Synthesis of new acridines and hydrazones derived from cyclic β-diketone for cytotoxic and antiviral evaluation

Article abstract of DOI:10.1016/j.ejmech.2009.04.001

Cyclic β-diketone namely, dimedone was utilized to prepare different chemical entities whether cyclic such as acridines, thiadiazole and triazole or acyclic systems as hydrazide, hydrazones, thiosemicarbazide and semicarbazide. The structures of the novel compounds were determined using elemental ana-lyses and various spectroscopic methods. Most acyclic derivatives especially semicarbazide 19, hydrazide 9 and thiosemicarbazide 16 showed a higher in vitro cytotoxic activity against hepatoma cell line (HepG2) than the cyclized acridine derivatives. The antiviral activity of the new compounds against Hepatitis A Virus (HAV) using the plague infectivity reduction assay revealed that the acridine 4 and the hydrazone 12 were more active than the reference drug amantadine.

Full text of DOI:10.1016/j.ejmech.2009.04.001

European Journal of Medicinal Chemistry 44 (2009) 3680–3686
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new acridines and hydrazones derived from
cyclic -diketone for cytotoxic and antiviral evaluation
b
,
b
Osama I. El-Sabbagh a , Hanaa M. Rady
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
b Department of Chemistry of Natural Compounds, National Research Centre, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclic b-diketone namely, dimedone was utilized to prepare different chemical entities whether cyclic
Received 2 September 2008
Received in revised form
18 March 2009
Accepted 2 April 2009
Available online 8 April 2009
such as acridines, thiadiazole and triazole or acyclic systems as hydrazide, hydrazones, thiosemicarbazide
and semicarbazide. The structures of the novel compounds were determined using elemental ana-
lyses and various spectroscopic methods. Most acyclic derivatives especially semicarbazide 19, hydrazide
9 and thiosemicarbazide 16 showed a higher in vitro cytotoxic activity against hepatoma cell line
(HepG2) than the cyclized acridine derivatives. The antiviral activity of the new compounds against
Hepatitis A Virus (HAV) using the plague infectivity reduction assay revealed that the acridine 4 and the
hydrazone 12 were more active than the reference drug amantadine.
Keywords:
Acridines
Hydrazones
Cytotoxic
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antiviral activity
1. Introduction
2. Results and discussions
Malignant tumor and viral infections are the most serious
threats against human health in the world. Hepatitis A Virus (HAV) is
a picornavirus, a common causative agent of acute self-limited
hepatitis that sometimes leads to fulminant hepatic failure [1,2].
Therefore, treatment of severe infections [1] is still an issue of
major concern especially the clinically used drugs remains rela-
tively poor.
Furthermore, there exist a number of acridine (amsacrine)
[3–7], thiadiazole [8,9] and triazole (ribavirin) [10–12] deriva-
tives having anticancer and antiviral activities. Moreover, some
hydrazones [13,14] and thiosemicarbazone [15] derivatives were
reported to exhibit anticancer and antiviral properties,
respectively.
2.1. Chemistry
In this work, the novel derivatives 4–19 were designed and
prepared as illustrated in Schemes 1 and 2.
The key intermediate ethyl 2-(5,5-dimethyl-3-oxocyclohex-1-
enyl amino) acetate (3) was prepared by condensation of equimolar
amounts of 5,5-dimethyl-1,3-cyclohexandione (1) with ethyl gly-
cinate HCl (2) in the presence of anhydrous sodium acetate
adopting two procedures either via heating the reactants under
reflux in toluene for 15 h to produce 65% yield [16–19] or in ethanol
for 2.5 h giving 85% yield. The latter method was preferred than the
former one due to it afforded pure product with higher yield in
shorter time.
Based on the above findings and coupled with our interest of the
The novel nonclassical acridine derivatives (4–8) were obtained
in one-pot synthesis reaction via cyclocondensation of enaminone
3 with half equivalent of aromatic aldehyde through heating the
reactants at reflux for 4 h in glacial acetic acid (Scheme 1). This
method gave the acridines in one step without passing through the
formation of bis intermediates. Moreover, acetic acid was found to
be the solvent of choice for conducting the cyclocondensation
whereas it provided the reaction with a higher temperature
necessary for its completion and also maintained the expelled
amine residue, thus it favors the formation of acridines.
chemistry of cyclic
b-diketone especially dimedone gave us the
opportunity for preparation of diverse heterocyclic ring systems
such as acridine, thiadiazole and triazole derivatives in addition to
acyclic systems e.g. hydrazone, thiosemicarbazide as well as sem-
icarbazide derivatives with the aim to evaluate their cytotoxic and
antiviral activities.
* Corresponding author. Tel.: þ20 109610300; fax: þ20 552303266.
E-mail addresses: elsabbagh_59@yahoo.com, elsabbagh_59@zu.edu.eg
(O.I. El-Sabbagh).
