Synthesis and evaluation of curcumin analogues as cytotoxic agents

Article abstract of DOI:10.1007/s00044-009-9199-3

Seventeen curcumin analogues were prepared and evaluated for in vitro and in vivo cytotoxicity against an Ehrlich ascites carcinoma (EAC). In vitro results revealed that compounds 10, 7, and 12 were the most potent analogues against EAC respectively. Howe

Full text of DOI:10.1007/s00044-009-9199-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:413–430
DOI 10.1007/s00044-009-9199-3
ORIGINAL RESEARCH
Synthesis and evaluation of curcumin analogues
as cytotoxic agents
Ahmed A. Fadda Æ Farid A. Badria Æ
Khaled M. El-Attar
Received: 29 April 2008 / Accepted: 20 March 2009 / Published online: 23 April 2009
¨
Ó Birkhauser Boston 2009
Abstract Seventeen curcumin analogues were prepared and evaluated for in vitro
and in vivo cytotoxicity against an Ehrlich ascites carcinoma (EAC). In vitro results
revealed that compounds 10, 7, and 12 were the most potent analogues against EAC
respectively. However, in vivo evaluation of compound 10 proved its capability to
normalize the blood picture compared with 5-fluorouracil, a well-known anticancer
drug.
Keywords Hydroxycurcuminoids ꢀ Synthesis ꢀ Ehrlich ascites in vitro ꢀ
Antitumor
Introduction
Curcumin (1) is a natural yellow-orange dye extracted from the rhizomes of the
plant Curcuma longa L. (zingiberaceae) (Jasim and Ali, 1988). A variety of
pharmacological properties, such as anticancer (Kuttan et al., 1985; Soudamini and
Kuttan, 1989) activities, have been reported. It also has been used as a
photodynamic agent for the destruction of bacteria and tumor cells (Dahl et al.,
1989; Pervaiz et al., 1990). From toxicological studies, curcumin is nontoxic even at
high dosage (Tonnesen, 1986). Attempts to use it as an antioxidant additive for
lubricants and motor oils, photoresists, and sunscreen compounds have been made
(Martin and Rajaratnam, 1988; Nambudiry and Natraj, 1991; Sharma, 1976). Well-
known anti-cancer drugs, e.g., tamoxifen, dexamethasone, cyclophosphamide, and
A. A. Fadda (&) ꢀ K. M. El-Attar
Chemistry Department, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
e-mail: afadda2@yahoo.com
F. A. Badria
Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt

414
Med Chem Res (2010) 19:413–430
methylprednisolone, have been reported to induce leukopenia and other toxic
manifestations to the host (Bergan et al., 1999; Steele, 2002; Varshavsky, 1998;
Wolff et al., 1998). Curcumin (diferuloylmethane), the active component of
turmeric (Curcuma longa L.), has been widely used as an anti-inflammatory, anti-
oxidant, and chemotherapeutic agent (Ammon and Wahl, 1991).
It has been recognized for chemopreventive properties in breast, skin, and fore
stomach carcinogenesis (Choudhuri et al., 2002; Kawamori et al., 1999). Interest-
ingly, turmeric has been used in a large quantity as a condiment for thousands of years
with no adverse reactions, thereby indicating its candidacy as probable nontoxic, anti-
cancer agent (Choudhuri et al., 2002). This active phytochemical has an immuno-
modulatory property and stimulates macrophage phagocytic activity (Antony et al.,
1999). Curcumin protects thymocytes from dexamethasone-induced apoptosis
(Jaruga et al., 1998). It also is able to effectively quench singlet oxygen at a very
low concentration in aqueous systems and is a powerful inhibitor of hydrogen
peroxide damage in human keratinocytes and fibroblasts (Das and Das, 2002; Phan
et al., 2001; Pal et al., 2005). In vivo and in vitro studies have demonstrated
curcumin’s ability to inhibit carcinogenesis at three stages: tumor promotion,
angiogenesis, and tumor growth. Curcumin suppresses mitogen-induced proliferation
of blood mononuclear cells, inhibits neutrophil activation and mixed lymphocyte
reaction, and inhibits both serum-induced and platelet-derived growth factor (PDGF)-
dependent mitogenesis of smooth muscle cells (Huang et al., 1992). It has been
reported to be a partial inhibitor of protein kinase (Liu et al., 1993; Reddy and
Aggarwal, 1994). The other salient feature of turmeric/curcumin is that despite being
consumed daily for centuries in Asian countries, it has not been shown to cause any
toxicity (Ammon and Wahl, 1991). Although a number of excellent reviews on
curcumin are available, this short review specifically focuses on the antioxidant,
wound healing, anti-angiogenic, and anti-cancer effects of turmeric/curcumin
(Maheshwari et al., 2006). Recently numerous studies have demonstrated the
remarkable cancer preventive properties of curcumin. The chemopreventive effects of
curcumin have been attributed to various biological properties, including neutraliza-
tion of carcinogenic free radicals and anti-angiogenesis action, which limits the blood
supply to rapidly growing malignant cells (Chandru et al., 2007; and references cited
therein). In a continuing search for potent and selective cytotoxic antitumor agents,
we synthesized 17 curcumin analogues (1–17) and evaluated their cytotoxic effects
against an Ehrlich ascites cells.
Chemistry
Results and discussion
The designed target compounds are depicted in Schemes (1, 2, 3, 4). Derivatives of
hydroxy arylazobenzene are well known as dyes (Daris and Muchowski, 1975;
Gilman and Stern, 1957; Gilman and Stern, 1960). Consequently, the authors
decided to couple the diazonium salt of different aromatic amines with curcumin (1)
with a view to synthesis new azodisperse dyes to explore the possibility of finding

