Zwitterionic pyrimidinium adducts as antioxidants with therapeutic potential as nitric oxide scavenger

Article abstract of DOI:10.1016/j.ejmech.2014.07.026

A variety of zwitterionic adducts were synthesized by using means green chemistry method. The products contain the biologically active barbituric acid moiety embedded in zwitterion products. Both features are pharmaceutically relevant. The chemical structures were deduced by ¹H-, ¹³C-, NMR and HRMS spectral analysis, and X-ray diffraction techniques. In vitro evaluation for the antioxidant activities were carried out towards the inhibition of nitric oxide (NO) radical, known to regulate a mechanism of signals for various cellular functions. NO also play an important role as a mediator of various pathological conditions responsible for cellular damages such as strokes, cancers, diabetes, chronic heart failure and inflammatory disease and various neurodegenerative disorders. All tested compounds were found to be more potent nitric oxide scavengers as compared to standard drug ascorbic acid (IC₅₀ = 618 ± 2.0 μM). Compounds 4c and e exhibiting several hundred fold more activity against nitric oxide radical with IC₅₀ values of 69 ± 1.66 and 70.1 ± 0.89 μM respectively, as compared to standard drug ascorbic acid (IC ₅₀ = 618 ± 2.0 μM).

Full text of DOI:10.1016/j.ejmech.2014.07.026

European Journal of Medicinal Chemistry 84 (2014) 146e154
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Zwitterionic pyrimidinium adducts as antioxidants with therapeutic
potential as nitric oxide scavenger
,
b
,
*
,
,
, 1
Assem Barakat a
1, Abdullah Mohammed Al-Majid a 1, Hany J. Al-Najjar a
,
Yahia Nasser Mabkhot a 1, Sumaira Javaid c 1, Sammer Yousuf c 1, M. Iqbal Choudhary a
,
,
,
, c, 1
a Department of Chemistry, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia
b Department of Chemistry, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 21321, Egypt
c H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
A variety of zwitterionic adducts were synthesized by using means green chemistry method. The
products contain the biologically active barbituric acid moiety embedded in zwitterion products. Both
features are pharmaceutically relevant. The chemical structures were deduced by 1H-, 13C-, NMR and
HRMS spectral analysis, and X-ray diffraction techniques. In vitro evaluation for the antioxidant activities
were carried out towards the inhibition of nitric oxide (NO) radical, known to regulate a mechanism of
signals for various cellular functions. NO also play an important role as a mediator of various pathological
conditions responsible for cellular damages such as strokes, cancers, diabetes, chronic heart failure and
inflammatory disease and various neurodegenerative disorders. All tested compounds were found to be
Received 19 May 2014
Received in revised form
4 July 2014
Accepted 8 July 2014
Available online 9 July 2014
Keywords:
Reactive oxygen species (ROS)
Nitric oxide (NO)
Zwitterion
more potent nitric oxide scavengers as compared to standard drug ascorbic acid (IC50 ¼ 618 2.0
mM).
Compounds 4c and e exhibiting several hundred fold more activity against nitric oxide radical with IC50
values of 69
(IC50 ¼ 618 2.0
1.66 and 70.1
M).
0.89
m
M respectively, as compared to standard drug ascorbic acid
Barbituric acids
Cancer
Diabetes
m
© 2014 Published by Elsevier Masson SAS.
Chronic inflammatory disease and heart
failure
1. Introduction
functional groups having potential antioxidant properties could be
useful in chemoprevention and chemotherapy. Several anti-
necrotic, anti-inflammatory, neuroprotective, digestive, and hep-
atoprotective drugs have recently been shown to act through anti-
radical and/or antioxidant scavenging mechanisms [9,10].
The recent literature supports that the excess of reactive nitro-
gen (RNS) and oxygen species (ROS) plays an important role in the
degenerative or pathological processes leading various diseases,
such as liver and lung damages, aging, Alzheimer's and coronary
heart disease, neurodegenerative disorders, atherosclerosis, as well
as breast cancers through damage to DNA and other vital bio-
molecules [1e7]
Antioxidants are therefore considered as significant factors for
the remediation and prevention of degenerative diseases including
cancer [8]. It is assumed that compounds functionalized with
Previous studies have suggested that chemopreventive and
antioxidant properties of numerous compounds such as ascorbic
acid,
a-tocopherol, carotenoids, proteins, peptides, amino acids,
flavonoids and polyphenols, are due to the presence of phenolic
hydroxyl group(s) [11e14], and heterocyclic systems [15].
The six membered pyrimidine-2,4,6(1H,3H,5H)-trione scaffold
is an interesting heterocyclic ring system present in a large number
of natural or synthetic compounds and have wide applications as
pharmaceuticals. Moreover, literature revealed that the 5-alkylated
barbituric acids have special significance in the design and syn-
thesis of novel chemotherapeutic agents with remarkable activities
such as anticancer, HIV-1 and HIV-2 protease inhibitors, anticon-
vulsant, sedative-hypnotic [16e19], etc.
* Corresponding author. Department of Chemistry, College of Science, King Saud
University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
E-mail address: ambarakat@ksu.edu.sa (A. Barakat).
Equal contributors.
1
http://dx.doi.org/10.1016/j.ejmech.2014.07.026
0223-5234/© 2014 Published by Elsevier Masson SAS.

A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
147
Similarly, the related pyrimidine-trione derivatives are the key
components of hypnotic and anti-inflammatory drugs, such as
seconal, veronal, sodium pentothal phenobarbital, and bucolome
(Fig. 1) [20e23].
In the light of the aforementioned facts, and in continuation for
our interest in the synthesis of biologically active heterocyclic
compounds, we describe here the synthesis of a series of previously
reported [24,25] and new zwitterionic adducts. These compounds
were subjected to biological evaluation to determine antioxidant
activity and inhibition of NO production.
technique. Crystallographic data for the structures were deposited
to the Cambridge Crystallographic Data Center as supplementary
publication No. CCDC-957026, CCDC-957025, CCDC-933624, CCDC-
1001798, CCDC-1001799 and CCDC-1001745 respectively [24].
The compounds of 4c, e, i were obtained as crystals by slow
diffusion of diethyl ether solution of pure compounds 4c, e, i in
dichloromethane at room temperature followed by allowed to
stand for 2 days. The structures were resolved by direct methods by
using the SHELXS97 program [29] in the SHELXTL-plus package,
and refined by a full-matrix least-squares procedure on F2 using
SHELXS97. Diffraction data were collected on a Bruker SMART
APEXII CCD diffractometer. The crystal structure and refinement
data of compounds 4c, e, i are listed in Table 1. The final atomic
coordinates for all atoms and a complete listing of bond distance
and angles are presented in Supplementary Tables 1e6. ORTEP
drawings of final X-ray model of compounds 4c, e, i with the atomic
numbering scheme is presented in Fig. 3, while crystal packing
presentation of compounds 4c, e, and i is shown in Fig. 4.