The structures of the novel acridines were confirmed using IR
spectra which showed the disappearance of NH stretching band at
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.001

O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
3681
O
H3N
+
OC2H5
Cl
OH
O
2
1
ethanol
reflux
AcONa
O
OC2H5
N
H
O
3
CHO
acetic acid
reflux
toluene
NH2NH2
warming
R
O
R
NHNH2
N
H
O
O
O
9
CHO
ethanol
reflux
R
N
O
R
O
OC2H5
NHN
O
N
H
4-8
4: R= H
5: R= Br
7: R= OH
10-15
6: R= Cl
8: R= OCH3
10: R= H
12: R= Cl
11: R= Br
13: R= OH
14: R=OCH3 15:R= NO2
Scheme 1. Synthetic pathway for compounds 3–15.
y
¼ 3260 cmꢀ1 for the starting enaminone. 1H NMR proved the
just warming them in toluene with continuous stirring for 10 min
only (83% yield). The latter method was characterized by affording
pure product in an excellent yield.
absence of vinylic H singlet peak around
NH singlet peak around
addition, the appearance of singlet signal at
able to 9-H of acridine nucleus.
d
¼ 4.992 ppm as well as
d
¼ 7.25 ppm for the starting enaminone. In
d
¼ 5.168 ppm assign-
Condensation of the hydrazide 9 with different aromatic alde-
hydes was conducted by heating the reactants in ethanol contain-
ing catalytic amount of glacial acetic acid to afford the novel
hydrazone derivatives 10–15.
An interesting observation is the appearance of geminal methyl
groups at different chemical shifts (0.954 and 1.065). This obser-
vation of the different values for geminal methyl groups may be due
to either the nonbonding interaction between the aromatic residue
at position 9 and the 1,8-dioxo functions.
This may allow the former to occupy angular position to the
tricyclic plane of the acridine skeleton. The other possibility for
the distinguished chemical shifts of the methyls may be due to the
limited free rotation of the phenyl group at position 9. One of
the geminal methyl may also be affected by the anisotropy of the
ring current. Thus, it appears at higher chemical shift value which
was supported by constructing models for these compounds.
Moreover, the acid hydrazide 9 was synthesized through the
reaction of enaminone ester 3 with hydrazine hydrate either in
ethanol by heating the reactants at reflux for 3 h (50% yield) or by
The structures of these hydrazones were confirmed using 1H
NMR spectra which showed the disappearance of NH2 singlet broad
peak around
the appearance of singlet signal at
to azomethine proton as well as multiplet peaks around
7.794 ppm due to aromatic residue. Furthermore, the presence of
vinylic H singlet around
¼ 4.7 ppm as well as NH broad singlet
around
d
¼ 4.322 ppm for the starting hydrazide. In addition,
¼ 7.939–8.031 ppm assignable
¼ 6.827–
d
d
d
d
¼ 7.0 ppm of enaminone system exclude the formation of
acridine nucleus under the reaction condition.
The formation of the novel thiosemicarbazide derivative 16 was
accomplished through the reaction of equimolar amounts of
hydrazide 9 and phenyl isothiocyanate in refluxing ethanol. In
addition, the thiosemicarbazide 16 was cyclized either under acidic

3682
O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
O
NHNH2
N
H
O
9
NCS
ethanol
rt
ethanol
reflux
NCO
Cl
O
O
O
H
S
H
N
N
Cl
N
H
N
H
N
H
N
H
N
H
N
H
O
O
19
16
8% NaOH
reflux
Conc. H2SO4
rt
O
O
N
N
N
H
N
S
N
H
N
NH
N
SH
17
18
Scheme 2. Synthetic pathway for compounds 16–19.
condition (conc. H2SO4) to afford the novel thiadiazole derivative 17
or in basic media (8% NaOH) to give the triazole derivative 18
(Scheme 2).
Furthermore, the acid hydrazide 9 was allowed to react with
2-chloroethylisocyanate by stirring the reactants together in
ethanol at room temperature for 12 h [20] in order to produce the
novel 1-(2-(5,5-dimethyl-1-oxo-2-cyclohexen-3-ylamino)acetyl)-
4-(2-chloroethyl)semicarbazide (19).
All tested compounds showed marked and significant cytotoxic
effect when the treated cells incubated for 24 h except compound 4.
Unfortunately, compounds 10, 12, 15 and 17 cannot be determined
due to solubility problems.
First of all, through analysis of the data presented in Table 1 and
illustrated in Fig. 1, it was observed that the ester 3 showed 50%
reduction in the cell growth. It can be modified to acyclic deriva-
tives or cyclized to nonclassical acridines.
The new compounds were characterized using melting points
and thin layer chromatography techniques in different solvent
systems. Moreover, the structures of the novel compounds were
determined using elemental analyses and various spectroscopic
methods.
Table 1
Effect of test compounds on cell growth of hepatoma cell line (HepG2) incubated for
24 h in vitro.
