharmacological studies
3.1. Anticancer activity
3.1.1. Cell culture
All cells were routinely cultured in DMEM (Dulbeco’s Modified
Eagle’s Medium) at 37 ꢁC in humidified air containing 5% CO2.
Media were supplemented with 10% fetal bovine serum (FBS),
2,3,4,6-tetra-O-acetyl-
4) in anhydrous DMF/acetone and potassium carbonate afforded 4-(4-
chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
-glucopyranosyloxy)-6-
(thien-2-yl)nicotinonitrile (5a) and 4-(4-chlorophenyl)-2-(20,30,40,
60-tetra-O-acetyl-
-galactopyranosyloxy)-6-(thien-2-yl)nicotinoni-
a-D-gluco- or galactopyranosyl bromide (3 and
b-D
b-D
trile (5b), respectively (Scheme 2).
The structure of compounds 5a and 5b were based on the
spectroscopic data. Thus, the
5b were supported by their 1H NMR spectra, which revealed the
anomeric proton as doublet at 6.47 and 6.36 ppm with coupling
constant J ¼ 8.20 and 8.40 Hz, respectively, corresponding to
a diaxial orientation of H-10and H-20. The formation of O-glycosides
5a and 5b and not the corresponding N-glycosides 6a and 6b
(Scheme 2) were proved by 13C NMR spectroscopy, which revealed
b
-configuration of compounds 5a and
2 mM L-glutamine, containing 100 units/mL penicillin G sodium,
100 units/mL streptomycin sulfate, and 250 mg/mL amphotericin B.
Monolayer cells were harvested by trypsin/EDTA treatment, while
leukemia cells were harvested by centrifugation. All experiments
were repeated four times, unless mentioned, and the data was
represented as (mean ꢂ S.D.). Cell culture material was obtained
from Cambrex BioScience (Copenhagen, Denmark) and all chem-
icals were from Sigma (USA).
d
the presence of the anomeric carbons at
d 93.8 and 94.7 ppm. The
absence of a carbonyl amide band at 1687 cmꢀ1 indicated that
glycosylation had taken place on the oxygen to give 5a and 5b and
not on the nitrogen to give 6a and 6b, respectively. This preference
can be due to the formation of the respective mesomeric anion of 2
that promote the reaction with electrophiles reagent to give the
O-glycosides.
3.1.2. Cytotoxicity assay
Cytotoxicity of tested samples against different types of cells
was measured using the MTT Cell Viability Assay. MTT (3-[4,
5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is
based on the ability of active mitochondrial dehydrogenase enzyme
of living cells to cleave the tetrazolium rings of the yellow MTT and
form a dark blue insoluble formazan crystals which is largely
impermeable to cell membranes, resulting in its accumulation
within healthy cells. Solubilization of the cells results in the liber-
ation of crystals, which are then solubilized. The number of viable
cells is directly proportional to the level of soluble formazan dark
blue color. The extent of the reduction of MTT was quantified by
measuring the absorbance at 570 nm [37].
4-(4-Chlorophenyl)-2-(20,30,50-tri-O-acetyl-
xy)-6-(thien-2-yl)nicotinonitrile (11) was obtained by the reaction
of 2,3,5-tri-O- -xylofuranosyl bromide (8) with pyridin-2(1H)-
b-D-xylofuranosylo-
a-D
one 2 under the conditions or with peracetylated xylose (9) under
microwave irradiation using silica gel as a solid support [32,36]
(Scheme 3). Similarly pyridin-2(1H)-one 2 was reacted with per-
acetylated ribose 10 under microwave irradiation [32,36] to give
the ribofuranosyl derivative 12 (Scheme 3).
The 1H NMR spectra of 11 and 12 showed signals at
d
2.10e2.25 ppm for the acetoxy groups and a doublet in the range at
6.79e6.63 ppm characteristic for the anomeric protons with
coupling constant J ¼ 3.6e3.8 Hz, which confirmed the
3.1.3. Reagents preparation
d
MTT solution: 5 mg/mL of MTT in 0.9% NaCl.
Acidified isopropanol: 0.04 N HCl in absolute isopropanol.
b
-

2950
H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
Ar
Ar
CN
K2CO3
CN
O
N
K
DMF/Acetone
O
N
H
S
S
2
OAc
R'
R"
O
AcO
AcO
Br
and 4)
Ar
Ar
(
3
CN
CN
N
O
O
N
S
OAc
S
OAc
O
R'
O
R'
OAc
R"
AcO
OAc
R"
Ac O
5a,b
6a,b
a, R' = H, R" = OAc
b, R' = OAc, R" = H
a, R' = H, R" = OAc
b, R' = OAc, R" = H
Et3N/H2O
CH3OH
Ar
N
CN
O
OH
S
O
R'
OH
R"
HO
7a,b
a, R' = H, R" = OH
b, R' = OH, R" = H
Ar = p.Cl-C6H4
Scheme 2. Glycosylated and galactosylated analogs.