Med Chem Res (2010) 19:413–430
415
O
O
O
O
ArN2+Clꢀ
pyridine
N NHAr
HO
HO
OH
OCH3
OH
OCH3
OCH3
OCH3
1
2a-e
2a , Ar = C6H4ꢀ OCH3ꢀp
2b , Ar = C6H4ꢀ CH3ꢀp
2c , Ar = C6H5
2d , Ar = C6H4ꢀ Clꢀp
2e , Ar = C6H4ꢀ NO2ꢀp
Scheme 1
some new azodyes capable of dyeing different types of fibers and have expected
wide spectrum of biological activity. Diazonium salts undergoes a coupling reaction
with curcumin (1) to give the corresponding 4-arylazo derivatives (2a–e). In
accordance with the nomenclature of arylazo curcumin, the general structure
formula for 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-4-arylazo-3,5-
dione (2a–e) should be as follows:
From the infrared measurements of the prepared arylazo derivatives (2a–e),
presence of strong bands in the region 1550–1580 cm-1 indicating the (–N=N–)
stretching frequency and the (NH) absorption was recorded at 3250 cm-1 besides
the (OH) stretching at 3400 cm-1, and in a tautomeric mixture (Daris and
Muchowski, 1975) as well. Furthermore, the a,b-unsaturated carbonyl function
appeared at 1690 cm-1
.
Generally, variation in color of these dyes results from the alternation in the
diazonium components. The UV spectra of the diazonium coupling products of (1)
provide additional evidence that such compounds have the tautomeric relation with
azo-hydrazo system. Most of the dyes show four absorption bands in the region
196–438 nm. The relatively small differences in kmax may be due to the polarity
change of the absorbing system caused by solvent interactions due to the general
solvent effect (Gilman and Stern, 1957). It has been reported that UV spectra of
monophenyl azo compounds differ from those of monophenyl hydrazones. The azo
compounds generally show two absorption bands at 400–410 and 290–300 nm
corresponding to n–p* and p–p* transitions respectively (Gilman and Stern, 1960).
On the other hand, monophenyl hydrazones show three intense bands at 220–230,
250–280, and 330–390 nm regions (Gilman and Stern, 1957). The UV spectra of
compounds (2a–e) investigated can be interpreted in terms of the tautomeric
mixture as well. It is clear that these dyes exhibit four bands; of these, the medium
and high wavelength bands seem to be affected by the nature of the polar
substituents in the arylazo group, and the low wavelength bands is unaffected. This
indicates the presence also of hydrazone structure where the resonance interactions
with the substituents in the diazo component are minimal due to steric factors.
Treatment of 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-4-[(4-nitro-phenyl)-hydraz-
ono]-hepta-1,6-diene-3,5-dione (2e) with bromine in the presence of glacial acetic
acid afforded the corresponding 6,7-dibromo-1,7-bis-[3-(4-nitrophenylazo)-4-
hydroxy-5-methoxy]hepta-1-ene-3,5-dione (3).

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Med Chem Res (2010) 19:413–430
Condensation of (2e) with thiourea was performed in molar ratio (1:2),
respectively, in boiling ethanolic sodium ethoxide to give the corresponding (Z)-4-
(4-hydroxy-3-methoxy-phenyl)-6-[{6-(4-hydroxy-3-methoxy-phenyl)-2-thioxo-1,2,
5,6-tetrahydro-pyrimidine-4-yl}(2-(4-nitrophenyl)hydrazono)methyl]-5,6-dihydro-
pyrimidine-2(1H)-thione (4). Structure (4) was confirmed by its IR spectrum, which
showed the absence of spectrum band in the region of 1600 and 1690 cm-1, i.e.,
a,b-unsaturated ketone was involved in the cyclocondensation process, and in the
same time the IR spectrum showed new bands (cf. experimental part).
Moreover, (1Z,3E)-4-(4-hydroxy-3-methoxy-phenyl)-1-[6-(4-hydroxy-3-meth-
oxy-phenyl)-2-thioxo-2,3,4,5-tetrahydro-pyrimidine-4-yl]-1-[2-(4-nitrophenyl)hy-
drazono]-but-3-ene-2-one (5) was prepared by refluxing compound (2e) with
thiourea (1:1) molar ratio in boiling ethanolic sodium ethoxide. Structure (5) was
deduced from both correct analytical and spectral data (cf. experimental part).
Similarly, it has been found that N-[bis-(5-(4-hydroxy-3-methoxy-phenyl)-2,5-
dihydro-isoxazol-3-yl)-methylene]-N0-(4-nitro-phenyl)-hydrazine (6) has been pre-
pared by reaction of (2e) with hydroxylamine hydrochloride in refluxing pyridine.
As an extension of our interest in the synthesis of new heterocycles incorporating
a pyrazole nucleus (Metwally et al., 1985), we report the behavior of (2e) toward
hydrazine hydrate and/or its derivatives as a facile and convenient route to some
heterocyclic derivatives containing a pyrazole moiety. Therefore, the reaction of
O
O
Br
Ar
Br2
excess
Ar
Ar'HN N Br
AcOH glacial
3
S
S
S
NH2CNH
2 (1:2)
HN
N
N
NH
Ar
EtONa
Ar
Ar'HN N
O
4
S
S
O
O
N
NH
Ar
NH2CNH
2 (1:1)
Ar
Ar
Ar
EtONa
Ar'HN N
Ar'HN N
2e
5
O
NH HN
O
NH2OH (1:2)
pyridine
Ar
Ar
Ar=
OH
,
OCH3
Ar'HN N
6
NO2
Ar'=
HN
Ar
N
N
NH
Ar
NH2NH2 (1:3)
EtOH :AcOH (1:1)
Ar'HN N
7
Scheme 2

