Design and synthesis of new barbituric- and thiobarbituric acid derivatives as potent urease inhibitors: Structure activity relationship and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2015.05.038

In this study 36 new compounds were synthesized by condensing barbituric acid or thiobarbituric acid and respective anilines (bearing different substituents) in the presence of triethyl orthoformate in good yields. In vitro urease inhibition studies again

Full text of DOI:10.1016/j.bmc.2015.05.038

Accepted Manuscript
Design and synthesis of new barbituric- and thiobarbituric acid derivatives as
potent urease inhibitors: structure activity relationship and molecular modeling
studies
Abdul Rauf, Sohail Shahzad, Marek Bajda, Muhammad Yar, Faiz Ahmed,
Nazar Hussain, Muhammad Nadeem Akhtar, Ajmal Khan, Jakub Jończyk
PII:
S0968-0896(15)00448-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.05.038
BMC 12334
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 April 2015
23 May 2015
25 May 2015
Please cite this article as: Rauf, A., Shahzad, S., Bajda, M., Yar, M., Ahmed, F., Hussain, N., Akhtar, M.N., Khan,
A., Jończyk, J., Design and synthesis of new barbituric- and thiobarbituric acid derivatives as potent urease
inhibitors: structure activity relationship and molecular modeling studies, Bioorganic & Medicinal Chemistry
(2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.05.038
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Design and synthesis of new barbituric- and thiobarbituric acid derivatives
as potent urease inhibitors: structure activity relationship and molecular
modeling studies
Abdul Raufa,*, Sohail Shahzada,b, Marek Bajdac, Muhammad Yarb,*, Faiz Ahmeda, Nazar
Hussaina, Muhammad Nadeem Akhtard, Ajmal Khane, Jakub Jończykc
aDepartment of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
bInterdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology,
Lahore,54000, Pakistan
cDepartment of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-688 Cracow, Poland
dFaculty of Industrial Sciences & Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak 26300, Kuantan
Pahang, Malaysia
eH.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of
Karachi, Karachi 75270, Pakistan
*Correspondence Address:
E-mail addresses: lecorganic@yahoo.com (A Rauf); drmyar@ciitlahore.edu.pk (M. Yar)
Abstract:
In this study 36 new compounds were synthesized by condensing barbituric acid or
thiobarbituric acid and respective anilines (bearing different substituents) in the presence
of triethyl orthoformate in good yields. In vitro urease inhibition studies against jack bean
urease revealed that barbituric acid derived compounds (1-9 and 19-27) were found to
exhibit low to moderate activity however thiobarbituric acid derived compounds (10-18
and 28-36) showed significant inhibition activity at low micro-molar concentrations.
Among the synthesized compounds, compounds (15), (12), (10), (36), (16) and (35)
showed excellent urease inhibition with IC50 values 8.53 0.027, 8.93 0.027, 12.96 0.13,
15 0.098, 18.9 0.027 and 19.7 0.63 μM, respectively, even better than the reference
compound thiourea (IC50 = 21 0.011). The compound (11) exhibited comparable activity
to the standard with IC50 value 21.83 0.19 μM. In silico molecular docking studies for
1

most active compounds (10), (12), (15), (16), (35) and (36) and two inactive compounds
(3) and (6) were performed to predict the binding patterns.
Key words: Urease, barbituric, thiobarbituric, jack bean, in silico, triethyl orthoformate
1. Introduction:
Urease (urea amidohydrolase, EC 3.5.1.5) is a nickel-based enzyme that catalyzes the
transformation of urea to carbon dioxide and ammonia. Urease is part of a range of living
organisms including plants, algae, fungi and bacteria [1-3]. Urease plays a key function in
nitrogen metabolism of plant during the germination process [4]. Even though having
different origin of urease, all of them constitute highly conserved tertiary structures,
common amino acid sequences in the active site and thus show same mechanism of action.
Among numerous ureases, jack bean (Canavalia ensiformis) urease, the first enzyme
crystallized [5] and best-characterized [6-8], has been widely employed in urease inhibition
studies [9, 10]. The production of ammonia from urease is responsible for harmful
complications in agriculture and health fields. During urea fertilization in agriculture, large
quantities of ammonia are emitted into the atmosphere by high urease activity, it causes
significant environmental and economic problems [11-13]. The enormous quantity of
ammonia further stimulates plant damage by soil pH increase and ammonia toxicity [14].
Some bacterial ureases served as a critical factor involved in the progression of kidney
stones, pyelonephritis, peptic ulcers, and other health complications [14, 15]. Urease in
Helicobacter pylori is now accepted as a major cause of peptic ulcers [16-18], and its
inhibition has been recognized as a potential therapeutic approach for peptic ulcers [19].
Therefore, urease inhibitors based anti-ulcer drugs have attracted great attention. Urease
inhibitors have been broadly classified into two categories: (i) substrate-like inhibitors,
such as hydroxyurea and hydroxamic acids, and (ii) mechanism-based inhibitors, such as
phosphorodiamidates and imidazoles [20].
Recently, a variety of compounds including phosphoramidates [21], hydroxamic acids [21,
22], boric and boronic acids [23], heavy metal ions [24], quinones [25] and imidazoles [26]
have been investigated for their urease inhibition activity. And more recently, barbiturates
[27] and thiobarbiturates [28, 29] have been discovered as potent urease inhibitors.
2