3.1.4. Procedure
3.1.5. Results
Cells (0.5 ꢃ 105 cells/well) in serum-free media were plated in
Chemotherapy is a major therapeutic approach for the both
localized and metastasized cancers. Seven selected newly synthe-
sized compounds 5a, 5b, 7a,11,12,14 and 16 were tested forcytotoxic
activity against the MCF7 (breast carcinoma cell line) in comparison
to the known anticancer drugs: 5-Fluorouracil and Doxorubicin as
reference drugs. All tested new compounds dissolved in DMSO in
a flat bottom 96-well microplate, and treated with 20
ml of
different concentrations of each tested compound for 48 h at
37 ꢁC, in a humidified 5% CO2 atmosphere. After incubation, media
were removed and 40
incubated for an additional 4 h. MTT crystals were solubilized by
adding 180 l of acidified isopropanol/well and plate was shacked
ml MTT solution/well were added and
m
different concentrations (25, 50 and 100 mg/mL). The tested
at room temperature. This has been followed by photometric
determination of the absorbance at 570 nm using microplate
ELISA reader. Triplicate measures were performed for each
concentration and the average was calculated. Data were
expressed as the percentage of relative viability compared with
the untreated cells.
compounds 7a and 14 were proven to have no cytotoxic activity
against the MCF7 at all drug concentrations (see Table 1). The 4-(4-
chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b
-
D
-glucopyranosyloxy)-
4-(4-chlorophenyl)-2-(20,30,
-ribofuranosyloxy)-6-(thien-2-yl)nicotinonitrile
4-(4-chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b-D-
6-(thien-2-yl)nicotinonitrile
50-tri-O-acetyl-
(12) and
(5a),
b-D

H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
2951
Table 1
Effect of selected 2-O-glycosides nicotinonitrile on MCF7 tumor cell lines.
Compound
%Inhibition of cell viability (MCF7) (breast
carcinoma cell line)
100
mg/mL
50
m
g/mL
25 mg/mL
5a
5b
7a
11
12
14
16
10%
20%
0%
8%
12%
0%
10%
7%
50%
5%
5%
0%
0%
7%
0%
5%
0%
15%
0%
0%
0%
0%
2%
0%
0%
0%
6%
5-Fluorouracil (5-Fu)
Doxorubicin (Dox)
activityexplained by the presence of the 4-chlorophenyl and acetoxy
groups in sugar moiety provided a good affinity toward the enzyme
on account of force of electrostatic attraction between the planar
4-chlorophenyl and electronegativity of acetoxy groups with the
target site pocket of the tumor cells. The antitumor activity of 4-(4-
chlorophenyl)-2-(20,30,50-tri-O-acetyl-
en-2-yl)nicotinonitrile (11) against MCF7 has higher activity at high
concentration (100 g/mL) compared to 5-Fluorouracil and low with
Doxorubicin, but lower activity at low concentrations (25 and
50
g/mL). Compound 4-(4-chlorophenyl)-2-(20,30,40,60-tetra-O-
acetyl- -galactopyranosyloxy)-6-(thien-2-yl)nicotinonitrile (5b)
exhibited increase in the antitumor activity against MCF7 at high
concentration (100 g/mL) compared to 5-Fluorouracil and signifi-
cant activities compared to Doxorubicin and very low effect at
concentration (50 g/mL). All of the tested derivatives showed
a significant and higher antitumor activity at high concentration
(100 g/mL) against MCF7 (breast carcinomacell line) compared to 5-
b-D-xylofuranosyloxy)-6-(thi-
m
m
b-D
m
m
m
Fluorouracil and have a significant and moderate activity compared
to Doxorubicin.
3.2. Antimicrobial activity
The antimicrobial activities of some synthesized compounds
were screened for their antibacterial activity against four species of
bacteria, namely Staphylococcus aureus and Bacillus subtilis as Gram-
positive bacteria as well as Escherichia coli and Pseudomonas aeur-
oginosa as Gram-negative bacteria, using a cup plate agar diffusion
method [38]. The tested compounds were dissolved in dimethyl
Scheme 3. Ribosyl and xylosyl derivatives.
galactopyranosyl-(1 / 4)-(20,30,40,60-tetra-O-acetyl-
b-D-glucopyr-
anosyloxy-6-(thiophen-2-yl)nicotinonitrile (16), have showed
higher antitumor activities compared to the reference drug
(5-Fluorouracil) while moderate compared with (Doxorubicin). This
sulfoxide to get of 1 mg/mL concentration. The inhibition zone were
measured in mm at the end of an incubation period of 48 h at 37 ꢁC.
Dimethyl sulfoxide showed no inhibition zones. Ampecillin was
used as a reference to evaluate the potency of tested compounds.
