Synthesis and antioxidant activity evaluation of new hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones obtained by Biginelli reaction in two steps

Article abstract of DOI:10.1016/j.ejmech.2007.07.012

New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-car

Full text of DOI:10.1016/j.ejmech.2007.07.012

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1270e1275
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antioxidant activity evaluation of new
hexahydropyrimido[5,4-c]quinoline-2,5-diones and
2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones
obtained by Biginelli reaction in two steps
Lhassane Ismaili a, Arulraj Nadaradjane a, Laurence Nicod b,
Catherine Guyon b, Alain Xicluna a, Jean-Franc¸ois Robert a, Bernard Refouvelet a,
*
a Equipe Sciences se´paratives et Biopharmaceutique (2SB), Laboratoire de chimie the´rapeutique,
Faculte´ de Medecine et de Pharmacie, 4, place Saint Jacques e 25030 Besanc¸on Cedex, France
b Equipe Sciences se´paratives et Biopharmaceutique (2SB), Laboratoire de biologie cellulaire,
Faculte´ de Medecine et de Pharmacie, 4, place Saint Jacques e 25030 Besanc¸on Cedex, France
Received 11 April 2007; received in revised form 10 July 2007; accepted 12 July 2007
Available online 2 August 2007
Abstract
New hexahydropyrimido[5,4-c]quinoline-2,5-diones and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-ones were prepared in two steps
from ethyl 4-phenyl-6-methyl-2-oxo tetrahydropyrimidine-5-carboxylates or 4-phenyl-6-methyl-2-thioxotetrahydropyrimidine-5-carboxylates,
previously prepared by Biginelli reaction using appropriate aldehyde, urea derivatives and ethyl acetoacetate.
Their antioxidant properties were evaluated by two methods: scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and scav-
enging effect on hydroxyl radicals. The results show that the compounds containing thiourea moiety have better activity.
Ó 2007 Published by Elsevier Masson SAS.
Keywords: Quinoline; Biginelli reaction; Antioxidants; DPPH; Hydroxyl radical
1. Introduction
formed in biological system by the partial reduction of mo-
lecular oxygen. Formation of the hydroxyl radical (HOꢁ), an-
other ROS, is thought to occur through the one-electron
reduction of H2O2, a reaction that is facilitated by transition
metals that are in a reduced valence state (e.g. reduced cop-
per or iron). Additionally, there are a large number of other
reactive species that are formed from the reaction of ROS
with biological molecules [e.g. polyunsaturated lipids, thiols
Free radicals play an important role in the pathogenesis of
many diseases, accounting for continuing interest in the iden-
tification and development of novel antioxidants that prevent
radical-induced damage.
In humans, several pathologies involve the overproduction of
reactive oxygen species (ROS): These species such as the super-
oxide radical anion (O2ꢀ ) and hydrogen peroxide (H2O2), are
ꢁ
ꢁ
and nitric oxide (NO)]. For example, Oꢀ2 readily reacts with
NO to form peroxynitrite anion (ONOOꢀ), which is unstable
at physiological pH and rapidly decomposes to form potent
nitrating and oxidizing species [1,2].
In addition a lot of heterocyclic compounds either natural
(phytoestrogens) or obtained by synthesis, having coumarin
or quinoline rings, were studied for their biological activities.
They are used specially as radicals scavengers like quercetol
* Corresponding author. Tel.: þ33 381665542; fax: þ33 381665606.
E-mail addresses: lhassane.ismaili@univ-fcomte.fr (L. Ismaili), arulraj.
nadaradjane@univ-fcomte.fr (A. Nadaradjane), laurence.nicod@univ-fcomte.
fr (L. Nicod), catherine.guyon@univ-fcomte.fr (C. Guyon), alain.xicluna@
univ-fcomte.fr (A. Xicluna), jean-francois.robert@univ-fcomte.fr (J.-F.
Robert), bernard.refouvelet@univ-fcomte.fr (B. Refouvelet).
0223-5234/$ - see front matter Ó 2007 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2007.07.012

L. Ismaili et al. / European Journal of Medicinal Chemistry 43 (2008) 1270e1275
1271
or coumestrol [3,4] or the copper or iron chelating molecules
such as clioquinol [5,6].
All new products were characterized by 1H NMR, IR spec-
1
tra and elementary analysis. The H NMR spectra of com-
After first biological studies realized in our laboratory [7e9] on
new compounds with quinoline and coumarin structures, we report
the synthesis of new hexahydropyrimido[5,4-c]quinoline-2,5-dio-
nes and 2-thioxohexahydropyrimido[5,4-c]quinoline-5-one. Their
pounds 5aej show the disappearance of the triplet and
quartet of ester group and appearance of additional NH signal.