Med Chem Res (2010) 19:413–430
417
diferuloyl-(4-nitrophenyl)methane (2e) with hydrazine hydrate in (1:3) molar ratio
in boiling mixture of ethanol-glacial acetic acid afforded the bis pyrazolyl derivative
(7). The structure of (7) was established by both elemental and spectral data. The
H1-NMR spectrum revealed the absence of signals at d 6.5 and 7.5 of olefinic
protons and instead appeared signal at d 3.9 due to the methylene protons of
pyrazole ring. The mass spectrum showed m/z 545 (M?).
In connection of our search toward the design and synthesis of heterocyclic
analogues of curcumin as potential chemotherapeutic agents, it was decided to take
4-[(4-chlorophenyl)-hydrazono]-1,7-bis-(4-hydroxy-3-methoxy-phenyl)hepta-1,6-
diene-3,5-dione (2d) as adaptable starting material for the synthesis of new
heterocyclic binary system with the hope that the new compounds may exert more
potent effect. We report the synthesis of a new bis pyridyl derivative. Thus, it was
found that compound (2d) reacted with ethyl acetoacetate in boiling ethanolic
sodium ethoxide in (1:2) molar ratio, respectively, to afford the Michael adduct
2,10-diacetyl-6-[(4-chloro-phenyl)-hydrazono]-3,9-bis-(4-hydroxy-3-methoxy-phe-
nyl)-5,7-dioxo-undecanedioic acid diethyl ester (8).
The Michael adduct (8) was then subjected to react with excess ammonium
acetate in boiling glacial acetic acid to give the corresponding (Z)-ethyl-6-{2-(4-
chloro-phenyl)hydrazono}[4-(4-hydroxy-3-methoxy-phenyl)-6-1,4-dihydropyridin-
2-yl]-4-(4-hydroxy-3-methoxy-phenyl)-2-methyl-1,4-dihydropyridine-3-carboxy-
late (9) (cf. experimental part).
In addition, it was found that diketone moieties of curcumin were replaced with
hydrazine hydrate in refluxing absolute methanol; triethylamine and catalytic
amounts of glacial acetic acid were added. One step coupling of (1) with
hydrazinium dihydrochloride gave compound (10) (Joong et al., 2002; Ishida et al.,
2002; Flynn et al., 1991). The compound was purified by crystallization from
ethanol to give dark red crystals, which was analyzed for C21H20N2O4 by elemental
analysis. 4,40-(1E,10E)-2,20-(1H-Pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-bis-[2-(4-
chloro-phenyl)]diazenyl-6-methoxy-phenol (10) was proven on the basis of both
analytical and spectral data.
COOEt
Ar
Ac
Ac
O
O
O
EtOOC
Ar
O
EAA (1:2)
EtONa
Ar
Ar
Ar''HN N
Ar''HN N
2d
8
AcNH4 (1:2)
CH3
AcOH
CH3
EtOOC
COOEt
Ar=
OH
,
NH HN
OCH3
Cl
Ar''=
Ar''HN N
9
Scheme 3

418
Med Chem Res (2010) 19:413–430
In continuation of our study, we report the use of compound (10) as a key
intermediate for the synthesis of new series of curcumin derivatives, which have not
been investigated so far. Selective N-bromination of (10) using N-bromosuccini-
mide in chloroform gave a gummy bromopyrazoline derivative (11), which was
converted directly into the corresponding 4,40-(1E,10E)-2,20-(1-hydrazinyl-1H-
pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-bis-(2-methoxy-phenol) (12). Structure of
compound (11) was elucidated by the formation of compound (12). Structure (12)
was established on the basis of both analytical and spectral data. The IR spectrum of
compound (11) showed the absence of band at 3230 cm-1 due to the absorption
frequency of (NH) and instead appeared new band at 720 cm-1 due to (N-Br)
absorption.
N-Nitrosopyrazoline derivative (13) also was obtained on treating (10) with
sodium nitrite and hydrochloric acid—the structure that was confirmed from IR, H1-
NMR, and mass spectra. The absence of absorption due to (NH) group and the
appearance of new absorption band at t9 (1580 cm-1) in the IR spectrum of (13)
substantiate the assigned structure of this product.
Moreover, it has been found that fusion of (10) with chloroacetyl chloride at
95°C for 2 h afforded the corresponding chloroacetyl derivative (14). The IR
spectrum of (14) showed absorption at 2900, 1685, and 760 cm-1 due to (CH2),
(CO), and (C–Cl) functions. The H1-NMR showed singlet signal at d 3.5 ppm of
(CH2) protons, in addition to the other expected signals (cf. experimental part).
It was found that the pyrazole derivative (10) was subjected to coupling reaction
with p-chlorophenyl diazonium chloride in (1:2) molar ratio afforded the
corresponding 3,5-bis-[b-(5-{4-chloro-phenylazo}-4-hydroxy-3-methoxy-phenyl)-
ethynyl]-1H-pyrazole (15). Structure of compound (15) was established on the
basis of both elemental and spectral analysis.
Reaction of 3,5-bis-[b-(5-{4-Chloro-phenylazo}-4-hydroxy-3-methoxy-phenyl)-
ethynyl]-1H-pyrazole (15) with formamide in methanol gave 3,5-bis-{b-[3-(4-
chloro-phenylazo)-4-hydroxy-5-methoxy-phenyl]-ethynyl}-pyrazole-1-formyl (16)
(cf. experimental part).
Treatment of 3,5-bis-[b-(5-{4-Chloro-phenylazo}-4-hydroxy-3-methoxy-phe-
nyl)-ethynyl]-1H-pyrazole (15) with formaldehyde in methanol afforded 3,5-bis-[b-
(5-{4-Chloro-phenylazo}-4-hydroxy-3-methoxy-phenyl)-ethynyl]-2-hydroxy-methyl-
pyrazole (17) (cf. experimental part).
Biological activity
Discussion
Effect of drugs on the viability of Ehrlich ascites cells in vitro
To examine whether these substances have a direct cytotoxic effect on Ehrlich
ascites cells (EAC) viability, the percentage of viable cells was estimated by the
trypan blue (Sheeja et al., 1997) exclusion test. The desired concentration of tumor
cells (2 9 106 cells per 0.2 ml) was obtained by dilution with saline (0.9% sodium

Med Chem Res (2010) 19:413–430
419
N-bromosuccinimide
Ar
Ar
chloroform
N
N Br
11
NH2NH2 (1:1)
Ar
EtOH
Ar
Ar
N
N NHNH2
12
NaNO2, HCl
3oC
Ar
Ar
Ar
N
NH
N
N NO
13
10
Ar
Ar
N C CH2
Ar=
OH
OCH3
ClCH2COCl (1:1)
95oC
N
O
Cl
14
Ar'N2+Clꢀ(1:2),
NaOH 10%
Ar''
Ar''
N
NH
15
HCONH2
MeOH
CH2O
MeOH
Ar''
Ar''
N CHO
Ar''
Ar''
N CH2OH
N N
OH
OCH3
Cl
N
N
Ar''=
16
17
Scheme 4
chloride solution). Viability of tumor cells obtained and used in this experiment was
always greater than 90%. Below this percentage, the cells were discarded and the
entire procedure was repeated. Seventeen 1,3-diketo-propane and related analogues
(1–17) were tested for cytotoxicity against EAC in vitro. Results for the ED50, ED25
and ED10 values of the active compounds are summarized in Table 1. Compounds 7
and 12 displayed moderate cytotoxicity, whereas (10) was the most potent with
ED25. The other rest of compounds showed weak to no activity. Thus, it would
appear that introducing pyrazole moiety enhances the cytotoxic properties. By
comparing the cytotoxicity results in Table 1, the following structure-activity
relationships (SARs) were drawn: (a) Converting the keto-enol moiety to the
corresponding pyrazole (10, 7, and 12) led to increase cytotoxicity against EAC.
Compound 10 was the most active, whereas compounds 7 and 12 exhibited good
activity. Thus, the ring substituents affected the activity in the pyrazole derivatives.
(b) All halogenated derivatives (2d, 3, 8, 14–17) were very weak or completely
inactive at (ED25). Thus, the position and nature of substituents on the structure of
curcumin seem to modulate antitumor activity. The reliable criteria for judging the
value of any anticancer drug are prolongation of life span and decrease of WBC
from blood (Clarkson and Burchenal, 1965; Oberling and Guerin, 1954). The results
of the present study showed an antitumor effect of compounds (1–17) against EAC
in Swiss albino mice (Table 2).

