New barbiturates and thiobarbiturates as potential enzyme inhibitors

Article abstract of DOI:10.3109/14756366.2014.895717

A series of 27 new barbiturates and thiobarbiturates have been synthesized by a convenient multi-component reaction in overall excellent yields (87-96%). All the synthesized compounds were characterized by 1H, 13C NMR, EIMS and elemental analysis (C, H, N and S). Furthermore, all compounds were screened for in vitro antioxidant (DPPH radical scavenging), lipoxygenase, chymotrypsin, α-glucosidase and anti-urease activities. Out of the series, 23 in DPPH, 14 in lipoxygenase, 2 in chymotrypsin have shown appreciable IC₅₀ values.

Full text of DOI:10.3109/14756366.2014.895717

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J Enzyme Inhib Med Chem, Early Online: 1–7
2014 Informa UK Ltd. DOI: 10.3109/14756366.2014.895717
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ORIGINAL ARTICLE
New barbiturates and thiobarbiturates as potential enzyme inhibitors
Ashfaq M. Qureshi1, Saira Mumtaz1, Abdul Rauf2, Muhammad Ashraf3, Rumana Nasar3, and Zahid H. Chohan1
2
3
1Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, Pakistan, Department of Chemistry, and Department of Biotechnology,
The Islamia University of Bahawalpur, Bahawalpur, Pakistan
Abstract
series of 27 new barbiturates and thiobarbiturates have been synthesized by
convenient multi-component reaction in overall excellent yields (87–96%). All the synthesized
compounds were characterized by 1H, 13C NMR, EIMS and elemental analysis (C, H, N and S).
Furthermore, all compounds were screened for in vitro antioxidant (DPPH radical scavenging),
lipoxygenase, chymotrypsin, a-glucosidase and anti-urease activities. Out of the series, 23 in
DPPH, 14 in lipoxygenase, 2 in chymotrypsin have shown appreciable IC50 values.
Keywords
A
a
ꢀ-Glucosidase and anti-urease, antioxidant
(DPPH radical scavenging), barbituric acid,
chymotrypsin, lipoxygenase, thiobarbituric
acid, thiosemicarbazone
History
Received 28 September 2013
Revised 3 February 2014
Accepted 7 February 2014
Published online 25 March 2014
Introduction
are synthetically afforded in the present series of compounds
(1–27) via an efficient one pot multi-component reaction in
an excellent yield. All the synthesized compounds were exten-
sively characterized and screened for in vitro antioxidant (DPPH
radical scavenging), lipoxygenase, chymotrypsin, a-glucosidase
and anti-urease activities.
Barbiturates and thiobarbiturates are cheap and readily available
synthon used in the manufacturing of diverse pharmaceutical
products1 due to their remarkable biological potentials for various
medical conditions. Barbiturates are well-known anesthetic and
sleep inducing agents2 by acting through central nervous system.
These are employed as hypnotic, sedative, anticonvulsant and
for anesthesia3,4. Furthermore, some are used for the treatment
Material and methods
of psychiatric disorders such as anxiety and epilepsy by posing General
effects on motor and sensory functions5,6 and are also reported
All reagents and solvents were used as supplied and/or
recrystallized/redistilled as necessary. Melting points were taken
on a Fisher–Johns melting point apparatus and are uncorrected.
Elemental analyses were performed on a Leco CHNS-9320
elemental analyzer. The NMR spectra were recorded in DMSO-d6
on Bruker (Rhenistetten-Forchheim, Germany) AM 300 spec-
trometers operating at 300 MHz, using TMS as an internal
standard. 1H chemical shifts are reported in (ppm) and coupling
constants in Hz. The electron impact mass spectra (EIMS) were
determined with a Finnigan MAT-312 and a JEOL MS Route
mass spectrometer. The progress of the reaction and purity of the
products were checked on TLC plates coated with Merck silica gel
60 GF254 and the spots were visualized under ultraviolet light
at 254 and 366 nm and/or spraying with iodine vapors. In vitro
enzyme inhibition and antibacterial assays of the synthesized
compounds were carried out at the Department of Biotechnology,
The Islamia University of Bahawalpur.
as bactericidal agent3,4,7
.
