Synthesis, anticoagulant and PIVKA-II induced by new 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.bmc.2010.04.009

The action of the coumarin-type drugs and related compounds is reviewed to their VKOR antagonistic effects. In our study, twenty 3-pyridinyl, pyrimidinyl and pyrazolyl-4-hydroxycoumarin derivatives were synthesized. A comparative in vivo (CT, PT determination) and in vitro (measurement of PIVKA-II levels) anticoagulant study with respect to warfarin showed that the synthesized compounds have different anticoagulant activities, the most prospective compounds were the 3-pyrazolyl-4-hydroxycoumarin derivatives.

Full text of DOI:10.1016/j.bmc.2010.04.009

Bioorganic & Medicinal Chemistry 18 (2010) 3371–3378
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, anticoagulant and PIVKA-II induced by new
4-hydroxycoumarin derivatives
b
a
c
d
Omaima M. Abdelhafez a, , Kamelia M. Amin , Rasha Z. Batran , Timothy J. Maher , Somaia A. Nada ,
*
Shalini Sethumadhavan c
a Department of Chemistry of Natural Products, National Research Center, Dokki, Cairo, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt
c Massachusettes College of Pharmacy and Health Science, Boston University, Massachusettes, Boston, USA
d Department of Pharmacology, National Research Center, Dokki, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The action of the coumarin-type drugs and related compounds is reviewed to their VKOR antagonistic
effects. In our study, twenty 3-pyridinyl, pyrimidinyl and pyrazolyl-4-hydroxycoumarin derivatives were
synthesized. A comparative in vivo (CT, PT determination) and in vitro (measurement of PIVKA-II levels)
anticoagulant study with respect to warfarin showed that the synthesized compounds have different anti-
coagulant activities, the most prospective compounds were the 3-pyrazolyl-4-hydroxycoumarin
derivatives.
Received 11 February 2010
Revised 1 April 2010
Accepted 2 April 2010
Available online 8 April 2010
Keywords:
4-Hydroxycoumarin
Pyridines
Ó 2010 Elsevier Ltd. All rights reserved.
Pyrimidines
Pyrazoles
Anticoagulant
PIVKA-II
1. Introduction
the effectiveness of anticoagulant therapy.13 PIVKA-II ELISA has
also been successfully used to detected uncarboxylated prothrom-
4-Hydroxycoumarin derivatives as warfarin, acenocoumarol
and phenprocoumon have been the mainstay of oral anticoagula-
tion therapy for 20 years,1 their interest as anticoagulant2–4 and
rodenticides5–8 has been documented in several reports.
Coumarin anticoagulants are considered as antivitamin K. Vita-
min K is an essential cofactor for c-carboxylation of key glutamic
acid residues in blood clotting proteins by vitamin K-dependent car-
boxylase. During catalysis, vitamin K is converted from the active
form to vitamin K 2,3-epoxide, which must be recycled to the active
form by vitamin K epoxide reductase (VKOR) to maintain the coagu-
lation cycle.9,10 4-Hydroxycoumarins antagonize VKOR, preventing
vitamin K recycling and resulting in an accumulation of abnormal
bin in culture supernatant of primary human liver cells.14
Several reports have provided evidence that human hepatoma
cell line, HepG2, secretes PIVKA-II in response to warfarin expo-
sure.15,16 It was reported in a previous study the development of
a detection system for anticoagulation coumarins17 using ELISA as-
say to detect PIVKA-II produced by HepG2 exposed to several cou-
marin compounds.
form of coagulation protein called des-c-carboxyprothrombin
(DCP) or proteins induced by vitamin K antagonism (PIVKA-II)11
(Fig. 1).
PIVKA-II has been identified as a marker of hepatocellular carci-
noma in humans. An enzyme-linked immunoassay (ELISA) con-
taining monoclonal antibody that specifically detected
uncarboxylated prothrombin12 was developed to diagnose the
hepatocellular carcinoma and is also used to clinically monitor
* Corresponding author. Tel.: +20 202 37608284; fax: +20 202 33370931.
E-mail address: ommryan@yahoo.com (O.M. Abdelhafez).
Figure 1. Coumarin anticoagulants antagonizing VKOR.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.009

3372
O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
On the other hand, Bhatia et al.,18 have studied the anticoagu-
2. Results and discussion
2.1. Chemistry
lant effects of some pyridyl chromene-2-one derivatives. In addi-
tion, condensed coumarinopyrimidines were considered useful
leads for the development of new gastric sparing antiplatelet drug
with combined anti ADP-TXA2 activities.19
We described here a convenient approach to the preparation of
4-aryl-2-amino-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-pyridin-3-
carbonitriles 4a–4c and 4-aryl-2-oxo-6-(4-hydroxy-2-oxo-2H-
chromen-3-yl)-pyridin-3-carbonitriles 5a–5c, 3-(6-aryl-1,2,5,6-
tetrahydro-2-thioxo pyrimidin-4-yl)-4-hydroxy-2H-chromen-2-
one 6a, 6b and pyrazol-3-yl-4-hydroxycoumarin derivatives 7a–
7c, 8a, 8b, 9a, 9b, 10a, 10b and 11a–11c.
The main objective of our present work depends on the hybrid-
ization of 4-hydroxycoumarin unit as anticoagulant scaffold and
some azaheterocyclic rings reported to have antithrombotic activ-
ity as pyridine20,21 pyrimidine19 and pyrazole22–24 in order to pre-
pare some new molecules (models A and B) to study their in vivo
and in vitro anticoagulant activity.
The key reactions involved the intermediate formation of chal-
cone compounds 3a–3d, a facile synthesis these synthesized
a,b-
Ar
unsaturated ketones involves condensation of 3-acetyl-4-hydroxy-
coumarin 225 with the appropriate aldehydes; 4-methoxybenzal-
dehyde, 3,4,5-trimethoxybenzaldehyde, 3-pyridincarboxaldehyde
and 2-furfural in alcohol and in the presence of piperidine.
4-Aryl-2-amino-6-(4-hydroxycoumarin-3yl)-pyridin-3-carbo-
nitriles 4a–4c were prepared by direct reaction of 3-acetyl-4-
hydroxycoumarin 2 with the appropriate aldehydes in the pres-
ence of malononitrile and ammonium acetate in one step, that is,
one pot reaction (method A) (Scheme 1) or by cyclization of the
corresponding chalcones with malononitrile and ammonium ace-
tate (method B) (Scheme 1).
X
OH
N
OH
O
O
O
A
; X=C,N
R
OH
O
N
N
Ar
4-Hydoxycoumarin Bioactive Scaffold
One the other hand when the above reactions were carried out
in the presence of ethylcyanoacetate instead of malononitrile, 4-
aryl-1,2-dihydro-6-(4-hydroxycoumarin-3yl)-2-pyridin-3-carbo-
nitriles 5a–5c were obtained (Scheme 1).