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Med Chem Res (2010) 19:413–430
Table 1 In vitro cytotoxicity of curcumin analogues using EAC assay
Compound
Dead (%)
ED50 9 103 lM
ED25 9 103 lM
ED10 9 103 lM
5-flu
1
96.3
100
100
100
94.1
100
100
96.1
100
100
100
100
100
100
100
28.6
100
100
100
100
0
4.8
3.4
0
0
3.4
0
2a
2b
2c
2d
2e
3
2.4
0
0
3.2
2.8
3.2
4.8
0
0
2.5
0
4
0
5
2.9
0
6
5.3
38.9
0
7
0
8
0
10
12
13
14
15
16
17
66.7
22.2
6.7
0
0
0
0
0
0
0
0
0
3.6
0
ED is the concentration of compounds in lM
Table 2 In vivo cytotoxicity of compound 10 using EAC assay
Test group
Hb 12–16 g/dl
HCT (hematocrit)
35–50%
WBCs 4,000–
11,000/cmm
Ehrlich cells
count mil/ml
MST/day
Normala
Controlb
13.9
8.7
10
53.6
35.5
42.3
41
8.4
38.6
13.8
13.8
–
–
220
9.1
5-fluorouracil
Compound (10)
a
123
16.7
14.6
9.4
169.6
Normal test: without EAC
b
Control test: with EAC
Effect of compound 10 on survival time
The mean survival time (MST) of each group consisting of seven mice was noted
(Sur and Ganguly, 1994). The antitumor efficacy was compared with that of
5-fluorouracil (5-FU) for 9 days. The MST of the treated groups was compared with
that of the control group using the following calculation: % Increase in life span

Med Chem Res (2010) 19:413–430
421
over control = (MST of treated group-MST of control group) 9 100-100, where
MST = survival time (days of each mouse in a group)/Total no. of mice (Table 2).
Effect of compounds (1–17) on hematological parameters
To examine the influence of (1–17) on the hematological status of EAC-bearing
mice, a comparison was made among the following groups (n = 7) of mice on the
14th day after inoculation. The groups were comprised of 1) tumor-bearing mice, 2)
tumor-bearing mice treated for the first 9 days, and 3) control mice (normal). Blood
was drawn from each mouse by the retro orbital plexus method and the white blood
cell count (WBC), red blood cell count (RBC), and hemoglobin was determined.
From each group, 100-ll sample of Ehrlich ascites cells (from three mice) was taken
and 20-fold dilutions in saline were made. The cells were stained by Giemsa stain
and the number of viable cells was measured under a microscope. The viable cells,
those that did not take up the stain, i.e., the dead cells (stained cells).
From Table 2, it was noticed that: (1) Mice received compound (10) and 5-FU
were more protected against ascites and increase in weight than control mice; (2)
Percentage increase of life span compared with control was high in mice treated
with total extracts; (3) Mice that received compound (10) showed comparable
results to 5-FU; (4) Hematological parameters of tumor-bearing mice on day 14
showed significant changes compared with normal mice; (5) Total WBC count
increased with a reduction in the hemoglobin content and increase of RBC; (6)
Compound (10) and 5-FU showed minimal ascites and slight increase in body
weight unlike control group.
Conclusions
Modification of curcumin produced compounds with potential for further develop-
ment as anticancer agents. Based on these preliminary screening results, compounds
(10), (7), and (12) showed significant activity in certain cancer cells and have been
targeted for further studies. Additional research, including mode of action studies, is
planned to accurately establish relative activity for (SAR) and rational design.
Recently, the cancer chemopreventive effects of curcumin have been intensively
investigated. Curcumin exhibited pronounced antitumor activity by triggering
apoptosis inhuman tumor cells (Ishida et al., 2002). Studies are underway to
investigate the apoptosis-inducing activity of compounds found to be cytotoxic in
this study.
Experimental
General
All melting points (uncorrected) are in degree centigrade and were determined on
Gallenkamp electric melting point apparatus. FTIR spectra (KBr disk) were