Previously we have reported and identified various pharmaco-
phores responsible for potent activity of barbiturate/thiobarbiturate
nucleus via molecular modeling studies8–11. It is evident that the
imide or b-amino a, b-unsaturated amide functionality is the basic
structural moiety in these compounds which has multidimensional
array of interaction sites resulting in either potential urease
inhibitors (PUI), potential antibacterial (PAB), antifungal
Inhibitors (PAF) and potential kinase inhibitors (PKI; Figure 1).
In case of PUI, PAB and PAF the mode of action is believed to be
mainly involving the scavenging or encapsulation of the metal ion
whereas in PKI the presence of various donors/receptors inter-
actions via hydrogen bonding, electronic interactions and metal ion
encapsulation seems to be the cause. Further studies are still
required to strengthen and verification of these findings.
Emphasizing on these valuable pharmacophores, various new
libraries of barbiturates/thiobarbiturates were identified and
Synthesis
Address for correspondence: Ashfaq M. Qureshi and Zahid H. Chohan,
Institute of Chemical Sciences, Bahauddin Zakariya University, Multan
60800, Pakistan. E-mail: amqureshi@bzu.edu.pk (A.M. Qureshi); dr.za-
hidchohan@gmail.com (Z.H. Chohan)
General procedure for the preparation of compounds (1–27)
To a hot stirred solution of barbituric/thiobarbituric acid
(2.00 mmoles) and ethylorthoformate (2.02 mmoles) in 2-butanol

2
A. M. Qureshi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
H C
3
H C
3
CH
3
H
N
X
O
δ
-
Ru
Leu-83
δ
O
O
+
H
N
Translator
δ
NH
N
H
S
Y
Z
+
NH
Oδ
-
δ
-
O
Kinase site
(PKI)
Glu-81
H
X
N
O
O
O
Y
HN
NH
S
Translator
NH
Z
O
H C
3
CH
3
Urease Site
-
δ
H C
3
H
H
N
X
N
O
X
O
Ni
δ
Ru
-
O
δ
-
O
O
O
δ -
HN
N
HN
N
Translator
Translator
S
Y
S
Y
Z
H
H
NH
NH
O
O
(PUI)
Z
(PAB) / (PAF)
δ
Figure 1. Pharmacophore site combination. (X ¼O, S). PUI: Potential urease inhibitors (Y, Z ¼ O, N, S); (PAB)/(PAF): potential antibacterial and
antifungal inhibitors (Y, Z ¼ O, N, S); (PKI): potential kinase inhibitors8.
(10 mL) was added the respective amine (2.00 mmoles). Then the 30 min. Absorbance was then measured at 517 nm. Quercetin
reaction mixture was refluxed till the completion of the reaction was used as standard antioxidants. Decrease in absorbance
(for &3 h). The precipitates formed during refluxing were indicated that the increased radical scavenging activity which
collected by suction filtration. Washing with hot ethanol afforded was determined by the following formula.
TLC pure products in good to excellent yield8 (Scheme 1).