2. Results and discussion
2.1. Chemistry
Green chemistry is the most robust and economical approach
for the generation of diverse classes of compounds. Recently
several, research groups are involved in employing green chemistry
for the generation of valuable scaffolds with interesting biological
activities. The chemistry which involves aqueous diethylamine
medium catalyzed formation of zwitterionic adduct 4, by 3 com-
ponents reactants is a powerful tool for the generation of privileged
therapeutic scaffolds, due to its efficiency, selectivity, and mild and
environmentally benign conditions.
The synthesis of compound 4 has been achieved earlier through
a very mild and one-pot synthesis [24e28] (Scheme 1). Products
were obtained by reacting barbituric acid 1, dimedone 2, with
aldehyde 3, for 1e5 h, in presence of aqueous diethylamine me-
dium. This one-pot three-component reaction completed sponta-
neously at room temperature. “Trimolecular adduct salts” 4ae4w
were obtained in quantitative yields by simple filtration.
2.3. Biological activity
Three different series of zwitterionic pyrimidinium adducts
were evaluated for their nitric oxide scavenging potential and re-
sults are presented in (Table 2). Series 1 (4ae4i) comprises of
compounds having bis(6-hydroxy-N,N-1,3-dimethyl pyrimidinium-
2,4,6-trione) rings linked by phenyl substituted methane bridge.
Whereas members of series 2 (4je4m) lack the N,N-dimethyl
groups. In compounds of series 3 (4oe4r) one of the (6-hydroxy-
N,N-1,3-dimethyl pyrimidinium-2,4,6-trione) ring is substituted by
dimethyl 6-oxocyclohexene ring. All of these compounds showed
several hundred times more nitric oxide scavenging activity
Products 4f, i, n, r, v are new products and obtained as described
for previous zwitterion derivatives [24].
(IC50 ¼ 75 2.0e466 4.0
(IC50 ¼ 681 2.0 M).
The electron withdrawing peNO2 substituted phenyl ring con-
M) was found to be the
mM), than the standard ascorbic acid
m
The structures of the compounds were deduced by 1H NMR, 13
C
NMR, MS and IR, spectroscopy and elemental analysis, and X-ray
crystallography.
taining compound 4c (IC50 ¼ 69 1.66
m
most active member of series 1. The NO scavenging potential found
to be several hundred fold more than the standard ascorbic acid
2.2. X-ray diffraction
(IC50 ¼ 681 2.0
when p-NO2 phenyl ring is replaced with p-chloro (4a,
IC50 ¼ 75 4.33 M), m-bromo (4e, IC50 ¼ 70.1 0.89 M) phenyl
and naphthalene (4h, IC50 ¼ 79.1 2.92 M) rings.
mM). A slight decrease in activity was observed
The structures of compounds 4a, b, o, c, e and i were unam-
biguously deduced by single-crystal X-ray diffraction (Fig. 2)
m
m
m
Fig. 1. Biologically active barbituric acids derivatives and ascorbic acid.

148
A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
Scheme 1. Synthesis of compounds 4.
Fig. 2. ORTEP representation of the structure of 4a, b, o.

A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
149
Table 1
The crystal and experimental data of compounds c and e, i.
Compound c
Compound e
Compound i
Empirical formula
Formula weight
Temperature
C24H29N5O8
515.52
273 (2)
C23H29BrN5O6
551.42
273 (2)
C24H30N5O7
500.53
273ꢀ
K
Wavelength
Crystal system
Space group
0.71073 Å
Monoclinic
P2 1/n
0.71073 Å
Monoclinic
P2 (1)/n
0.71073 Å
Monoclinic
P2 (1)/n
a
b
c
a
11.6202 (10) Å
14.1044 (13) Å
15.5263 (14)Å
90ꢀ
9.9073 (11) Å
14.5554 (16) Å
17.8380 (18) Å
90ꢀ
11.4427 (15) Å
14.229 (2) Å
15.643 (2) Å
90ꢀ
b
92.511 (2)ꢀ
90ꢀ
98.696 (3)ꢀ
90ꢀ
91.800 (4)ꢀ
90ꢀ
g
Volume
Z
Calculated density
Absorption coefficient
F (000)
2542.3 (4) A3
4
2542.8 (5) A3
4
2545.7 (6) A3
4
1.347 mg/m3
0.103 mmꢁ1
1088
1.440 mg/m3
1.664 mmꢁ1
1140
1.306 mg/m3
0.097 mmꢁ1
1060
Crystal size
0.52 ꢂ 0.38 ꢂ 0.35 mm
1.95e25.49ꢀ
14,821
0.26 ꢂ 0.14 ꢂ 0.11 mm
1.81e25.50ꢀ
14,853
0.54 ꢂ 0.34 ꢂ 0.28 mm
1.94e25.50ꢀ
14,914
q
range
Reflections collected
Reflections unique
4629
4621
4610
(Rint
)
0.0182
0.0639
0.0452
R1 with I > 2
R2 with I > 2
s
(I)
(I)
0.0624
0.1790
0.0515
0.1096
0.1051
0.3109
s
R1 for all data
R2 for all data
0.0774
0.1962
0.1117
0.1316
0.1748
0.3786
Goodness of fitꢁ3
1.041
1.014
1.165
max/min
r
eAꢀ
0.427 and ꢁ0.316
0.332 and ꢁ0.368
1.442 and ꢁ0.453
CCDC number
1001798
1001799
1001945
p-Methoxy
(IC50 ¼ 206.5 1.0
phenyl
M) was the most active member of series 2
(4je4m) with several hundred times more scavenging potential
than the standard, ascorbic acid (IC50 ¼ 681 2.0 M). The anti-
oxidant potential significantly decreased when methoxy group is
4.5 M), napthyl (4m,
M) and methyl (4j, IC50 ¼ 377 .21 M)
ring
containing
compound
4I
potentially as important bioactive compounds. All compounds
were found to be potent antioxidant against NO free radicals and
more active than the standard ascorbic acid. The NO radicals play a
key role in pathogenesis of various types of cancers, diabetes,
strokes, cardiovascular complications associated to chronic in-
flammatory disease and heart failure, various neurodegenerative
disorders and atherosclerosis and its inhibition has therapeutic
significance. The promising results indicate that synthesized
zwitterionic compounds can be potential candidates to use as
antioxidant in future to overcome associated health disorders.
m
m
replaced with chloro (4k, IC50 ¼ 304
m
IC50 ¼ 308
2.0
m
m
substituents.