ꢁ
antioxidant properties were evaluated by hydroxyl radical OH
scavenging activity and by their reducing power 2,2-diphenyl-1-
picrylhydrazyl radical (DPPH).
2.2. Antioxidant activity studies
Free radical scavenging is one of the best known mechanisms
by which antioxidants inhibit lipid oxidation. DPPH and hy-
droxyl radical scavenging activity evaluation are standard as-
says in antioxidant activity studies and offer rapid techniques
for screening the radical scavenging activity (RSA) of specific
compounds. The RSAs of hexahydropyrimido[5,4-c]quino-
line-2,5-diones 5aef and 2-thioxohexahydropyrimido[5,4-c]-
quinoline-5-ones 5gei were estimated using these two methods.
2. Results and discussion
2.1. Synthesis
Differently substituted 4-methyl 1,2,3,5,6,10b-hexahydropyri-
mido[5,4-c]quinoline-2,5-diones and 4-methyl-2-thioxo-1,2,3,5,6,
10b-hexahydropyrimido[5,4-c]quinoline-5-ones were obtained
easily in two steps. The first step (Scheme 1) consists of a Biginelli
reaction [10,11] and the second step is the cyclisation in the pres-
ence of ammonia [12e14].
2.2.1. DPPH radical scavenging
A freshly prepared DPPH solution exhibits a deep purple
colour with an absorption maximum at 517 nm. This purple
colour generally disappears when an antioxidant is present
in the medium as shown in the Scheme 3. Thus, antioxidant
molecules can quench DPPH free radicals (by providing hy-
drogen atoms or by electron donation, conceivably via a free
radical attack on the DPPH molecule) and convert them to col-
ourless/bleached product [17,18].
The RSA DPPH values of methanolic solutions of
pyrimido[5,4-c]quinoline-2,5-diones 5aef and 2-thioxopyri-
mido[5,4-c]quinoline-5-ones 5gej were examined and com-
pared (Table 3). Results are expressed as a percentage of the
ratio of the decrease in absorbance at 517 nm, to the absorbance
of DPPH solutions in the absence of compounds 5aej at
517 nm.
From analysis of Table 3, we can conclude that the pyrimido-
quinoline 5aej scavenging effects on DPPH radicals increase
with the concentration and were suitable for 2-thioxopyrimido
[5,4-c]quinoline-5-ones 5gej with a thiourea moiety (ꢂ25%
at 250 mmol Lꢀ1) comparable with RSA DPPH values for the
standard ascorbic acid (ꢂ24.6% at 25 mmol Lꢀ1). The RSA
DPPH value decreased between 8 and 18% at 250 mmol Lꢀ1
for pyrimido[5,4-c]quinoline-2,5-diones 5aef with a urea
moiety. The presence of a Cl group in 7 or 8 position seems to
increase this activity (compounds 5d, 5e and 5h).
As shown in Scheme 1, the condensation of the derivatives of
2-chlorobenzaldehyde 1, ethyl acetoacetate 2, urea or thiourea
and their derivatives 3 and boric acid according to Biginelli
reaction gives ethyl 4-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylates 4aef or 4-phenyl-6-methyl-2-
thia-1,2,3,4-tetrahydropyrimidine-5-carboxylates 4gej. For all
compoundsprepared, weexclusivelyobtainedtheN1-substituted
regioisomer 4-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropy-
rimidine-5-carboxylate or N1-substituted regioisomer 4-phenyl-
6-methyl-2-thia-1,2,3,4-tetrahydropyrimidine-5-carboxylate
according to the mechanisms described by Sweet and Fissekis
[15] or described by Kappe [16]. The measurement around
3 ppm of H doublet coupling constant for the C4 of compounds
4aej and C10b of compounds 5aej confirmed this regiochem-
istry. All the compounds summarized in Table 1 were obtained
in moderate to good yields ranging from 56% to 94%. All these
products were isolated from reaction mixture by recrystallisa-
tion from ethanol, and their structures were characterized by
1H NMR, IR spectra and elementary analysis.
Scheme 2 reports cyclisation procedure for the formation of
5aej from compounds 4aej. This cyclisation is realized with
ammonia under pressure.
Products 5aej obtained with good yields from 65% to 90%
are presented in the (Table 2).