Test compounds
Absorbance
Cell growth
%Age
reduction
Control
T 3
T 4
T 5
T 6
T 7
T 8
T 9
T 11
T 13
T 14
T 16
T 18
T 19
0.724
0.360
0.700
0.368
0.332
0.328
0.449
0.307
0.434
0.432
0.601
0.327
0.466
0.302
100
50
0.00
50
3.32
49.17
54.14
54.7
37.99
57.4
40.06
40.32
16.99
54.84
35.64
58.29
2.2. Biological study
96.68
50.83
45.86
45.3
62.01
42.6
59.94
59.68
83.01
45.16
64.36
41.71
Compounds 3–19 were evaluated in vitro to explore their cyto-
toxic effect using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide] assay and also their antiviral activities were
studied adopting plaque reduction assay.
2.2.1. Cytotoxicity test
Cytotoxicity of the new compounds was tested against hepa-
toma cell line (HepG2) at 10
mmol/ml using MTT assay [21,22]. The
results were calculated and expressed as percentage reduction in
cell proliferation (Table 1).

O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
3683
70
60
50
40
30
20
10
0
10ug
20ug
70
60
50
40
30
20
10
0
T 3
T 4
T 5
T 6
T 7
T 8
T 9
T 11 T 13 T 14 T 16 T 18 T 19
Amantadine
4
5
Compounds
10
12
Test compounds
Fig. 1. Effect of new compounds on cell growth of hepatoma cell line (HepG2) incu-
bated for 24 h in vitro expressed as %age reduction.
Fig. 2. Effect of some compounds (4, 5, 10, 12) on Hepatitis A Virus in comparison with
the reference drug amantadine at concentrations 10 and 20 g/ml.
m
ꢁ It was also noted that cyclization of the thiosemicarbazide 16 to
the triazole 18 led to decrease in the cytotoxic activity from
54.84 to 35.64% (Table 1 and Fig. 1).
ꢁ Regarding the acyclic derivatives (9, 11, 13, 14, 16 and19), it was
observed that semicarbazide (19), hydrazide (9) and thio-
semicarbazide (16) showed higher cytotoxic activity (58.29,
57.4 and 54.84%, respectively) than the hydrazone derivatives
(11,13,14). The higher activity of the semicarbazide (19) may be
attributed to the presence of chloroethylurea moiety in the
semicarbazide which can form aziridinium cation alkylating
DNA inhibiting cell growth [20].
ꢁ It was found also that the acyclic semicarbazide 19 was the
most cytotoxic derivative (58.29%) while the cyclized parent
acridine 4 bearing 9-unsubstitutedphenyl group (3.32%) was
the least active one. Replacement of the phenyl group of acri-
dine 4 by p-substituted phenyl led to the enhancement of the
cytotoxic activity in general to reach 54.7% for compound 7
(Table 1). These acridines may act either through binding
tightly but reversibly to DNA by intercalation between the base
pairs of the double helix or by blocking topoisomerase binding
to DNA. The latter mechanism of action may be of great interest
in the development of anticancer or antiviral agents [4–6].
Table 2
Antiviral activity for amantadine and compounds 3–19 against Hepatitis A Virus at
concentrations 10 and 20
m
g/ml.
Compound No
Concentration
Initial virus count
(PFU/ml)a
Virus count
(PFU/ml)
%Age
reduction
(m
g/ml)
Amantadineb
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
m
m
mg
m
m
m
mg
m
m
m
mg
m
mg
m
mg
m
mg
m
m
m
m
m
m
m
m
m
m
m
m
m
m
m
g
g
1.06 ꢂ 107
0.74 ꢂ 107
0.58 ꢂ 107
1.00 ꢂ 107
0.9 ꢂ 107
30.1
45.2
5.6
Thus, it was noted that the acyclic derivatives (9, 16, 19) were
relatively more active than the cyclized acridine derivatives (4–8).
3
g
g
g
15
2.2.2. Evaluation of the antiviral activity
4
0.66 ꢂ 107
0.42 ꢂ 107
0.56 ꢂ 107
0.5 ꢂ 107
37.7
60.3
47.1
52.8
30.1
47.1
30.1
48.1
24.5
45.2
33.9
34.9
56.6
58.4
43.3
27.3
57.5
60.3
9.4
All the synthesized compounds 3–19 were examined in vitro
against Hepatitis A Virus (HAV) to evaluate their antiviral activity
using plague infectivity reduction assay [23]. The known drug,
amantadine, commonly utilized for therapeutic treatment of HAV
was used as a reference drug. The results of the antiviral evaluation
recorded in Table 2 and illustrated in Fig. 2 revealed the following:
The ester 3 showed very weak antiviral activity which was
improved through its cyclization to nonclassical acridines or its
conversion to acyclic derivatives.
5
g
g
g
6
0.74 ꢂ 107
0.56 ꢂ 107
0.74 ꢂ 107
0.55 ꢂ 107
0.8 ꢂ 107
7
g
8
g
0.58 ꢂ 107
0.7 ꢂ 107
9
g
0.69 ꢂ 107
0.46 ꢂ 107
0.44 ꢂ 107
0.6 ꢂ 107
10
11
12
13
14
15
16
17
18
19
1. For the acridine derivatives 4–8, the most active one at a dose
of 20 mg was the parent compound 4 bearing unsub-
stitutedphenyl group whereas it caused 60.3% reduction in the
virus titer (Fig. 2) while compound 8 bearing a p-methox-
yphenyl group showed the least antiviral activity at both conc.