422
Med Chem Res (2010) 19:413–430
1
recorded on a Nicolet Magna. IR model 550 spectrophotometers, H-NMR spectra
in DMSO, were determined on Brucker Wpsy 200 MHz spectrometer with TMS as
internal standard and the chemical shifts are in d ppm. Mass spectra were recorded
at 70 ev with a varian MAT 311. C, H and N elemental analyses are satisfactory for
all synthesized compounds (1–17).
Synthesis of curcumin derivatives
Synthesis of 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-
4-arylazo-3,5-dione; (2a–e)
General procedure Preparation of the diazonium salt: A solution of sodium nitrite
(0.2 g in 2 ml water) was gradually added to a well-cooled solution of different
aromatic amines (0.01 mol) in concentrated HCl (3.0 ml). The diazonium salt
solution was added drop wise with continuous stirring to cold solution of curcumin
(1) in pyridine (20 ml). The reaction mixture was stirred at 0–5°C for 2 h and left to
stand at room temperature. The solid product that obtained was filtered off, dried,
and recrystallized from ethanol to give compounds (2a–e).
4-(4-methoxy-phenylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-
1,6-diene-3,5-dione (2a)
Yield 58%; m.p. 153°C; IR (KBr): m/cm-1 = 3400 (OH), 3250 (NH), 1688 (C=O),
1618 (C=C), 1550 (N=N). H1-NMR (DMSO-d6): d/ppm = 3.73 (s, 3H, OCH3), 3.95
(s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H,
2 9 CH=CH), 6.91–8.02 (m, 10H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH),
11.03 (s, 1H, NH). MS: m/z (%) = 505 (M??1, 0.81), Anal. for C28H26N2O7
(502.52): Calcd. C, 66.92; H, 5.22; N, 5.57%. Found: C, 66.88; H, 5.20; N, 5.62%.
4-(p-tolylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-
1,6-diene-3,5-dione (2b)
Yield 68%; m.p. 142°C; IR (KBr): m/cm-1 = 3400 (OH), 3250 (NH), 1690 (C=O),
1620 (C=C), 1560 (N=N). H1-NMR (DMSO-d6): d/ppm = 2.35 (s, 3H, CH3), 3.95
(s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H,
2 9 CH=CH), 6.91–7.94 (m, 11H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH),
11.03 (s, 1H, NH). MS: m/z (%) = 486 (M?, 7.31), Anal. for C28H26N2O6 (486.52);
Calcd. C, 69.12; H, 5.39; N, 5.76%; Found: C, 69.20; H, 5.29; N, 5.50%.
4-(phenylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-
1,6-diene-3,5-dione (2c)
Yield 93%; m.p. 132°C;IR (KBr): m/cm-1 = 3400 (OH), 3250 (NH), 1690 (C=O),
1605 (C=C), 1580 (N = N). H1-NMR (DMSO-d6): d/ppm = 3.95 (s, 6H,
2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H,
2 9 CH=CH), 6.91–8.12 (m, 11H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH),

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11.03 (s, 1H, NH). MS: m/z (%) = 472 (M?, 0.32), Anal. for C27H24N2O6 (472.49):
Calcd. C, 68.63; H, 5.12; N, 5.93%; Found: C, 68.58; H, 5.15; N, 5.98%.
4-(4-Chloro-phenylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-
1,6-diene-3,5-dione (2d)
Yield 90%; m.p. 150°C; IR (KBr): m/cm-1 = 3400 (OH), 3250 (NH), 1690 (C=O),
1615 (C=C), 1575 (N=N). H1-NMR (DMSO-d6): d/ppm = 3.95 (s, 6H, 2 9 OCH3),
5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H, 2 9 CH=CH), 6.91–
7.93 (m, 10H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH), 11.03 (s, 1H, NH).
MS: m/z (%) = 507 (M?, 8.85), Anal. for C27H23ClN2O6 (506.93): Calcd. C, 63.97;
H, 4.57; N, 5.53%; Found: C, 63.90; H, 4.63; N, 5.57%.
1,7-Bis-(4-hydroxy-3-methoxy-phenyl)-4-[(4-nitro-phenyl)-hydrazono]-
hepta-1,6-diene-3,5-dione (2e)
Yield 49%; m.p. 152°C; IR (KBr): m/cm-1 = 3400 (OH), 3250 (NH), 1690 (C=O),
1600 (C=C), 1565 (N=N). H1-NMR (DMSO-d6): d/ppm = 3.95 (s, 6H, 2 9 OCH3),
5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H, 2 9 CH=CH), 6.91–
7.98 (m, 10H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH), 11.03 (s, 1H, NH).
MS: m/z (%) = 517 (M?, 15.2), Anal. for C27H23N3O8 (517.49): Calcd. C, 62.67;
H, 4.48; N, 8.12%; Found: C, 62.60; H, 4.40; N, 8.18%.
Synthesis of 6,7-dibromo-1,7-bis-[3-(4-nitrophenylazo)-4-hydroxy-5-
methoxy]hepta-1-ene-3,5-dione; (3)
To 4-(4-nitro-phenylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-
3,5-dione (2e), (0.01 mol) in acetic acid (22 ml) was added slowly. With stirring
a solution of bromine (0.02 mol) in acetic acid (20 ml). The reaction mixture was
left overnight and crude product filtered, washed with water, dried, and recrystal-
lized from ethanol to give compound (3). Yield 65%; m.p. 140°C; IR (KBr):
m/cm-1 = 3425 (OH), 1665 (C=O), 1581 (N=N), 700 (C–Br). MS: m/z (%) = 678
(M??1, 1.50), 671 (2.06), 578 (0.37), 368 (16.76), 356 (9.64), 79 (100.0), Anal. for
C27H23Br2N3O8 (677.29): Calcd. C, 47.88; H, 3.42; N, 6.20%; Found: C, 47.95; H,
3.48; N, 6.15%.
Synthesis of (Z)-4-(4-hydroxy-3-methoxy-phenyl)-6-[{6-(4-hydroxy-
3-methoxy-phenyl)-2-thioxo-1,2,5,6-tetrahydro-pyrimidine-4-yl}(2-(4-
nitrophenyl)hydrazono)methyl]-5,6-dihydropyrimidine-2(1H)-thione; (4)
A mixture of compound (2e) (0.01 mol) thiourea (0.02 mol) and sodium ethoxide
(0.02 mol sodium, in 50 ml ethanol) was refluxed for 6 h, left to cool, dilute, and
acidified with dilute acetic acid. The precipitated solids were filtered and
crystallized from ethanol to give product (4). Yield 35%; m.p. 225°C; IR (KBr):
m/cm-1 = 3420 (OH), 3220 (NH), 1620 (C=N), 1575 (N=N), 1273 (C=S). H1-NMR
(DMSO-d6): d/ppm = 1.5, 1.7 (m, 2H, CH2), 1.8, 2.0 (m, 2H, CH2), 2.6 (t, 1H, CH),