X
R2
HN
N
O
X
O
O
H
H3BO3, AcOH
reflux, 3h
H
O
R1
R1
H2N
NH
R2
+
OEt
+
OEt
Cl
Cl
1
2
3
4a - f X = O
4g - j X = S
Scheme 1.

1272
L. Ismaili et al. / European Journal of Medicinal Chemistry 43 (2008) 1270e1275
Table 1
Table 2
Compounds
R1
R2
X
Yields (%)
Compounds
R1
R2
X
Yields (%)
4a
4b
4c
4d
4e
4f
H
CH3
C2H5
C2H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
O
O
O
O
O
O
S
70
73
75
90
56
88
58
60
64
71
5a
5b
5c
5d
5e
5f
H
CH3
C2H5
C2H5
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
O
O
O
O
O
O
S
70
73
80
81
73
90
82
65
72
88
H
H
6-F
3-Cl
3-Cl
6-Cl
H
10-F
7-Cl
7-Cl
10-Cl
H
4g
4h
4i
5g
5h
5i
4-Cl
6-F
6-Cl
S
8-Cl
10-F
10-Cl
S
S
S
4j
S
5j
S
2.2.2. OHꢁ radical scavenging
4. Experimental
We have used the benzoic acid hydroxylation method [19].
The benzoic acid is hydroxylated by OHꢁ formed by Fenton re-
action at C3 or C4 position of the aromatic ring and the fluo-
rescence was measured at 407 nm emission with excitation at
305 nm. This fluorescence generally decreases when an anti-
oxidant is present in the medium. Antioxidant molecules by
providing hydrogen atom prevent the hydroxylation of benzoic
acid.
4.1. General
All compounds were characterized using the methods of el-
ementary analyses, IR spectroscopy and NMR spectroscopy.
Infrared spectra were recorded on a Shimadzu FTIR-8201
PC spectrometer in KBr (n in cmꢀ1). Proton NMR spectra
were recorded on a Bruker AC 300 spectrometer. Chemical
shift values and IR data for all compounds are summarized
in Section 4 and are in agreement with the proposed structures.
Melting points (mp) were obtained with a Kofler apparatus
and were not corrected. All reactions were monitored with
Thin Layer Chromatography (TLC) and carried out on Alugram
Sil G/UV254 plate with appropriate solvents. Microanalyses
were carried out by the service Central d’Analyses, Centre
National de la Recherche Scientifique, Vernaison, France.
The RSA OHꢁ result of molecules 5aej are summarized in
(Table 4), the results are expressed as:
RSA OHꢁ% ¼ Absorbance in the presence of sample/absor-
bance in the absence of sample ꢃ 100 and compared to querce-
tol. We can conclude that the OHꢁ radical scavenging activity for
all compounds increase with the concentration and were excel-
lent for 5h and 5j with thiourea moiety (90.2% and 92.3% at
200 mmol Lꢀ1) and comparable to quercetol at 100 mmol Lꢀ1
(93.5%).
4.2. Synthesis
3. Conclusion
4.2.1. Ethyl 4-(2-chlorophenyl)-1,6-dimethyl-2-oxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (4a)
Several new hexahydropyrimido[5,4-c]quinoline-2,5-dio-
nes and 2-thioxohexahydropyrimido [5,4-c]quinoline-5-ones
were prepared by Biginelli reaction of ethyl 4-phenyl-6-
methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylates or
4-phenyl-6-methyl-2-thia-1,2,3,4-tetra hydropyrimidine-5-car-
boxylates, respectively, in moderate to good yields.
The antioxidant properties of these molecules were evalu-
ated by two methods. The compounds with thiourea moiety
seem to give good results.
A solution of o-chlorobenzaldehyde (3 mmol), ethyl ace-
toacetate (3 mmol), urea derivatives (3.6 mmol) and H3BO3
(0.6 mmol), in glacial acetic acid (10 mL) is heated at
100 ꢄC, while stirring for 2 h. Then it is cooled to rt, and
poured into ice water (50 mL). The product collected by filtra-
tion was recrystallised from EtOH (95%), to give the pure
product, 4a in 70% yield, mp 215 ꢄC, IR n(NH) 2990,
1
n(CaO) ester 1708, n(CaO) urea 1615. H NMR (DMSO-d6)
X
X
N
NH
N
N°
R2
R2
HN
O
N
HN
N
O
O2N
NO2
R°
NO2
O2N
+
RH
+
NH3, 16h
R1
R1
10 bars, 250°C
OEt
N
H
Cl
NO2
NO2
DPPH
517 nm
4a - j
5
5a - e X = O
5f - j X = S
Scheme 2.
Scheme 3.

