Antimicrobial, antitumor and 5α-reductase inhibitor activities of some hydrazonoyl substituted pyrimidinones

Article abstract of DOI:10.1016/j.ejmech.2010.09.026

A series of 2-[N-aryl-2-oxo-2-(4-chlorophenyl)ethanehydrazonoyl]-6-methyl- 4(3H)-pyrimidinones 5 were prepared by coupling the diazonium salt of aniline derivatives with 2-(4-chlorobenzoylmethylene)-6-methyl-4(3H)-pyrimidinone 4 in sodium hydroxide soluti

Full text of DOI:10.1016/j.ejmech.2010.09.026

European Journal of Medicinal Chemistry 45 (2010) 5702e5707
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antimicrobial, antitumor and 5a-reductase inhibitor activities of some
hydrazonoyl substituted pyrimidinones
,
b
c
d
Mastoura M. Edrees a , Thoraya A. Farghaly , Fatma A.A. El-Hag , Mohamed M. Abdalla
*
a National Organization for Drug Control & Research, P.O. Box: 29, Cairo, Egypt
b Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
c Department of Chemistry of Natural and Microbial Products, National Research Center, Dokki, Cairo, Egypt
d MAPCO Pharmaceuticals Ltd., Balteem, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of 2-[N-aryl-2-oxo-2-(4-chlorophenyl)ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinones 5
Received 10 May 2010
Received in revised form
31 August 2010
Accepted 1 September 2010
Available online 17 September 2010
were prepared by coupling the diazonium salt of aniline derivatives with 2-(4-chlorobenzoylmethylene)-
6-methyl-4(3H)-pyrimidinone 4 in sodium hydroxide solution. The structures of these newly synthesized
compounds were confirmed by IR, NMR, mass spectrometry and elemental analyses and the tautomeric
structure of these compounds was discussed. All the newly synthesized compounds were screened for
their antibacterial and antifungal activities, some of which exhibited moderate activity. Also, the above
compounds were evaluated for their antitumor activity against a panel of 60 human tumor cell lines by
the National Cancer Institute (NCI), USA. Compounds 5b, 5d and 5i showed good cytotoxic activities
against the tested cell lines. In addition, the newly synthesized compounds were screened for their 5a-
reductase inhibitor activity and all the tested compounds showed activities in descending order as
follows 5b, 5c, 5g, 5j, 5d, 5h, 5f, 5e and 5i.
Keywords:
Antimicrobial activity
Antitumor activity
Hydrazones
Pyrimidinone
5a-Reductase inhibitor activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
activities. We design the compounds in this study based on the
synthesis of new pyrimidinone derivatives with certain co-
The pyrimidinone skeleton exists in many natural and
synthetic biologically active materials and its derivatives are
applied in various pharmaceutical and biochemical fields [1,2]. It is
of great interest that specifically functionalized pyrimidinones
may possess specific biological properties. For example, it was
recently found that some of pyrimidinones carrying an arylidene
moiety have potential as antitumor agents [3], some of these
analogues also showed, besides broad-spectrum antitumor
activity, a distinctive pattern of selectivity toward individual cell
lines, such as that of leukemia. In addition, hydrazones are known
as very important class of organic compounds, some of which
show significant biological activities, such as analgesic, anticancer,
immunomodulating, antitumor, anti-inflammatory, antibacterial,
and antifungal activities [4e9]. In continuation to our previous
work [10], we wish to report herein synthesis of a new series
of hydrazones namely, 2-{[N-aryl-2-oxo-2-(4-chlorophenyl)]
ethanehydrazonoyl}]-6-methyl-pyrimidin-4(3H)-ones 5 and study
their tautomeric structures and their biological activities like
planarity characters so that they can bind and interact with the
grooves in the DNA and RNA-transcriptase interfering with the
cancer gene coding and preventing cancer cell replication.
Also, we aimed to replace the triazine moiety in the clinical
worldwide used anstrazoleÒ, a 5
a-reductase inhibitor, by its bio-
isoster pyrimidine and try to assemble its electronic and inter-
atomic distance in the synthesized compounds 5aej.
2. Results and discussion
The required compound 4 was prepared from 6-methyl-2-
thiouracil as described by Hurst et al. [11]. Reaction of compound 4
with diazotized anilines in ethanolic sodium hydroxide solution at
0e5 ꢀC afforded the corresponding arylhydrazono derivatives 5aej
(Scheme 1). The mass spectra of products 5aej revealed the
molecular ion peaks at the expected m/z values, with relative
intensities varying from 37 to 80%. Compound 5 can exist in one or
more of five tautomeric structures AeE (Chart 1). Infrared spectral
data (see Experimental) appear to be more consistent with the
hydrazone structure (A or B) rather than the keto-azo form C, enol-
azo tautomeric form D or the CH-azo form E (Chart 1). For example,
all compounds exhibit two carbonyl bands in the regions
antibacterial, antifungal, antitumor and 5a-reductase inhibitor
* Corresponding author. Tel.: þ20 2 237125836.
E-mail address: mmohamededrees@yahoo.com (M.M. Edrees).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.026

M.M. Edrees et al. / European Journal of Medicinal Chemistry 45 (2010) 5702e5707
5703
Table 1
O
O
CO-CH2-Br
NaOH
UV spectra of coupling compounds 5aej.