O
O
B
OH
O
O
1
CH3COCl
pyridine/piperidine
OH
COCH
3
ArCHO/CH2(CN)2/
ArCHO,/NCCH2COOC2H5/
CH3COONH4/EtOH, reflux
CH3COONH4/EtOH/reflux
O
O
2
Ar
N
Ar
Ar
CN
NH
CN
OH
OH
CN
OH
ArCHO/
2
N
H
O
piperidine
EtOH, reflux
N
OH
O
O
O
O
O
O
4a-c
5a-c
Ar=C6H2-3,4,5-(OCH3)3 , 3-pyridiyl, 2- furyl
OH
O
Ar=C6H2-3,4,5-(OCH3)3, 3-pyridiyl, 2- furyl
Ar
NCCH2COOC2H5/
CH2(CN)2/
CH3COONH4/EtOH, reflux
CH3COONH4/EtOH/reflux
O
O
3a-d
Ar=C6H4-4-(OCH3),C6H2-3,4,5-(OCH3)3 ,3-pyridiyl, 2- furyl
S
3b,c
H
N
NH
2
2
Ar
N
/KOH/EtOH, reflux
Ar
OH
O
NH
OH
O
N
S
N
SH
O
O
6a,b
Ar = C6H2-3,4,5-(OCH3)3, 3-pyridiyl
Scheme 1.

O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
3373
The target pyrimidin-2-thiones 6a, 6b were obtained in a good
yield by cyclocondensation of the ,b-unsaturated ketones 3b, 3c
200 ng/ml represents a high positive result (1.982 nm). Under the
same experimental conditions, exposure of HepG2 cells to 0.03,
a
with thiourea in 5% ethanolic potassium hydroxide solution
0.3, 3, 10 and 30 lM warfarin resulted in an approximate concen-
(Scheme 1).
tration-dependent increase in PIVKA-II in cell supernatants (0.577,
1.1135, 1.137, 2.282 and 2.342 nm, respectively). In contrast, a va-
lue of less than 2 ng/ml PIVKA-II (0.063 nm) was detected follow-
Heterocyclization of the appropriate hydrazines with electro-
philic species such as a,b-unsaturated ketones afforded the corre-
sponding pyrazolines; cyclocondensation of the key intermediates
3a–3c with hydrazine hydrate in ethyl alcohol gave pyrazolines
7a–7c. Similarly, N-acetylpyrazolines 8a, 8b were obtained by
refluxing the key chalcones 3b, 3c with hydrazine hydrate in gla-
cial acetic acid. N-Phenylpyrazolines 9a, 9b were also prepared
from the proper chalcones 3b, 3c and phenylhydrazine (Scheme 2).
Moreover, the target compounds 10a, 10b were prepared by
refluxing a mixture of chalcones 3a, 3c and thiosemicarbazide in
ethyl alcohol in the presence of glacial acetic acid (Scheme 2).
The target thioureas 11a–11c were prepared by the reaction of pyr-
azolines 7a–7c with phenyl isothiocyanate in dry acetone at room
temperature (Scheme 2). The proposed structures of all new pre-
pared compounds were confirmed by elemental analysis, IR, 1H
NMR, 13C NMR and mass spectra (Section 3).
ing exposure to 0.003 lM warfarin.
The anticoagulant activity of the synthesized coumarin deriva-
tives (4c, 5a–5c, 6a, 6b, 7a–7c, 8b, 9a, 9b, 10a, 10b and 11a–11c)
was determined at different concentrations according to their sol-
ubility using an ELISA assay that detects proteins induced by vita-
min K antagonism-II (PIVKA-II) in HepG2 cells, compounds 4a, 4b
and 8a were not tested due to their low solubility (Table 1).
PIVKA-II production was clearly detected in supernatants after
treatment with compounds 4c (30
7a (30 M), 10a (30 M), 10b (30
These tested compounds while active, were less potent than war-
farin at 0.003 M (0.577 nm). The other tested compounds gave
l
M), 5b (10
l
M), 6a (30
lM),
l
l
l
M) and 11c (30
lM) (Table 1).
l
values below the 2 ng/ml reference standard and thus are consid-
ered void of significant anticoagulant activity.
Although the relation between the structure of the tested cou-
marin-C3-linked azaheterocycles and their in vitro anticoagulant
activity results is not very clear, the following points can still be
concluded. The pyrazolyl coumarin derivatives 11c (R.P. 19.4%),
7a (R.P. 17.3%) and 10a (R.P. 18.1%) were proved to be the most ac-
tive of the tested compounds. The relative anticoagulant potency of
compound 11c was decreased to 11.2%, 11.45% and 8.7% when the
phenylcarbothioamide group in position 1 of the pyrazolyl func-
tionality was replaced with carbothioamide group (compound
10b), acetyl function (compound 8b) or phenyl nucleus (compound
9b), respectively. Pyrazolyl coumarins that contained 3,4,5-trime-
thoxyphenyl substituent in position 5 of the pyrazolyl moiety were
inactive; for example, the activity of compounds 11c and 7a was
lost by substituting the C-5 pyridine function or C-5 4-methoxy-
phenyl group on the pyrazole ring, respectively, with 3,4,5-trime-
2.2. Pharmacological screening
2.2.1. In vitro anticoagulant activity
In this study, a generic biological screening assay was designed
to detect the accumulation of under-carboxylated prothrombin or
proteins induced by vitamin K antagonism (PIVKA-II). A combined
cell culture/ELISA assay was optimized to measure PIVKA-II pro-
duction by the human hepatoma HepG2 cell line cultured in the
presence of anticoagulant coumarins.
On each ELISA plate, four reference standards (2, 50, 120 and
200 ng/ml) provided with the commercially-available kit were
tested at the same dilutions as the new chemical entities. PIVKA-
II production by HepG2 cells is considered negative if assay values
are below the 2 ng/ml reference standard (0.158 nm). A value of
OH
O
S
S
NH2CNHNH2
NH2NH2
Ar
NH
2
CH3COOH/ reflux
EtOH/ reflux
O
O
N
OH
N
Ar
NH
OH
N
Ar
O
O
10a,b
NH2NH2
O
O
Ar=C6H4-4-OCH3, 3-Pyridyl
7a-c
CH3COOH
PhNHNH2
Ph
Ar
= C6H4-4-OCH3,
C6H2-3,4,5-(OCH3)3,
3-pyridyl
EtOH/ reflux
N
N
OH
Ar
COCH
3
N
OH
N
Ar
O
O
9a,b
PhNCS
acetone/R.T.
O
O
Ar = C6H2-3,4,5-(OCH3)3,
3-pyridyl
S
8a,b
NHPh
Ar
Ar = C6H2-3,4,5-(OCH3)3,
3-pyridyl
N
OH
N
O
O
11a-c
Ar = C6H4-4-OCH3,
C6H2-3,4,5-(OCH3)3,
3-pyridyl
Scheme 2.