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3.8 (s, 6H, 2 9 OCH3), 3.9 (t, 1H, CH), 6.68–8.0 (m, 10H, Ar–H), 9.8 (s, 2H,
2 9 OH), 10.6 (s, 1H, NHhydrazo). MS: m/z (%) = 632 (M?-1, 4.13), Anal. for
C29H27N7O6S2 (633.7): Calcd. C, 54.96; H, 4.29; N, 15.47%; Found: C, 54.85; H,
4.30; N, 15.49%.
Synthesis of (1Z,3E)-4-(4-hydroxy-3-methoxy-phenyl)-1-[6-(4-hydroxy-
3-methoxy-phenyl)-2-thioxo-2,3,4,5-tetrahydro-pyrimidine-4-yl]-1-[2-
(4-nitrophenyl)hydrazono]-but-3-ene-2-one; (5)
A mixture of compound (2e) (0.01 mol) thiourea (0.01 mol) and sodium ethoxide
(0.02 mol sodium, in 50 ml ethanol) was refluxed for 4 h, left to cool, dilute, and
acidified with dilute acetic acid. The precipitated solids were filtered and
crystallized from ethanol to give product (5). Yield 54%; m.p. 235°C; IR (KBr):
m/cm-1 = 3400 (OH), 3230 (NH), 3210 (NH), 2930 (CH2), 1690 (CO), 1620
(C=N), 1580 (N=N), 1275 (C=S). H1-NMR (DMSO-d6): d/ppm = 1.5, 1.7 (m, 2H,
CH2), 2.0 (s, 1H, NH), 2.6 (t, 1H, CH), 3.8 (s, 6H, 2 9 OCH3), 6.43 (d, j = 13 Hz,
1H, CH=CH), 6.68–8.0 (m, 10H, Ar–H), 7.55 (d, j = 13 Hz, 1H, CH=CH), 9.8 (s,
2H, 2 9 OH), 10.6 (s, 1H, NHhydrazo). MS: m/z (%) = 575 (M?, 3.93), Anal. for
C28H25N5O7S (575.59): Calcd. C, 58.43; H, 4.38; N, 12.17%; Found: C, 58.55; H,
4.44; N, 12.20%.
Synthesis of N-[bis-(5-(4-hydroxy-3-methoxy-phenyl)-2,5-dihydro-isoxazol-
3-yl)-methylene]-N0-(4-nitro-phenyl)-hydrazine (6)
A mixture of compound (2e) (0.01 mol) hydroxyl amine hydrochloride (0.02 mol)
in pyridine (30 ml) was refluxed for 6 h, left to cool, diluted with water, and the
obtained solid products were crystallized from ethanol to give product (6). Yield
47%; m.p. 185°C; IR (KBr): m/cm-1 = 3400 (OH), 3240 (NH), 3220 (NH), 2929
(CH), 1594 (N=N). MS: m/z (%) = 546 (M?-1, 1.34), Anal. for C27H25N5O8
(547.52): Calcd. C, 59.23; H, 4.60; N, 12.79%; Found: C, 59.30; H, 4.52; N,
12.84%.
Synthesis of 5-(4-hydroxy-3-methoxy-phenyl)-3-{[5-(4-hydroxy-3-
methoxy-phenyl)-4,5-dihydro-1H-pyrazol-3-yl](4-nitro-phenylazo)
methyl}-4,5-dihydro-1H-pyrazole; (7)
A mixture of compound (2e) (0.01 mol) hydrazine hydrate (0.03 mol) in ethanol,
acetic acid (1:1), was refluxed for 4 h, left to cool, diluted with water, and the
obtained solid products were crystallized from ethanol to give product (7). Yield
54%; m.p. 220°C; IR (KBr): m/cm-1 = 3400 (OH), 3230 (NH), 3210 (NH), 2900
(CH), 1690 (CO), 1620 (C=N), 1600 (C=C). H1-NMR (DMSO-d6): d/ppm = 1.5,
1.7 (m, 2H, CH2), 1.8, 2.0 (m, 2H, CH2), 2.6 (t, 1H, CH), 3.8 (s, 6H, 2 9 OCH3),
3.9 (t, 1H, CH), 6.68–8.0 (m, 10H, Ar–H), 9.8 (s, 2H, 2 9 OH), 10.6 (s, 1H,
NHhydrazo). MS: m/z (%) = 545 (M?, 3.03), Anal. for C27H27N7O6 (545.51): Calcd.
C, 59.44; H, 4.99; N, 17.97%; Found: C, 59.35; H, 4.94; N, 17.92%.

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Synthesis of 2,10-diacetyl-6-[(4-chloro-phenyl)-hydrazono]-3,9-bis-(4-
hydroxy-3-methoxy-phenyl)-5,7-dioxo-undecanedioic acid diethyl ester; (8)
A mixture of 4-(4-Chloro-phenylazo)-1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-
1,6-diene-3,5-dione (2d) (0.01 mol), ethyl acetoacetate (0.022 mol), was refluxed in
absolute ethanol for 6 h in the presence of sodium ethoxide (3%). The reaction
mixture was allowed to stand overnight and then acidified by dilute HCl. The solid
products obtained was crystallized from ethanol to give the corresponding Michael
adduct (8). Yield 48%; m.p. 142°C; IR (KBr): m/cm-1 = 3424 (OH), 3220 (NH),
3100 (CH3), 2900 (CH2), 1735 (CH), 1730 (carbonyl acetyl), 1710 (carbonyl ester),
1513 (N=N). MS: m/z (%) = 768 (M??1, 3.47), Anal. for C39H43ClN2O12
(767.22): Calcd. C, 61.05; H, 5.65; N, 3.65%; Found: C, 61.15; H, 5.60; N, 3.71%.
Synthesis of (Z)-ethyl-6-{2-(4-chloro-phenyl)hydrazono}[4-(4-hydroxy-
3-methoxy-phenyl)-6-1,4-dihydropyridin-2-yl]-4-(4-hydroxy -3-
methoxy-phenyl)-2-methyl-1,4-dihydropyridine-3-carboxylate; (9)
A mixture of 2,10-diacetyl-6-[(4-chloro-phenyl)-hydrazono]-3,9-bis-(4-hydroxy-3-
methoxy-phenyl)-5,7-dioxo-undecanedioic acid diethyl ester (8) (0.01 mol) and
ammonium acetate (0.022 mol) was refluxed in boiling acetic acid (30 ml) for
4–6 h. The reaction mixture was cooled and diluted with water to give product (9).
Yield 39%; m.p. 235°C; IR (KBr): m/cm-1 = 3400 (OH), 3300 (NH), 3000 (CH),
2900 (CH3), 1720 (carbonyl ester), 1595 (C=C), 1580 (N=N). H1-NMR (DMSO-d6):
d/ppm = 1.3 (t, 6H, 2 9 CH3CH2), 2.26 (s, 6H, 2 9 CH3), 3.83 (s, 6H,
2 9 OCH3), 4.2 (q, 4H, CH2CH3), 4.4 (s, 2H, 2 9 CH), 6.68–8.0 (m, 10H,
Ar–H), 9.83 (s, 2H, 2 9 OH), 13.2 (s, 1H, NHhydrazo). MS: m/z (%) = 729 (M?,
24.00), Anal. for C39H41ClN4O8 (728.5): Calcd. C, 64.24; H, 5.67; N, 7.68%;
Found: C, 64.31; H, 5.70; N, 7.62%.
Synthesis of 4,40-(1E,10E)-2,20-(1H-pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-
bis-[2-(4-chloro-phenyl)]diazenyl-6-methoxy-phenol; (10)
Purified curcumin (1) (10 mg, 0.027 mol) was dissolved in methanol (2 ml), and
hydraziniumdihydrochloride (14 mg, 0.135 mol) and triethylamine (18.8 ml,
0.135 mol) were added to the solution. In the presence of catalytic amount of
glacial acetic acid, the reaction mixture was refluxed for 4 h, left to cool, the solvent
was evaporated in vacuo, and the solid products obtained were crystallized from
ethanol to give product (10). Yield 83%; m.p. 218°C; IR (KBr): m/cm-1 = 3400
(OH), 3230 (NH), 1590 (C=C), 1620 (C=N). H1-NMR (DMSO-d6): d/ppm = 3.95
(s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d, j = 13 Hz, 2H,
2 9 CH=CH), 6.91–8.12 (m, 6H, Ar–H), 7.55 (d, j = 13 Hz, 2H, 2 9 CH=CH), 9.6
(s, 1H, NH). MS: m/z (%) = 364 (M?, 70.46), Anal. for C21H20N2O4 (364.39):
Calcd. C, 69.22; H, 5.53; N, 7.69%; Found: C, 69.31; H, 5.56; N, 7.60%.