H
H
S
N
N
O
+
Compd. no.
lmax (log 3)
\
20min.
SH
H3C
N
1
H3C
N
5a
5b
5c
5da
5e
5f
393 (4.52), 305 (4.00)
401 (4.21), 307 (3.81)
399 (4.01), 306 (3.65)
392 (3.99), 304 (3.80)
393 (4.57), 299 (3.85)
389 (4.22), 298 (3.74)
403 (3.01), 311 (4.23)
390 (4.30), 305 (4.00)
390 (4.12), 305 (4.05)
394 (4.15), 285 (4.20)
2
Cl
3
Cl
_
+
Ph2O
N=N Cl
O
H
N
O
O
H
H3C
N
N
5g
5h
5i
X
O
N
o
NaOH \ 0 C
NH
H3C
N
Cl
4
5j
5
Cl
a
Solvent: lmax (log
3
): acetic acid 396 (4.11), 300 (4.25); chlo-
X
roform 394 (4.50), 300 (4.60); ethanol 392 (4.27), 302 (4.44);
cyclohexane 394 (4.00), 304 (3.98).
X: a, 4-OCH3; b, 4-CH3; c, 3-CH3; d, H; e,4-Cl; f,3-Cl; g, 3-NO2;
h, 4-NO2; i, 4-COCH3; j, 4-COOEt
a representative example of the two series studied (aec: electron
donating substituents X; eej: electron-withdrawing substituents
X) was recorded in solvents of different polarities. The spectra
showed little, if any, difference in shift (Table 1). The small shifts in
lmax of 5d in different solvents are due to soluteesolvent interac-
tions. In agreement with this conclusion, is the observation that the
spectra of aryl hydrazones derived from the reaction of quinones
with N-alkyl-N-phenylhydrazines, are largely independent of the
solvent polarity, unlike those of o- and p-hydroxyazo compounds
[12]. This finding, while it excludes the azo tautomeric forms C, D
and E, indicates that each of compounds 5aej exists in one tauto-
meric form, namely A or B (Chart 1).
Scheme 1. Synthesis of compounds 5aej.
1690e1660 and 1659e1617 cmꢁ1 corresponding to the stretching
vibrations of the pyrimidinone and the benzoyl carbonyl groups,
respectively. The low value of the wave number assigned for the
latter CO stretching band in compounds 5aej seems to result from
strong chelation with NH and conjugation with the C]N double
bond as required by hydrazone form A or B [12]. This finding
excludes the enol-azo form D.
To elucidate the actual tautomeric form of the studied
compounds 5aej, the electronic absorption spectra were measured.
The data are summarized in Table 1. As shown, each of compounds
5aej (in dioxane) exhibits two characteristic absorption bands in
the regions 403e389 and 311e285 nm. Such an absorption pattern
is similar to that of some typical hydrazone chromophores
[10a,13,14]. The spectrum of 5d (unsubstituted phenyl ring), as
Finally, to distinguish between the two forms A and B, 13C NMR
of compounds 5aed and 5gej were recorded. The data showed
signals for the carbonyl carbon resonance at
d
¼ 160e164 ppm. This
chemical shift suggests that N-(3) near to C]O is sp3-hybridized
(pyrrole type as in compound 6) and different from the sp2-
hybridized carbon C-4, which appears at 170e175 ppm (compound
7) (Chart 2) [15e19]. Based on the above finding, we conclude that
the isolated products are found in each case in one tautomeric form
namely, A rather than B.
O
O
H
N
O
3. Pharmacology
N
O
H3C
N
H3C
N
H
3.1. Antimicrobial and antifungal activities
N
NH
Cl
N
NH
Cl
The novel compounds 5 were screened for their antibacterial
5A
and antifungal properties using agar well diffusion method [20].
5B
X
O
X
160 - 164
O
H
O
N
H
O
H
H
190.33 - 192.53
N
O
O
N
H3C
N
H
H3C
N
N
N
Ar
H
Me
N
Cl
N
N
N
Cl
5D
5C
5
N
Ar'
O
X
H
O
X
N
H
161-164
O
170-175
O
Me
R'
H3C
N
N
N
N
N
Cl
N
5E
N
Me
7
R'
6
X
Chart 1. Tautomeric structures of compound 5.
Chart 2. The 13C NMR chemical shift.

5704
M.M. Edrees et al. / European Journal of Medicinal Chemistry 45 (2010) 5702e5707
Table 2
Table 3
Antimicrobial activity of compounds 5.
Developmental therapeutics program one-dose mean graph.
Compd. no.
Micro-organism/IZD (cm)a
Panel/cell line
Growth percent
Growth percent
Growth percent
AF
PI
SR
CA
SA
PA
BS
EC
5b
5d
5i
5a
5b
5c
5d
5e
5f
5g
5h
5i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
þþ
þþ
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
þþ
ꢁ15.08
108.59
91.74
98.62
85.08
84.35
66.03
50.74
52.73
39.84
96.57
nd
nd
0
115.14
87.61
84.16
91.91
62.16
þþ
0
þ
þ
þ
0
þ
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
0
0
0
0
0
100.03
91 .46
94.24
92.54
103.39
104.07
88.71
108.67
105.18
107.24
108.76
nd
94.61
105.04
116.72
97.73
111.00
86.10
100.21
103.13
84.76
5j
0
þþ
0
þþ
CAb
TEc
þþþ
þþþ
þþþ
þþþ
þþþ
þþ
90.99
a
107.41
111 .33
106.17
105.30
nd
50 mL of solution in DMSO whose concentration 5, 2.5, 1.0
Chloramphenicol as standard antibacterial agent.