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
0.77 ꢂ 107
0.45 ꢂ 107
0.42 ꢂ 107
0.96 ꢂ 107
0.9 ꢂ 107
10
mg and 20
mg. The order of antiviral activity for acridines at
15
0.8 ꢂ 107
24.5
36.2
39.6
43.3
18.8
39.6
45.2
49
28.3
30.1
42.4
40.5
a dose of 20
mg/ml is 4 > 5 > 6 > 7 > 8.
0.65 ꢂ 107
0.64 ꢂ 107
0.6 ꢂ 107
2. Regarding the acyclic derivatives, the hydrazone bearing the
p-chlorophenyl moiety (12) was the most active one whereas it
caused 60.3% reduction in the virus titer at 20 mg/ml (Fig. 2)
while most derivatives showed moderate antiviral activity.
0.86 ꢂ 107
0.64 ꢂ 107
0.58 ꢂ 107
0.54 ꢂ 107
0.76 ꢂ 107
0.74 ꢂ 107
0.61 ꢂ 107
0.63 ꢂ 107
The rank order of antiviral activity is 12 > 4 >10 > 5. The parent
acridine 4 bearing 9-unsubstitutedphenyl group showed very weak
in vitro cytotoxic activity (3.32%) against hepatoma cell line (HepG2)
and in the same time it exhibited the highest antiviral activity (60.3%)
m
m
m
m
g
g
g
a
at concentration 20
mg/ml against Hepatitis A Virus, so, it can be
PFU: Plaque forming unit.
Amantadine: positive control.
b
subjected to future optimization to be used as a safe antiviral agent.

3684
O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
3. Conclusion
4.1.2.2. Ethyl 2-[9-(4-bromophenyl)-1,2,3,4,5,6,7,8-octahydro-3,3,6,6-
tetramethyl-1,8-dioxoacridin-10(9H)-yl]acetate (5). Yield: 53%; m.p.:
208–210 ꢃC; MS: m/z (rel. int.) ¼ 516 (Mþ þ 1, 4.7), 515 (Mþ, 5.6), 458
(3.9), 426 (9.9), 358 (64.4), 270 (16.3), 201 (14.6), 154 (69.5), 90
(25.3), 55 (100.0). Anal. calcd for C27H32BrNO4: C, 63.04; H, 6.27; N,
2.72. Found: C, 63.07; H, 6.33; N, 2.74%.
From cyclic
b-diketone, heterocyclic derivatives such as acri-
dines and thiadiazole or acyclic systems as hydrazones and semi-
carbazide were synthesized. The acyclic semicarbazide 19 showed
a higher in vitro cytotoxic activity against hepatoma cell line
(HepG2) than the cyclized acridine derivatives. The antiviral study
revealed that the acridine 4 and the hydrazone 12 were more active
against Hepatitis A Virus than the reference drug amantadine upon
using the plague infectivity reduction assay.
4.1.2.3. Ethyl 2-[9-(4-chlorophenyl)-1,2,3,4,5,6,7,8-octahydro-3,3,6,6-
tetramethyl-1,8-dioxoacridin-10(9H)-yl]acetate (6). Yield: 50%; m.p.:
195–197 ꢃC; 1H NMR (CDCl3):
d
¼ 0.954 (s, 6H, 2CH3), 1.065 (s, 6H,
2CH3), 1.319 (t, 3H, CH2CH3), 2.187 (s, 4H, 2CH2), 2.309 (d, 4H, 2CH2),
4.275–4.385 (m, 4H, CH2CH3 þ CH2CO), 5.168 (s, 1H, C9-acridine H),
7.115 (d, 2H, J ¼ 8.6 Hz, ArH), 7.291 (d, 2H, J ¼ 8.4 Hz, ArH) ppm. Anal.
calcd for C27H32ClNO4: C, 69.00; H, 6.86; N, 2.98. Found: C, 69.13; H,
6.75; N, 3.11%.
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Gallenkamp digital
melting point apparatus in open capillaries and are uncorrected. IR
spectra (KBr) were recorded using Bruker spectrophotometer.1H
NMR spectra were determined on Varian Gemini 200 MHz using
4.1.2.4. Ethyl 2-[9-(4-hydroxyphenyl)-1,2,3,4,5,6,7,8-octahydro-3,3,6,6-
tetra methyl-1,8-dioxoacridin-10(9H)-yl]acetate (7). Yield: 52%; m.p.:
252–254 ꢃC; IR:
y
¼ 3335 (OH), 3022 (CH, aromatic), 2960 (CH,
aliphatic), 1738 (CO, ester), 1628 (CO) cmꢀ1 1H NMR (CDCl3):
;
TMS as an internal standard (chemical shifts in
d
, ppm). Mass
d
¼ 0.966 (s, 6H, 2CH3),1.055 (s, 6H, 2CH3), 1.316 (t, 3H, CH2CH3), 1.714
spectra were measured on a GCMS-QP1000EX-SHIMADZU with
ionization energy 70 eV. Elemental analyses were determined
using Heraeus (Elemntar), CHNS analyzer (Germany) at Microana-
lytical Center, Faculty of Science, University of Cairo, Giza, Egypt.