Third series of compounds 4oe4r, having cyclohexene and N,N-
dimethyl pyrimidone rings, also showed a potent nitric oxide
scavenging ability and found to be more active than ascorbic acid.
The most active o,p-dicholoro (IC50 ¼ 174.9 3.65
mM) phenyl ring
4. Experimental
containing compound 4p showed a five hundred times more
antioxidant potential than ascorbic acid.
General: All the chemicals were purchased from Aldrich, Sig-
maeAldrich, Fluka, etc, and used without further purification, un-
less otherwise stated. All melting points were measured on a
Gallenkamp apparatus in open glass capillaries and are uncorrec-
ted. IR Spectra were measured as KBr pellets on a Nicolet 6700 FT-IR
spectrophotometer. The NMR spectra were recorded on a Jeol-400
NMR spectrometer. 1H NMR (400 MHz), and 13C NMR (100 MHz)
were recorded in either deuterated dimethylsulphoxide (DMSO-d6)
or deuterated chloroform (CDCl3) on Bruker 400 MHz. Chemical
However, when we compared the activities of p-nitro
substituted
phenyl
ring
containing
compounds
4q
(IC50 ¼ 466 1.40
m
M) and 4c (IC50 ¼ 69 1.66
m
M), several fold
more potent activity of 4c indicated the significant role of 6-
hydroxy N,N-dimethyl pyrimidone ring to enhance the nitric ox-
ide scavenging potential. The observation further supported by the
decrease activity of 4r (IC50 ¼ 380
4.08 mM), having dimethyl
cyclohexene and N,N-dimethyl pyrimidone rings, as compared to
bis-6-hydroxy N,N-dimethyl pyrimidone ring containing com-
shifts (d) are referred in terms of ppm and J-coupling constants are
pound 4i (IC50 ¼ 113 0.53
mM).
given in Hz. Mass spectra were recorded on a Jeol of JMS-600H.
Elemental analysis was carried out on Elmer 2400 Elemental
Analyzer; CHN mode.
3. Conclusion
In conclusion, we have developed an efficient methodology for
the construction of zwitterionic adducts through tandem Aldol/
Michael addition reactions between barbituric acid and dimedone
with aldehydes catalyzed by aqueous diethylamine. The final
products were obtained in high yields. This provides a rapid means
of synthesizing a variety of trimolecular adducts. Generality and
applicability of a wide range of substrates is the key advantage of
our method for the synthesis of zwitterionic adduct derivatives,
4.1. General procedure for Aldol condensation Michael addition for
the synthesis of 4 (GP1)
A mixture of aldehyde 3 (1.5 mmol), 1 and 2 (3 mmol) as well as
Et2NH (1.5 mmol, 155 mL) in 3 mL of degassed H2O (bubbling ni-
trogen through the water) was stirred at room temperature for
1e5 h until TLC showed complete disappearance of the reactants.

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A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
Fig. 3. ORTEP representation of the structure of 4c, e, i.
The precipitate was removed by filtration and washed with ether
(3 ꢂ 20 mL). The solid was dried to afford pure products 4aew.
4.1.2. 5,50-(p-Tolylmethylene)bis(1,3-dimethylpyrimidine-
2,4,6(1H,3H,5H)-trione) diethylaminium salt (4b)
4b was prepared from 1,3-dimethylbarbituric acid 1a, and p-tol-
ualdehyde according to the general procedure (GP1) yielding color-
less needle materials (1.41 g, 2.91 mmol, 97%). m.p.: 152 ꢀC; IR (KBr,
cmꢁ1): 3455, 3210, 2984, 2820, 1560,1449, 1359; 1H NMR (400 MHz,
4.1.1. 5,50-((4-Chlorophenyl)methylene)bis(1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione) diethylaminium salt
(4a)
CDCl3): d 17.64 (s,1H, OH), 6.99e6.96 (m, 4H, Ph), 5.80 (s, 1H, benzyl-
H), 3.32 (s, 12H, 4CH3), 3.03 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 2.25 (s, 3H,
4a was prepared from 1,3-dimethylbarbituric acid 1a, and p-
chlorobenzaldehyde according to the general procedure (GP1)
yielding colorless crystals (1.44 g, 2.85 mmol, 95%). m.p.: 103 ꢀC; IR
(KBr, cmꢁ1): 3450, 3198, 2988, 1698, 1603, 1479, 1385; 1H NMR
CH3), 1.28 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 165.3, 164.3, 151.8, 138.6, 134.8, 128.9, 126.3, 92.1, 42.0, 34.2, 28.9,
28.6, 21.0,11.4; LC/MS (ESI): 487[M]þ; Anal. for C24H35N5O6; Calcd: C,
59.12; H, 6.82; N, 14.36; Found: C, 59.13; H, 6.81; N, 14.35.
(400 MHz, CDCl3):
d
17.66 (s, 1H, OH), 7.18 (d, 2H, J ¼ 8.8 Hz, Ph),
The structure of 4b was confirmed by X-ray crystal structure
analysis (Bruker AXS GmbH). CCDC-957025 contains the supple-
mentary crystallographic data for this compound. These data can be
obtained free of charge from the Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif. A colorless crystal
suitable for X-ray analysis was obtained from recrystallization the
compound from DCM/Et2O at room temperature after 2 days.
7.05 (d, 2H, J ¼ 8.8 Hz, Ph), 5.80 (s, 1H, benzyl-H), 3.34 (s, 12H,
4CH3), 3.06 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.30 (t, 6H, J ¼ 7.3 Hz,
CH2CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 165.3, 164.3, 151.7, 138.6,
134.8, 128.2, 126.3, 91.7, 42.1, 34.2, 28.9, 28.7, 11.5; LC/MS (ESI): 507
[M]þ; Anal. for C23H30ClN5O6; Calcd: C, 54.38; H, 5.95; Cl, 6.98; N,
13.79; Found: C, 54.38; H, 5.94; Cl, 7.01; N, 13.81.
The structure of 4a was unambiguously deduced by single-
crystal X-ray diffraction structure analysis (Bruker AXS GmbH).
CCDC-957026 contains the supplementary crystallographic data for
this compound. These data can be obtained free of charge from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif. A colorless crystal suitable for X-ray analysis was
obtained from recrystallization the compound from DCM/Et2O at
room temperature after 2 days.
4.1.3. 5,50-((4-Nitrophenyl)methylene)bis(1,3-dimethylpyrimidine-
2,4,6(1H,3H,5H)-trione) diethylaminium salt (4c)
4c was prepared from 1,3-dimethylbarbutric acid 1a, and p-
nitrobenzaldehyde according to the general procedure (GP1)
yielding a yellow powder (1.35 g, 2.61 mmol, 87%); m.p.: 195 ꢀC; IR
(KBr, cmꢁ1): 3453, 3205, 2987, 2904, 1675, 1608, 1576, 1511, 1438,

A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
151
Fig. 4. Crystal packing of 4c, e, i.