3374
O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
Table 1
Table 3
Screening measurement of proteins induced by vitamin K antagonism-II (PIVKA-II) as
indicator of anticoagulation activity
Effect of tested compounds and warfarin (150 mg/kg, po) on clotting time (s) and
prothrombine time (min) in mice after three days of daily treatments (mean SE,
n = 6/group)
Tested compounds
ABS (490 nm) SD
PIVKA-II
Tested compounds 150 mg/kg
CT/s
8.17 0.60
PT/min
4c-30
5a-30
5b-10
5c-30
6a-30
6b-30
7a-30
7b-30
7c-30
8b-3.3
9a-3.3
9b-30
10a-30
10b-30
11a-30
l
l
l
l
l
l
l
l
l
l
l
l
M
M
M
M
M
M
M
M
M
M
M
M
0.23425 0.003536
0.057 0.003
Yes
No
Control
4a
5a
6a
7c
8b
9b
10b
11c
Warfarin
LSD*
1.283 0.069
0.193 0.036
0.192 0.010
0.173 0.007
0.530 0.020
3.373 0.257
0.827 0.141
4.017 0.292
3.505 0.226
4.445 0.314
0.5
0.1865 0.008
0.207 0.0045
0.30725 0.00495
0.20625 0.0047
0.40625 0.012728
0.071 0.001
0.07 0.007
0.205 0.00432
0.059 0.008
0.2025 0.0058
0.4235 0.008991
0.26175 0.0047
0.0535 0.006
0.05 0.000
Weak
Weak
Yes
Weak
Yes
No
No
Weak
No
Weak
Yes
9.50 0.62
15.67 1.05
15.83 0.95
17.67 0.80
20.83 0.79
22.50 0.96
16.67 0.71
12.17 0.79
18.17 0.79
2.26
l
M
*
LSD: least significant difference.
lM
Yes
No
No
l
M
l
11b-3.3
M
11c-30
lM
0.4535 0.01879
Yes
Table 4
LD50 in male mice after oral administration of 9b
thoxyphenyl group, compounds 11b and 7b, respectively (Tables 1
and 2).
Group
No.
Oral doses (mg/
kg. b. wt.)
No. of dead
animals
Dose
difference
Meana Productb
—
Concerning the coumarinyl pyridines derivatives, compound 5b
(R.P. 10%) and 4c (R.P. 10%) showed anticoagulant activity higher
than that of the reference standard at 2 ng/ml (0.158 nm). replace-
ment of the 4-pyridinyl substituent of the pyridine functionality in
compound 5b with furan nucleus or 3,4,5-trimethoxyphenyl group
resulted in a decrease (compound 5c) or complete loss (compound
5a) of the activity.
1
2
3
4
5
6
7
Total
1200
1400
1600
1800
2000
2200
2400
—
2
4
5
6
200
200
200
200
200
200
200
1
3
200
600
4.5
5.5
7.5
9
900
1100
1500
1800
6100
8
10
The coumarinopyrimidine derivatives 6a (R.P. 13%) and 6b (R.P.
8%) showed anticoagulant activity value in PIVKA-II assay compa-
rable to that of the pyridinyl coumarin 5b and 4c, respectively (Ta-
bles 1 and 2).
No of animals/group = 10 mice.
LD50 = 2400 À (6100/10) = 1790 mg/kg. b.wt.
a
Interval mean of the number of dead animals (mice).
Product of the interval mean and the dose difference.
b
2.2.2. In vivo anticoagulant screening
This study was conducted to evaluate the in vivo effects of the
new synthesized compounds 4a, 5a, 6a, 7c, 8b, 9b, 10b and 11c
on blood coagulation time (CT) and prothrombin time (PT) in mice,
warfarin, the reference compound, was tested under the same con-
ditions. From the anticoagulant values obtained in Table 3, modest
to high activity over warfarin was observed for the pyrazolyl couma-
rin derivatives 7c, 8b, 9b, 10c and 11c. The effect of compound 7c
(CT; 17.67 s) increased by introduction of acetyl group or phenyl nu-
cleus at position 1 of the pyrazolyl functionality, compounds 8b (CT;
20.83 s) and 9b (CT; 22.50 s), respectively.
Table 2
Additionally, compounds 8b (PT; 3.373 min), 10b (PT;
4.017 min) and 11c (PT; 3.505 min) with acetyl, carbothioamide
and phenylcarbothioamide, respectively, in the pyrazolyl moiety
showed pronounced prolongation of PT with anticoagulant values
similar to that of warfarin (PT; 4.445 min) (Table3).
On the other hand, the tested pyridyl and pyrimidinyl coumarin
derivatives 5a and 6a had relatively moderate in vivo clotting
activity with CT values 15.67 and 15.83 s, both compounds and
4a showed no effect on PT.
Relative potency of positive and Weak anticoagulants
Test
compounds
Concd
Avg. Abs. SD
Relative potency% in relation
to warfarin (R.P. %)
Reference
2 ng/ml
0.158
4c
30
10
10
l
l
l
M
M
M
0.23425 0.003536 10%
0.169 0.00495
0.24875 0.009
0.1355 0.004243
0.207 0.0045
0.2055 0.0034
0.30725 0.00495
7.4%
5b
10%
*
3.33
l
M
—
5c
30
10
30
10
30
10
30
10
3.3
1.1
30
10
30
10
30
10
30
10
lM
lM
lM
lM
lM
lM
lM
lM
8.8%
9%
13%
LD50 (po) of the most active compound 9b (CT; 22.50 0.96)
was determined as 1790 mg/kg. (Table 4)26(cf. LD50 (po) of warfa-
rin 760 mg/kg).
6a
0.119 0.000707 5.2%
6b
0.20625 0.0047
0.117 0.006364
8%
—
*
7a
0.40625 0.012728 17.3%
0.4055 0.018385 17.76%
2.2.3. Conclusion
In conclusion, a series of 4-hydroxycoumarins incorporating
pyridine, pyrimidine and pyrazole nucleus linked at C-3 has been
prepared as potential anticoagulant agents. These azaheterocycles
were selected because they have historically provided improved
in vitro and/or in vivo antithrombotic potency. We have identified
that the hybrid pyrazolyl coumarins, 11c, 10b and 7a, are the most
active tested compounds. The ambiguity between in vitro (low)
and in vivo (high) anticoagulant values may be attributed to their
in vivo dual mechanism of action of coumarin pharmacophore and
pyrazole pharmacophore group. The in vitro assay abundant repre-
sents a single step in anticoagulant cascade. However, in vivo assay
8b
l
l
M
M
0.205 0.00432
0.0075 0.001
0.2025 0.0058
0.192 0.00428
11.45%
—
8.7%
8.4%
*
9b
lM
lM
lM
lM
lM
lM
lM
lM
10a
10b
11c
0.4235 0.008991 18.1%
0.3135 0.00495
0.26175 0.0047
13.7%
11.2%
0.207 0.005657 9.1%
0.4535 0.01879 19.4%
0.441 0.007778 19.3%
*
Compound showed no activity.