Their results corresponded to the calculated values within experi-
mental error. TLC was performed on silica gel G (Fluka) and spots
were visualized by iodine vapors or irradiation with UV light
(254 nm). The starting materials were purchased from Sigma–
Aldrich.
(br s,1H, OH, exch.), 2.198 (s, 4H, 2CH2), 2.295–2.367 (m, 4H, 2CH2),
4.257–4.386 (m, 4H, CH2CH3 þ CH2CO), 5.125 (s, 1H, C9-acridine H),
6.514 (d, 2H, J ¼ 8.4 Hz ArH), 7.095 (d, 2H, J ¼ 8.6 Hz ArH) ppm. Anal.
calcd for C27 H33N O5: C, 71.82; H, 7.37; N, 3.10. Found: C, 71.66; H,
7.16; N, 3.13%.
4.1.2.5. Ethyl 2-[9-(4-methoxyphenyl)-1,2,3,4,5,6,7,8-octahydro-3,3,6,6-
tetramethyl-1,8-dioxoacridin-10(9H)-yl]acetate (8). Yield: 58%; m.p.:
239–241 ꢃC; IR:
y
¼ 3066 (CH, aromatic), 2959 (CH, aliphatic), 1744
(CO, ester), 1629 (CO) cmꢀ1; 1H NMR (CDCl3):
d
¼ 0.969 (s, 6H, 2CH3),
4.1.1. Ethyl 2-(5,5-dimethyl-3-oxocyclohex-1-enylamino)acetate (3)
A mixture of equimolar amounts (21.42 mmol) of 5,5-dimethyl-
1,3-cyclohexanedione (dimedone, 1), ethyl glycinate HCl (2) and
anhydrous sodium acetate was heated under reflux either in
toluene (50 ml) for 15 h (method 1) or in ethanol (50 ml) for 2.5 h
(method 2). The reaction mixture was filtered, concentrated and
then allowed to cool to room temperature. The reaction mixture
was mixed with petroleum ether (60–80) with continuous stirring.
The obtained crystalline product was filtered, dried and crystallized
from petroleum ether (60–80).
1.067 (s, 6H, 2CH3), 1.319 (t, 3H, CH2CH3), 2.195 (s, 4H, 2CH2), 2.305 (d,
4H, 2CH2), 3.716 (s, 3H, OCH3), 4.278–4.372 (m, 4H, CH2CH3 þ CH2CO),
5.163 (s, 1H, C9-acridine H), 6.695 (d, 2H, J ¼ 8.6 Hz, ArH), 7.242 (d, 2H,
J ¼ 8.4 Hz, ArH) ppm. Anal. calcd for C28H35NO5: C, 72.23; H, 7.58;
N,3.01. Found: C, 72.22; H, 7.36; N, 3.08%.
4.1.3. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)aceto-
hydrazide (9)
4.1.3.1. Method 1. A mixture of ester 3 (10 mmol) and hydrazine
hydrate (15 mmol) in ethanol (50 ml) was refluxed for 3 h. The
solvent was removed by distillation under reduced pressure and the
separated product was filtered, and then crystallized from ethanol.
Yield: 65% (method 1); 85% (method 2); m.p.: 101–103 ꢃC; IR:
y
¼ 3282, 3260 (NH), 3093 (CH, aromatic), 2953 (CH, aliphatic),1743
(C]O, ester), 1602 (C]O, cyclic) cmꢀ1 1H NMR (CDCl3):
;
d
¼ 1.067
(s, 6H, 2CH3), 1.272 (t, 3H, CH2CH3), 2.182 (s, 2H, CH2), 2.238 (s, 2H,
CH2), 3.801 (s, 1H, CH2CO), 3.824 (s, 1H, CH2CO), 4.243–4.279 (q, 2H,
CH2CH3), 4.992 (s, 1H, vinylic H), 7.25 (s, 1H, NH, exch.) ppm. Anal.
calcd for C12H19NO3:C, 63.98; H, 8.50; N, 6.22. Found: C, 63.93; H,
8.45; N, 6.42%.
4.1.3.2. Method 2. A mixture of ester 3 (10 mmol) and hydrazine
hydrate (15 mmol) in toluene (50 ml) was just warmed with
continuous stirring for 10 min only. The reaction mixture was
allowed to cool to room temperature and the separated product
was filtered, washed with toluene and then crystallized.