1343, 1254; 1H NMR (400 MHz, CDCl3):
d
17.58 (s, 1H, OH), 8.08 (d,
4.1.4. 5,50-((4-Methoxyphenyl)methylene)bis(1,3-
2H, J ¼ 8.8 Hz, Ph), 7.29 (d, 2H, J ¼ 8.8 Hz, Ph), 5.95 (s, 1H, benzyl-H),
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione) diethylaminium salt
(4d)
3.34 (s, 12H, 4CH3), 3.07 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.29 (t, 6H,
J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 165.2, 164.4,
4d was prepared from 1,3-dimethylbarbutric acid 1a, and p-
methoxybenzaldehyde according to the general procedure (GP1)
yielding rose-colored crystalline materials (1.35 g, 2.7 mmol, 90%).
m.p.: 160 ꢀC; IR (KBr, cmꢁ1): 3445, 3195, 2977, 2836, 1689, 1664,
151.6, 150.8, 146.1, 127.5, 123.5, 91.4, 42.2, 34.9, 28.9, 28.7, 11.5; LC/
MS (ESI): 518[M]þ; Anal. for C23H30N6O8; Calcd: C, 53.28; H, 5.83; N,
16.21; Found: C, 53.29; H, 5.85; N, 16.23.
The structure of 4c was confirmed by X-ray crystal structure
analysis (Bruker SMART APEXII CCD diffractometer). CCDC-1001798
contains the supplementary crystallographic data for this com-
pound. These data can be obtained free of charge from the Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif. A colorless crystal suitable for X-ray analysis was ob-
tained from recrystallization the compound from DCM/Et2O at
room temperature after 2 days.
1613, 1504, 1447, 1378, 1242; 1H NMR (400 MHz, CDCl3):
d 17.67 (s,
1H, OH), 7.01 (d, 2H, J ¼ 8.8 Hz, Ph), 6.75 (d, 2H, J ¼ 8.8 Hz, Ph), 5.79
(s, 1H, benzyl-H), 3.33 (s, 12H, 4CH3), 2.99 (q, 4H, J ¼ 7.3 Hz,
CH2CH3), 1.26 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz,
CDCl3):
d
¼ 165.3, 164.3, 157.4, 151.7, 133.6, 132.0, 127.4, 114.3, 92.1,
55.6, 42.1, 33.8, 28.9, 11.5; LC/MS (ESI): 503[M]þ; Anal. for
24H33N5O7; Calcd: C, 57.25; H, 6.61; N, 13.91; Found: C, 57.26; H,
6.61; N, 13.90.
C

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A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
4.1.7. 5,50-(3-Tolylmethylene)bis(1,3-dimethylpyrimidine-
2,4,6(1H,3H,5H)-trione) diethylaminium salt (4g)
Table 2
Result of nitric oxide scavenging assay.
4g was prepared from 1,3-dimethylbarbituric acid 1a, and m-
tolualdehyde according to the general procedure (GP1) yielding
rose-colored crystalline materials (1.41 g, 2.91 mmol, 97%). m.p.:
135 ꢀC; IR (KBr, cmꢁ1): 3455, 3201, 2988, 1693, 1667, 1611, 1573,
S. no.
Compounds
Nitric oxide scavenging assay IC50 SEM [mM]
1
2
3
4
5
6
7
8
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
75 4.33
230 4.95
69 1.66
1443; 1H NMR (400 MHz, CDCl3):
d 17.62 (s, 1H, OH), 7.10 (t, 1H,
234 2.30
70.1 0.89
310 3.67
174.5 4.80
79.1 2.92
113 0.53
377 2.21
304 4.5
206.5 1.0
308 2.0
330 2.02
290 4.0
174.9 3.65
466 4.20
380 4.08
361 4.89
432 3.17
439.2 3.7
281 2.48
618 2.00
J ¼ 7.3 Hz, Ph), 6.92 (d, 1H, J ¼ 7.3 Hz, Ph), 6.88 (d, 1H, J ¼ 7.3 Hz, Ph),
5.82 (s, 1H, benzyl-H), 3.32 (s, 12H, 4CH3), 3.01 (q, 4H, J ¼ 7.3 Hz,
CH2CH3), 2.25 (s, 3H, CH3), 1.26 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR
9
(100 MHz, CDCl3):
d
¼ 165.3, 164.4, 151.8, 141.7, 137.4, 127.9, 127.1,
10
11
12
13
14
15
16
17
18
19
20
21
22
STD
126.4, 123.6, 92.1, 42.0, 34.4, 28.9, 28.6, 21.8, 11.4; LC/MS (ESI): 487
[M]þ; Anal. for C24H35N5O6; Calcd: C, 59.12; H, 6.82; N, 14.36;
Found: C, 59.13; H, 6.81; N, 14.35.
4.1.8. 5,50-(Naphthalen-2-ylmethylene)bis(1,3-dimethylpyrimidine-
2,4,6(1H,3H,5H)-trione) diethylaminium salt (4h)
4h was prepared from 1,3-dimethylbarbutric acid 1a, and 2-
naphthaldehyde 2i according to the general procedure (GP1)
yielding beige powder (1.47 g, 2.82 mmol, 94%). m.p.: 146 ꢀC; IR
(KBr, cmꢁ1): 3454, 3200, 2967, 1668, 1585, 1438, 1250; 1H NMR
4q
4r
4s
4t
V
4w
(400 MHz, CDCl3):
d
17.33 (s, 1H, OH), 8.10 (d, 2H, J ¼ 8.8 Hz,
Ascorbic acid
naphthyl-H), 7.99 (d, 2H, J ¼ 8.8 Hz, naphthyl-H), 7.92 (d, 2H,
J ¼ 8.8 Hz, naphthyl-H), 7.90 (d, 2H, J ¼ 8.8 Hz, naphthyl-H), 7.84 (d,
2H, J ¼ 8.8 Hz, naphthyl-H), 7.68e7.38 (m, 3H, naphthyl-H), 6.37 (s,
1H, benzyl-H), 3.39 (s, 12H, 4CH3), 3.01 (q, 4H, J ¼ 7.3 Hz, CH2CH3),
1.30 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3):
4.1.5. 5,50-((3-Bromophenyl)methylene)bis(1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione) diethylaminium salt
(4e)
d
¼ 164.9, 151.7, 136.8, 135.3, 134.3, 131.5, 129.1, 128.5, 127.0, 125.2,
124.9, 123.8, 93.2, 41.8, 33.2, 28.8, 11.4; LC/MS (ESI): 523 [M]þ; Anal.
for C27H33N5O6; Calcd: C, 61.94; H, 6.35; N, 13.38; Found: C, 61.95;
H, 6.34; N, 13.40.