O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
3375
represents the total anticoagulant activity, so the in vitro data do
not represent the real state of the anticoagulation activity. We
hope that the efficient synthesis approach disclosed herein can
lead to the rapid output of a series of pyrazolylcoumarins for eval-
uation and anticoagulant mechanism studies.
or 2-furfural (5 mmol) in ethyl alcohol (20 ml), malononitrile
(0.33 g, 5 mmol) and ammonium acetate (0.75 g, 10 mmol) were
added. The reaction mixture was refluxed for 7–9 h. The obtained
solid was filtered off, washed with absolute ethyl alcohol and
recrystallized from methyl alcohol to give the desired compounds.
Method (B): An ethanolic mixture of the selected chalcones 3b–
3d (5 mmol), and malononitrile (0.33 g, 5 mmol) in the presence of
ammonium acetate (0.75 g, 10 mmol) was refluxed for 5–6 h, after
cooling, the obtained solid was filtered off, washed with ethyl alco-
hol and recrystallized from methyl alcohol to give the title
compounds.
3. Experimental
3.1. Synthesis
Melting pointswere determinedonElectrothermalIA 9000 appa-
ratus and were uncorrected. Elemental microanalysis was per-
formed on Elementar, Vario EL, at the microanalytical center,
National Research Center. The infrared (IR) spectra were recorded
on Nexus 670 FT-IR FT-Raman spectrometer as potassium bromide
discs, at National Research Center. The proton nuclear magnetic res-
onance (1H NMR) spectra were determined on Varian mercury
300 MHzspectrometer, usingtetramethylsilane (TMS)asan internal
standard, at Faculty of Science—Cairo University and National Re-
search Center. 13C NMR was carried out using 1D–13C NMR Bruker
Jeol-EX 125 MHz spectrometer using TMS as an internal standard.
The mass spectra (MS) were performed on Jeol JMS-AX500 mass
spectrometer at National Research Center. The reactions were fol-
lowed by TLC (silica gel, aluminum sheets 60 F 254, Merk) using ben-
zene/ethyl acetate (8:2 v/v) as eluent and sprayed with iodine–
potassium iodide reagent.
3.1.2.1. 2-Amino-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-4-
(3,4, 5-trimethoxy phenyl)pyridine-3-carbonitrile (4a)27
.
Mol.
for.: C24H19N3O6, M.wt. 445.42, yield 90% (method A), 83% (method
B), mp >300 °C.
3.1.2.2. 2-Amino-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-4-(pyr-
idine-3-yl)
pyridin-3-carbonitrile
(4b).
Mol.
for.:
C20H12N4O3, M.wt. 356.33, yield 88% (method A), 78% (method B),
mp >300 °C; Anal. Calcd: C, 67.41; H, 3.39; N, 15.72. Found: C,
67.21; H, 3.22; N, 15.61. IR (KBr, cmÀ1): 3392 (OH, br s), 3118,
3099 (NH2), 2210 (C„N), 1722 (C@O,
a
-pyrone), 1653 (C@N). 1H
NMR (DMSO-d6, d, ppm): 4.31 (2H, s, NH2, D2O exchangeable),
7.33–8.86 (9H, m, Ar-H and pyridine protons), 8.92 (1H, s, OH,
D2O exchangeable). 13C NMR (DMSO, d, ppm): 88.62, 98.06,
104.28, 117.40, 117.95, 121.95, 124.40, 125.95, 127.89, 128.48,
133.62, 134.92, 148.40, 149.92, 150.62, 152.31, 159.95, 162.17,
162.40, 166.73; MS; m/z (R.A. %): M+ 356 (10%), 340 (54%), 339
(48%), 338 (54%), 311 (100%).
3.1.1. Preparation of 1-(4-hydroxy-2-oxo-2H-chromen-3-yl)-3-
aryl-2-propen-1-ones (3a–3d)
A solution of 3-acetyl-4-hydroxycoumarin25 (1 g, 5 mmol) in
ethyl alcohol (10 ml) and the selected aromatic aldehyde, namely,
4-methoxybenzaldehyde, 3,4,5-trimethoxybenzaldehyde, 3-pyrid-
incarboxaldehyde or 2-furfural (5 mmol) in the presence of piper-
idine (1 ml) was refluxed for 10–12 h. The solution was cooled and
water was added to precipitate the desired chalcone compounds.
3.1.2.3. 2-Amino-4-(furan-2-yl)-6-(4-hydroxy-2-oxo-2H-chro-
men-3-yl)-pyridin-3-carbonitrile
(4c).
Mol.
for.:
C19H11N3O4, M.wt. 345.31, yield 74% (method A), 69% (method B),
mp >300 °C; Anal. Calcd: C, 66.09; H, 3.21; N, 12.17. Found: C,
65.96; H, 3.17; N, 12.15. IR (KBr, cmÀ1): 3316 (OH, br s), 3208,
3057 (NH2), 2206 (C„N), 1680 (C@O,
a
-pyrone), 1643 (C@N). 1H
3.1.1.1. 1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(4-methoxy-
NMR (DMSO-d6, d, ppm): 4.01 (2H, s, NH2, D2O exchangeable),
6.35–7.41 (3H, m, furyl protons), 7.01–7.65 (4H, m, AR-H), 7.5
(1H, s, CH-pyridine proton), 13.92 (1H, s, OH, D2O exchangeable);
13C NMR (DMSO, d, ppm): 88.62, 98.06, 104.28, 105.3, 107.52,
117.40, 117.95, 121.95, 125.95, 127.89, 128.48, 143.29, 150.62,
154.30, 152.31, 159.95, 162.17, 162.40, 166.73; MS; m/z (R.A. %):
(M++1) 345 (6%), 239 (10%), 202 (31%), 144 (12%), 92 (15%), 77
(70%), 62 (100%), 45 (64%).
phenyl)-2-propen-1-one (3a)27
.
Mol. for.: C19H14O5, M.wt.
322.31, yield 91%, mp 148–150 °C.
3.1.1.2. 1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(3,4,5-trime-
thoxyphenyl)-2-propen-1-one (3b)27
Mol. for.: C21H18O7,
M.wt. 382.36, yield 93%, mp 157–159 °C.
.