Percent scavenging activity ¼ ½100
ꢀ
ꢁ
Abs of test compound
Abs of control
Enzyme inhibition (in vitro)
ꢁ
DPPH radical scavenging activity
ꢂ 100
Stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) was used for
determining antioxidant activity according to the reported
method12. Different concentrations of compounds in respective
solvents were added at an equal volume (10 mL) to 90 mL of
100 mM methanolic DPPH in a total volume of 100 mL in 96-well
plates. After mixing contents, these were incubated at 37 ꢀC for
Lipoxygenase
Lipoxygenase (LOX) activity was assayed according to the
reported method13–15 with slight modifications. A total volume
of 200 mL assay mixture contained 140 mL sodium phosphate

DOI: 10.3109/14756366.2014.895717
New barbiturates and thiobarbiturates
3
O
S
S
S
H N
N
H
H N
N
H
R=
2
2
N
H
N
H
3 X= O
4 X=S
5 X=S
1 X= O
2 X=S
6 X=O
F
S
F
S
S
S
R=
N
H
N
N
H
N
H
F
N
H
N
H
H
N
H
N
H
F
F
9 X= O
10 X= O
11 X= O
7 X= O
8 X=S
Cl
Cl
Cl
Cl
S
S
S
S
R=
N
H
N
H
N
H
N
H
N
H
N
H
Cl
N
N
H
Cl
H
Cl
14 X= O
13 X= O
12 X= O
15 X= O
Br
S
S
S
S
R=
N
H
N
H
N
N
N
H
N
N
H
N
H
H
H
H
Br
Cl
19 X=O
18 X=O
16 X=O
17 X=O
I
S
S
S
F C
3
S
N
H
N
R=
R=
N
H
N
N
H
N
H
N
H
N
H
H
H
OCH
3
CF
3
23 X=O
22 X=O
20 X=O
21 X=O
OCH
3
H CO
S
3
S
S
S
NC
N
H
N
H
N
N
N
H
N
H
H
H
N
N
CN
H
H
27 X=O
26 X=O
24 X=O
25 X=O
Scheme 1. Synthesis of 1–27.
buffer (100 mM, pH 8.0), 20 mL test compound and 15 mL (600 U) 410 nm. The reaction was initiated by the addition of 15 mL
purified lipoxygenase enzyme (Sigma, St. Louis, MO). Contents (1.3 mM) substrate (N-succinyl phenyl-alanine-p-nitroanilide).
were mixed, pre-read at 234 nm and pre-incubated for 10 min The change in absorbance was observed after 30 min at 410 nm.
at 25 ꢀC. Reaction was initiated by addition of 25 mL substrate Chymostatin (0.5 mM wellꢁ1) was used as a positive control.
solution. Change in absorbance was observed after 6 min
at 234 nm. Baicalein (0.5 mM wellꢁ1) was used as a positive ꢀ-Glucosidase
control.
The a-glucosidase inhibition activity was performed according to
the slightly modified method of Pierre et al17. Total volume of the
Chymotrypsin
reaction mixture of 100 mL contained 70 mL 50 mM phosphate
a-Chymotrypsin inhibition activity was performed according to buffer saline, pH 6.8, 10 mL (0.5 mM) test compound, followed
slightly modified method of Rehman et al16. A total volume of by the addition of 10 mL (0.057 units) enzyme. The contents were
100 mL assay mixture contained 60 mL Tris–HCl buffer (50 mM mixed, pre-incubated for 10 min at 37 ꢀC and pre-read at 400 nm.
pH 7.6), 10 mL test compound and 15 mL (0.9 units) purified The reaction was initiated by the addition of 10 mL of 0.5 mM
a-chymotrypsin enzyme (Sigma, St. Louis, MO). The contents substrate (p-nitrophenyl glucopyranoside). Acarbose was used as
were mixed and incubated for 20 min at 37 ꢀC and pre-read at positive control. After 30 min of incubation at 37 ꢀC, absorbance

4
A. M. Qureshi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
was measured at 400 nm using Synergy HT microplate reader. 160 (6), 144 (10), 127 (4), 113 (5), 101 (8), 84 (3), 59 (14), 43
All experiments were carried out in triplicates.
(14); Anal. Calcd. for C7H7N2O2S2: C, 39.07; H, 3.26; N, 13.02;
S, 29.77; Found: C, 39.09; H, 3.28; N, 13.00; S, 29.79.
Urease
2-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
hydrazincarboxamide (3)
The enzyme assay is the modified form of the commonly known
Berthelot assay. A total volume of 85 mL assay mixture contained
10 mL of phosphate buffer of pH 7.0 in each well in the 96-well 1H-NMR (300 MHZ, DMSO-d6, d, ppm): 6.56 (2H, s, NH2),
plate followed by the addition of 10 mL of sample solution 8.83 (1H, s, ¼CH), 9.93 (2H, s, NHNH), 11.12 (2H, s, NHBA);
and 25 mL of enzyme solution (0.1347 units). Contents were pre- 13C-NMR (300 MHZ, DMSO-d6, d, ppm): 167, 157, 151,
incubated at 37 ꢀC for 5 min. Then, 40 mL of urease stock solution 99; EIMS (70 eV) m/z (%): ([M+], 213 (1)), 199 (3), 128
(20 mM) was added to each well and incubation continued (100), 100 (10), 85 (49), 69 (9), 42 (85); Anal. Calcd. for
at 37 ꢀC for further 10 min. After given time, 115 mL phenol C6H7N5O4: C, 33.80; H, 3.29; N, 32.86; Found: C, 33.82; H, 3.28;
hypochlorite was added in each well (freshly prepared by mixing N, 32.87.