4e was prepared from 1,3-dimethylbarbutric acid 1a, and m-
bromobenzaldehyde according to the general procedure (GP1)
yielding colorless crystalline materials (1.5 g, 2.76 mmol, 92%).
m.p.: 169 ꢀC; IR (KBr, cmꢁ1): 3450, 3120, 2982, 1694, 1667, 1615,
4.1.9. 4-(bis(6-Hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl)benzaldehyde diethylaminium
salt (4i)
1577, 1445, 1250; 1H NMR (400 MHz, CDCl3):
d 17.63 (s, 1H, OH),
7.22 (d, 1H, J ¼ 7.3 Hz, Ph), 7.19 (s, 1H, Ph), 7.07 (d, 1H, J ¼ 7.3 Hz,
Ph), 7.05 (d, 1H, J ¼ 7.3 Hz, Ph), 5.84 (s, 1H, benzyl-H), 3.34 (s, 6H,
2CH3), 3.32 (s, 6H, 2CH3), 3.02 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.27 (t,
Pure product 4i was obtained according to GP1 as colorless
crystal (1.5 g, 2.76 mmol, 92%). IR (cmꢁ1): 3450, 3000, 2872, 1670,
1582, 1510, 1466, 1384, 1339; 1H NMR (CDCl3, 400 MHz) 17.58 (s, 1H,
OH), 9.90 (s, 1H, CHO), 7.73 (d, 2H, J ¼ 8.0 Hz, Ph), 7.29 (d, 2H,
J ¼ 8.0 Hz, Ph), 5.93 (s, 1H, benzyl-H), 3.33 (s, 12H, 4CH3), 3.06 (q,
4H, J ¼ 7.3 Hz, CH2CH3), 1.27 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR
6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 165.2,
164.4, 151.7, 144.7, 129.7, 129.6, 128.7, 125.3, 91.5, 42.1, 34.4, 28.9,
28.7, 11.5; LC/MS (ESI): 552[M]þ; Anal. for C23H30BrN5O6; Calcd: C,
50.01; H, 5.47; Br, 14.46; N, 12.68; Found: C, 50.03; H, 5.48; Br,
14.47; N, 12.71.
(100 MHz, CDCl3):
d
¼ 192.2, 165.3, 164.4, 151.7, 150.3, 134.3, 129.9,
The structure of 4e was confirmed by X-ray crystal structure
analysis (Bruker SMART APEXII CCD diffractometer). CCDC-1001799
contains the supplementary crystallographic data for this com-
pound. These data can be obtained free of charge from the Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif. A colorless crystal suitable for X-ray analysis was ob-
tained from recrystallization the compound from DCM/Et2O at
room temperature after 2 days.
127.3, 91.7, 42.2, 35.1, 29.0, 28.7, 11.5; LC/MS (ESI): 501.53 [M]þ;
Anal. for C24H31N5O7; Calcd: C, 57.48; H, 6.23; N, 13.96; Found: C,
57.50; H, 6.25; N, 14.00.
4.1.10. 5,50-(p-Tolylmethylene)bis(6-hydroxypyrimidine-
2,4(1H,3H)-dione) diethylaminium salt (4j)
4j was prepared from barbituric acid 1b, and p-tolualdehyde
according to the general procedure (GP1) yielding white powder
(1.22 g, 2.85 mmol, 95%); m.p.: 205 ꢀC; IR (KBr, cmꢁ1): 3459, 3120,
2978, 2811, 1689, 1612, 1325, 1252; 1H NMR (400 MHz, DMSO-d6):
4.1.6. 5,50-((4-hydroxyphenyl)methylene)bis(6-hydroxy-1,3-
dimethylpyrimidine-2,4(1H,3H)-dione) diethylaminium salt (4f)
4f was prepared from 1,3-dimethylbarbutric acid 1a, and p-
hydroxybenzaldehyde according to the general procedure (GP1)
yielding a yellow powder (1.3 g, 2.64 mmol, 88%); m.p.: 180 ꢀC; IR
(KBr, cmꢁ1): 3458, 3200, 2980, 2904, 1677, 1620, 1572, 1511, 1438,
d 17.18 (s, 1H, OH), 10.09 (bs, 4H, NH), 6.93 (m, 4H, Ph), 5.90 (s, 1H,
benzyl-H), 2.79 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 2.20 (s, 3H, CH3), 1.07 (t,
6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 164.8,
164.1, 151.3, 142.1, 133.5, 128.5, 127.1, 91.6, 42.6, 30.6, 21.1, 13.0; LC/
MS (ESI): 431[M]þ; Anal. for C20H25N5O6; Calcd: C, 55.68; H, 5.84;
N, 16.23; Found: C, 55.67; H, 5.83; N, 16.22.
1343, 1254; 1H NMR (400 MHz, CDCl3):
d 17.62 (s, 1H, OH), 7.31
(d, 2H, J ¼ 8.8 Hz, Ph), 6.99 (d, 2H, J ¼ 8.8 Hz, Ph), 5.79 (s, 1H,
benzyl-H), 3.33 (s, 12H, 4CH3), 3.03 (q, 4H, J ¼ 7.3 Hz, CH2CH3),
1.27 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3):
4.1.11. 5,50-((4-Chlorophenyl)methylene)bis(6-hydroxypyrimidine-
2,4(1H,3H)-dione) diethylaminium salt (4k)
4k was prepared from barbituric acid 1b, and p-chlor-
obenzaldehyde according to the general procedure (GP1) yielding a
white powder (1.28 g, 2.85 mmol, 95%); m.p.: 221 ꢀC; IR (KBr,
cmꢁ1): 3435, 3185, 2978, 2830, 1677, 1548, 1448, 1345, 1250; 1H
d
¼ 165.3, 164.4, 151.7, 141.1, 131.2, 128.5, 119.3, 91.7, 42.1, 34.2,
28.9, 28.7, 11.5; LC/MS (ESI): 489.52 [M]þ; Anal. for C23H31N5O7;
Calcd: C, 56.43; H, 6.38; N, 14.31; Found: C, 56.44; H, 6.36; N,
14.30.

A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
153
NMR (400 MHz, DMSO-d6):
d
17.17 (s, 1H, OH), 10.00 (bs, 4H, NH),
MS (ESI): 457 [M]þ; Anal. for C25H35N3O5; calcd: C, 65.62; H, 7.71; N,
9.18; Found: C, 65.61; H, 7.73; N, 9.20.