The newlysynthesized glycosides 5a,b, 7a,b,12,13,14 and17 were
tested for their in vitro antibacterial activity against a panel of stan-
dard strains of the Gram-positive bacteria (S. aureus and B. subtilis)
and the Gram-negative bacteria (E. coli and P. aeuroginosa).
Compounds 5b, 12 and 14 showed higher antibacterial activity than
OAc
O
OAc
OAc
O
K2CO3
O
2
+
AcO
AcO
DMF/Acetone
AcO
AcO
Br
15
Ar
Table 2
Ar
Antimicrobial activity of tested compounds (Inhibition zones mm, Minimum
Inhibitory Concentration mg/mL).
CN
CN
O
N
Compound No.
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Pseudomonas
aeuroginosa
Et3N/H2O
CH3OH
O
N
S
OAc
O
S
OH
O
5a
5b
7a
7b
12
13
14
17
8
12
3
0
9
3
10
5
11
10
1
0
8
3.5
7
3
3
5
0
0
3
2
6
0
2
13
26
0
0
25
9
28
10
23
AcO
AcO
OAc
O
HO
O
OH
O
O
AcO AcO
HO HO
HO
AcO
HO
16
17
Ar = p.Cl-C6H4
Ampecillin
7
6
Scheme 4. Lactosyl derivatives.

2952
H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
the standard drug (ampecillin). Compound 5a showed moderate
antibacterial activity against Gram (þve) bacteria and lower activity
against Gram (ꢀve) bacteria comparing to ampecillin. While
compounds 7a and 17 showed lower activity against Gram (þve)
bacteria and inactive against Gram (ꢀve) bacteria. Compound 13
showed lower activity against Gram (þve) and Gram (ꢀve) bacteria
compared to standard drug. Compound 7b did not show any activity
against tested micro-organisms as shown in Table 2.
bromide (0.011 mol) was added. The reaction mixture was stirred at
room temperature overnight then refluxed for 3e5 h, filtered off
and the solvent was then evaporated under reduced pressure. The
product was dried and crystallized from the proper solvent or
chromatographed on silica gel column.
Method B: A mixture of pyridin-2-(1H)-one (2) (0.01 mol) and
(0.01 mol) of peracetylated sugar, were dissolved in ethanol/CHCl3
(70/30)then1.0gofsilicagel(200e400mesh)wasadded.Thesolvent
was removed by evaporation. The dried residue was transferred into
a glass beaker and irradiated (5 min) in a domestic microwave oven.
The product was chromatographed on a silica gel column.
4. Conclusions
The glycosylation of pyridin-2-(1H)-one 2 gave the respective
glycosyloxy derivatives 5a, 5b, 11, 12, 16 and not the respective
nucleosides. The in vitro growth inhibitory activities of 5a, 5b, 7a,11,
12, 14 and 16 against (MCF7) cell lines revealed significant potential
antitumor activity. Best results were gained by compound 5b at
5.3.1. 4-(4-Chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b-D-glucopy-
ranos-yloxy)-6-(thien-2-yl)nicotinonitrile (5a)
Pale yellow crystals from ethanol, Method A: yield 59%, mp
180e182 ꢁC. Anal. Calcd. for C30H27ClN2O10S (643.06): C, 56.03; H,
4.23; N, 4.36; Found: C, 55.89; H, 4.16; N, 4.40. IR (KBr, cmꢀ1): 2229
higher used concentration (100 mg/mL), compared to 5-Fluorouracil
and Doxorubicin as known anticancer reference drugs. Although
significant results were obtained when evaluation the antibacterial
activity of glycosides 5a,b, 7a,b, 12, 13, 14 and 17.