Barbiturates and thiobarbiturates are medicinally imperative class of heterocyclic
compounds, Barbiturates exhibites a wide range of biological activities including
antibacterial, hypotensive, tranquilizing [30, 31], antioxidants [32], anticonvulsant and
anesthetic [33], antiepileptic [34], sedatives and hypnotics [33-35], anticancer [36, 37],
immuno-modulating [37], radio-sensitizing [38] and gelatinase inhibitors [39].
Similarly thiobarbiturates behave as HIV integrase inhibitors [40], anticonvulsant and
anesthetic [33], antibacterial [41, 42], antifungal [43], antiviral [44] antitumor activities
[45], tyrosinase inhibitors [46, 47] and anticancer with anti-inflammatory activities [48].
Literature review also revealed that barbiturates and thiobarbiturates show anti-
tuberculosis [49], anti-diabetic and anti-bacterial properties [50].
In view of these observations, it was thought worthwhile to synthesize a range of new and
more potent derivatives of barbituric and thiobarbituric acids by incorporating substituted
anilines at C-5 position of barbituric- or thiobarbituric acids. Research in the field of
enzyme inhibition has enormous potential to introduce new drug candidates against
different clinical conditions. Keeping in view the therapeutic importance of ureases, we
have selected them as synthetic targets and to evaluate their urease inhibition potential to
discover new lead molecules for the treatment of gastric ulcers, and other urease associated
disorders. This method afforded the efficient synthesis of required compounds and also
tolerated a range of functional groups including halo, -COOH, -CF3, -SH, -OH, and NO2.
2. Results and discussion
2.1. Chemistry
The desired substituted anilines based barbiturates and thiobarbiturates (1-36) were
prepared in good to excellent yields (70–97%) according to the procedure given in the
Scheme 1. The preparation of (1-36) was achieved from condensation of barbituric or
thiobarbituric acid and the corresponding susbstituted anilines (Table 1), in the presence of
triethyl orthoformate and 2-butanol. Thus, a variety of functional groups such as
carboxylic acid, hydroxyl, nitro, chloro, iodo, trifluoromethyl and thiol were tolerated
under our reaction protocol. The chemical structures of all the synthesized compounds
were establishe
3

d with the aid of spectroscopic and physical methods.
H
O
H
H
H
O
N
N
2-Butanol
N
N
N R
4
X
R NH2
CH(OEt)3
X
5
Reflux 3 hours
H
O
O
70 - 97 %
Compounds 1 - 36
X = O or S and R= substituted aryls
Scheme 1: Synthetic protocol for substituted anilines based barbiturates and thio-
barbiturates; X = O or S and R= substituted aryls
2.2. Urease inhibition assay
Ureases are among the few enzymes that require nickel for activity. It is known that
binding of nickel to urease is very specific and tight and the removal of metal ions can be
achieved only by harsh treatment with denaturants or acids [51-53]. Literature studies
revealed that biological activities of barbiturates and thiobarbiturates are different from
each other [54]. The mechanism involved for the biological activities of barbiturates and
thiobarbiturates is still not completely known, but recently, quantitative–structure–
activity–relationship (QSAR) study has proposed that electronic factors play an important
role in imparting biological activities to barbiturates and thiobarbiturates [55]. The
thiobarbiturates were proved to possess better biological activities than barbiturates due to
their better hydrogen bond acceptance ability which helps binding to the enzyme receptor.
The reason for better hydrogen bond acceptance of sulfur atom was due to lower ionization
energies and subsequently better electron donating abilities [54]. It has also been
experimentally determined that sulfur atom in C=S group forms hydrogen bonds with
enzymes (e.g. uridine phosphorylase) and biologically important thioureas [56, 57].
Recently, synthesis and urease inhibition activities of various thiobarbiturates have been
reported and they showed excellent urease inhibition activity. The reason explained for the
better activity was the possible involvement of two hydroxyl groups in the formation of
hydrogen bonds with nickel atoms present in the enzyme. It was further reported that the
4