7.18 (m, 4H, Ph), 5.93 (s, 1H, benzyl-H), 2.88 (q, 4H, J ¼ 7.3 Hz,
CH2CH3),1.12 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR (100 MHz, DMSO-
The structure of 4o was confirmed by X-ray crystal structure
analysis. CCDC-933624 contains the supplementary crystallo-
graphic data for this compound. This data can be obtained free of
charge from the Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif. A colorless crystal suitable for X-
ray analysis was obtained from recrystallization of the compound
from CHCl3/Et2O at room temperature after 2 days.
d6):
d
¼ 164.7, 164.0, 151.2, 144.6, 133.5, 129.9, 129.1, 127.8, 91.3, 42.1,
30.7, 11.8; LC/MS (ESI): 451[M]þ; Anal. for C19H22ClN5O6; Calcd C,
50.50; H, 4.91; Cl, 7.85; N,15.50; Found: C, 50.51; H, 4.90; Cl, 7.83; N,
15.51.
4.1.12. 5,50-((4-Methoxyphenyl)methylene)bis(6-
hydroxypyrimidine-2,4(1H,3H)-dione) diethylaminium salt (4l)
4l was prepared from barbituric acid 1b, and p-methox-
ybenzaldehyde according to the general procedure (GP1) yielding a
beige powder (1.22 g, 2.73 mmol, 91%); m.p.: 195 ꢀC; IR (KBr, cmꢁ1):
3449, 3190, 2991, 2835, 1688, 1592, 1505, 1383, 1247; 1H NMR
4.1.16. 5-((2,4-Dichlorophenyl)(2-hydroxy-4,4-dimethyl-6-
oxocyclohex-1-en-1-yl)methyl)-1,3-dimethyl-2,6-dioxo-1,2,3,6-
tetrahydropyrimidin-4-olate (4p)
4p was prepared from 1,3-dimethylbarbituric acid 1a, dimedone
2 and 2,4-dichlorobenzaldehyde according to the general proce-
dure (GP1) yielding a beige solid material (710 mg,1.35 mmol, 90%).
m.p: 164 ꢀC; IR (KBr, cmꢁ1): 3059, 2995, 2867, 2114,1741,1658,1591,
(400 MHz, DMSO-d6): d 17.26 (s, 1H, OH), 9.99 (bs, 4H, NH), 6.92 (d,
2H, J ¼ 8.0 Hz, Ph), 6.72 (d, 2H, J ¼ 8.0 Hz, Ph), 5.88 (s, 1H, benzyl-H),
2.90 (q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.14 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13
C
NMR (100 MHz, DMSO-d6):
d
¼ 164.6, 164.0, 157.0, 151.2, 137.2,
1463,1429,1370,1341,1256,12011H NMR (400 MHz, CDCl3):
d 14.80
132.4, 115.1, 91.7, 55.4, 42.1, 30.7, 11.6; LC/MS (ESI): 447[M]þ; Anal.
for C20H25N5O7; Calcd C, 53.69; H, 5.63; N,15.65; Found: C, 53.69; H,
5.63; N, 15.66.
(s, 1H, OH), 7.29 (d, 1H, J ¼ 8.0 Hz, Ph), 7.19 (s, 1H, Ph), 7.12(d, 2H,
J ¼ 8.0 Hz, Ph), 5.76 (s,1H, benzyl-H), 3.28 (s,12H, 4CH3), 3.07(q, 4H,
J ¼ 7.3 Hz, CH2CH3), 2.37 (s, 2H, CH2), 2.27 (d, 2H, J ¼ 5.1 Hz, CH2),
1.34(t, 6H, J ¼ 7.3 Hz, CH2CH3), 1.04(s, 3H, CH3), 1.01(s, 3H, CH3); 13
C
4.1.13. 5,50-(Naphthalen-2-ylmethylene)bis(6-hydroxypyrimidine-
2,4(1H,3H)-dione) diethylaminium salt (4m)
4m was prepared from barbituric acid 1b, and 2-naphthaldehyde
according to the general procedure (GP1) yielding a beige powder
(1.3 g, 2.79 mmol, 93%); m.p.: 192 ꢀC; IR (KBr, cmꢁ1): 3459, 3208,
2994, 1677, 1579, 1448, 1386, 1354; 1H NMR (400 MHz, DMSO-d6):
NMR (100 MHz, CDCl3):
d
¼ 199.1, 165.4, 164.4, 152.5, 139.8, 133.6,
131.7, 131.2, 129.3, 126.4, 115.7, 89.8, 51.2, 45.7, 41.9, 32.4, 31.2, 28.3,
28.2, 11.3; LC/MS (ESI): 526 [M]þ; Anal. for C25H33Cl2N3O5; calcd: C,
57.04; H, 6.32; Cl, 13.47; N, 7.98; Found: C, 57.09; H, 6.31; Cl, 13.44;
N, 8.01.
d
16.92 (s, 1H, OH), 10.41 (bs, 4H, NH), 8.13 (d, 1H, J ¼ 8.8 Hz,
4.1.17. 5-((2-Hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)(4-
nitrophenyl)methyl)-1,3-dimethyl-2,6-dioxo-1,2,3,6-
tetrahydropyrimidin-4-olate (4q)
naphthyl), 7.81(d, 1H, J ¼ 8.8 Hz, naphthyl), 7.63 (d, 1H, J ¼ 8.8 Hz,
naphthyl), 7.38e7.32 (m, 4H, naphthyl), 6.46 (s, 1H, benzyl-H), 2.79
(q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.08 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR
4q was prepared from 1,3-dimethylbarbituric acid 1a, dimedone
2 and p-nitrobenzaldehyde according to the general procedure
(GP1) yielding a beige material (700 mg, 1.39 mmol, 93%). m.p:
148 ꢀC; IR (KBr, cmꢁ1): 3050, 2950, 2865, 2500, 1669, 1580, 1510,
(100 MHz, DMSO-d6):
d
¼ 164.9, 151.1, 141.5, 135.8, 134.0, 132.4,
129.3, 128.7, 126.0, 125.8, 125.5, 125.2, 124.9, 123.8, 92.3, 42.5, 29.7,
12.7; LC/MS (ESI): 467[M]þ; Anal. for C23H25N5O6; Calcd C, 59.09; H,
5.39; N, 14.98; Found: C, 59.12; H, 5.40; N, 15.01.