Yield: 50% (method 1); 83% (method 2); m.p.: 214–216 ꢃC; IR:
4.1.2. General procedure for preparation of compounds 4–8
A solution of enaminone 3 (10 mmol) and half equivalent of
aromatic aldehyde (5 mmol) in glacial acetic acid (15 ml) were
heated at reflux for 4 h. The reaction mixture was allowed to cool to
room temperature and then diluted with water. The separated
product was filtered, washed with water, dried and crystallized
with ethanol/H2O (5:2).
y
¼ 3304, 3249 (NH2, 2NH), 3086 (CH, aromatic), 2954
(CH, aliphatic), 1658 (CONH), 1609 (C]O, cyclic) cmꢀ1;1H NMR
(DMSO-d6):
d
¼ 0.980 (s, 6H, 2CH3), 1.969 (s, 2H, CH2), 2.237 (s, 2H,
CH2), 3.599 (s, 1H, CH2CO), 3.628 (s, 1H, CH2CO), 4.322 (br s, 2H,
NH2, exch.), 4.750 (s, 1H, vinylic H), 7.115 (br s, 1H, NH, exch.), 9.188
(br s, 1H, NH, exch.) ppm. Anal. calcd for C10H17N3O2: C, 56.85; H,
8.11; N, 19.89. Found: C, 57.00; H, 8.10; N, 19.98%.
4.1.2.1. Ethyl
2-[9-(phenyl)-1,2,3,4,5,6,7,8-octahydro-3,3,6,6-tetra-
4.1.4. General procedure for preparation of compounds 10–15
A mixture of equimolar amounts (5 mmol) of hydrazide 9 and
the appropriate aldehyde was refluxed in ethanol (30 ml) con-
taining acetic acid (1 ml) for 2 h. After cooling, the reaction mixture
was treated with least amount of cold H2O and the separated
product was filtered and then crystallized from the appropriate
solvent.
methyl-1,8-dioxoacridin-10(9H)-yl]acetate (4). Yield: 56%; m.p.:
202–204 ꢃC; IR:
y
¼ 3063 (CH, aromatic), 2960 (CH, aliphatic), 1750
(CO, ester), 1632 (CO, cyclic) cmꢀ1; MS: m/z (rel. int.) ¼ 435
(Mþ, 19.8), 358 (100.0), 330 (23.0), 274 (4.7), 218 (2.0), 165 (2.0), 115
(1.9), 77 (9.8). Anal. calcd for C27H33NO4: C, 74.45; H, 7.64; N, 3.22.
Found: C, 74.55; H, 7.40; N, 3.25%.

O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
3685
4.1.4.1. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-benzyliden-
Yield: 90%; m.p.: 192–193 ꢃC; IR:
y
¼ 3286, 3255, 3228 (NH),
eacetohydrazide (10). Yield: 66%; m.p.: 211–213 ꢃC; crystallized
from ethanol/H2O; MS: m/z (rel. int.) ¼ 299 (Mþ, 39.1), 284 (53.0),
269 (35.7), 207 (20.0), 180 (20.9), 152 (46.1), 138 (20.0), 124 (27.8),
106 (53.0), 95 (32.2), 83 (59.1), 67 (100.0).Anal. calcd for
C17H21N3O2:C, 68.20; H, 7.07; N, 14.04. Found: C, 68.40; H, 6.92; N,
14.24%.
3019 (CH, aromatic), 2956 (CH, aliphatic), 1701, 1680 (CO) cmꢀ1; 1H
NMR (DMSO-d6):
d
¼ 1.001 (s, 6H, 2CH3), 1.996 (s, 2H, CH2), 2.289
(s, 2H, CH2), 3.826 (d, 2H, CH2CO), 4.841 (s,1H, vinylic H), 7.100 (br s,
1H, NHCH2 exch.), 7.169–7.471 (m, 5H, ArH), 9.613 (br s, 1H, NHCS,
exch.), 9.723 (s, 1H, NHPh, exch.), 10.170 (s,1H, NHCO, exch.) ppm.
Anal. calcd for C17H22N4O2S: C, 58.94; H, 6.40; N, 16.17. Found: C,
58.89; H, 6.55; N, 16.41%.
4.1.4.2. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-(4-bromob-
enzylidene)acetohydrazide (11). Yield: 85%; m.p.: 245–247 ꢃC;
4.1.6. 5,5-Dimethyl-3-[(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-
yl)methylamino] cyclohex-2-enone (17)
crystallized from dioxane/H2O; IR:
aromatic), 2952 (CH, aliphatic),1693 (CO) cmꢀ1
d6):
3.808 (s, 1H, CH2CO), 4.216 (s, 1H, CH2CO), 4.743 (s, 1H, vinylic H),
7.082 (br s, 1H, NHCH2, exch.), 7.648–7.655 (m, H, ArH), 7.980 (s, 1H,
CH]N), 11.666 (s,1H, NHCO exch.) ppm. Anal. calcd for
C17H20BrN3O2: C, 53.98; H, 5.33; N, 11.11. Found: C, 53.97; H, 5.22; N,
11.42%.
y
¼ 3264 (NH), 3088 (CH,
;
1H NMR (DMSO-
Thiosemicarbazide 16 (5 mmol) was heated at reflux in NaOH
(8%, 20 ml) for 2 h. The solution was cooled, filtered and acidified
with 2 N HCl. The separated solid was filtered, dried and crystal-
lized from ethanol/H2O.
d
¼ 0.974 (s, 6H, 2CH3), 1.967 (s, 2H, CH2), 2.279 (s, 2H, CH2),
Yield: 60%; m.p.: 296–297 ꢃC; IR:
aromatic), 2949 (CH, aliphatic), 1600 (CO, cyclic) cmꢀ1
(DMSO-d6):
y
¼ 3269 (NH), 3064 (CH,
.