presence of one extra sulfur atom on the phenyl ring might also helpful in making
coordination bonds with nickel [58].
Khan et. al., synthesized and evaluated the in vitro urease inhibition activities of various
arylidene barbiturates. It was suggested that the presence of highly electronegative fluoro
group at ortho, meta or para positions enhanced the activity but the most activity was
found when the fluoro group was at para position. Furthermore, molecular docking
analysis showed that in most of the molecules, one of the carbonyl groups coordinates with
both Ni atoms, while the other one is involved in the formation of hydrogen bonds with
important active site residues, e.g., Ala170, Gly280 and Arg339 [27]. Nickel coordination
might be one of the important factors for the activities of these compounds, as mostly
known urease inhibitors also interact with nickel ions [59-62].
Previously we reported novel sulphonamides based barbiturates and thiobarbiturates and
determined their urease inhibition activities. The thiobarbiturates (% inhibition 88.3-99.9
%) were found to be more potent than barbiturates. It was suggested that some
coordinating sites such as, C=O group of thiobarbiturates ring and N-H groups, have been
involved in chelating Ni (II) atoms of urease enzyme [29].
In current paper, we report the synthesis of structurally more diverse substituted aniline
based barbiturate and thiobarbiturate derivatives. Khan et al reported arylidene barbiturates
the present research include aniline derived compounds which provide an amino
derivatives (=C-NHR). All the synthesized substituted anilines based barbiturates and
thiobarbiturates (1–36) were evaluated for urease inhibitory activity. The compounds (15),
(12), (10), (36), (16) and (35) showed excellent urease inhibition with IC50 values of
8.53 0.027, 8.93 0.027, 12.96 0.13, 15 0.098, 18.9 0.027 and 19.7 0.63 μM,
respectively, much better than the standard urease inhibitor thiourea (IC50 = 21 0.011)
(Table 2). The compound (11) also exhibited comparable activities to standard with IC50
value of 21.83 0.19 μM respectively. The compounds which showed less than 50%
inhibition, their IC50 values were not calculated.
5

2.3. Structure activity relationship
In order to explain the structure–activity relationship and to get optimized urease
inhibitors, both barbituric and thiobarbituric acids and substituted anilines were used to
furnish the synthesis of substituted anilines based barbiturates and thiobarbiturates (1-36).
These substituents include electron donating group such as hydroxyl, thiol and electron
withdrawing groups such as carboxylic, nitro, trifluoromethyl, chloro, dichloro, trichloro
and iodo. Furthermore, the positions of these groups were also changed in the phenyl ring
to elaborate more detailed inhibition potential. The present investigation shows that
thiobarbiturates (10-18 and 28-36) are more active than barbiturates (1-9 and 19-27). This
is attributed to the presence of sulfur atom in thiobarbituric acid instead of oxygen atom in
the barbituric acid, which plays an important role in imparting urease inhibition to these
compounds. It is proposed that sulphur is less electronegative and bigger in size as
compared to oxygen and make more feasible transfer or shifting of electrons from
tetrahydropyrimidine ring to chelate the nickel ion present in the enzyme of bacteria, which
is responsible for the activity of bacteria. Apart from this, other factors such as
hydrophobic, steric and electronic properties may also amongst the possible reasons
causing enhancement of the urease inhibition of these compounds.
Amongst thiobarbiturates, (10-18) containing carboxyl group (-COOH) in their respective
substituted anilines, inhibition potential varies due to changing position of carboxyl group
(-COOH) and absence or presence of different other substituents in the phenyl ring.
Compound (15), bearing carboxyl group (–COOH) at C-2 position and nitro group (-NO2)
at C-5 position in the phenyl ring, was proved to be the most active with an IC50 value of
8.53 0.027 µM. Removing the nitro group (-NO2) from the C-5 position of (15), producing
(10) (IC50 = 12.96 0.13) slightly diminished the activity, but it is still more active than
standard thiourea. Changing the position of carboxyl group (-COOH) in the phenyl ring
from C-2 to C-4, producing (12) (IC50 = 8.93 0.027 µM) again enhanced the activity.
Thus, from the activity pattern it can be ascertained that the presence of carboxyl group (-
COOH) at C-2 and C-4 positions in the phenyl ring plays a significant role in the high
activities of these compounds and the activity is highest when nitro group (-NO2) is also
present at C-5 position along with carboxyl group (-COOH) at C-2 (Table 1). The possible
6