1427, 1373, 1255, 1214;1H NMR (400 MHz, CDCl3):
d 15.26 (s, 1H,
OH), 6.99 (d, 2H, J ¼ 8.0 Hz, Ph), 6.72 (d, 2H, J ¼ 8.8 Hz, Ph), 5.69 (s,
1H, benzyl-H), 3.71 (s, 12H, 4CH3), 2.85(q, 4H, J ¼ 7.3 Hz, CH2CH3),
2.31(d,4H, J ¼ 14.7 Hz, CH2), 1.19(t, 6H, J ¼ 7.3 Hz, CH2CH3), 1.12(s,
4.1.14. 4-((6-Hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)(6-hydroxy-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl)benzaldehyde diethylaminium
salt (4n)
3H, CH3), 1.03(s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 161.6,
153.2, 145.5, 141.6, 129.1, 128.2, 127.8, 125.8, 88.5, 49.1, 41.9, 27.5,
11.5; LC/MS (ESI): 502[M]þ; Anal. for C25H34N4O7; calcd: C, 59.75; H,
6.82; N, 11.15; Found: C, 59.73; H, 6.81; N, 11.17.
Pure product 4n was obtained according to GP1 as white solid
(1.20 g, 88%). IR (cmꢁ1): 3455, 3305, 3000, 2910, 1677, 1582, 1510,
1466, 1384, 1339; 1H NMR (CDCl3, 400 MHz) 17.30 (s, 1H, OH), 9.90
(s, 1H, CHO), 8.23 (brs, 2H, NH), 7.56 (d, 2H, J ¼ 8.0 Hz, Ph), 7.11 (d,
2H, J ¼ 8.0 Hz, Ph), 5.85 (s, 1H, benzyl-H), 3.34 (s, 12H, 4CH3), 3.03
(q, 4H, J ¼ 7.3 Hz, CH2CH3), 1.25 (t, 6H, J ¼ 7.3 Hz, CH2CH3); 13C NMR
4.1.18. 4-((6-Hydroxy-1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)(2-hydroxy-4,4-dimethyl-6-oxocyclohex-
1-en-1-yl)methyl)benzaldehyde diethylaminium salt (4r)
(100 MHz, CDCl3):
d
¼ 192.1, 165.2, 164.1, 151.2, 150.0, 134.1, 129.5,
Pure product 4r was obtained according to GP1 as solid (1.26 g,
90%). IR (cmꢁ1): 3156, 2950, 2872,1678, 1590,1508,1375,1256,1232,
1167; 1H NMR (CDCl3, 400 MHz): 14.16 (s, 1H, OH), 9.80 (s, 1H, CHO),
8.01 (brs, 2H, NH), 6.98 (d, 2H, J ¼ 7.3 Hz, Ph), 6.75 (d, 2H, J ¼ 7.3 Hz,
Ph), 5.61 (s, 1H, benzyl-H), 3.73 (s, 6H, CH3), 2.92 (q, 4H, J ¼ 7.3 Hz,
CH2CH3), 2.31 (m, 4H, 2CH2), 1.26 (t, 6H, J ¼ 7.3 Hz, CH2CH3), 1.05 (s,
127.5, 91.6, 42.2, 35.1, 29.0, 28.7, 11.5; LC/MS (ESI): 473.48 [M]þ;
Anal. for C22H27N5O7; Calcd: C, 55.81; H, 5.75; N, 14.79; Found: C,
55.83; H, 5.76; N, 14.81.
4.1.15. 5-((2-Hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-
yl)(phenyl)methyl)-1,3-dimethyl-2,6-dioxo-1,2,3,6-
tetrahydropyrimidin-4-olate (4o)
3H, CH3), 1.00 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
188.1, 165.0, 157.2, 127.8, 115.7, 113.8, 91.6, 55.2, 48.8, 48.6, 42.4, 31.5,
29.4, 27.7, 11.7; LC/MS (ESI): 485.57 [M]þ; Anal. for C26H35N3O6;
Calcd: C, 64.31; H, 7.27; N, 8.65; Found: C, 64.30; H, 7.26; N, 8.63.
d
¼ 193.0,
4o was prepared from 1,3-dimethylbarbituric acid 1a, dimedone
2 and benzaldehyde according to the general procedure (GP1)
yielding colorless crystalline material (671 mg, 1.47 mmol, 98%).
m.p: 159 ꢀC; IR (KBr, cmꢁ1): 3150, 2959,1667,1617,1585,1422,1256,
4.1.19. 5-((2-Hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-
yl)(phenyl)methyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate
(4s)
1227; 1H NMR (400 MHz, CDCl3):
d 15.28 (s, 1H, OH), 7.17e7.04(m,
5H, Ph), 5.85 (s, 1H, benzyl-H), 3.29 (s, 12H, 4CH3), 2.96(q, 4H,
J ¼ 7.3 Hz, CH2CH3), 2.42 (d, 2H, J ¼ 5.1 Hz, CH2), 2.29 (m, 2H, CH2),
4s was prepared from barbituric acid 1b, dimedone 2 and
benzaldehyde according to the general procedure (GP1) yielding a
white solid material (598 mg, 1.39 mmol, 93%). m.p: 215 ꢀC; IR (KBr,
cmꢁ1): 3027, 2948, 2867, 2156, 1683, 1593, 1451, 1374, 1291, 1257,
1.24(t, 6H, J ¼ 7.3 Hz, CH2CH3), 1.14(s, 3H, CH3), 1.05(s, 3H, CH3); 13
C
NMR (100 MHz, CDCl3):
d
¼ 192.5, 180.8, 152.5, 142.5, 128.0, 126.7,
125.1, 116.3, 90.9, 51.4, 45.9, 42.2, 33.0, 31.5, 29.6, 28.4, 27.6, 11.4; LC/

154
A. Barakat et al. / European Journal of Medicinal Chemistry 84 (2014) 146e154
1141; 1H NMR (400 MHz, CDCl3):
NH), 7.12(m, 5H, Ph), 5.52 (s, 1H, benzyl-H), 2.99(q, 4H, J ¼ 7.3 Hz,
CH2CH3), 2.45 (d, 4H, J ¼ 5.1 Hz, CH2), 1.24(t, 6H, J ¼ 7.3 Hz, CH2CH3),
1.09(s, 3H, CH3), 1.03(s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
12.26 (s, 1H, OH), 9.31(brs, 2H,
After incubation, 50
glacial acetic acid) was added and allowed to stand for 5 min for
completion of diazotisation. Then 50 L of N-(1-naphthyl)ethyl-
mL of sulphanalic acid reagent (0.33% in 20%
m
enediamine dihydrochloride (0.1% w/v) was added and stirred, and
allowed to stand for 1e2 min. A pink-colored chromophore was
formed in diffused light. The absorbance of the solution was
measured at 546 nm against the corresponding blank solution.