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Synthesis of 4,40-(1E,10E)-2,20-(1-bromo-1H-pyrazole-3,5-diyl)-bis-
(ethane-2,1-diyl)-bis-(2-methoxy-phenol); (11)
A mixture of 4,40-(1E,10E)-2,20-(1H-pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-bis-[2-
(4-chloro-phenyl)]diazenyl-6-methoxy-phenol (10) 0.005 mol and N-bromosuccin-
imide 0.005 mol in chloroform. The mixture was stirred in direct sun light for 2 h.
The solvent was evaporated to give product (11). Yield 63%; m.p. 250°C; IR (KBr):
m/cm-1 = 3400 (OH), 3230 (NH), 1590 (C=C), 1620 (C=N), 720 (N–Br). H1-NMR
(DMSO-d6): d/ppm = 3.95 (s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H,
2 9 OH), 6.43 (d, j = 13 Hz, 2H, 2 9 CH=CH), 6.91–8.12 (m, 6H, Ar–H), 7.55 (d,
j = 13 Hz, 2H, 2 9 CH=CH), Anal. for C21H19BrN2O4 (443.29): Calcd. C, 56.90;
H, 4.32; N, 6.32%; Found: C, 55.70; H, 4.90; N, 6.74%.
Synthesis of 4,40-(1E,10E)-2,20-(1-hydrazinyl-1H-pyrazole-3,5-diyl)-bis-
(ethane-2,1-diyl)-bis-(2-methoxy-phenol); (12)
An equimolar mixture (0.005 mol) of 4,40-(1E,10E)-2,20-(1H-pyrazole-3,5-diyl)-bis-
(ethane-2,1-diyl)-bis-[2-(4-chloro-phenyl)]diazenyl-6-methoxy-phenol (11) and
hydrazine hydrate in ethanol (30 ml) was heated at reflux for 2 h, and cooled.
The resulting solid was collected, washed with dilute ammonia, air-dried, and
recrystallized from ethanol afforded the corresponding product (12). Yield 90%;
m.p. 200°C; IR (KBr): m/cm-1 = 3450 (NH2), 3420 (OH), 3200 (NH), 1590 (C=C),
1620 (C=N). H1-NMR (DMSO-d6): d/ppm = 3.95 (s, 6H, 2 9 OCH3), 4.6 (s, 1H,
NH), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.2 (s, 1H, NH), 6.43 (d, j = 13 Hz,
2H, 2 9 CH=CH), 6.91–8.12 (m, 6H, Ar–H), 7.55 (d, j = 13 Hz, 2H,
2 9 CH=CH). MS: m/z (%) = 395 (M??1, 1.01), Anal. for C21H22N4O4
(394.42): Calcd. C, 63.95; H, 5.62; N, 14.20%; Found: C, 64.05; H, 5.68; N,
14.10%.
Synthesis of 4,40-(1E,10E)-2,20-(1-nitrosyl-1H-pyrazole-3,5-diyl)-bis-
(ethane-2,1-diyl)-bis-(2-methoxy-phenol); (13)
A mixture of (10, 0.005 mol) in 1:1 hydrochloric acid (20 ml) was treated at 3°C
with a cooled concentrated solution of sodium nitrite (0.02 mol in 10 ml water). The
mixture was stirred in the cold for 1 hour and left in the refrigerator for 12 h. The
separated solid was recrystallized from ethanol to give product (13). Yield 75%;
m.p. over 280°C; IR (KBr): m/cm-1 = 3425 (OH), 3200 (NH), 1580 (N=O). MS:
m/z (%) = 393 (M?, 4.24), 368 (10.10), 313 (27.7), 264 (25.92), 236 (24.69), 185
(19.73), 135 (38.78), 97 (89.48), 69 (100.00); Anal. for C21H19N3O5 (393.39):
Calcd. C, 64.12; H, 4.87; N, 10.68%; Found: C, 64.22; H, 4.90; N, 10.74%.
Synthesis of 4,40-(1E,10E)-2,20-(1-chloroacetyl-1H-pyrazole-3,5-diyl)-
bis-(ethane-2,1-diyl)-bis-(2-methoxy-phenol); (14)
To a solution of 4,40-(1E,10E)-2,20-(1H-pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-bis-
[2-(4-chloro-phenyl)]diazenyl-6-methoxy-phenol (10), 0.01 mol was added to