45 mL phenol reagent with 70 mL of alkali reagent). For color
development, incubation was done at 37 ꢀC for another 10 min. 2[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
Absorbance was measured at 625 nm using the 96-well plate hydrazinecarboxamide (4)
reader Synergy HT.
1H-NMR (300 MHZ, DMSO-d6, d, ppm): 6.49 (2H, s, NH2), 8.06
(1H, s, ¼CH), 9.90 (2H, s, NHNH), 11.38 (1H, s,
Statistical analysis
NHCOBA),11.92 (1H, s, CSNHBA); 13C-NMR (300 MHZ,
Statistical analysis was performed by Microsoft Excel 2003. DMSO-d6, d, ppm) 177, 174, 163, 157, 155, 90; EIMS (70 eV)
Results are presented as mean SEM. The percent inhibition was m/z (%): ([M+], 229 (1)), 199 (7), 186 (47), 144 (19), 116 (8), 127
calculated by the help of following equation.
(6), 110 (11), 69 (20), 59 (12), 44 (100); Anal. Calcd. for
C6H7N5O2S2: C, 29.39; H, 2.86; N, 28.57; S, 26.12; Found: C,
29.37; H, 2.85; N, 28.58; S, 26.11.
Control ꢁ Test
Inhibition ð%Þ ¼
ꢂ 100
Control
2[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
hydrazinecarbothioamide (5)
IC50 values (concentration at which there is 50% enzyme
inhibition) of compounds were calculated using EZ-Fit Enzyme
kinetics software (Perrella Scientific Inc., Amherst, NH). All
the measurements were done in triplicate. Synergy HT (BioTek,
Winooski, VT) 96-well plate reader was used in all experiments.
The positive and negative controls were included in the assay.
1H-NMR (300 MHZ, DMSO-d6, d, ppm): 7.05 (2H, s, NH2),
8.07 (1H, s, ¼CH), 11.44 (2H, s, NHNH), 11.97 (2H, s, NHBA);
13C-NMR (300 MHZ, DMSO-d6, d, ppm) 181, 177, 162, 91;
EIMS (70 eV) m/z (%): ([M+], 245 (100)), 186 (61), 158 (48),
144 (22), 136 (22), 127 (6), 115 (20), 101 (5), 89 (25), 77 (11),
63 (14), 50 (8), 44 (17); Anal. Calcd. for C6H7N5O2S2: C, 29.39;
H, 2.86; N, 28.57; S, 26.12; Found: C, 29.37; H, 2.87; N, 28.58;
S, 26.11).
Results and discussion
This study comprises of synthesis and in vitro evaluation of
enzyme inhibitory activities of 27 new barbiturates and
thiobarbiturates.
N-(phenyl-2-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]hydrazinecarbothio amide (6)
Chemistry
1H-NMR (300 MHZ, DMSO-d6, d, ppm): 5.67 (1H, s, NHAr),
7.01 (1H, d, J 9, ArH), 7.28 (2H, t, J 6,15, ArH), 7.61 (2H, d, J 6,
ArH), 8.00 (1H, s, ¼CH), 10.51 (2H, s, NHNH), 10.94 (2H, s,
NHBA); 13C-NMR (300 MHZ, DMSO-d6, d, ppm) 179, 167,
154, 151, 150, 128, 128, 86; EIMS (70 eV) m/z (%): ([M+], 305
(4)), 231 (100), 187 (3), 170 (67), 154 (58), 128 (90), 118 (22),
77 (49), 44 (31); Anal. Calcd. for C12H11N5O3S: C, 47.21;
H, 3.61; N, 22.95; S, 10.49; Found: C, 47.22; H, 3.60; N, 22.94;
S, 10.50.