(C^N) and 1750 (C]O, acetoxy). 1H NMR (DMSO-d6,
d ppm): 1.96,
0
0
1.99, 2.00 and 2.03 (4s, 12H, 4 CH3CO), 4.05 (dd, 1H, J5 ,6 ¼ 4.67,
0
0
00
0
00
0
00
J6 ,6 ¼ 12.22 Hz, H-6 ), 4.20 (dd, 1H, J5 ,6 ¼ 4.94, J6 ,6 ¼0 12.22 Hz, H-
600), 4.38 (m, 1 H, H-50), 5.03 (t, 1H, J3 ,4 ¼ 9.40 Hz, H-4 ), 5.22 (t, 1H,
0
0
J1 ,2 ¼ 8.23, J2 ,3 ¼ 9.60 Hz, H-20), 5.66 (dd, 1H, J2 ,3 ¼ 9.60,
0
0
0
0
0
0
5. Experimental
J3 ,4 ¼ 9.30 Hz, H-30), 6.47 (d, 1H, J1 ,2 ¼ 8.20 Hz, H-10), 7.26 (t,
1H, J ¼ 5.10, 3.60 Hz, thiophene-H), 7.69 (s,1H, pyridine-H5), 7.75 (d,
2H, J ¼ 7.8 Hz, Ar-H), 7.79 (d, 2H, J ¼ 7.8 Hz, Ar-H), 7.91 (d, 1H,
J ¼ 5.10 Hz, thiophene-H), 8.18 (d, 1H, J ¼ 3.60 Hz, thiophene-H). 13C
0
0
0
0
5.1. Chemistry
All melting points are uncorrected and were measured using an
Electro thermal IA 9100 apparatus. TLC was performed on Merck
Silica Gel 60F254 with detection by UV light and by charring after
seperating with 10% EtOH solution of H2SO4. The IR spectra (KBr
disc) were recorded on a Pye Unicam Sp-3-300 or a Shimadzu FTIR
8101 PC infrared spectrophotometer. The 1H and 13C NMR spectra
were determined with JEOL-JNM-LA 300 MHz spectrometer. The
NMR (DMSO-d6, d ppm): 20.25, 20.28, 20.3 and 20.4 (4 CH3CO), 61.5
(C-60), 68.0(C-40), 68.9(C-30), 71.4 (C-20), 72.2(C-50), 93.8(C-10),114.0
(C^N),128.3,128.9,129.0,129.3,130.5,131.0,131.9,134.1,135.1,141.9,
152.8, 155.3, 161.5 (Ar-C) and 169.0, 169.3, 170.0, 170.1 (4 C]O).
5.3.2. 4-(4-Chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b-D-galacto-
chemical shifts are expressed on the
d
(ppm) scale using TMS as the
pyran-osyloxy)-6-(thien-2-yl)nicotinonitrile (5b)
standard reference. Elemental analysis determined on a Perkin
Elmer 240 (microanalysis). The antitumor activity was performed
at Pharmacology unit, National Cancer Institute, Cairo University,
Cairo, Egypt. Antibacterial activity was carried out in Microbiolog-
ical Center at Faculty of Science, Zagazig University, Egypt.
Method A: As pale yellow crystals chromatographed by using
(CH2Cl2/MeOH, 9.9: 0.1) as eluent, yield 67%, mp 139e140 ꢁC. Anal.
Calcd. for C30H27ClN2O10S (643.06): C, 56.03; H, 4.23; N, 4.36; Found:
C, 56.24; H, 4.20; N, 4.38. IR (KBr, cmꢀ1): 2226 (C^N) and 1752 (C]
O, acetoxy). 1H NMR (DMSO-d6,
d ppm): 1.87, 1.96, 2.01 and 2.17 (4s,
0
0
0
0
00
12 H, 4 CH3CO), 4.03 (dd, 1 H, J5 ,6 ¼ 6.0, J6 ,6 ¼ 11.06 Hz, H-6 ), 4.16
00
0
0
0
0
00
5.2. 4-(4-Chlorophenyl)-3-cyano-6-(thien-2-yl)-1H-pyridin-2-one (2)
(dd,1 H, J5 ,6 ¼ 6.30, J6 ,6 ¼11.06 Hz, H-6 ), 4.59 (m,1 H, H-5 ), 5.32 (t,
1H, J3 ,2 ¼ 10.23, J3 ,4 ¼ 2.80 Hz, H-30), 5.40 (t, 1H, J2 ,1 ¼ 8.40,
0
0
0
0
0
0
J2 ,3 ¼ 10.30 Hz, H-20), 5.59 (t, 1H, J4 ,3 ¼ 2.81, J4 ,5 ¼ 2.78 Hz, H-40),
0
0
0
0
0
0
General procedure: Method A: A mixture of chalcone 1 (0.01 mol),
ethyl cyanoacetate (0.01 mol) and ammonium acetate (0.08 mol) in
ethanol (40 mL) was refluxed for 10 h. The precipitatewas filtered off
and crystallized from absolute ethanol or acetic acid.
6.36 (d, 1H, J1 ,2 ¼ 8.40 Hz, H-10), 7.26 (t, 1H, J ¼ 5.7, 4.80 Hz,
thiophene-H), 7.67 (d, 2H, J ¼ 8.47 Hz, Ar-H), 7.75 (d, 2H, J ¼ 8.47 Hz,
Ar-H), 7.82 (s,1H, pyridine-H5), 7.89 (d,1H, J ¼ 5.7 Hz, thiophene-H),
0
0
Method B:
A
mixture of 2-acetyl thiophene (0.01 mol),
8.15 (d, 1H, J ¼ 4.80 Hz, thiophene-H). 13C NMR (DMSO-d6,
d ppm):
4-chlorobenzaldehyde (0.01 mol), ethyl cyanoacetate (0.01 mol),
and ammonium acetate (0.08 mol), in ethanol (40 mL) was refluxed
for 3 h. The obtained precipitate was filtered off, dried and crys-
tallized as above. Method A: yield 30%, method B: yield 52%, as
yellow crystals, mp 295e297 ꢁC. Anal. Calcd. for C16H9ClN2OS
(312.77): C, 61.44; H, 2.90; N, 8.96. Found: C, 61.23; H, 3.06; N, 8.95.