L. Ismaili et al. / European Journal of Medicinal Chemistry 43 (2008) 1270e1275
1273
Table 3
Decreasing absorbance (%) DPPH of compounds 5aej
CH), 7.2e7.4 ppm (2H, m, aromH), 7.5 ppm (1H, m, aromH),
8 ppm (1H, s, NH). Anal. Calcd for C16H18ClFN2O3: C, 56.39;
H, 5.32; Cl, 10.40; F, 5.58; N, 8.22. Found: C, 56.42; H, 5.27;
Cl, 10.37; F, 5.52; N, 8.27.
Compounds
Concentration (mmol Lꢀ1
)
50
100
150
200
250
5a
5b
5c
5d
5e
5f
2.5
3.4
3.0
3.1
1.9
1.5
4.0
9.5
3.3
7.7
6.6
4.8
4.6
5.4
2.3
2.1
8.2
11.2
8.6
8.2
9.1
6.7
9.8
9.1
12.0
10.5
9.8
4.2.4. Ethyl 4-(2,3-dichlorophenyl)-1,6-dimethyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4d)
6.0
8.7
10.2
4.3
16.2
8.1
17.4
11.8
7.6
Compound 4d was prepared with the same procedure as 4a
in 90% yield, mp 118 ꢄC, IR n(NH) 3350, n(CaO) ester 1672,
n(CaO) urea 1621. 1H NMR (DMSO-d6) 0.9 ppm (3H, t, CH3
ester), 2.5 ppm (3H, s, CH3), 3.1 ppm (3H, s, NeCH3),
3.9 ppm (2H, q, CH2 ester), 5.6 ppm (1H, d, J ¼ 3.2 Hz,
CH), 7.2e7.5 ppm (3H, m, aromH), 7.9 ppm (1H, s, NH).
Anal. Calcd for C15H16Cl2N2O3: C, 52.49; H, 4.70; Cl,
20.66; N, 8.16. Found: C, 52.37; H, 4.73; Cl, 20.69; N, 8.19.
5.0
5.4
5g
5h
5i
14.1
17.8
12.4
9.5
17.9
23.8
17.4
15.3
26.2
30.6
25.4
24.9
5j
Ascorbic acid: 25 mmol Lꢀ1, 24.6%; 50 mmol Lꢀ1, 36.9%.
1.15 ppm (3H, t, CH3 ester), 2.6 ppm (3H, s, CH3), 3.2 ppm
(3H, s, NeCH3), 3.9 ppm (2H, q, CH2 ester), 5.7 ppm (1H, d,
J ¼ 2.9 Hz, CH), 7.3e7.5 ppm (4H, m, aromH), 7.9 ppm (1H,
s, NH). Anal. Calcd for C15H17ClN2O3: C, 58.35; H, 5.55; Cl,
11.48; N, 9.07. Found: C, 58.24; H, 5.59; Cl, 11.49; N, 9.12.
4.2.5. 4.2.5. Ethyl 4-(2,3-dichlorophenyl)-1-ethyl-6-methyl-
2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4e)
Compound 4e was prepared with the same procedure as 4a
in 56% yield, mp 116 ꢄC, IR n(NH) 3344, n(CaO) ester 1678,
n(CaO) urea 1624. 1H NMR (DMSO-d6) 1.0 ppm (3H, t, CH3
ethyl), 1.2 ppm (3H, t, CH3 ester), 2.5 ppm (3H, s, CH3),
3.7 ppm (1H, m, CH ethyl), 3.8 ppm (1H, m, CH ethyl),
3.9 ppm (2H, q, CH2 ester), 5.6 ppm (1H, d, J ¼ 3.0 Hz,
CH), 7.3e7.4 ppm (2H, m, aromH), 7.6 ppm (1H, d, aromH),
7.9 ppm (1H, s, NH). Anal. Calcd for C16H18Cl2N2O3: C,
53.79; H, 5.08; Cl, 19.85; N, 7.84. Found: C, 53.75; H,
5.12; Cl, 19.77; N, 7.88.
4.2.2. Ethyl 4-(2-chlorophenyl)-1-ethyl-6-methyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4b)
Compound 4b was prepared with the same procedure as 4a
in 52% yield, mp 210 ꢄC, IR n(NH) 3300, n(CaO) ester 1705,
n(CaO) urea 1670. 1H NMR (DMSO-d6) 1.1 ppm (3H, t, CH3
ethyl), 1.2 ppm (3H, t, CH3 ester), 2.6 ppm (3H, s, CH3),
3.8 ppm (1H, m, CH ethyl), 3.9 ppm (1H, m, CH ethyl),
3.95 ppm (2H, q, CH2 ester), 5.7 ppm (1H, d, J ¼ 2.9 Hz,
CH), 7.3e7.4 ppm (4H, m, aromH), 7.9 ppm (1H, s, NH).