Terbinafin as standard antifungal agent. þþþ, inhibition value 1.5e1.1 cm; þþ,
mg/mL was tested.
b
c
inhibition value 0.6e1.0 cm; þ, inhibition value 0.1e0.5 cm beyond control; 0, no
inhibition detected.
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
116.23
115.84
86.94
110.86
110.66
114.61
93.70
105.71
109.10
86.88
112.06
105.27
101.95
94.56
121.73
111.21
90.39
The antibacterial activity of the testing compounds was evaluated
against four Gram-Positive and Gram-Negative bacterial species;
Staphylococcus aureus SA, Pseudomonas aeruginosa PA, Bacillus
subtilis BS, and Escherichia coli EC. Chloramphenicol was used as
a reference to evaluate the potency of the testing compounds under
the same conditions. Antifungal activity was screened against four
fungal species; Aspergillus fumigatus AF, Penicillium italicum PI,
Syncephalastrum racemosum SR and Candida albicans CA, using
Terbinafin as the reference antifungal agent. All the compounds
96.72
101.59
100.14
98.45
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
107.28
90.05
85.29
103.64
115.66
103.37
117.24
75.72
92.33
95.47
128.46
106.82
95.91
95.38
90.87
95.91
91.94
96.78
were dissolved in DMSO to concentrations of 5, 2.5, and 1.0 mg/mL,
using inhibition zone diameter (IZD, cm) as a measure for the
antimicrobial activity. The antibacterial screening revealed that
compounds 5f, 5g, and 5h showed moderate activity against EC,
however, compounds 5b and 5d are highly active against EC, and
compounds 5g and 5h are active against SA. Antifungal results
indicated that all compounds showed no activity against chosen
fungal species. All the data are shown in Table 2.
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
92.62
126.09
82.73
123.17
95.33
117.97
82.48
104.52
90.80
98.95
113.86
97.08
107.78
102.42
110.14
92.44
90.57
110.47
88.06
95.80
99.06
106.93
88.40
99.94
80.29
102.52
91.99
3.2. Anticancer assay for preliminary in vitro testing
Ovarian cancer
IGROV1
The human tumor cell lines of the cancer screening panel were
grown in RPMI 1640 medium containing 5% fetal bovine serum and
7.59
95.76
17.65
97.53
110.81
117.08
92.85
94.20
104.16
99.20
91.69
92.95
93.99
95.27
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
116.35
121.85
100.70
102.57
115.86
2 mM
L-glutamine. For a typical screening experiment, cells were
inoculated in 96 well microtiter plates in 100
mL assay volume, at
plating densities ranging from 5000 to 40,000 cell/well. After cell
inoculation, the microtiter plates were incubated at 37 ꢀC, under an
atmosphere of 5:95 CO2:air (v/v) at 100% relative humidity, for 24 h
prior to addition of drugs under assessment. Following drug addi-
tion, the plates were incubated for an additional 48 h, under the
same conditions. Sulfarohodamine B (SRB) solution (100 mL, 0e4%
w/v in 1% aq acetic acid) is added to each well and plates were
incubated for 10 min at room temperature [21,22].
105.88
98.31
Renal cancer
786-0
A498
82.91
107.77
99.08
112.71
40.73
99.97
136.08
nd
82.24
83.96
110.63
103.86
28.48
93.24
65.49
116.06
103.17
97.59
90.63
95.00
94.35
95.54
120.15
75.87
ACHN
CAKI-1
RXF393
SN12C
TK-10
UO-31
3.2.1. In vitro assay
Evaluation of anticancer activity was performed on all
compounds by NCI, USA. The recorded data demonstrates that
among all tested compounds possessing a pyrimidinone moiety;
three compounds produced the highest growth inhibition
percentage. These three compounds were tested against 60 human
tumor cell lines, from 9 cancer types. Initially, a single high dose
was used (1.0 M) in the full NCI 60-cell panel. Only compounds
which satisfied predetermined threshold inhibition criteria were
progressed to the 5-dose screen, which was designed to efficiently
capture compounds with anti-proliferate activity. This screen was
Prostate cancer
PC-3
DU-145
95.01
102.09
92.58
104.88
90.50
95.47
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
91.01
95.90
99.88
117.79
88.32
nd
90.11
101.67
83.59
97.44
77.02
nd
89.39
85.96
90.12
99.42
75.56
94.65
MDA-MB-468
nd ¼ not done.

M.M. Edrees et al. / European Journal of Medicinal Chemistry 45 (2010) 5702e5707
5705
Table 4
ꢂ The hydrazonoyl group with nitro substituents contributes to
Evaluation of ED50, LD50, and 5a-reductase inhibitor activities relative to anastrozole.
add antimicrobial activities to the tested compounds.
a
b
c
Compd.
No.
ED50
LD50
g kgꢁ1
LD90
g kgꢁ1
)
Potency relative to
anastrozole
(
m
g kgꢁ1
)
(
m
)
(
m
It is worth to mention that all the tested compounds have high
safety margin and broad therapeutic windows.