1H NMR
d
¼ 0.887 (s, 6H, 2CH3), 1.908 (s, 2H, CH2), 1.980 (s, 2H,
CH2), 4.106 (d, 2H, N–CH2), 4.713 (s, 1H, vinylic H), 7.057–7.533
(m, 7H, ArH þ 2NH) ppm. Anal. calcd for C17H20N4OS: C, 62.17; H,
6.14; N, 17.06. Found: C, 61.85; H, 5.77; N, 17.52%.
4.1.4.3. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-(4-chlorobe-
nzylidene)acetohydrazide (12). Yield: 90%; m.p.: 236–237 ꢃC; crys-
tallized from ethanol/H2O; IR:
y
¼ 3259 (NH), 3087 (CH, aromatic),
2953 (CH, aliphatic), 1692 (CO) cmꢀ1
;
1H NMR (DMSO-d6):
4.1.7. 5,5-Dimethyl-3-[(5-(phenylamino)-1,3,4-thiadiazol-2-yl)methy-
lamino] cyclohex-2-enone (18)
d
¼ 1.007 (s, 6H, 2CH3), 1.999 (s, 2H, CH2), 2.309 (s, 2H, CH2), 3.809
(d, 1H, J ¼ 5.6 Hz, CH2CO), 4.231 (d, 1H, J ¼ 5.4 Hz, CH2 CO), 4.782
(s, 1H, vinylic H), 7.066 (s, 1H, NHCH2, exch.), 7.506 (d, 2H,
J ¼ 8.6 Hz, ArH), 7.752 (d, 2H, J ¼ 8.4 Hz, ArH), 8.031 (s, 1H,
CH]N), 11.652 (s, 1H, NHCO exch.) ppm. Anal. calcd for
C17H20ClN3O2: C, 61.17; H, 6.04; N, 12.59. Found: C, 61.18; H, 6.11;
N, 12.65%.
Thiosemicarbazide 16 (3 mmol) was dissolved in 10 ml
concentrated H2SO4 while cooling and then allowed to stand for
1 h. The reaction mixture was poured on crushed ice with contin-
uous stirring and then neutralized with NH4OH. The separated
product was filtered, washed with water and crystallized from
dioxane/H2O.
Yield: 50%; m.p.: 252–254 ꢃC; MS: m/z (rel. int.) ¼ 330 (Mþ þ 2,
5.5), 329 (Mþ þ 1, 23.3), 328 (Mþ, 64.1), 210 (11.2), 190 (56.5), 136
(41.1), 118 (28.7), 77 (100.0). Anal. calcd for C17H20N4OS: C, 62.17; H,
6.14; N, 17.06. Found: C, 62.17; H, 6.21; N, 17.40%.
4.1.4.4. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-(4-hydrox-
ybenzylidene)acetohydrazide (13). Yield: 80%; m.p.: 247–249 ꢃC;
crystallized from ethanol; IR:
(CH, aromatic), 2960 (CH, aliphatic), 1686 (CONH), 1609 (C]O,
cyclic) cmꢀ1. 1H NMR (DMSO-d6):
2H, CH2), 2.286 (d, 2H, CH2), 3.773 (d, 1H, J ¼ 5.4 Hz, CH2CO), 4.189
(d, 1H, J ¼ 5.2 Hz, CH2CO), 4.789 (s, 1H, vinylic H), 6.827 (d, 2H,
J ¼ 7.8 Hz, ArH), 7.037 (br s, 1H, NH exch.), 7.535 (d, 2H, J ¼ 8.6 Hz,
ArH), 7.939 (s, 1H, N]CH), 9.919 (s, 1H, OH, exch.), 11.393 (s, 1H, NH,
exch.) ppm. Anal. calcd for C17H21N3O3: C, 64.74; H, 6.71; N, 13.32.
Found: C, 64.88; H, 6.90; N, 13.42%.
y
¼ 3397 (OH), 3300, 3175 (NH), 3078
d
¼ 1.007 (s, 6H, 2CH3), 2.002 (s,
4.1.8. N1-[2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)acetyl]-N4-
(2-chlor oethyl)semicarbazide (19)
A
mixture of the hydrazide 9 (5 mmol) and 2-chlor-
oethylisocyanate (5 mmol) in ethanol (25 ml) was stirred at room
temperature for 12 h. The separated product was filtered and
crystallized from ethanol.