Anal. Calcd for C16H19ClN2O3: C, 59.54; H, 5.93; Cl,
10.98; N, 8.68. Found: C, 60.03; H, 5.87; Cl, 10.92; N, 8.60.
4.2.6. Ethyl 4-(2,6-dichlorophenyl)-1,6-dimethyl-2-
oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4f)
Compound 4f was prepared with the same procedure as 4a
in 88% yield, mp 130 ꢄC, IR n(NH) 3322, n(CaO) ester 1683,
n(CaO) urea 1630. 1H NMR (DMSO-d6) 0.9 ppm (3H, t, CH3
ester), 2.4 ppm (3H, s, CH3), 3.2 ppm (3H, s, NeCH3),
3.9 ppm (2H, q, CH2 ester), 6.2 ppm (1H, d, J ¼ 3.0 Hz,
CH), 7.2e7.6 ppm (3H, m, aromH), 7.8 ppm (1H, s, NH).
Anal. Calcd for C15H16Cl2N2O3: C, 52.49; H, 4.70; Cl,
20.66; N, 8.16. Found: C, 52.34; H, 4.74; Cl, 20.48; N, 8.22.
4.2.3. Ethyl 4-(2-chloro-6-fluorophenyl)-1-ethyl-6-methyl-2-
oxo-1,2,3,4-tetrahydro pyrimidine-5-carboxylate (4c)
Compound 4c was prepared with the same procedure as 4a
in 75% yield, mp 158 ꢄC, IR n(NH) 3342, n(CaO) ester 1703,
n(CaO) urea 1670. 1H NMR (DMSO-d6) 1.0 ppm (3H, t, CH3
ethyl), 1.2 ppm (3H, t, CH3 ester), 2.6 ppm (3H, s, CH3),
3.7 ppm (1H, m, CH ethyl), 3.8 ppm (1H, m, CH ethyl),
3.9 ppm (2H, q, CH2 ester), 5.7 ppm (1H, d, J ¼ 3.1 Hz,
4.2.7. Ethyl 4-(2-chlorophenyl)-1,6-dimethyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (4g)
Compound 4g was prepared with the same procedure as 4a
in 58% yield, mp 116 ꢄC, IR n(NH) 3186, n(CaO) ester 1705,
n(CaS) thiourea 1635. H NMR (DMSO-d6) 1.1 ppm (3H, t,
CH3 ester), 2.6 ppm (3H, s, CH3), 3.6 ppm (3H, s, NeCH3),
4 ppm (2H, q, CH2 ester), 5.8 ppm (1H, d, J ¼ 2.9 Hz, CH),
7.3e7.5 ppm (4H, m, aromH), 10 ppm (1H, s, NH). Anal.
Calcd for C15H17ClN2O2S: C, 55.46; H, 5.28; Cl, 10.91; N,
8.62; S, 9.87. Found: C, 55.40; H, 5.31; Cl, 10.95; N, 8.54;
S, 9.95.
Table 4
Decreasing fluorescence of 5aej
1
Compounds
Concentration (mmol Lꢀ1
)
20
50
100
150
200
5a
5b
5c
5d
5e
5f
58.4
58.1
12.5
66.0
63.8
67.1
56.3
68.7
70.6
68.0
71.8
70.9
58.4
77.3
74.1
72.8
69.4
76.5
78.8
82.7
80.8
81.1
72.4
82.9
80.6
80.9
77.2
87.5
80.2
87.4
83.7
86.5
80.2
87.3
84.4
83.2
82.3
88.5
81.5
89.8
89.7
89.1
83.1
89.2
86.7
83.3
88.2
90.2
83.3
92.3
4.2.8. Ethyl 4-(2,4-dichlorophenyl)-1,6-dimethyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (4h)
5g
5h
5i
Compound 4h was prepared with the same procedure as
4a in 60% yield, mp 125 ꢄC, IR n(NH) 3205, n(CaO) ester
5j
1
Quercetol: 50 mmol Lꢀ1, 87.9%; 100 mmol Lꢀ1, 92.3%.