reason might be the involvement of carboxyl group (-COOH) to form hydrogen bonds with
nickel atom present in the urease enzyme.
The presence of hydroxyl groups (-OH) at C-3 or C-4 positions in the phenyl ring slightly
decreased the activities as compounds (13) and (14). Similarly, the presence of nitro (-
NO2) at C-4 and chloro (-Cl) at C-6 positions in the phenyl ring also decreased the
activities as in the compounds (17) and (18) respectively.
Amongst thiobarbiturates, (28-36) which are without carboxyl group (-COOH) in their
respective substituted anilines, the compound (36) showed better urease inhibition activity
with an IC50 = 15 0.098. It was due to the presence of thiol (-SH) group at C-2 position.
Replacing the thiol (-SH) group from C-2 position with hydroxyl group (-OH) slightly
decreased the activity, producing (35) with an IC50 value of 19.7 0.63 µM. From the
activity pattern it can be ascertained that the presence of the thiol (-SH) or hydroxyl group
(-OH) at C-2 position in the phenyl ring plays a significant role in the high activities of
these compounds and the activity is higher when thiol group (-SH) is present at C-2
position instead of hydroxyl group (compounds 36 vs. 35). The possible reason might be
the involvement of thiol and hydroxyl groups to form hydrogen bonds with nickel atom
present in the urease enzyme. However, the presence of trifluoromethyl group (-CF3),
dichloro, trichloro and iodo groups in the phenyl ring decreased the activities as in the
compounds (28-34).
7

Table 1. Chemical structures, urease inhibition activity and IC50 values (mean SEM, n=3)
of substituted anilines based barbiturates and thiobarbiturates (ND, not determined).
H
H
O
N
N
N R
X
H
O
(Compounds 1-36)
IC50
IC50
No
1
X
O
O
R
No
19
X
R
HOOC
ND
ND
F3C
O
ND
ND
ND
COOH
CF3
O
O
2
3
20
21
ND
ND
COOH
CF3
O
O
F3C
88.3 1.22
OH
COOH
O
O
O
4
5
6
22
23
24
NO2
Cl
ND
ND
ND
ND
COOH
OH
O
O
Cl
HOOC
Cl
Cl
Cl
NO2
8

COOH
ND
ND
I
O
O
O
7
25
26
27
28
HO
COOH
NO2
25.8 0.47
ND
ND
HO
HS
O
O
8
COOH
52.6 0.29
O
S
9
Cl
HOOC
12.96 0.13
21.83 0.19
65.3 0.57
F3C
S
10
COOH
24.56 0.098
CF3
S
S
S
S
11
12
29
30
136.4 0.50
28.7 0.32
8.93 0.027
COOH
CF3
F3C
32.3 0.16
OH
COOH
S
S
S
S
S
S
13
14
15
31
32
33
NO2
Cl
ND
89.16 1.00
COOH
OH
Cl
HOOC
ND
8.53 0.027
Cl
Cl
Cl
NO2
9