Yield: 75%; m.p.: 168–169 ꢃC; 1H NMR (DMSO-d6):
d
¼ 0.983
(s, 6H, 2CH3), 1.979 (s, 2H, cyclic 4-CH2), 2.256 (s, 2H, cyclic 6-CH2),
3.320–3.350 (m, 2H, CH2NH), 3.549–3.611 (m, 2H, CH2CO), 3.707–
3.737 (m, 2H, CH2Cl), 4.765 (s, 1H, vinylic H), 6.617 (t, 1H, NHCH2,
exch.), 7.124 (br s, 1H, NH, exch.), 8.07 (s, 1H, NH, exch.) 9.756 (s, 1H,
NH, exch.) ppm. Anal. calcd for C13H21ClN4O3: C, 49.29; H, 6.68; N,
17.69. Found: C, 49.40; H, 6.69; N, 17.80%.
4.1.4.5. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-(4-methox-
ybenzylidene) acetohydrazide (14). Yield: 66%; m.p.: 235–236 ꢃC;
crystallized from ethanol; IR:
y
¼ 3371, 3206 (NH), 3068
(CH, aromatic), 2957 (CH, aliphatic), 1688, 1607 (CO) cmꢀ1; MS: m/z
(rel. int.) ¼ 330 (Mþ þ 1, 2.2), 329 (Mþ, 2.6), 134 (38.0), 67 (100.0).
Anal. calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76. Found: C,
65.78; H, 7.02; N, 12.66%.
4.2. Biological study
4.1.4.6. 2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)-N0-(4-nitrobe-
nzylidene) acetohydrazide (15). Yield: 85%; m.p.: 260–261 ꢃC;
4.2.1. Cytotoxicity test
crystallized from ethanol; IR:
y
¼ 3366, 3256 (NH), 3082
4.2.1.1. Reagents. MTT solution (0.5 mg/ml) in complete medium
RPMI-1640; Phosphate buffered saline (PBS, 0.15 mM pH 7.4);
Dimethyl sulphoxide (DMSO).
(CH, aromatic), 2957 (CH, aliphatic), 1695 (CO), 1520, 1343 (NO2)
cmꢀ1. MS: m/z (rel. int.) ¼ 345 (Mþ, 7.2), 344 (26), 343 (9.9), 330
(5.3), 330 (5.3), 329 (26.3), 67 (100.0). Anal. calcd for C17H20N4O4: C,
59.29; H, 5.58; N, 16.27. Found: C, 59.42; H, 5.70; N, 16.37%.
4.2.1.2. Cell culture. Tumor Liver cell line (HepG2), was supplied by
Naval American Research Unit – Egypt (NAmRU). Cells were prop-
agated and maintained in RPMI-1640 medium with L-glutamine
(Sigma) and supplemented with 10% fetal calf serum (Sigma) for
growth and 2% for maintenance medium; 1% of 4% sodium bicar-
bonate (Merck) and 1% antibiotic mixture (1,000,000 units of
4.1.5. N1-[2-(5,5-Dimethyl-3-oxocyclohex-1-enylamino)acetyl]-N4-
phenyl thiosemicarbazide (16)
A mixture of the hydrazide 9 (5 mmol) and phenyl iso-
thiocyanate (5 mmol) in 40 ml ethanol was heated at reflux for
1.5 h. After cooling, the separated product was filtered and
crystallized from dioxane/H2O.
penicillin G sodium and 1,000,000
100 ml deionized water.
mg streptomycin sulfate); in

3686
O.I. El-Sabbagh, H.M. Rady / European Journal of Medicinal Chemistry 44 (2009) 3680–3686
4.2.1.3. MTT test. The cytotoxic effect of compounds 3–19 was
done according to Mosmann [21]. The cells at approximately
80% confluence (i.e., logarithmically growing cells) were
selected for trypsinization. The cell suspension was prepared
and cells were seeded in 96-well microplates (3 ꢂ103 cells/well)
sheets. The plates were left to solidify and incubated at 37 ꢃC until
the development of virus plaques. Cell sheets were fixed in 10%
formalin solution for 2 h, and stained with crystal violet. Control
virus and cells were treated identically without the test
compound. Virus plaques were counted and the % age reduction
was calculated.
in 100 m
l RPMI-1640 culture medium and incubated at 37 ꢃC
and 5% CO2 overnight. After overnight incubation, the cells were
treated with the corresponding newly synthesized compounds
and then incubated for further 24 h. The medium was discarded
Acknowledgements
and the cells were washed with sterile PBS and then 100 ml of
the MTT (0.5 mg/ml) solution was added to each well. Cells
were incubated for 4 h and then the blue crystals were dis-
The authors would like to express their thanks to Dr. Ahmed
Mostafa, Chemistry of Natural Compounds Department., National
Research Centre Cairo, Egypt, for performing the antiviral study.
solved in 100 ml DMSO and the optical density (OD) was
measured at 570 nm (reference filter 690 nm) using an auto-
matic microplate reader. The results were recorded and
expressed as %age reduction in cell growth of hepatoma cell
line (HepG2) compared with the control (Table 1).
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