For synthesis, to a refluxing solution of thio/barbituric acid and
triethylorthoformate in 2-butanol was added respective amine.
The precipitates formed were collected and washed with hot
ethanol offering pure products in excellent yields (87–96%). The
structures of the synthesized compounds were deduced by analyt-
ical and spectroscopic (1H-NMR, 13CNMR and EIMS) data
(supplementary material). Satisfactory elemental analyses ( 0.4%
of calculated values) were obtained for all the compounds.
N-[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
ethanethioamide (1)
The 1H-NMR spectra of these compounds exhibited three
separate singlets at d ¼ 4.30–6.56, 9.21–11.45 and 10.00–12.81
for the thiosemicarbazones N4–H, thiosemicarbazone N2–H and
N3–H and barbiturate NH, respectively. Whereas ¼CH appeared
as a singlet at d 7.84–9.84. Peaks in APT 13CNMR appeared at d
175–200 for C ¼ S, 155–170 for C¼O, 140–150 for C ¼ CH–NH,
118–140 for aromatic C, 110–70 for C ¼ CH.
1H-NMR (300 MHZ, DMSO-d6, d, ppm): 2.94 (3H, s, CH3), 5.31
(1H, s, NH), 8.00 (1H, s, ¼CH), 11.51 (2H, s, NHBA); 13C-NMR
(300 MHZ, DMSO-d6, d, ppm): 208, 178, 150, 100, 90, 23; EIMS
(70 eV) m/z (%): ([M+], 213 (77)), 197 (14), 185 (17), 155 (100),
144 (32), 128 (37), 114 (14), 99 (13), 85 (11), 69 (25), 59 (44), 44
(41); Anal. Calcd. for C7H7N3O3S: C, 39.44; H, 3.29; N, 19.72; S,
15.02; Found: C, 39.43; H, 3.28; N, 19.69; S, 15.05.
In EI mass spectra, all the compounds showed molecular ions
of different intensity, which confirmed their molecular weights.
The major fragmentation pathway involved the cleavage of the
exocyclic N–N, NH–CS and endocyclic NH–CO bonds. Few of
the compounds did not show the molecular ion peaks in their
spectra. However, the fragments corresponding to thiosemicarba-
zone moiety, formed by the cleavage of N–N and NH–CS bonds
confirmed their structures.
N-[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]ethanethioamide (2)
1H-NMR (300 MHZ, DMSO-d6, d, ppm): 2.81 (3H, s, CH3), 5.11
(1H, s, NHCS), 8.20 (1H, s, ¼CH), 11.45 (2H, s, CONH); 13C-
NMR (300 MHZ, DMSO-d6, d, ppm): 208, 178, 158, 91, 23;
EIMS (70 eV) m/z (%): ([M+], 229 (35)), 213 (23), 171 (100),
The proposed fragmentation pattern of 8 is presented in
Scheme 2.

DOI: 10.3109/14756366.2014.895717
New barbiturates and thiobarbiturates
5
+
.
+
F
F
HN
N
S
m/z =95
m/z =168
.
- NH
+ H
3
+
.
+
- NH
3
+
+
.
F
O
F
F
N
S
H N
2
- NH NH
N
C
HN
S
N
2
2
NH NH
S
H N
+
2
S
N
H
O
m/z =111
m/z =153
m/z =185
m/z =227
.
- H
+
.
+
+
.
F
O
O
F
O
- H S
NH
2
N
N
HN
HN
NH
N
C
HN
S
- H.
S
S
N
H
O
N
H
O
S
N
H
O
m/z = 304
m/z = 339
m/z =144
- H CS
2
- N2
+
.
F
O
- HF
NH
O
HN
- H S
2
S
N
H
m/z = 265
O
+
.
N
N
N
C
HN
S
N
H
O
m/z =285
O
O
R
NH
HN
2 - Butanol, CH(OEt)
reflux for 3 hours
3
HN
H N–R
+
2
X
N
H
O
X
N
H
O
Scheme 2. Proposed fragmentation pattern of 8.