3.1.1.3. 1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(pyridin-3-yl)-
2-propen-1-one (3c). Mol. for.: C17H11NO4, M.wt. 293.27,
3.1.3. Preparation of 4-aryl-1,2-dihydro-6-(4-hydroxy-2-oxo-
2H-chromen-3-yl)-2-oxopyridin-3-carbonitriles (5a–5c)
General procedure. Method (A): To a mixture of compound 2 (1 g,
5 mmol) and the appropriate aldehyde, namely; 3,4,5-trimethoxy-
benzaldehyde, 3-pyridincarboxaldehyde or 2-furfural (5 mmol) in
ethyl alcohol (20 ml), ethyl cyanoacetate (0.57 ml, 5 mmol) and
ammonium acetate (0.75 g, 10 mmol) were added. The reaction
mixture was refluxed for 7–9 h. The obtained solid was filtered
off, washed with absolute ethyl alcohol and recrystallized from
methanol to give the desired compounds.
yield 88%, mp 190–192 °C; Anal. Calcd: C, 69.62; H, 3.78; N, 4.78.
Found: C, 69.50; H, 3.63; N, 4.58; IR (KBr, cmÀ1): 3339 (OH, br s),
3099 (–CH aliphatic stretching), 1677 (C@O,
a-pyrone), 1648
(C@O), 1607 (C@N), 1574 (C@C); 1H NMR (DMSO-d6, d, ppm):
7.03, 7.08 (2H, d, CH@CH), 7.13–8.66 (8H, m, Ar-H and pyridine
protons), 12.92 (1H, s, OH, D2O exchangeable). 13C NMR (DMSO,
d, ppm):98.92, 117.43, 122.52, 123.74, 125.11, 127.01, 128.42,
129.63, 132.92, 133.43, 148.92, 150.10, 150.32, 152.71, 160.03,
179.75, 183.84; MS; m/z (R.A. %): (M++1) 292 (100%).
Method (B): An ethanolic mixture of chalcones 3b–3d (5 mmol),
and ethyl cyanoacetate (0.57 ml, 5 mmol) in the presence of
ammonium acetate (0.7 g, 10 mmol) was refluxed for 5–6 h, after
cooling, the obtained solid was filtered off, washed with ethyl alco-
hol and recrystallized from methyl alcohol to give the title
compounds.
3.1.1.4. 1-(4-Hydroxy-2-oxo-2H-chromen-3-yl)-3-(furan-2-yl)-
2-propen-1-one (3d)28
.
Mol. for.: C16H10O5, M.wt. 282.25,
yield 65%, mp 204–205 °C.
3.1.2. Preparation of 4-aryl-2-amino-6-(4-hydroxy-2-oxo-2H-
chromen-3-yl)-pyridin-3-carbonitriles (4a–4c)
General procedure. Method (A): To a mixture of 3-acetyl-4-
hydroxycoumarin (1 g, 5 mmol) and the appropriate aldehyde,
namely; 3,4,5-trimethoxybenzaldehyde, 3-pyridincarboxaldehyde
3.1.3.1. 1, 2-Dihydro-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-4-
(3,4,5-trimethoxyphenyl)-2-oxopyridin-3-carbonitrile (5a). Mol.
for.: C24H18N2O7, M.wt. 446.41, yield 75% (method A), 63% (method

3376
O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
B), mp 223–225 °C; Anal. Calcd: C, 64.57; H, 4.06; N, 6.28. Found: C,
MS; m/z (R.A. %):M+ 440 (28%), 395 (16%), 290 (17%), 244 (36%),
121 (100%).
64.34; H, 3.99; N, 6.09. IR (KBr, cmÀ1): 3264 (OH, br s), 3043 (NH),
2364 (C„N), 1697 (C@O, a
-pyrone), 1612 (C@O). 1H NMR (DMSO-
d6, d, ppm): 3.82 (9H, s, CH3), 5.91 (1H, s, oxopyridine proton),
6.83–7.56 (6H, m, AR-H), 8.01 (1H, s, NH, D2O exchangeable), 11.81
(1H, s, OH, D2O exchangeable); 13C NMR (DMSO, d, ppm): 56.22,
56.22, 56.75, 95.22, 99.19, 104.19, 104.19, 115.43, 115.71, 117.75,
121.75, 125.75, 126.80, 127.19, 128.64, 138.19, 138.63, 150.23,
150.70, 150.70, 158.53, 159.54, 161.86, 166, 92; MS; m/z (R.A. %):
(M++1) 447 (12%), 379 (18%), 281 (25%), 181 (61%), 121 (33%), 78
(83%), 63 (75%), 43 (100%).
3.1.4.2. 3-[1,2,5,6-Tetrahydro-6-(pyridin-3-yl)-2-thioxopyrimi-
din-4-yl]-4-hydroxy-2H-chromen-2-one (6b).
Mol. for.:
C18H13N3O3S, M.wt. 351.38, yield 80%, mp 235–237 °C; Anal. Calcd:
C, 61.53; H, 3.73; N, 11.96; S, 9.13. Found: C, 61.03; H, 3.33; N,
11.77; S, 9.07. IR (KBr, cmÀ1): 3331 (OH, br s), 3208 (NH), 1714
(C@O,
a
-pyrone), 1299 (C@S). 1H NMR (DMSO-d6, d, ppm): 2.00
(1H, s, NH, D2O exchangeable), 3.81 (1H, dd, CH-pyrimidine),
4.42 (1H, dd, CH-pyrimidine), 4.75 (1H, dd, CH-pyrimidine),
7.20–8.63 (8H, m, Ar-H and pyridinyl protons), 15.31 (1H, s, OH,
D2O exchangeable); 13C NMR (DMSO, d, ppm): 35.06, 52.31,
81.81, 117.23, 121.65, 123.15, 125.15, 126.56, 128.22, 133.32,
140.84, 147.06, 148.35, 150.02, 159.13, 165.03, 181.05, 186.56;
MS; m/z (R.A. %): (M++1) 352 (100%), 337 (7%), 290 (47%), 245
(52%), 235 (43%), 213 (31%), 119 (71%), 76 (100%).
3.1.3.2. 1,2-Dihydro-6-(4-hydroxy-2-oxo-2H-chromen-3-yl)-2-
oxo-4-(pyridine-3-yl) pyridin-3-carbonitrile (5b).
Mol. for.:
C20H11N3O4, M.wt. 357.32, yield 70% (method A), 68% (method B),
mp 219–222 °C; Anal. Calcd: C, 67.23; H, 3.10; N, 11.76. Found:
C, 67.21; H, 3.02; N, 11.58. IR (KBr, cmÀ1): 3449 (OH, br s), 3057
(NH), 2739 (C„N), 1739 (C@O,
a
-pyrone), 1692 (C@O). 1H NMR
(DMSO-d6, d, ppm): 5.99 (1H, s, oxopyridine proton), 7.33–8.41
(7H, m, AR-H and pyridinyl protons), 8.50 (1H, s, CH-pyridine pro-
ton), 8.61 (1H, s, NH, D2O exchangeable), 10.71 (1H, s, OH, D2O
exchangeable); 13C NMR (DMSO, d, ppm): 95.22, 99.19, 115.43,
117.75, 121.32, 121.75, 124.15, 125.75, 126.90, 127.19, 133.42,
133.72, 138.19, 149.81, 150.01, 150.25, 158.53, 159.64, 161.86,
166.92; MS; m/z (R.A. %): (M++1) 358 (0.9%), 201 (33%), 146
(19%), 120.5 (21%), 61 (24%), 60 (98%), 62 (100%), 43 (84%).