5b
5c
5d
5e
5f
5g
5h
5i
0.173
0.174
0.211
0.611
0.5911
0.188
0.478
0.698
0.198
1.09
2663
2555
4356.87
3986.54
3245.92
3123.76
2980.32
3300.12
3290.98
2250.87
3217.17
6.300578
6.264368
5.165877
1.783961
1.84402
5.797872
2.280335
1.561605
5.505051
1.00
2111.22
2345.37
2133.45
2553.48
2345.23
1870.34
2341.33
4. Conclusion
In this context, we synthesized a new series of hydrazones and
investigated their biological activities. For the activity against fungi
and Bacteria, four compounds exhibited moderate activity and
three have low activity against some kinds of bacteria. For what
concern the antitumor activity, three compounds namely, 5b, 5d
and 5i showed significant inhibition of tumor cells at various
5j
Anastrozole
2.415
4.314
a
ED50: Dose caused 50% of pharmacological response in the test.
LD50: Dose killed 50% of the tested animals.
LD90: Dose killed 90% of the tested animals.
b
c
cell lines. Regarding the 5a-reductase inhibitor, all the tested
compounds except compound 5a are highly enough to provide
good therapeutic windows and soft profile margin activities. Also,
the relation between the structure of the tested compounds and
their biological activities was discussed and showed that replacing
based on careful analysis of historical DTP screening data, and the
data set was reported as a mean graph giving the growth percent
for each compound. The growth percent data is presented in Table
3. The activity of the compounds is measured according to a value
of 100 means no growth inhibition. A value of 40 would mean 60%
growth inhibition. A value of 0 means no net growth over the
course of the experiment. A value of ꢁ40 would mean 40% lethality.
A value of ꢁ100 means all cells are dead, this means that compound
5b exhibits its highest activity against leukemia CCRF-CEM, ovarian
IGROV1 cell lines and moderate activity against renal RXF393 cell
line. Compound 5d exhibits the highest activity against ovarian
IGROV1 and renal RXF393 cell lines, and moderate activity against
leukemia CCRF-CEM, HL-60(TB), K-562, MOLT-4 cell lines renal
TK-10 cell lines. Compound 5i exhibits moderate activity against
leukemia SR, and the less activity against renal UO-31, and breast
T-47D cell lines which is presented in Table 3.
the triazine moiety in the clinical used 5a-reductase inhibitor
anstrazoleÒ by its bioisoster pyrimidine highly increases the
activity. The hydrazonoyl group is essential for the anticancer
activity, while the substituents do not play a great role. Moreover,
the hydrazonoyl group with nitro substituents contributes to add
antimicrobial activities to the tested compounds.
5. Experimental protocols
5.1. Chemistry
Melting points were determined on a Gallenkamp apparatus and
are uncorrected. IR spectra were recorded in potassium bromide
using Perkin Elmer FTIR 1650 and Pye-Unicam SP300 infrared
spectrophotometers. 1H and 13C NMR spectra were recorded on
a varian mercury VXR-300 spectrophotometer (300 MHz for 1H and
75 MHz for 13C) and the chemical shifts were related to that of the
solvent DMSO-d6. Mass spectra were recorded on a GCMS-Q 1000
EX Shimadzu and GCMS 5988-A HP spectrometers, the ionizing
voltage was 70 eV. Electronic absorption spectra were recorded on
PerkineElmer Lambada 40 spectrophotometer. Elemental analyses
were carried out at the Microanalytical Laboratory of Cairo
University, Giza, Egypt. 2-(4-chlorobenzoylmethylthio)-1,2-dihy-
dro-6-methyl-4(3H)-pyrimidinones 3 was prepared as previously
described [11].
3.3. Pharmacological screening
Circulating testosterone and dihydrotestosterone hormone
levels or tissue concentrations were measured after administration
of 5
All synthesized compounds were tested for their 5
inhibitor activity in vivo; the ED50, and LD50 data were determined
and are given in Table 4. All the tested compounds showed 5
a-reductase inhibitor radioimmuno assays.
a
-reductase
a
-
reductase inhibitor activities with good LD50, the order of activity in
descending order is 5b, 5c, 5g, 5j, 5d, 5h, 5f, 5e and 5i. All
compounds were highly enough to provide good therapeutic
windows and soft profile margin (Table 4).
5.2. 2-[N-aryl-2-oxo-2-(4-chlorophenyl)-ethanehydrazonoyl]-6-
From the data in Table 4, we concluded that:
methyl-4(3H)-pyrimidinone (5)
ꢂ The influence of the substituent group toward decreasing the
activity was as follow: methyl, nitro, ethyl ester, hydrogen,
chloro and acetyl substituents.
ꢂ The methyl substituent on the diazo entity highly increases the
activity (the para substituent provides more effect than the
meta substituent).
To a stirred solution of 2-(4-chlorobenzoylmethylene)-6-
methyl-4(3H)-pyrimidinone 4 (0.65 g, 5 mmole) in ethanol (25 mL)
was added sodium hydroxide (0.2 g, 5 mmole) and the mixture was
cooled in an ice bath to 0e5 ꢀC. To the resulting solution, while
being stirred, was added dropwise over a period of 20 min, a solu-
tion of the appropriate arenediazonium chloride, prepared as usual
by diazotizing the respective aniline (5 mmole) in hydrochloric acid
(6 M, 3 mL) with sodium nitrite (1 M, 5 mL). The whole mixture was
then left in a refrigerator overnight. The precipitated solid was
collected, washed with water and finally crystallized from ethanol
to give the respective hydrazone 5.