Bioactivities
compound 18 found to be most active inhibitor with % inhibition
of 78.62.
In DPPH activity p-F is most effective inhibitor. Comparing
Enzyme assays
Results for enzyme inhibition are presented in Table 1. All the DPPH IC50 values of 4 and 6, it appeared that thiobarbiturate with
compounds are antioxidants except 1, 5, 13 and 19. Compound 10 NH2 is more effective than barbiturate with unsubstituted phenyl
showed maximum inhibition with IC50 value 63.11 at 0.5 mM and ring but less effective than the same with a para Fluro substitution
% inhibition of 92.23. 17 Compounds 9, 17, 15, 20, 14, 8, 23, 25, 10. Whereas same Fluro at meta 9, is less inhibitory than 10. Also,
27, 21, 12, 24, 7 and 11 showed IC50 value of 138.71 at 0.5 mM, from IC50 values of 14 and 15, it is apparent that electron
145.25 at 0.5 mM, 196.41 at 0.5 mM, 198.21 at 0.5 mM, 200.31 at withdrawing substitution at ortho also effects negatively on
0.5 mM, 204.8 at 0.5 mM, 205.91 at 0.25 mM, 229.1 at 0.25 mM, inhibition activity of such compounds. Order of lipoxygenase
303 at 0.25 mM, 329.71 at 0.5 mM, 376 at 0.25 mM, 378.11 at activity with respect to substitution position on phenyl ring is
0.5 mM, 407.11 at 0.5 mM, 407.11 at 0.5 mM, 5500 at 0.5 mM, m-F4p-CH34diortho-p-triCl4p-I4o,p-diCl4o-F4o-OCH3
5500 at 0.25 mM and 5500 at 0.5 mM against lipoxygenase. 4p-OCH3 4m-CN4m-OCH3 4m-Cl4o–F. From IC50 values
Against Chymotrypsin almost all the compounds are active but of 9 and 7 (i.e. m-F and o-F) and 9 and 8 (i.e. X ¼O and X ¼ S),
only 18 and 13 showed reportable IC50 value of 78.4 at 0.25 mM, it can be inferred that electron withdrawing fluoro-substituent
12.87 at 0.25 mM and 159.3 at 0.5 mM, respectively. Hence at meta position inhibits more than at ortho position and also

6
A. M. Qureshi et al.
J Enzyme Inhib Med Chem, Early Online: 1–7

DOI: 10.3109/14756366.2014.895717
New barbiturates and thiobarbiturates
7
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thoibarbiturate is more effective in this typical activity than
barbiturate. In chymotrypsin inhibition only o-Br and p-Cl
compounds have shown some IC50 values. Therefore, this typical
activity can be attributed to the halogens at ortho, para position of
such compounds. Comparing IC50 values, it is speculated that
electrostatic effects of substituents (translator, Figure 1) play an
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This study successfully afforded the synthesis of targeted 27 new
barbiturates/thiobarbiturates derivatives in good to excellent yield
via an efficient one pot three component reaction. All the
synthesized compounds were extensively characterized and
screened for biological activities. All the compounds exhibited
appreciable % inhibition at each instance strengthening the idea
that structural motifs such as imide/b-amino a,b-unsaturated
amide play a vital role as pharmacophore–enzyme interaction site
and presence of suitable substituents (translator) eventually
magnify inhibition of enzymes in general (Figure 1). In vitro
evaluation for antioxidant (DPPH radical scavenging), lipoxygen-
ase, chymotrypsin, a-glucosidase and anti-urease activities
yielded 23 compounds in DPPH, 14 in lipoxygenase and two in
chymotrypsin exhibiting appreciable IC50 values.
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of metal-barbiturate conjugates against pathogenic strains of
Escherichia coli and Staphylococcus aureus. Lett Drug Dis Des
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Declaration of interest
There is no declaration of interest. One of the authors Saira Mumtaz likes
to acknowledge Higher Education Commission of Pakistan for providing
funding and support throughout this study.
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Supplementary material available online
Supplementary materials