3.1.5. Preparation of 3-(5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-4-
hydroxy-2H-chromen-2-one derivatives (7a–7c)
A mixture of the appropriate chalcones 3a–3c (1 mmol) and
hydrazine hydrate 99% (2 mmol) in ethyl alcohol (30 ml) was re-
fluxed for 1 h. The reaction mixture was cooled and the formed
precipitate was filtered off, washed and recrystallized from methyl
alcohol to give 7a–7c compounds.
3.1.5.1. 3-[4,5-Dihydro-5-(4-methoxyphenyl)-1H-pyrazol-3-yl]-
3.1.3.3.
4-(Furan-2-yl)-1,2-dihydro-6-(4-hydroxy-2-oxo-2H-
Mol. for.:
4-hydroxy-2H-chromen-2-one
C19H16N2O4, M.wt. 336.34, yield 81%, mp 196–198 °C.
(7a)27
.
Mol.
for.:
chromen-3-yl)-2-oxopyridin-3-carbonitrile (5c).
C19H10N2O5, M.wt. 346.29, yield 74% (method A), 69% (method
B), mp 228–230 °C; Anal. Calcd: C, 65.90; H, 2.91; N, 8.09. Found:
C, 65.82; H, 2.78; N, 8.11. IR (KBr, cmÀ1): 3262 (OH, br s), 3098
3.1.5.2. 3-[4,5-Dihydro-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-
3-yl]-4-hydroxy-2H-chromen-2-one Mol. for.:
(7b)27
C21H20N2O6, M.wt. 396.39, yield 80%, mp 185–187 °C.
.
(NH), 2354 (C„N), 1696 (C@O,
a
-pyrone), 1658 (C@O). 1H NMR
(DMSO-d6, d, ppm): 5.09 (1H, s, oxopyridine proton), 6.73–8.01
(7H, m, AR-H and furyl protons), 8.63 (1H, s, NH, D2O exchange-
able), 12.71 (1H, s, OH, D2O exchangeable); 13C NMR (DMSO, d,
ppm): 95.22, 99.19, 111.54, 112.97, 115.43, 117.75, 121.32,
121.75, 125.75, 126.90, 127.19, 138.19, 145.91, 149.82, 150.21,
158.53, 159.64, 161.86, 166.42; MS; m/z (R.A. %): (M++2) 348
(1%), 322 (19%), 279 (4%), 187 (25%), 163 (85%), 146 (61%).
3.1.5.3.
3-[4,5-Dihydro-5-(pyridine-3-yl)-1H-pyrazol-3-yl]-4-
Mol. for.: C17H13N3O3,
hydroxy-2H-chromen-2-one (7c).
M.wt. 307.30, yield 79%, mp 233–235 °C; Anal. Calcd: C, 66.44; H,
4.26; N, 13.67. Found: C, 66.13; H, 4.06; N, 13.62. IR (KBr, cmÀ1):
3430 (OH, br s), 3187 (NH), 1675 (C@O,
a-pyrone), 1603 (C@N).
1H NMR (DMSO-d6, d, ppm): 3.81 (1H, dd, H-pyrazoline), 4.04
(1H, dd, H-pyrazoline), 5.36 (1H, dd, H-pyrazoline), 6.86 (1H,
s,NH), 7.02–8.84 (8H, m, Ar-H and pyridinyl protons), 14.23 (1H,
s, OH, D2O exchangeable); 13C NMR (DMSO, d, ppm): 41.42,
49.23, 88.05, 117.56, 121.65, 123.15, 125.05, 126.56, 128.42,
133.35, 140.24, 147.03, 148.31, 150.22, 156.02 159.13, 165.09;
MS; m/z (R.A. %): (M++1) 308 (100%).
3.1.4. Preparation of 4–3-(6-aryl-1,2,5,6-tetrahydro-2-
thioxopyrimidin-4-yl)-4-hydroxy-2H-chromen-2-one
derivatives (6a, 6b)
To a mixture of the selected chalcones 3b, 3c (1 mmol) and thio-
urea (0.3 g, 4 mmol) in ethyl alcohol (25 ml), a solution of potas-
sium hydroxide (0.4 g) in water (4 ml) was added and the
reaction mixture was refluxed for 10–12 h. The solvent was evap-
orated under vacuum till dryness and the residue was acidified
with dilute hydrochloric acid, the formed solid was filtered off
and crystallized from methyl alcohol to give compounds 6a, 6b.
3.1.6. Preparation of 3-(5-aryl-1-acetyl-4,5-dihydro-1H-pyrazol-
3-yl)-4-hydroxy-2H-chromen-2-ones (8a, 8b)
A mixture of chalcones 3b, 3c (1 mmol) and hydrazine hydrate
99% (2 mmol) in glacial acetic acid (30 ml) was refluxed for 30–
45 min. The reaction mixture was cooled and diluted with water;
the formed precipitate was filtered off, washed and recrystallized
from ethyl alcohol to give compounds 8a, 8b.
3.1.4.1. 3-[1,2,5,6-Tetrahydro-6-(3,4,5-trimethoxyphenyl)-2-thi-
oxopyrimidin-4-yl]-4-hydroxy-2H-chromen-2-one
(6a).
Mol. for.: C22H20N2O6S, M.wt. 440.47, yield 75%, mp
3.1.6.1. 3-[1-Acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1H-
pyrazol-3-yl]-4-hydroxy-2H-chromen-2-one
(8a)27
.
Mol.
242–245 °C; Anal. Calcd: C, 59.99; H, 4.58; N, 6.36; S, 7.28. Found:
C, 59.68; H, 4.55; N, 6.11; S, 7.20. IR (KBr, cmÀ1): 3558 (OH, br s),
for.: C23H22N2O7, M.wt. 438.43, yield 82%, mp 203–204 °C.