ꢂ The chloride atom at position-4 increases the activity more
than that at position-3 in the benzene ring.
ꢂ The decrease in the degree of intramolecular hydrogen
bonding greatly increases the 5a-reductase inhibitor activity.
ꢂ Replacing the triazine moiety in the clinical used 5
a-reductase
inhibitor anstrazoleÒ by its bioisoster pyrimidine highly
increases the activity.
5.2.1. 2-[N-(4-Methoxyphenyl)-2-oxo-2-(4-chlorophenyl)-
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5a)
Orange crystals, Yield 1.63 g, 82%, m.p. 206e208 ꢀC. 1H NMR
ꢂ The hydrazonoyl group is essential for the anticancer activity,
while the substituents do not play a great role, hence þM
substituents slightly increase the activity with respect to ꢁM
ones.
(DMSO-d6):
d 2.33 (s, 3H, 6-CH3), 3.79 (s, 3H, OCH3), 6.24 (s, 1H,
5-CH), 6.78 (d, J ¼ 8 Hz, 2H, ArH), 6.84e7.22 (m, 4H, ArH), 7.69

5706
M.M. Edrees et al. / European Journal of Medicinal Chemistry 45 (2010) 5702e5707
(d, J ¼ 8 Hz, 2H, ArH), 12.85 (s, 1H, NH), 16.28 (s, 1H, NH); 13C NMR
5.2.7. 2-[N-(3-Nitrophenyl)-2-oxo-2-(4-chlorophenyl)
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5g)
Yellow crystals, yield (1.63 g, 79%), m.p. 280 ꢀC. 1H NMR
(DMSO-d6):
d 21.94, 58.21, 117.37, 120.21, 126.58, 129.0, 129.20,
130.40, 131.31, 132.84, 135.98, 137.99, 143.55, 148.04, 162.11, 190.49.
IR (KBr)
n
3423, 3172, 1681, 1658 cmꢁ1. MS m/z (%) 396 (Mþ, 7), 152
(CDCl3): d 2.36 (s, 3H, 6-CH3), 6.35 (s, 1H, 5-CH), 7.53e7.81,
(100), 111 (42). Anal. Calcd for C20H17ClN4O3 (396.84): C, 60.53; H,
4.32; N, 14.12. Found: C, 60.19; H, 4.12; N, 13.91%.
8.02e8.33 (m, 4H, ArH), 7.82 (d, J ¼ 8 Hz, 2H, ArH), 7.87 (d,
J ¼ 8 Hz, 2H, ArH), 12.37 (s, 1H, NH), 13.10 (s, 1H, NH). 13C NMR
(DMSO-d6):
d 22.75, 117.82, 121.46, 127.84, 128.28, 129.07, 129.19,
5.2.2. 2-[N-(4-Methylphenyl)-2-oxo-2-(4-chlorophenyl)-
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5b)
Yellow crystals, Yield 1.62 g, 85%, m.p 190 ꢀC (ethanol). 1H NMR
130.31, 130.71, 131.61, 133.28, 136.17, 138.03, 143.61, 148.56, 164.0,
190.33. IR (KBr)
n
3423, 3172, 1681, 1658 cmꢁ1. MS m/z (%) 411
(Mþ, 64), 410 (100), 111 (30). Anal. Calcd for C19H14ClN5O4
(411.81): C, 55.42; H, 3.43; N, 17.01. Found: C, 55.66; H, 3.51; N,
17.36%.
(DMSO-d6):
d 2.27 (s, 3H, Ar-CH3), 2.37 (s, 3H, 6-CH3), 6.23 (s, 1H,
CH), 7.32 (d, J ¼ 8 Hz, 2H, ArH), 7.58 (d, J ¼ 8 Hz, 2H, ArH), 7.78
(d, J ¼ 8 Hz, 2H, ArH), 7.93 (d, J ¼ 8 Hz, 2H, ArH), 12.45 (s, 1H, NH),
14.64 (s, 1H, NH).13C NMR (DMSO-d6):
d
20.51, 21.98, 116.80, 120.60,
5.2.8. 2-[N-(4-Nitrophenyl)-2-oxo-2-(4-chlorophenyl)
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5h)