COOH
18.9 0.027
41.33 0.29
I
S
S
S
S
S
16
17
18
34
35
36
HO
COOH
NO2
39.76 0.072
26.66 0.95
19.7 0.63
15 0.098
HO
HS
COOH
S
Cl
Thio-ureaa
21 0.011
a Thiourea standard inhibitor for antiurease activity
2.4. Molecular Modelling
We performed molecular modelling studies for all compounds which were more active
than reference - thiourea (10, 12, 15, 16, 35 and 36) as well as for two inactive compounds
(3 and 6) which were the analogues of the two most potent inhibitors (12) and (15). The
urease from Jack bean was selected for the analysis among a group of ureases from a few
different sources because our biological assay was based on such enzyme. Selection of the
most active and inactive compounds for docking enabled us to reveal their binding mode
and to show the important structural features responsible for the activity.
All analyzed derivatives of barbituric and thiobarbituric acid displayed similar, converged
binding mode. Even the general orientation of each ligand in the active site was almost the
same, the small differences appeared and were responsible for the large variations in the
activity. The (thio)urea fragment of (thio)barbituric acid was engaged in complexation of
nickel ions (Figure 1). However, thiobarbiturates were better complexing agents due to the
easier thiolactam – thiolactim tautomerization, stronger acidic properties and easier
subsequent ionization in comparison with barbiturates. Moreover, the scoring function
obtained higher values for thiobarbiturates and therefore, derivatives containing sulfur
10

atom were potent while oxygen analogues were inactive. The NH group from
(thio)barbituric moiety created hydrogen bond with C=O from backbone of Gly550 while
the neighbouring carbonyl group formed H-bond with guanidine fragment of side chain of
Arg609. In case of the most active compound (15) (Figure 1) carboxyl group from phenyl
ring created salt bridge with the same arginine, and nitro group was orientated toward
His594 and was able to form weak hydrogen bond. The phenyl ring could interact with
imidazole ring of His593 by π- π stacking. In case of inactive compound (6) which was the
analogue of inhibitor (15) all interactions were preserved (Figure 2). However, barbiturate
– nickel complex was less stable than thiobarbiturate – nickel one and this led to inactivity.
Inhibitor (10) which was devoid of nitro group was a little bit less active than compound
(15) because of no interactions with His594. Compound (12) (Figure 3) contained only
carboxyl substituent in the para position of the phenyl ring but it was sufficient to produce
almost the same activity as in case of derivative (15) due to the creation of the hydrogen
bond with His594. The difference between compounds (3) and (12) was exactly the same
as between (6) and (15), and derivative (3) was also inactive. Inhibitor (16) due to the
presence of hydroxyl substituent in position 2 of the phenyl ring and carboxyl in position 5
gave hydrogen bonds with Asp494 and His594, respectively. The second H-bond was
weak and this might lead to decreased potency in comparison with compound (15) or (12).
The change of carboxyl position from 5 to 4 could improve the potency because in position
4 carboxylate could form stronger hydrogen bond as in case of compound (12). Derivatives
(35) and (36) possessed only one substituent in the phenyl ring in position 2 – hydroxyl
and thiol group, respectively. In case of inhibitor (35) the phenyl ring was a little bit
rotated and this hydroxyl group formed hydrogen bond with Asp494. Thiol group, present
in compound (36), could ionize and form ionic interaction with Arg609. Such interaction
was more profitable than hydrogen bond for derivative (35), and inhibitor (36) was a little
bit more potent, however both compounds were still less active than (15) and (12).
The reference compound – thiourea was also able to interact with nickel cations and it mimicked
the substrate of the enzyme. It was located a little bit farther from metal ions than our compounds
but the distance was still good to obtain the inhibitory effect on the urease.
11

Figure 1. The binding mode of the most active compound (15) within the active site of Jack bean
urease. The most important feature of the bonding mode is formation of the complex with nickel
ions. The violet dashed lines represent interactions with nickel cations while the grey ones ionic
interactions and hydrogen bonds.
12

Figure 2. The binding mode of the inactive compound (6) within the active site of Jack bean urease.
The interactions scheme is quite the same as for the potent compound (15) but the complexing
ability and stability of complex are lower. The violet dashed lines represent interactions with nickel
cations while the grey ones ionic interactions and hydrogen bonds.
13

Figure 3. The binding mode of compound (12) within the active site of Jack bean urease. The violet
dashed lines represent interactions with nickel cations while the grey ones ionic interactions and
hydrogen bonds.
3. Conclusion
In conclusion, a facile and high yield synthesis of novel substituted anilines based
barbiturates and thiobarbiturates derivatives has been achieved. The in-vitro urease
inhibition activities of these compounds revealed that thiobarbiturate derivatives were
more active than the barbiturates, thus the thio-compounds (10), (12), (15), (16), (35) and
(36) were found to be significant active among this tested series. The docking studies were
also performed for the compounds (10, 12, 15, 16, 35 and 36) which were more active than
reference - thiourea as well as for two inactive compounds (3 and 6) to investigate the
binding patterns of these compounds with target enzyme. Our current findings may lead to
thiobarbiturates based future urease inhibitors to treat ulcers and other urease related
problems.
14