1708, n(CaS) thiourea 1651. H NMR (DMSO-d6) 1.0 ppm

1274
L. Ismaili et al. / European Journal of Medicinal Chemistry 43 (2008) 1270e1275
(3H, t, CH3 ester), 2.6 ppm (3H, s, CH3), 3.6 ppm (3H, s, Ne
CH3), 3.9 ppm (2H, q, CH2 ester), 5.6 ppm (1H, d, J ¼ 2.6 Hz,
CH), 7.2 ppm (1H, d, aromH), 7.4 ppm (1H, d, aromH),
7.6 ppm (1H, d, aromH), 9.8 ppm (1H, s, NH). Anal. Calcd
for C15H16Cl2N2O2S: C, 50.15; H, 4.49; Cl, 19.74; N, 7.80;
S, 8.93. Found: C, 50.22; H, 4.41; Cl, 19.28; N, 7.91; S, 8.90.
C14H15N3O2: C, 65.35; H, 5.88; N, 16.33. Found: C, 65.31;
H, 5.91; N, 16.35.
4.2.13. 3-Ethyl-10-fluoro-4-methyl-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-2,5-diones (5c)
Compound 5c was prepared with the same procedure as 5a
in 80% yield, mp 228 ꢄC, IR n(NH) 3100, n(CaO) lactam
1683, n(CaO) urea 1630. 1H NMR (DMSO-d6) 1.1 ppm
(3H, t, CH3 ethyl), 2.5 ppm (3H, s, CH3), 3.7 ppm (1H, m,
NeCH ethyl), 3.8 ppm (1H, m, NeCH ethyl), 5.9 ppm (1H,
d, J ¼ 3.6 Hz, CH), 7.3e7.5 ppm (3H, m, aromH), 7.6 ppm
(1H, s, NH), 11.9 ppm (1H, s, NH). Anal. Calcd for
C14H14FN3O2: C, 61.08; H, 5.13; F, 6.90; N, 15.26. Found:
C, 61.12; H, 5.06; F, 6.94; N, 15.18.
4.2.9. Ethyl 4-(2-chloro-6-fluorophenyl)-1,6-dimethyl-2-
thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4i)
Compound 4i was prepared with the same procedure as 4a
in 64% yield, mp 173 ꢄC, IR n(NH) 3193, n(CaO) ester 1710,
1
n(CaS) thiourea 1643. H NMR (DMSO-d6) 0.9 ppm (3H, t,
CH3 ester), 2.5 ppm (3H, s, CH3), 3.6 ppm (3H, s, NeCH3),
3.9 ppm (2H, q, CH2 ester), 5.8 ppm (1H, d, J ¼ 2.8 Hz,
CH), 7.1e7.5 ppm (3H, m, aromH), 9.7 ppm (1H, s, NH).
Anal. Calcd for C15H16ClFN2O2S: C, 52.55; H, 4.70; Cl,
10.34; F, 5.54; N, 8.17; S, 9.35. Found: C, 52.70; H, 4.65;
Cl, 10.31; F, 5.48; N, 8.05; S, 9.28.
4.2.14. 7-Chloro-3,4-dimethyl-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-2,5-diones (5d)
Compound 5d was prepared with the same procedure as 5a
in 81% yield, mp 254 ꢄC, IR n(NH) 2990, n(CaO) lactam
1673, n(CaO) urea 1622. 1H NMR (DMSO-d6) 2.6 ppm
(3H, s, CH3), 3.2 ppm (3H, s, NeCH3), 5.6 ppm (1H, d,
J ¼ 3.5 Hz, CH), 7.2e7.6 ppm (3H, m, aromH), 7.8 ppm
(1H, s, NH), 11.9 ppm (1H, s, NH). Anal. Calcd for
C13H12ClN3O2: C, 56.22; H, 4.36; Cl, 12.77; N, 15.13. Found:
C, 56.16; H, 4.39; Cl, 12.70; N, 15.24.
4.2.10. Ethyl 4-(2,6-dichlorophenyl)-1,6-dimethyl-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carboxylate (4j)
Compound 4j was prepared with the same procedure as 4a
in 70% yield, mp 166 ꢄC, IR n(NH) 3202, n(CaO) ester 1702,
1
n(CaS) thiourea 1665. H NMR (DMSO-d6) 1.0 ppm (3H, t,
CH3 ester), 2.6 ppm (3H, s, CH3), 3.5 ppm (3H, s, NeCH3),
3.9 ppm (2H, q, CH2 ester), 5.7 ppm (1H, d, J ¼ 2.9 Hz,
CH), 7.2e7.4 ppm (3H, m, aromH), 9.8 ppm (1H, s, NH).