Orange crystals, yield 1.69 g, 82%, m.p. 130e132 ꢀC. 1H NMR
126.51, 128.29, 129.21, 129.90, 131.32, 132.11, 135.61, 137.75, 143.65,
147.58, 163.14, 191.48. IR (KBr) n
3424, 3129, 1690, 1659 cmꢁ1. MS m/
z (%) 382 (Mþ þ 2, 35), 381 (Mþ þ 1, 48), 380 (Mþ, 51), 379 (100), 111
(53). Anal. Calcd for C20H17ClN4O2 (380.84): C, 63.08; H, 4.50; N,
14.71. Found: C, 63.00; H, 4.70; N, 14.93%.
(CDCl3):
d
2.06 (s, 3H, CH3), 6.25 (s, 1H, 5-CH), 7.62 (d, J ¼ 9 Hz, 2H,
ArH), 7.63 (d, J ¼ 9 Hz, 2H, ArH), 7.81 (d, J ¼ 9 Hz, 2H, ArH), 7.93 (d,
J ¼ 9 Hz, 2H, ArH), 12.83 (s, 1H, NH), 12.82 (s, 1H, NH). 13C NMR
(DMSO-d6):
d 22.66, 118.15, 120.44, 127.74, 128.38, 129.48, 130.33,
5.2.3. 2-[N-(3-Methylphenyl)-2-oxo-2-(4-chlorophenyl)-
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5c)
Dark orange crystals, yield 1.48 g, 78%, m.p. 234e236 ꢀC. 1H
131.96, 133.78, 136.0, 139.47,143.05, 146.24,162.15,189.27. IR (KBr) n
3400, 3308, 1673, 1666 cmꢁ1. MS m/z (%) 413 (Mþ þ 2, 1), 412
(Mþ þ 1, 2), 411 (Mþ, 2), 152 (100), 111 (44). Anal. Calcd for
C19H14ClN5O4 (411.81): C, 55.42; H, 3.43; N, 17.01. Found: C, 55.27;
H, 3.60; N, 17.35%.
NMR(DMSO-d6):
d 2.35 (s, 3H, Ar-CH3), 2.41 (s, 3H, 6-CH3), 6.31
(s,1H, 5-CH), 7.06e7.31 (m, 4H, ArH), 7.58 (d, J ¼ 8 Hz, 2H, ArH), 7.78
(d, J ¼ 8 Hz, 2H, ArH), 12.42 (s, 1H, NH), 15.32 (s, 1H, NH). 13C NMR
(DMSO-d6):
d
20.97, 22.66, 116.28, 120.94, 127.81, 127.64, 129.18,
5.2.9. 2-[N-(4-Acetylyphenyl)-2-oxo-2-(4-chlorophenyl)
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5i)
Redish brown solid, Yield (1.49 g, 73%), m.p. 182 ꢀC. 1H NMR
130.46, 131.0, 132.43, 134.25, 135.28, 136.43, 138.11, 142.02, 147.09,
160.36, 191.07. IR (KBr)
n
3424, 3105, 1683, 1617 cmꢁ1. MS m/z (%)
382 (Mþ þ 2, 42), 381 (Mþ þ 1, 38), 380 (Mþ, 54), 111 (43), 91 (100).
Anal. Calcd for C20H17ClN4O2 (380.84): C, 63.08; H, 4.50; N, 14.71.
Found: C, 63.20; H, 4.65; N, 14.81%.
(CDCl3): d 2.06 (s, 3H, 6-CH3), 2.36 (s, 3H, COCH3), 6.34 (s, 1H, 5-CH),
7.28 (d, J ¼ 9 Hz, 2H, ArH), 7.62 (d, J ¼ 9 Hz, 2H, ArH), 7.86 (d,
J ¼ 9 Hz, 2H, ArH), 7.95 (d, J ¼ 8 Hz, 2H, ArH), 11.18 (s, 1H, NH), 12.86
(s, 1H, NH). 13C NMR (DMSO-d6):
d 22.36, 26.24, 115.32, 118.56,
5.2.4. 2-[N-Phenyl-2-oxo-2-(4-chlorophenyl)-ethanehydrazonoyl]-
6-methyl-4(3H)-pyrimidinone (5d)
123.23, 126.80, 128.45, 132.62, 137.46, 141.23, 148.44, 150.21, 154.05,
159.43, 161.66, 164.0, 192.53. IR (KBr)
n 3429, 3062, 1708, 1658,
Orange solid, yield 1.52 g, 83%, m.p. 198 ꢀC. 1H NMR (DMSO-d6):
1627 cmꢁ1. MS m/z (%) 410 (Mþ þ 2, 12), 409 (Mþ þ 1, 30), 408 (Mþ,
37), 111 (81). Anal. Calcd for C21H17ClN4O3 (408.85): C, 61.69; H,
4.19; N, 13.70. Found: C, 61.90; H, 4.00; N, 14.01%.
d
2.38 (s, 3H, 6-CH3), 6.29 (s, 1H, 5-CH), 7.23 (d, J ¼ 8 Hz, 2H, ArH),
7.38e7.79 (m, 5H, ArH), 7.82 (d, J ¼ 8 Hz, 2H, ArH), 12.43 (s, 1H, NH),
14.32 (s, 1H, NH). 13C NMR (DMSO-d6):
21.35, 118.02, 121.21,
126.50, 128.38, 129.37, 132.01, 133.60, 134.51, 138.37, 142.25, 147.18,
d
5.2.10. 2-[N-(Ethoxycarbonylphenyl)-2-oxo-2-(4-chlorophenyl)-
148.56,163.01,190.78. IR (KBr)
n
3421, 3090,1670,1631 cmꢁ1. MS m/
ethanehydraz-onoyl]-6-methyl-4(3H)-pyrimidinone (5j)
z (%) 368 (Mþ2, 36), 367 (Mþ þ 1, 43), 366 (Mþ, 62), 111 (63). Anal.
Calcd for C19H15ClN4O2 (366.81): C, 62.22; H, 4. 12 ; N, 15.27. Found:
C, 62.30; H, 4.35; N, 15.34%.