IR (KBr, cmꢀ1): 3212 (NH), 2216 (C^N) and 1687 (C]O, amide). 1H
20.23, 20.31, 20.84 and 20.88 (4 CH3CO), 61.9 (C-60), 68.0 (C-40), 68.1
(C-20), 70.7 (C-30), 71.8 (C-50), 94.7 (C-10), 114.5 (C^N), 128.9, 129.3,
129.6,129.8,130.5,131.0,132.3,134.6,135.6,142.4,152.3,155.9,162.1
(Ar-C) and 169.4, 169.9, 170.3, 170.5 (4 C]O).
5.3.3. 4-(4-Chlorophenyl)-2-(20,30,50-tri-O-acetyl-
b-D-xylofurano-
syloxy)-6-(thien-2-yl)nicotinonitrile (11)
NMR (DMSO-d6,
d
ppm): 7.25 (t, 1H, J ¼ 4.50, 4.2 Hz, thiophene-H),
As pale yellow crystals chromatographed by using (CH2Cl2/
MeOH, 9.9: 0.1), Method A: Yield 52%, method B: Yield 58%, mp
158e159 ꢁC. Anal. Calcd. for C27H23ClN2O8S (571.00): C, 56.79; H,
4.06; N, 4.91. Found: C, 56.82; H, 3.98; N, 4.84. IR (KBr, cmꢀ1): 2224
7.60 (d, 2H, J ¼ 8.70 Hz, Ar-H), 7.65 (d, 2H, J ¼ 8.70 Hz, Ar-H), 7.73 (s,
1H, pyridone-H-5), 7.88 (d, 1H, J ¼ 4.50 Hz, thiophene-H), 8.06 (d,
1H, J ¼ 4.20 Hz, thiophene-H),12.85 (s,1H, NH). 13C NMR (DMSO-d6,
d
ppm): 116.6 (C^N), 128.1, 128.9, 129.0, 129.3, 129.4, 130.2, 130.5,
(C^N) and 1759 (C]O, acetoxy). 1H NMR (DMSO-d6,
d ppm): 2.10,
0
0
131.0, 132.3, 132.6, 132.7, 135.6 (Ar-C) and 160.4 (C]O).
2.15 and 2.25 (3s, 9H, 3 CH3CO), 3.76, (dd, 1H, J4 ,5 ¼ 6.0,
J5 ,5 ¼ 12.0 Hz, H-50), 3.96 (dd, 1H, J4 ,5 ¼ 5.80, J5 ,5 ¼ 12.0 Hz, H-
0
00
0
00
0
00
500), 5.10 (m, 1H, H-40), 5.25 (t, 1H, J1 ,2 ¼ 3.0, J2 ,3 ¼ 9.0 Hz, H-20),
0
0
0
0
5.3. General procedure for synthesis of glycosides
0
0
0
0
0
0
0
5.50 (t, 1H, J2 ,3 ¼ 9.0, J3 ,4 ¼ 9.0 Hz, H-3 ), 6.79 (d, 1H, J1 ,2 ¼ 3.6 Hz,
H-10), 7.22 (t, 1H, J ¼ 5.32, 4.10 Hz, thiophene-H), 7.70 (d, 2H,
J ¼ 8.42 Hz, Ar-H), 7.85 (d, 2H, J ¼ 8.42 Hz, Ar-H), 7.90 (s, 1H,
pyridine-H5), 7.94 (d, 1H, J ¼ 5.32 Hz, thiophene-H), 8.13 (d, 1H,
Method A: A mixture of pyridin-2-(1H)-one 2 (0.01 mol) and
(0.01 mol) potassium carbonate was stirred in dry acetone/DMF
(15 mL) for 1 h, then glycosyl, galactosyl, xylosyl and lactosyl

H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
2953
J ¼ 4.10 Hz, thiophene-H). 13C NMR (DMSO-d6,
d
ppm): 20.68, 20.91
5.4.2. 4-(4-Chlorophenyl)-2-(b-D-galactopyranosyloxy)-6-(thien-2-
and 21.04 (3 CH3CO), 62.19 (C-50), 71.20 (C-20), 73.33 (C-30), 78.56
(C-40) and 99.9 (C-10), 116.0 (C^N), 129.4, 129.5, 129.9, 130.6, 131.0,
131.4, 132.2, 134.9, 135.3, 140.8, 151.0, 153.1, 161.3 (Ar-C) and 169.9,
170.8, 171.0 (3 C]O).
yl)nic-otinonitrile (7b)
Pale yellow crystals from ethanol, mp 190e191 ꢁC. Anal. Calcd.
for C24H21ClN2O7S (643.06): C, 55.76; H, 4.09; N, 5.42. Found: C,
55.69; H, 4.10; N, 5.38. IR (KBr, cmꢀ1): 3389 (broad, 4OH) and 2217
(CN). 1H NMR (DMSO-d6,
d
ppm): 3.48 (m, 3H, H-30, H-60, H-600),
5.3.4. 4-(4-Chlorophenyl)-2-(20,30,50-tri-O-acetyl-
yloxy)-6-(thien-2-yl)nicotinonitrile (12)
Method B: As pale yellow crystals chromatographed by using
(CH2Cl2/MeOH, 9.9: 0.1) as eluent, yield 55%, mp 155e157 ꢁC. Anal.