Anal. Calcd for C15H16Cl2N2O2S: C, 50.15; H, 4.49; Cl,
19.74; N, 7.80; S, 8.93. Found: C, 50.20; H, 4.42; Cl, 19.70;
N, 7.91; S, 8.90.
4.2.15. 7-Chloro-3-ethyl-4-methyl-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-2,5-diones (5e)
Compound 5e was prepared with the same procedure as 5a
in 73% yield, mp 250 ꢄC, IR n(NH) 2998, n(CaO) lactam
1671, n(CaO) urea 1618. 1H NMR (DMSO-d6) 1.0 ppm
(3H, t, CH3 ethyl), 2.5 ppm (3H, s, CH3), 3.7 ppm (1H, m,
NeCH ethyl), 3.8 ppm (1H, m, NeCH ethyl), 5.6 ppm (1H, d,
J ¼ 3.3 Hz, CH), 7.2e7.4 ppm (3H, m, aromH), 7.6 ppm
(1H, s, NH), 11.9 ppm (1H, s, NH). Anal. Calcd for
C14H14ClN3O2: C, 57.64; H, 4.84; Cl, 12.15; N, 14.40. Found:
C, 57.42; H, 4.88; Cl, 12.10; N, 14.51.
4.2.11. 3,4-Dimethyl-1,2,3,5,6,10b-hexahydropyrimido
[5,4-c]quinoline-2,5-diones (5a)
A solution of o-chloro-3,4-dihydropyrimidin-2-(1H )-one,
4a (1 g, 3.2 mmol) in 80 mL of ammonia at 32% was heated
at 250 ꢄC under 10 bars for 16 h. The solvent was removed
in vacuum to afford a solid which was dissolved in water
and neutralized with 5 M hydrochloric acid. The precipitate
obtained was recuperated by filtration and recrystallised
from ethanol to give the pure product 6a in 70% yield, mp
236 ꢄC, IR n(NH) 3120, n(CaO) lactam 1680, n(CaO) urea
4.2.16. 10-Chloro-3,4-dimethyl-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-2,5-diones (5f)
Compound 5f was prepared with the same procedure as 5a
in 90% yield, mp 258 ꢄC, IR n(NH) 3005, n(CaO) lactam
1666, n(CaO) urea 1619. 1H NMR (DMSO-d6) 2.5 ppm
(3H, s, CH3), 3.2 ppm (3H, s, NeCH3), 6.1 ppm (1H, d,
J ¼ 3.2 Hz, CH), 7.2e7.5 ppm (3H, m, aromH), 7.6 ppm
(1H, s, NH), 11.6 ppm (1H, s, NH). Anal. Calcd for
C13H12ClN3O2: C, 56.22; H, 4.36; Cl, 12.77; N, 15.13. Found:
C, 56.25; H, 4.30; Cl, 12.65; N, 15.22.
1
1636. H NMR (DMSO-d6) 2.6 ppm (3H, s, CH3), 3.1 ppm
(3H, s, NeCH3), 5.6 ppm (1H, d, J ¼ 3.2 Hz, CH), 7.2e
7.6 ppm (4H, m, aromH), 7.9 ppm (1H, s, NH), 12 ppm
(1H, s, NH). Anal. Calcd for C13H13N3O2: C, 64.19; H, 5.39;
N, 17.27. Found: C, 64.25; H, 5.34; N, 17.21.
4.2.12. 3-Ethyl-4-methyl-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-2,5-diones (5b)
4.2.17. 3,4-Dimethyl-2-thioxo-1,2,3,5,6,10b-
Compound 5b was prepared with the same procedure as 5a
in 73% yield, mp 244 ꢄC, IR n(NH) 2990, n(CaO) lactam
1679, n(CaO) urea 1622. 1H NMR (DMSO-d6) 1.1 ppm
(3H, t, CH3 ethyl), 2.6 ppm (3H, s, CH3), 3.6 ppm (1H, m,
NeCH ethyl), 3.8 ppm (1H, m, NeCH ethyl), 5.7 ppm (1H,
d, J ¼ 3.3 Hz, CH), 7.2e7.5 ppm (4H, m, aromH), 7.8 ppm
(1H, s, NH), 12 ppm (1H, s, NH). Anal. Calcd for
hexahydropyrimido[5,4-c]quinoline-5-one (5g)
Compound 5g was prepared with the same procedure as 5a
in 82% yield, mp 262 ꢄC, IR n(NH) 3190, n(CaO) lactam
1
1672, n(CaS) thiourea 1646. H NMR (DMSO-d6) 2.5 ppm
(3H, s, CH3), 3.6 ppm (3H, s, NeCH3), 5.6 ppm (1H, d,
J ¼ 2.9 Hz, CH), 7.3e7.5 ppm (4H, m, aromH), 9.6 ppm
(1H, s, NH), 12.4 ppm (1H, s, NH). Anal. Calcd for

L. Ismaili et al. / European Journal of Medicinal Chemistry 43 (2008) 1270e1275
1275
C13H13N3OS: C, 60.21; H, 5.05; N, 16.20; S, 12.36. Found: C,
60.25; H, 5.01; N, 16.18; S, 12.45.