Red solid, Yield (1.64 g, 75%), m.p. 238 ꢀC. 1H NMR (CDCl3):
d 1.29
(t, J ¼ 7 Hz, 3H, CH3), 2.06 (s, 3H, 6-CH3), 4.25 (q, J ¼ 7 Hz, 2H, CH2),
6.26 (s, 1H, 5-CH), 7.31 (d, J ¼ 9 Hz, 2H, ArH), 7.64 (d, J ¼ 8 Hz, 2H,
ArH), 7.80 (d, J ¼ 9 Hz, 2H, ArH), 7.87 (d, J ¼ 8 Hz, 2H, ArH), 11.20 (s,
5.2.5. 2-[N-(4-Chlorophenyl)-2-oxo-2-(4-chlorophenyl)-
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5e)
Orange crystals, yield 1.70 g, 85%, m.p. 145 ꢀC. 1H NMR (CDCl3):
1H, NH), 12.95 (s, 1H, NH); 13C NMR (DMSO-d6):
d 15.21, 20.93,
59.21, 116.99, 119.82, 121.63, 128.38, 131.26, 132.89, 133.05, 135.09,
139.0, 149.56, 152.68, 154.0, 158.21, 161.83, 190.56. IR (KBr)
n 3413,
d
2.36 (s, 3H, 6-CH3), 6.30 (s,1H, 5-CH), 7.22 (d, J ¼ 9 Hz, 2H, ArH), 7.52
(d, J ¼ 9 Hz, 2H, ArH), 7.63 (d, J ¼ 8 Hz, 2H, ArH), 7.80 (d, J ¼ 8 Hz, 2H,
ArH),11.04 (s,1H, NH),12.84 (s,1H, NH).13C NMR (DMSO-d6):
22.63,
117.22, 127.71, 128.42, 129.17, 129.30, 130.29, 131.62, 133.26, 136.50,
3120, 1728, 1689,1631 cmꢁ1. MS m/z (%) 440 (Mþ þ 2, 18), 439
(Mþ þ 1, 36), 438 (Mþ, 51), 437 (100), 111 (40). Anal. Calcd for
C22H19ClN4O4 (438.87): C, 60.21; H, 4.36; N, 12.77. Found: C, 60.43;
H, 4.72; N, 12.89%.
d
136.56, 139.13, 140.93, 162.00. 190.95. IR (KBr)
n 3419, 3186, 1667,
1620 cmꢁ1. MS m/z (%) 403 (Mþ þ 2, 20), 402 (Mþ þ 1, 32), 401 (Mþþ,
78), 136 (100), 111 (95). Anal. Calcd for C19H14Cl2N4O2 (401.26): C,
56.87; H, 3.52; N, 13.96. Found C, 56.52; H, 3.29; N, 13.70%.
5.3. Biological assay
5.3.1. Treatment of animals
Animals were obtained from the animal house colony of the
company. All animals were allowed free access to water and were
kept on a constant standard diet. Twenty three groups, each of 12
male SpragueeDawley rats in the postnatal third days, were treated
5.2.6. 2-[N-(3-Chlorophenyl)-2-oxo-2-(4-chlorophenyl)
ethanehydrazonoyl]-6-methyl-4(3H)-pyrimidinone (5f)
Orange solid, yield 1.5 g, 75%, m.p. 222 ꢀC. 1H NMR (CDCl3):
d
2.35 (s, 3H, 6-CH3), 6.34 (s, 1H, 5-CH), 7.14e7.54 (m, 4H, ArH), 7.58
subcutaneously with the 5a-reductase inhibitor (tested compound
(d, J ¼ 8 Hz, 2H, ArH), 7.81 (d, J ¼ 8Hz, 2H, ArH), 12.33 (s,1H, NH),
15.32 (S, 1H, NH). MS m/z (%) 403 (Mþ þ 2, 17), 402 (Mþ þ 1, 29), 401
(Mþ, 69), 111 (78). Anal. Calcd for C19H14Cl2N4O2 (401.26): C, 56.87;
H, 3.52; N, 13.96. Found: C, 57.05; H, 3.82; N, 13.71%.
or reference standard). The tested compounds were dissolved in 5%.
Tween 80 in water. The solvent was used for both standard and
negative control group, beginning on the postnatal third day until
the age of seven weeks.

M.M. Edrees et al. / European Journal of Medicinal Chemistry 45 (2010) 5702e5707
5707
Twenty-one groups were used to test the activities, of which
Acknowledgements
one was used as the positive control for anastrozole and another
served as the negative control group. After scarifying blood was
withdrawn for testosterone and dihydrotestosterone (DHT) deter-
mination [23]. Moreover, intraprostatic concentrations of testos-
terone and DHT were determined [24].
The authors would like to thank NCI team for antitumor
screening studies of the newly synthesized compounds. The tests
were performed under the Developmental Therapeutics Program at
NCI USA.
The biological experiments were performed according to the
official standards.
References
[1] D.J. Brown, E.C. Taylor, A. Weissberger (Eds.), Wiley, New York, 1994.
[2] Y.S. Sadnandam, M.M. Shetty, P.V. Diwan, Eur. J. Med. Chem. 27 (1992) 87e92.
[3] P. Singh, P. Kaur, V. Luxami, S. Kaur, S. Kumar, Bioorg. Med. Chem. 15 (2007)
2386e2395.