Calcd. for C27H23ClN2O8S (571.00): C, 56.79; H, 4.06; N, 4.91; Found:
C, 56.78; H, 4.11; N, 5.01. IR (KBr, cmꢀ1): 2225 (C^N) and 1751 (C]
b-
D-ribofuranos-
3.59 (m, 3H, H-20, H-40, H-50), 4.64 (m, 2H, OH-40, OH-60), 4.95 (d,
1H, J ¼ 5.10 Hz, OH-30), 5.28 (d, 1H, J ¼ 4.80 Hz, OH-20), 5.96 (d, 1H,
J1 ,2 ¼ 8.10 Hz, H-10), 7.24 (t, 1H, J ¼ 4.99, 3.78 Hz, thiophene-H),
7.64 (d, 2H, J ¼ 8.50 Hz, Ar-H), 7.70 (d, 2H, J ¼ 8.50 Hz, Ar-H),
7.72 (s, 1H, pyridine-H-5), 7.76 (d, 1H, J ¼ 4.99 Hz, thiophene-H),
8.10 (d, 1H, J ¼ 3.78 Hz, thiophene-H). 13C NMR (DMSO-d6,
0
0
O, acetoxy). 1H NMR (DMSO-d6,
d
ppm): 2.01, 2.06 and 2.18 (3s, 9H,
d
ppm): 60.49 (C-60), 68.40 (C-40), 70.23 (C-20), 74.13 (C-30), 76.80
3 CH3CO), 4.16, (dd, 1H, J4 ,5 ¼ 4.5, J5 ,5 ¼ 11.02 Hz, H-500), 4.33 (dd,
(C-50) and 97.89 (C-10), 115.3 (C^N), 129.2, 129.3, 129.5, 130.6,
130.9, 131.4, 132.1, 134.9, 135.5, 142.8, 153.3, 155.7 (Ar-C) and 163.3
(C]N).
0
0
0
00
00
0
00
0
00
1H, J4 ,5 ¼ 4.23, J5 ,5 ¼ 11.02 Hz, H-5 ), 4.49 (m, 1H, H-4 ), 5.42 (dd,
1H, J2 ,3 ¼ 3.10, J3 ,4 ¼ 6.18 Hz, H-30), 5.51 (dd, 1H, J1 ,2 ¼ 3.8,
0
0
0
0
0
0
J2 ,3 ¼ 3.12 Hz, H-20), 6.63 (d, 1H, J1 ,2 ¼ 3.8 Hz, H-10), 7.24 (t, 1H,
J ¼ 5.02, 3.90 Hz, thiophene-H), 7.70 (d, 2H, J ¼ 8.42 Hz, Ar-H), 7.72
(d, 2H, J ¼ 8.42 Hz, Ar-H), 7.74 (s, 1H, pyridine-H5), 7.87 (d, 1H,
J ¼ 5.02 Hz, thiophene-H), 8.16 (d, 1H, J ¼ 3.90 Hz, thiophene-H).
0
0
0
0
5.4.3. 4-(4-Chlorophenyl)-2-(b-D-xylofuranosyloxy)-6-(thien-2-yl)
nico-tinonitrile (13)
Pale yellow crystals from ethanol, mp 188e189 ꢁC. Anal. Calcd.
for C21H17ClN2O5S (444.89): C, 56.69; H, 3.85; N, 6.30. Found: C,
56.68; H, 4.10; N, 6.22. IR (KBr, cmꢀ1): 3423 (broad, 4OH) and 2217
5.3.5. 4-(4-Chlorophenyl)-2-(20,30,40,60-tetra-O-acetyl-
b
-D
-galacto-
pyran-osyl-(1 / 4))-(20,30,40,60-tetra-O-acetyl-
loxy)-6-(thien-2-yl)nicotinonitrile (16)
b
-
D-glucopyranosy-
(CN). 1H NMR (DMSO-d6, ppm) 3.31 (m, 2H, H-50, H-500), 3.37 (m,
d
1H, H-40), 3.44e3.51 (2H, H-30, H-20), 4.40 (t, 1H, J ¼ 6.9 Hz, OH-50,
D2O exchangeable), 4.63 (d, 1H, J ¼ 4.5 Hz, OH-30, D2O exchange-
able), 4.85 (d, 1H, J ¼ 3.9 Hz, OH-20, D2O exchangeable), 6.09 (d, 1H,
Method A: As pale yellow syrup from ethanol, yield 56%. Anal.