Ascorbic acid and DMSO was used as the positive control and blank
respectively. The IC50 value was calculated by using the kinetic
program Ezi-Fit Enzyme Kinetic Program (Perrella Scientific Inc.,
Amherst, U.S.A.).
d
¼ 198.5, 180.8, 152.5, 142.5, 128.0, 126.7, 125.1, 116.3, 90.9, 51.4,
45.9, 42.2, 33.0, 28.4, 27.6, 11.3; LC/MS (ESI): 429[M]þ; Anal. for
23H31N3O5; calcd: C, 64.32; H, 7.27; N, 9.78; Found: C, 64.29; H,
C
7.29; N, 9.80.
4.1.20. 5-((2-Hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)(p-
tolyl)methyl)-2,6-dioxo-1,2,3,6-tetrahydropyrimidin-4-olate (4t)
4t was prepared from barbituric acid 1a, dimedone 2 and tol-
ualdehyde according to the general procedure (GP1) yielding a white
solid material (604 mg, 1.36 mmol, 91%). m.p: 213 ꢀC; IR (KBr, cmꢁ1):
3150, 2955, 2867, 1690, 1592, 1508, 1375, 1256, 1232, 1167; 1H NMR
The % radical scavenging activity (RSA) was calculated according
to the following formula:
% RSA ¼ 1 ꢁ [A of test compound/A of control) ꢁ 100].
Where; RSA is radical scavenging activity and A is absorbance.
(400 MHz, CDCl3): d 13.31 (s, 1H, OH), 8.83 (brs, 2H, NH), 7.27(d, 2H,
J ¼ 8.0 Hz, Ph), 7.00(d, 2H, J ¼ 8.0 Hz, Ph), 5.88 (s, 1H, benzyl-H),
2.83(q, 4H, J ¼ 7.3 Hz, CH2CH3), 2.31 (d, 4H, J ¼ 5.1 Hz, CH2), 2.23
(s, 3H, CH3),1.19(t, 6H, J ¼ 7.3 Hz, CH2CH3),1.04(s, 3H, CH3),1.02(s, 3H,
Acknowledgments
The authors extend their appreciation to the Deanship of Sci-
entific Research at King Saud University for funding the work
through the research group project Number RGP-VPP-257.
CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 196.5, 180.1, 152.8, 140.5, 131.4,
130.7, 128.7, 128.6, 118.5, 115.6, 91.0, 50.9, 42.8, 31.6, 31.5, 29.2, 28.3,
27.8, 20.9,11.3; LC/MS (ESI): 443 [M]þ; Anal. for C24H33N3O5; calcd: C,
64.99; H, 7.50; N, 9.47; Found: C, 64.95; H, 7.49; N, 9.50.
Appendix A. Supplementary data
4.1.21. 4-((6-Hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)methyl)
benzaldehyde diethylaminium salt (4v)
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.026.
Pure product 4v was obtained according to GP1 as solid (1.35 g,
95%). IR (cmꢁ1): 3150, 2955, 2867, 1690, 1592, 1508, 1375, 1256,
1232, 1167; 1H NMR (CDCl3, 400 MHz) 15.16 (s, 1H, OH), 9.93 (s, 1H,
CHO), 9.66 (brs, 2H, NH), 7.71 (d, 2H, J ¼ 7.3 Hz, Ph), 7.23 (d, 2H,
J ¼ 7.3 Hz, Ph), 5.93 (s, 1H, benzyl-H), 2.92 (q, 4H, J ¼ 7.3 Hz,
CH2CH3), 2.2 (m, 4H, 2CH2), 1.15 (t, 6H, J ¼ 7.3 Hz, CH2CH3), 1.02 (s,
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3H, CH3), 1.00 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 193.0,
166.0, 154.6, 151.8, 134.5, 129.5, 128.1, 126.3, 115.9, 89.6, 51.1, 42.7,
31.9, 31.6, 29.8, 27.9, 11.7; LC/MS (ESI): 457.52 [M]þ; Anal. for
C
24H31N3O6; Calcd: C, 63.00; H, 6.83; N, 9.18; Found: C, 63.03; H,
6.86; N, 9.21.
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4.1.22. Diethylammonium 2-((2-hydroxy-4,4-dimethyl-6-
oxocyclohex-1-en-1-yl)(4-nitrophenyl)methyl)-5,5-dimethyl-3-
oxocyclohex-1-enolate (4w)
Pure product 4w was obtained according to GP1 as solid (1.26 g,
90%). IR (cmꢁ1): 2872 (s), 1582 (s), 1510 (s), 1466 (s), 1384 (s), 1339
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(s), 757 (s), 487 (s); 1H NMR (CDCl3, 400 MHz)
d 0.91e1.06 (m, 12H,
ꢀ
[15] L. Salerno, N. Maria, M.N. Modica, G. Romeo, V. Pittala, M.A. Siracusa,
CH3), 1.21 (t, J ¼ 7.32 Hz, 6H, NH2CH2CH3),2.29 (s, 8H, CHþ2 COCH2),
2.94 (q, J ¼ 7.32 Hz, 4H, NHCH2CH3), 5.92 (s, 1H, PhCH), 7.21 (d,
J ¼ 8.80 Hz, 2H, ArH), 8.01 (m, J ¼ 8.80 Hz, 2H.ArH),8.32 (bs,2H.
M.E. Amato, R. Acquaviva, C. Di Giacomo, V. Sorrenti, Eur. J. Med. Chem. 49
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NH2), 15.12 (s, OH); 13C NMR (CDCl3, 100 MHz):
d
11.4 (CH3CH2NH),
31.6{C(CH3)2}, 32.2 (CH3)2, 34.1 (Ph-C), 42.5 (CH3CH2NH), 45.2,
50.3, 114.8, 123.2 (PhC3), 127.7 (PhC2), 145.5 (PhC4), 151.9 (PhC1),
186.8 (CeOH), 194.9 (C]O); Anal. Calcd. for C27H38N2O6: C, 66.74;
H, 7.98; N, 5.55; O, 19.91; Found: C, 66.64; H, 7.87; N, 5.76; O, 19.73:
LC/MS (ESI): m/z ¼ 468.27 [M]þ.
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4.2. Procedure for nitric oxide scavenging assay
Nitric oxide scavenging activities of compounds were deter-
mined by using the following method.
In the present investigation GriesseIlosavay methodology was
modified by using naphthylethylenediamine dihydrochloride (0.1%
w/v). The reaction mixture (250
test sample (in DMSO), 38 L of potassium phosphate buffer
(10 mM, pH 7.4) and 100 L of sodium nitropruside (10 mM), was
incubated at 25 ꢀC for 150 min for the formation of nitrite ions.
mL), containing 12 mL of 1.0 mM of
m
m
[29] G.M. Sheldrick, Acta Cryst. A64 (2008) 112e122.