3174 (NH), 2930 (–CH aliphatic stretching), 1716 (C@O, a-pyrone),
1240 (C@S). 1H NMR (DMSO-d6, d, ppm): 1.71 (1H, s, NH, D2O
exchangeable), 3.42 (1H, dd, CH-pyrimidine), 3.98 (9H, s, 3OCH3),
4.34 (1H, dd CH-pyrimidine), 4.76 (1H, dd, CH-pyrimidine), 6.62–
8.23 (6H, m, Ar-H), 15.31 (1H, s, OH, D2O exchangeable); 13C
NMR (DMSO, d, ppm): 35.02, 53.41 56.23, 56.23, 56.75, 82.21,
104.31, 104.31, 117.53, 121.75, 125.45, 126.80, 128.24, 137.84,
138.21, 150.72, 150, 72, 151.01, 159.43, 165.33, 181.01, 188.06;
3.1.6.2. 3-[1-Acetyl-4,5-dihydro-5-(pyridin-3-yl)-1H-pyrazol-3-
yl]-4-hydroxy-2H-chromen-2-one (8b). Mol. for.:
C19H15N3O4, M.wt. 349.34, yield 87%, mp 218–220 °C; Anal. Calcd:
C, 65.32; H, 4.33; N, 12.03. Found: C, 65.11; H, 4.31; N, 12.24. IR
(KBr, cmÀ1): 3410 (OH, br s), 2941 (–CH aliphatic stretching),
1718 (C@O,
(DMSO-d6, d, ppm): 2.21 (3H, s, COCH3), 3.85 (1H, dd, H-pyrazo-
a
-pyrone), 1663 (C@O), 1617 (C@N). 1H NMR

O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
3377
line), 4.64 (1H, dd, H-pyrazoline), 5.56 (1H, dd, H-pyrazoline),
7.08–8.55 (8H, m, Ar-H and pyridinyl protons), 14.93 (1H, s, OH,
D2O exchangeable); 13C NMR (DMSO, d, ppm): 23.08, 38.32,
59.34, 88.21, 117.49, 121.15, 123.35, 125.45, 126.52, 128.41,
133.39, 140.84, 147.02, 148.51, 150.04, 156.12 159.83, 166.19,
168.32; MS; m/z (R.A. %): (M++1) 350 (100%).
(8H, m, Ar-H), 14.61 (1H, s, OH, D2O exchangeable); 13C NMR
(DMSO, d, ppm): 38.55, 66.74, 88.15, 117.53, 121.50, 123.43,
125.52, 126.82, 128.42, 133.71, 140.77, 147.04, 148.54, 150.23,
155.64, 159.53, 161.19, 175.96; MS; m/z (R.A. %): (M+) 366 (6%),
365 (6%), 311 (100%), 301 (6%), 285 (87%), 284 (63%), 182 (32%).
3.1.9. Preparation of 5-Aryl-4,5-dihydro-3-(4-hydroxy-2-oxo-
2H-chromen-3-yl)-N-phenyl-pyrazol-1-carbothioamide (11a–
11c)
A mixture of 7a–7c (2 mmol) and phenylisothiocyanate (0.13 g,
2 mmol) in dry acetone and few drops triethylamine was stirred at
room temperature for 10–12 h. The formed precipitate was col-
lected by filtration, washed with dry acetone and crystallized from
methyl alcohol to give the desired compounds.
3.1.7. Preparation of 3-(5-aryl-4,5-dihydro-1-phenyl-1H-
pyrazol-3-yl)-4-hydroxy-2H-chromen-2-one derivatives (9a, 9b)
A mixture of the selected chalcones 3b, 3c (1 mmol) and phenyl
hydrazine (0.1 ml, 1 mmol) in ethyl alcohol (30 ml) was refluxed
for 1 h. The reaction mixture was cooled and the formed precipi-
tate was filtered off, washed and recrystallized from methyl alco-
hol to give the desired compounds.
3.1.7.1.
1H-pyrazol-3-yl]-4-hydroxy-2H-chromen-2-one
(9a)27
Mol. for.: C27H24N2O6, M.wt. 472.49, yield 95%, mp
191–192 °C.
3-[4,5-Dihydro-5-(3,4,5-trimethoxyphenyl)-1-phenyl-
3.1.9.1. 4,5-Dihydro-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-
5-(4-methoxyphenyl)-N-phenylpyrazol-1-carbothioamide
.
(11a).
Mol. for.: C26H21N3O4S, M.wt. 471.53, yield 88%, mp
208–210 °C; Anal. Calcd: C, 66.23; H, 4.49; N, 8.91; S, 6.80. Found:
C, 66.09; H, 4.41; N, 8.41; S, 6.56. IR (KBr, cmÀ1): 3433 (OH, br s),
3.1.7.2. 3-[4,5-Dihydro-1-phenyl-5-(pyridin-3-yl)-1H-pyrazol-3-
yl]-4-hydroxy-2H-chromen-2-one (9b). Mol. for.:
C23H17N3O3, M.wt. 383.4, yield 97%, mp 218–220 °C; Anal. Calcd:
3300 (NH), 2926 (–CH aliphatic stretching), 1721 (C@O,
a-pyr-
one), 1611 (C@N), 1249 (C@S). 1H NMR (DMSO-d6, d, ppm):
3.62 (1H, dd, H-pyrazoline), 3.89 (3H, s, OCH3), 4.33 (1H, dd, H-
pyrazoline), 5.94 (1H, dd, H-pyrazoline), 6.64–7.45 (13H, m, Ar-
H), 8.01 (1H, s, NH, D2O exchangeable), 12.05 (1H, s, OH, D2O
exchangeable); 13C NMR (DMSO, d, ppm): 38.56, 55.93, 66.97,
88.01, 114.12, 114.12, 117.52, 121.53, 124.84, 125.54, 126.53,
126.53, 126.86, 128.01, 128.01, 128.45, 129.12, 129.12, 135.85,
137.17, 150.24, 155.65, 158.72, 159.51, 166.13, 179.6; MS; m/z
(R.A. %): (M+) 471 (0.1%), 336 (31%), 335 (38%), 229 (100%), 135
(94%), 121 (82%).
C, 72.05; H, 4.47; N, 10.96. Found: C, 72.35; H, 4.39; N, 10.82. IR
(KBr, cmÀ1): 3421 (OH, br s), 1715 (C@O,
a-pyrone), 1605 (C@N).
1H NMR (DMSO-d6, d, ppm): 3.83 (1H, dd, H-pyrazoline), 4.24
(1H, dd, H-pyrazoline), 5.36 (1H, dd, H-pyrazoline), 6.98–8.15
(13H, m, Ar-H), 14.03 (1H, s, OH, D2O exchangeable); 13C NMR
(DMSO, d, ppm): 38.35, 53.82, 88.05, 113.46, 113.46 117.27,
117.53, 121.65, 123.45, 125.55, 126.76, 128.42, 129.70, 129.70,
133.45, 140.73, 143.90, 147.42, 148.66, 150.21, 155.72, 159.63,
166.18; MS; m/z (R.A. %): (M+) 383 (35%), 381 (23%), 307 (17%),
241 (17%), 183 (22%), 76 (67%), 69 (52%), 63 (100%).