Calcd. for C42H43ClN2O18S (931.31): C, 54.17; H, 4.65; N, 3.01. Found:
C, 54.39; H, 4.51; N, 3.18. IR (KBr, cmꢀ1): 2228 (C^N) and 1742 (C]
J1 ,2 ¼ 3.9 Hz, H-10), 7.23 (t, 1H, J ¼ 5.0, 3.94 Hz, thiophene-H), 7.64
(d, 2H, J ¼ 8.12 Hz, Ar-H), 7.71 (d, 2H, J ¼ 8.12 Hz, Ar-H), 7.78 (s, 1H,
pyridine-H5), 7.92 (d, 1H, J ¼ 5.0 Hz, thiophene-H), 8.10 (d, 1H,
J ¼ 3.94 Hz, thiophene-H).
0
0
O, acetoxy). 1H NMR (DMSO-d6, d ppm):
d 1.90, 1.93, 1.95, 1.97, 2.00,
2.06 and 2.10 (7s, 21H, 7 CH3CO). 3.98e4.03 (m, 3H, H-20b, H-60a, H-
60b), 4.14 (dd, 1H, J6 a,6 a ¼ 11.4, J6 a,5 a ¼ 5.6 Hz, H-600a), 4.35 (m, 1H,
00
0
00
0
H-50b), 4.69 (dd, 1H, J6 b,5 b ¼ 6.3 Hz, H-600b), 4.85 (m, 1H, H-50a),
00
0
4.91 (dd, 1H, J1 b,2 b ¼ 7.6 Hz, H-10b), 5.03 (1H, J4 b,3 b ¼ 3.1,
5.4.4. 4-(4-Chlorophenyl)-2-(b-D-ribofuranosyloxy)-6-(thien-2-yl)
0
0
0
0
J4 b,5 b ¼ 3.8 Hz, H-40b), 5.20 (dd, 1H, J2 a,1 a ¼ 8.7, J2 a,3 a ¼ 8.4 Hz, H-
nicotinonitrile (14)
0
0
0
0
0
0
20a), 5.25 (dd, 1H, J4 a,3 a ¼ 9.1, J4 a,5 a ¼ 9.9 Hz, H-40a), 5.32 (d, 1H,
Pale yellow crystals from ethanol, mp 183e185 ꢁC. Anal. Calcd.
for C21H17ClN2O5S (444.89): C, 56.69; H, 3.85; N, 6.30. Found: C,
56.65; H, 3.90; N, 6.33. IR (KBr, cmꢀ1): 3424 (broad, 4OH) and 2219
0
0
0
0
J3 b,4 b ¼ 3.1 Hz, H-30b), 5.39 (dd, 1H, J3 a,2 a ¼ 8.4, J3 a,4 a ¼ 9.1 Hz, H-
0
0
0
0
0
0
30a), 6.35 (d, 1H, J1 a,2 a ¼ 8.7 Hz, H-10a) 7.10 (t, 1H, J ¼ 5.25, 4.11 Hz,
thiophene-H), 7.23 (d, 2H, J ¼ 8.12 Hz, Ar-H), 7.65 (d, 2H, J ¼ 8.12 Hz,
Ar-H), 7.70 (s, 1H, pyridine-H5), 7.84 (d, 1H, J ¼ 5.25 Hz, thiophene-
H), 8.05 (d, 1H, J ¼ 4.11 Hz, thiophene-H).
0
0
(CN). 1H NMR (DMSO-d6,
d
ppm) 3.12 (m, 2H, H-50, H-500), 3.25 (m,
1H, H-40), 3.44e3.5 (2H, H-30, H-20), 4.28 (t, 1H, J ¼ 5.4 Hz, OH-50,
D2O exchangeable), 4.55 (d, 1H,
J
¼
4.81 Hz, OH-30, D2O
exchangeable), 4.77 (d, 1H, J ¼ 5.1 Hz, OH-20, D2O exchangeable),
6.19 (d, 1H, J1 ,2 ¼ 3.62 Hz, H-10), 7.24 (t, 1H, J ¼ 4.8, 3.90 Hz,
thiophene-H), 7.64 (d, 2H, J ¼ 8.10 Hz, Ar-H), 7.70 (d, 2H, J ¼ 8.12 Hz,
Ar-H), 7.72 (s, 1H, pyridine-H5), 7.74 (d, 1H, J ¼ 4.8 Hz, thiophene-
H), 8.05 (d, 1 H, J ¼ 3.90 Hz, thiophene-H).
0
0
5.4. General procedure for deprotection
Triethylamine (1 mL) was added to a solution of glycoside
(0.01 mol) in MeOH (20 mL) and 3 drops of water. The mixture was
stirred overnight at room temperature, evaporated under reduced
pressure and the residue was co-evaporated with MeOH until the
triethylamine was removed. The residue was crystallized from
proper solvent, yield >85%.