[4] A.B.A. El-Gazzar, M.M. El-Enany, M.N. Mahmoud, Bioorg. Med. Chem. 16
(2008) 3261e3273.
5.3.2. Radioimmuno assay for testosterone and dihydrotestosterone
Serum testosterone and dihydrotestosterone were measured by
radioimmuno assay in serum extracts using specific antisera
without prior chromatography. Serum samples of 0.5 mL were
extracted with 2 mL of freshly purified peroxide-free diethyl ether
by shaking for 60 s on a Vortex mixer. The aqueous phase was
frozen at ꢁ70 ꢀC, the ether phase containing steroids was trans-
ferred to a conical test tube and evaporated in BSA/phosphate
buffer (pH ¼ 7.4) containing (1,2,6,7-3H)-testosterone or (1,2,6,7-
3H)-dihydrotestosterone and then specific antisera were added and
incubated over a period of 24 h at 4 ꢀC under non-equilibrium
conditions. Bound hormone and free hormone were separated by
adsorption on dextran-coated charcoal. The activity of each sample
was determined in a Beckman-counter (USA) using a commercially
available scintillation cocktail (Mini-RIA, Zinsser, Spain).
[5] A.M. Cuadro, J. Valenciano, J.J. Vaquero, J. Alvarez-Builla, C. Sunkel, M.F. de
Casa-Juana, M.P. Ortega, Bioorg. Med. Chem. 6 (1998) 173e180.
[6] A.K. Ganguly, R.J. Doll, V.M. Girijavallabhan, Curr. Med. Chem.
8 (2001)
1419e1436.
[7] N.G. Kandile, M.I. Mohamed, H. Zaky, H.M. Mohamed, Eur. J. Med. Chem. 44 (5)
(2009) 1989e1996.
[8] P. Vicini, M. Incerti, P. La Colla, R. Loddo, Eur. J. Med. Chem. 44 (4) (2009)
1801e1807.
[9] B. Modzelewska-Banachiewicz, T. Kaminska, Eur. J. Med. Chem. 36 (1) (2001)
93e99.
[10] (a) A.S. Shawali, T.A. Farghaly, Tetrahedron 60 (13) (2004) 3051e3057;
(b) A.S. Shawali, T.A. Farghaly, M.M. Edrees, Int. J. Pure Appl. Chem. 1 (4)
(2006) 531e537.
[11] D.T. Hurst, C. Beaumont, D.T.E. Jones, D.A. Kingsley, J.D. Patridge,
T.J. Rutherford, Aust. J. Chem. 41 (1988) 1209e1219.
[12] R. Jones, A.J. Ryan, S. Sternhell, S.E. Wright, Tetrahedron 19 (1963)
1497e1507.
[13] A.S. Shawali, M.A. Abdallah, M.A.N. Mosselhi, Y.F. Mohamed, Z. Naturforsch.
57b (2002) 552e556.
[14] S.A. Khattab, A.S. Shawali, A.M. Farag, J. Chem. Eng. Data 22 (1977) 104e110.
[15] T.A. Farghaly, S.M. Riyadh, Arkivoc x (2009) 54e64.
[16] J. Reiter, L. Bongo, P. Dyortsok, Tetrahedron 43 (1987) 2497e2504.
[17] J.C. Jung, E.B. Watkins, M.A. Avery, Heterocycles 65 (2005) 77e94.
[18] G.R. Bedford, P.J. Taylor, G.A. Webb, Mag. Res. Chem. 33 (1995) 389e394.
[19] A.B.A. El-Gazzar, H.N. Hafez, G.A.M. Nawwar, Eur. J. Med. Chem. 44 (2009)
1427e1436.
As for other steroid hormones, commercially available KIA-kits,
e.g., Biermann GmbH, Germany, can be used.
The hormone level in the sample was calculated from a stan-
dard curve by means of a computer program (KIA-Calc, LKB,
Canada), using appropriate control sera. Steroid levels of rats
treated with different doses of 5a-reductase inhibitors were
compared with vehicle-treated controls (Table 4). The relative
potency was calculated by dividing the ED50 (dose that causes 50%
of pharmacological response in the test) of anastrozole by that of
a tested compound.
[20] A.M. Revol-Junelles, R. Mathis, F. Krier, Y. Fleury, A. Delfour, G. Lefebvre, Lett.
Appl. Microbiol. 23 (1996) 120e124.
[21] (a) P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyd, J. Nat. Cancer Inst. 82 (1990)
1107e1112;
5.4. Determination of acute toxicity
(b) M.R. Grever, S.A. Schepartz, B.A. Chabner, Semin. Oncol. 19 (1992)
622e638.
[22] M.R. Boyd, K.D. Paull, Drug Dev. Res. 34 (1995) 91e109.
[23] F.W. George, L. Johnson, J.D. Wilson, Endocrinology 125 (1989) 2434e2438.
[24] E. Disalle, D. Gindicen, G. Bricitico, G. Ornati, A. Panzer, A. Ornati, J. Steroid
Biochem. 46 (1993) 549e555.
LD50 and LD90 were determined by using male albino rats
and injecting them with different increasing doses of agents.
Doses that killed 50% and 90% of the tested animals, respec-
tively, were calculated according to Austen and Brocklehurst [25]
(Table 4).
[25] K.F. Austen, W.E. Brocklehurst, J. Exp. Med. 113 (1961) 521e539.