3.1.9.2. 4,5-Dihydro-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-5-
(3,4,5-trimethoxyphenyl)-N-phenylpyrazol-1-carbothioamide
3.1.8. Preparation of 5-aryl-4,5-dihydro-3-(4-hydroxy-2-oxo-
2H-chromen-3-yl)-pyrazol-1-carbothioamide (10a, 10b)
A mixture of the selected chalcones 3a, 3c (1 mmol) in absolute
ethyl alcohol (20 ml) and thiosemicarbazide (0.09 g, 1 mmol) in gla-
cial acetic acid (5 ml) was refluxed for 24 h. The reaction mixture
was cooled and poured onto crushed ice; the formed solid was fil-
tered off, washed and crystallized from ethyl alcohol to give the title
compounds.
(11b).
Mol. for.: C28H25N3O6S, M.wt. 531.58, yield 82%, mp
215–217 °C; Anal. Calcd: C, 63.26; H, 4.74; N, 7.90; S, 6.03. Found:
C, 63.11; H, 4.71; N, 7.66; S, 6.13. IR (KBr, cmÀ1): 3435 (OH, br s),
3290 (NH), 2936 (–CH aliphatic stretching), 1721 (C@O, a-pyrone),
1614 (C@N), 1235 (C@S). 1H NMR (DMSO-d6, d, ppm): 3.42 (1H, dd,
H-pyrazoline), 3.89 (9H, s, OCH3), 4.03 (1H, dd, H-pyrazoline), 5.44
(1H, dd, H-pyrazoline), 6.54–7.05 (11H, m, Ar-H), 7.76 (1H, s, NH,
D2O exchangeable), 13.35 (1H, s, OH, D2O exchangeable); 13C
NMR (DMSO, d, ppm): 38.51, 56.53, 56.53, 56.53, 67.57, 88.01,
114.23, 114.23, 117.52, 121.54, 124.72, 125.52, 126.53, 126.53,
126.76, 128.31, 129.12, 129.12, 137.17, 137.24, 137.84, 150.25,
150.62, 150.62, 155.64, 159.56, 166.12, 179.6; MS; m/z (R.A. %):
(M+À2) 529 (0.8%), 392 (4%), 226 (9%), 174 (5%), 106 (15%), 77
(28%), 44 (100%).
3.1.8.1. 4,5-Dihydro-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-5-
(4-methoxyphenyl) pyrazol-1-carbothioamide (10a).
Mol.
for.: C20H17N3O4S, M.wt. 395.43, yield 60%, mp 133–135 °C; Anal.
Calcd: C, 60.75; H, 4.33; N, 10.63; S, 8.11. Found: C, 60.39; H,
4.15; N, 10.61; S, 8.09. IR (KBr, cmÀ1): 3359 (OH, br s), 3171,
3046 (NH2), 1698 (C@O,
a
-pyrone), 1618 (C@N), 1243 (C@S); 1H
NMR (DMSO-d6, d, ppm): 2.10 (2H, s, NH2, D2O exchangeable),
3.70 (3H, s, OCH3), 3.74 (1H, dd, H-pyrazoline), 4.04 (1H, dd, H-pyr-
azoline), 5.32 (1H, dd, H-pyrazoline), 6.78–7.15 (8H, m, Ar-H),
14.30 (1H, s, OH, D2O exchangeable); 13C NMR (DMSO, d, ppm):
38.55, 56.02, 66.72, 88.05, 114.16, 114.16, 117.57, 121.60, 125.56,
126.76, 128.02, 128.02, 128.43, 134.95, 150.21, 155.62, 158.71,
159.63, 161.16, 175.96; MS; m/z (R.A. %): (M+) 395 (100%).
3.1.9.3. 4,5-Dihydro-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-N-
phenyl-5-(pyridin-3-yl)
pyrazol-1-carbothioamide
(11c). Mol. for.: C24H18N4O3S, M.wt. 442.49, yield 89%, mp
210–213 °C; Anal. Calcd: C, 65.14; H, 4.10; N, 12.66; S, 7.25. Found:
C, 65.11; H, 3.99; N, 12.44; S, 7.15. IR (KBr, cmÀ1): 3397 (OH, br s),
3061 (NH), 1703 (C@O,
a
-pyrone), 1610 (C@N), 1229 (C@S). 1H
NMR (DMSO-d6, d, ppm): 3.62 (1H, dd, H-pyrazoline), 4.23 (1H,
dd, H-pyrazoline), 6.15 (1H, dd, H-pyrazoline), 7.23–8.44 (13H,
m, Ar-H), 8.61 (1H, s, NH, D2O exchangeable), 10.45 (1H, s, OH,
D2O exchangeable); 13C NMR (DMSO, d, ppm): 38.56, 66.97,
88.01, 117.52, 117.52, 121.53, 123.84, 125.54, 126.53, 126.53,
126.86, 128.4, 128.4, 129.12, 129.12, 133.85, 137.17, 140.4, 148.5,
150.24, 155.65, 159.72, 166.13, 179.6; MS; m/z (R.A. %): (M++1)
443 (0.18%), 228 (11%), 194 (18%), 135 (55%), 93 (90%), 77
(100%), 51 (56%).
3.1.8.2. 4,5-Dihydro-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-5-
(pyridin-3-yl) pyrazol-1-carbothioamide (10b).
Mol. for.:
C18H14N4O3S, M.wt. 366.39, yield 82%, mp 215–217 °C; Anal.
Calcd: C, 59.01; H, 3.85; N, 15.29; S, 8.75. Found: C, 58.98; H,
3.81; N, 15.18; S, 8.55. IR (KBr, cmÀ1): 3412 (OH, br s), 3168,
3004 (NH2), 2928 (–CH aliphatic stretching), 1703 (C@O,
a-pyr-
one), 1609 (C@N), 1250 (C@S); 1H NMR (DMSO-d6, d, ppm):
2.121 (2H, s, NH2, D2O exchangeable), 3.75 (1H, dd, H-pyrazoline),
4.14 (1H, dd, H-pyrazoline), 5.36 (1H, dd, H-pyrazoline), 6.68–8.85

3378
O. M. Abdelhafez et al. / Bioorg. Med. Chem. 18 (2010) 3371–3378
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Seventeen 4-
hydroxycoumarin derivatives were tested for their PIVKA-II pro-
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3.2.1.2. In vivo anticoagulant activity.
The experiment was
carried out on 60 albino male mice weighing from 20 to 25 g.
The mice were divided into 10 equal groups of six mice each. Mice
within group (1) were kept as control and received propylene gly-
col in distilled water only, whereas those of groups (2) to (9) were
given the tested compounds 4a, 5a, 6a, 7c, 8b, 9b, 10b and 11c and
mice in the last group (10) were given warfarin as the standard
control. The tested compounds and warfarin suspended in propyl-
ene glycol were administered orally with daily dose 150 mg/kg for
three successive days. Blood samples were taken after 24 h from
the last injection and coagulation time (CT)29 and prothrombin
time (PT)30 were determined.
References and notes
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