Med Chem Res (2010) 19:413–430
427
chloroacetyl chloride (0.012 mol).The reaction mixture was subjected to fusion at
95°C for 2 h. The solid products obtained recrystallized from ethanol and washed by
pet-ether. Yield 83%; m.p. 190°C; IR (KBr): m/cm-1 = 3420 (OH), 2900 (CH2),
1685 (C=O), 1620 (C=N), 760 (C–Cl). H1-NMR (DMSO-d6): d/ppm = 3.5 (s, 2H,
CH2), 3.95 (s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H, 2 9 OH), 6.43 (d,
j = 13 Hz, 2H, 2 9 CH=CH), 6.91–8.12 (m, 6H, Ar–H), 7.55 (d, j = 13 Hz, 2H,
2 9 CH=CH). MS: m/z (%) = 440 (M?, 2.46); Anal. for C23H21ClN2O5 (440.88):
Calcd. C, 62.66; H, 4.80; N, 6.35%; Found: C, 62.49; H, 4.85; N, 6.30%.
Synthesis of 3,5-bis-[b-(5-{4-chloro-phenylazo}-4-hydroxy-3-methoxy-phenyl)-
ethynyl]-1H-pyrazole; (15)
General procedure Preparation of the diazonium salt: A solution of sodium nitrite
(0.2 gm in 2 ml water) was gradually added to a well-cooled solution of p-chloro
aniline (aromatic amine) (0.01 mol) in concentrated HCl (3.0 ml). The diazonium
salt solution was added drop wise with continuous stirring to (0.005 mol) cold
solution of 4,40-(1E,10E)-2,20-(1H-pyrazole-3,5-diyl)-bis-(ethane-2,1-diyl)-bis-[2-
(4-chloro-phenyl)]diazenyl-6-methoxy-phenol (10) in sodium hydroxide 10%
(20 ml). The reaction mixture was stirred at 0–5°C for 2 h and left to stand at
room temperature. The solid product that was obtained was filtered off, dried, and
recrystallized from ethanol to give product (15). Yield 63%; m.p. 270°C; IR (KBr):
m/cm-1 = 3423 (OH), 3230 (NH), 1598 (C=C), 1585 (N=N). MS: m/z (%) = 641
(M?, 11.49), 633 (17.24), 616 (13.79), 583 (33.33), 553 (36.78), 474 (88.51), 415
(64.37), 342 (60.92), 207 (36.78), 126 (100.00), 74 (70.11), Anal. for
C33H26Cl2N6O4 (641.50): Calcd. C, 61.79; H, 4.09; N, 13.10%; Found: C, 61.85;
H, 4.15; N, 13.02%.
Synthesis of 3,5-bis-{b-[3-(4-chloro-phenylazo)-4-hydroxy-5-methoxy-
phenyl]-ethynyl}-pyrazole-1-formyl; (16)
Compound (15, 0.005 mol) was suspended in mixture of formamide (30 ml) and
methanol (20 ml), stirred under reflux for 30 min, cooled to 0°C, and the desired
product (16) filtered off and recrystallized from methanol. Yield 65%; m.p. 240°C;
IR (KBr): m/cm-1 = 3423 (OH), 1690 (C=O), 1595 (C=C), 1580 (N = N). H1-
NMR (DMSO-d6): d/ppm = 3.95 (s, 6H, 2 9 OCH3), 5.8 (s, 1H, CH), 5.86 (s, 2H,
2 9 OH), 6.43 (d, j = 13 Hz, 2H, 2 9 CH=CH), 6.91–8.12 (m, 12H, Ar–H), 7.55
(d, j = 13 Hz, 2H, 2 9 CH=CH), 8.8 (s, 1H, CH). MS: m/z (%) = 670 (M??2,
2.01), Anal. for C34H26Cl2N6O5 (669.51): Calcd. C, 60.99; H, 3.91; N, 12.55%;
Found: C, 61.09; H, 3.87; N, 12.61%.
Synthesis of 3,5-bis-[b-(5-{4-Chloro-phenylazo}-4-hydroxy-3-methoxy-phenyl)-
ethynyl]-2-hydroxy-methyl-pyrazole; (17)
A mixture of (15) (0.005 mol) and formaldehyde (40%; 2 ml) in methanol (30 ml)
was stirred at room temperature for 1 hour, cooled (0°C) for 12 h, and the
precipitated solid filtered and recrystallized from methanol to yield product (17).

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Med Chem Res (2010) 19:413–430
Yield 61%; m.p. 255°C; IR (KBr): m/cm-1 = 3425 (OH), 1600 (C=N), 1582 (N=N).
H1-NMR (DMSO-d6): d/ppm = 2.0 (s, 1H, OH), 3.83 (s, 6H, 2 9 OCH3), 6.2 (s,
1H, CH), 5.97 (s, 2H, CH2), 6.9 (d, j = 13 Hz, 2H, 2 9 CH=CH), 7.06–7.94 (m,
12H, Ar–H), 7.5 (d, j = 13 Hz, 2H, 2 9 CH = CH), 9.8 (s, 2H, 2 9 OH), MS: m/z
(%) = 671 (M?, 4.67), Anal. for C34H28Cl2N6O5 (671.53): Calcd. C, 60.81; H,
4.20; N, 12.51%; Found: C, 60.95; H, 4.28; N, 12.44%.
Antitumor activity
Cells of Ehrlich ascites tumor were obtained from National Cancer Institute, Cairo,
Egypt. Animals, adult Swiss male albino mice (20–25 g) were procured from
Pharmacology Faculty, Mansoura University, Egypt, and used throughout this
study. They were housed in microlon boxes in a controlled environment
(temperature 25 ? 2°C and 12-h dark/light cycle) with standard laboratory diet
and water ad libitum (5-fluorouracil ? 20 mg/kg).
Animals were divided into seven groups as follows: All animals were inoculated
with 2 3 106 cells/mouse on day 0 except normal group and treatment started 24 h
after inoculation, at 2 doses of 50 and 250 mg/kg/day, i.p. The control group was
treated with the same volume of 0.9% sodium chloride solution. All the treatments
were given for 9 days.
Tumor-reducing activity
Ehrlich ascites tumor cells (0.2 ml) were injected into the mice peritoneal cavity and
different concentrations (1 mg/ml to 0.1 ml/mice on day) of the drug (5 mice/group)
were injected from day 1 to day 9 every day. Animals were observed for the
development as ascites tumor and death due to tumor burden. Life span % I
LS = (T-C)/C 3 100, where T is the number of days the treated animals survived
and C is the number of days control animals survived. % I LS [ 25% was
considered significant (Kuttan et al., 1985).
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