Hatano et al. [20]. Various concentrations of methanolic com-
pounds solutions (0.3 mL) were mixed with methanolic solu-
tion containing DPPH radicals (1.5 ꢃ 10ꢀ4 mol Lꢀ1, 2.7 mL).
The mixture was shaken vigorously and left to stand for 2 h
in the dark (until stable absorption values were obtained).
The reduction of the DPPH radical was determined by measur-
ing the absorption at 517 nm. The RSAwas calculated as a per-
centage of DPPH discolouration using the equation:
4.2.18. 8-Chloro-3,4-dimethyl-2-thioxo-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-5-one (5h)
Compound 5h was prepared with the same procedure as 5a
in 65% yield, mp 222 ꢄC, IR n(NH) 3001, n(CaO) lactam
1
1670, n(CaS) thiourea 1639. H NMR (DMSO-d6) 2.4 ppm
(3H, s, CH3), 3.4 ppm (3H, s, NeCH3), 5.5 ppm (1H, d,
J ¼ 2.7 Hz, CH), 7.1e7.4 ppm (3H, m, aromH), 7.7 ppm
(1H, s, NH), 9.6 ppm (1H, s, NH). Anal. Calcd for
C13H12ClN3OS: C, 53.15; H, 4.12; Cl, 12.07; N, 14.30; S,
10.91. Found: C, 53.11; H, 4.18; Cl, 12.12; N, 14.15; S, 10.85.
%RSA ¼ [(ADPPH ꢀ AS)/ADPPH] ꢃ 100, where AS is the ab-
sorbance of the solution when the compound has been added
at a particular level and ADPPH is the absorbance of the
DPPH solution. Mean values from three independent samples
were calculated for each compound and standard deviations
were less than 5%. Ascorbic acid was used as standard.
4.2.19. 10-Fluoro-3,4-dimethyl-2-thioxo-1,2,3,5,6,10b-
hexahydropyrimido[5,4-c]quinoline-5-one (5i)
Compound 5i was prepared with the same procedure as 5a
in 72% yield, mp 244 ꢄC, IR n(NH) 3108, n(CaO) lactam
1682, n(CaS) thiourea 1649. H NMR (DMSO-d6) 2.5 ppm
References
1
(3H, s, CH3), 3.6 ppm (3H, s, NeCH3), 5.8 ppm (1H, d,
J ¼ 2.6 Hz, CH), 7.1e7.4 ppm (3H, m, aromH), 7.5 ppm
(1H, s, NH), 9.6 ppm (1H, s, NH). Anal. Calcd for
C13H12FN3OS: C, 56.30; H, 4.36; F, 6.85; N, 15.15; S,
11.56. Found: C, 56.25; H, 4.40; F, 6.91; N, 15.21; S, 11.38.
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Compound 5j was prepared with the same procedure as 5a
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1673, n(CaS) thiourea 1627. H NMR (DMSO-d6) 2.4 ppm
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(3H, s, CH3), 3.7 ppm (3H, s, NeCH3), 6.2 ppm (1H, d,
J ¼ 2.9 Hz, CH), 7.0e7.4 ppm (3H, m, aromH), 7.5 ppm
(1H, s, NH), 9.5 ppm (1H, s, NH). Anal. Calcd for
C13H12ClN3OS: C, 53.15; H, 4.12; Cl, 12.07; N, 14.30; S,
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In a screw-capped test tube, 0.2 mL of sodium benzoate
(10 mmol) and 0.2 mL of FeSO4$7H20 (10 mmol) and EDTA
(10 mmol) were added. Then the sample solution and a phos-
phate buffer (pH 7.4, 0.1 mol) were mixed to give a total volume
of 1.8 mL. Finally, 0.2 mL of H2O2 solution (10 mmol) was
added, and the whole incubated at 37 ꢄC for 2 h. After incuba-
tion, thefluorescencewas measured at 407 nm emission with ex-
citation at 305 nm.
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The capacity of compounds to scavenge the ‘‘stable’’ free
radical DPPH was monitored according to the method of
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