5.4.5. 4-(4-Chlorophenyl)-2-(b-D-galactopyranosyl-(1 / 4))-(b-D-
glucopy ranosyloxy)-6-(thien-2-yl)nicotinonitrile (17)
Pale yellow crystals from ethanol/H2O, mp 165e167 ꢁC. Anal.
Calcd. for C28H29ClN2O11S (637.05): C, 52.79; H, 4.59; N, 4.40.
Found: C, 52.74; H, 4.60; N, 4.38. IR (KBr, cmꢀ1): 3420 (broad, 7
5.4.1. 4-(4-Chlorophenyl)-2-(b-D-glucopyranosyloxy)-6-(thien-2-yl)
nico-tinonitrile (7a)
OH) and 2224 (CN). 1H NMR (DMSO-d6,
d ppm): 3.42e3.55 (4 m,
Pale yellowcrystals from ethanol, mp 205e206 ꢁC. Anal. Calcd. for
C24H21ClN2O7S (643.06): C, 55.76; H, 4.09; N, 5.42. Found: C, 55.81; H,
4.23; N, 5.35. IR (KBr, cmꢀ1): 3423 (broad, 4OH) and 2212 (CN). 1H
12H, H-20b, H-30b, H-40b, H-50b, H-60b, H-600b, H-20a, H-30a, H-
40a, H-50a, H-60a, H-600a), 4.40 (d, 1H, OH-40b), 4.48 (d, 1H, OH-
60b), 4.80 (d, 1H, J ¼ 4.20 Hz, OH-30b), 4.92 (d, 1H, OH-20b), 5.02
(d, 1H, OH-60a), 5.10 (d, 1H, OH-30a), 5.29 (d, 1H, OH-20a), 5.82
NMR (DMSO-d6, d
ppm): 3.35 (m, 6H, H-60, H-600, H-50, H-40, H-30 and
H-20), 3.99 (t, 1H, J ¼ 3.50 Hz, OH-60, D2O exchangeable), 4.57 (d, 1H,
J ¼ 4.24 Hz, OH-40, D2O exchangeable), 5.19 (d,1H, J ¼ 4.21 Hz, OH-30,
D2O exchangeable), 5.44 (d, 1H, J ¼ 4.86 Hz, OH-20, D2O exchange-
(d, 1H, J1 b,2 b ¼ 7.76 Hz, H-10b), 5.87 (d, 1H, J1 a,2 a ¼ 8.91 Hz,
H-10a), 7.11 (t, 1H, J ¼ 4.60, 4.01 Hz, thiophene-H), 7.54 (d, 2H,
J ¼ 8.12 Hz, Ar-H), 7.58 (s, 1H, pyridine-H5), 7.61 (d, 2H,
J ¼ 8.12 Hz, Ar-H), 7.70 (d, 1H, J ¼ 4.60 Hz, thiophene-H), 7.97 (d,
0
0
0
0
able), 6.01 (d, 1H, J1 ,2 ¼ 8.13 Hz, H-10), 7.25 (t, 1H, J ¼ 4.99, 3.75 Hz,
thiophene-H), 7.70 (d, 2H, J ¼ 8.44 Hz, Ar-H), 7.75 (d, 2H, J ¼ 8.37 Hz,
Ar-H),7.94(s,1H, pyridine-H5), 8.09(d,1H, J ¼ 4.99 Hz, thiophene-H),
0
0
1H, J ¼ 4.01 Hz, thiophene-H). 13C NMR (DMSO-d6,
d ppm): 60.7
(C-60a), 61.3 (C-60b), 68.1 (C-30a), 69.8 (C-20b), 70.0 (C-20a), 73.0
(C-30b), 73.1 (C-40a), 73.5 (C-40b), 77.6 (C-50b), 78.5 (C-50a), 97.3
(C-10b), 99.3 (C-10a). 115.2 (C^N), 128.9, 129.3, 129.5, 130.9,
131.4, 132.1, 133.0, 134.8, 135.6, 142.7, 153.3, 155.7 (Ar-C) and
163.2 (C]N).
8.38 (d, 1H, J ¼ 3.75 Hz, thiophene-H). 13C NMR (DMSO-d6,
d ppm):
62.7 (C-60), 62.8 (C-20), 69.4 (C-30), 76.7 (C-40), 79.8 (C-50), 96.3 (C-10),
116.4 (C^N),128.5,129.1,129.4,130.4,131.0,132.9,133.8,135.4,135.8,
140.3, 152.2, 155.6 (Ar-C) and 162.7 (C]N).

2954
H.A. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 2948e2954
J. Balzarini, A. Brancale, T. Sarkar, E. Hamel, Bioorg. Med. Chem. 16 (2008)
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