4. Experimental
4.1. General methods
1H-NMR spectra were recorded in d6-DMSO with Bruker AM 300 and AM 400
spectrometers (Rheinstetten – Forchheim, Germany) operating at 300MHz for compounds
(2, 3, 9, 15, 18, 21, 22, 23, 28, 32) and at 400 MHz for compounds (1, 4-8, 10-14, 16-17,
1
19-20, 24-27, 29-31, 33-36). H chemical shifts are reported in δ (ppm) and coupling
constants in Hz. Splitting patterns were as follows s (singlet), d (doublet), dt (doublet of
triplet), dd (double doublets), t (triplet), and m (multiplet). All J values are given in Hz and
13
chemical shifts in δ-units. The CNMR spectra were recorded in d6-DMSO with Bruker
AM 100 spectrometer (Rheinstetten – Forchheim, Germany) operating at 75 and 100 MHz.
Tetramethylsilane (TMS) was taken as internal standard. Mass spectra were recorded with
JEOL JMS600 and MAT312 operated with Electron Ionization mode. The progress of all
reactions was monitored by TLC, which was performed on 2.0 × 5.0 cm aluminum sheets
precoated with silica gel 60F254 to a thickness of 0.25 mm (Merck). The chromatograms
were visualized under ultraviolet light (254–366 nm). The melting points were taken on
Gallen Kamp apparatus and are uncorrected. Chemical reagents were purchased from the
Merck Chemical Company in high purity. All the reagents were of commercial grade and
used as obtained from supplier or redistilled as necessary.
4.2. General procedure for the synthesis of compounds (1-36)
0.5g (0.0039mol) of barbituric acid or thiobarbituric acid was added in 2-butanol (8-10 ml)
and warmed till the solution become clear then added suitable substituted anilines
(0.0039mol), viz. 2-amino benzoic acid, 3-amino benzoic acid, 4-amino benzoic acid, 4-
amino salicylic acid, 5-amino salicylic acid, 2-amino-4-nitro benzoic acid, 3-amino-4-
hydroxy benzoic acid, 5-amino-2-nitro benzoic acid, 3-amino-4-chloro benzoic acid, in the
presence of slight excess of triethyl orthoformate (1ml). The resultant reaction mixture was
heated under reflux for 3-4 hours. The solid product was collected by suction filtration in
hot state. Then the product was washed with hot ethanol and dried. The products were
obtained in good to excellent yields. The purity of the synthesized compounds was
15

checked by thin layer chromatography using appropriate solvent systems
(Methanol:Chloroform; 1:1).
2-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic acid (1)
1
Yield 72%, off-white solid, m.p. 310-314°C (decompose); H NMR (d6-DMSO, δ, ppm);
13.78 (1H, s, COOH), 13.36 (1H, d, J 13.8, =CHNH-), 10.95 (1H, s, CONHCO), 10.87
(1H, s, CONHCO), 8.61 (1H, d, J 13.8, =CH-N), 8.01 (1H, d, J7.6, ArH), 7.8 (1H, d, J
8.4, ArH), 7.69 (1H, t, J 7.6 ArH), 7.31 (1H, t, J 7.6ArH); 13C NMR (d6-DMSO, δ, ppm):
167.6(C), 165(C), 163.7(C), 150.6(C), 149.8(CH), 139.9(C), 134.5(CH), 131.6(CH),
124.9(CH), 118.5(C), 116.8(CH), 94.2(C); MS (EI) m/z 275(M+ 88%), 257(67), 229(80),
170(18), 143(100), 119(31).
3-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic acid (2)
1
Yield 76%, off-white solid, m.p. 345-348°C (decompose); H NMR (d6-DMSO, δ, ppm);
13.19 (1H, s, COOH), 11.86 (1H, d, J 13.8, =CHNH-), 11.00 (1H, s, CONHCO), 10.87
(1H, s, CONHCO), 8.53 (1H, d, J 14.1, =CH-N), 7.94 (1H, s, ArH), 7.79 (1H, d, J 7.8,
13
ArH), 7.77 (1H, d, J 7.8, ArH), 7.54 (1H, t, J 7.8,ArH); C NMR (d6-DMSO, δ, ppm):
166.6(C), 165.9(C), 163.5(C), 151.5(CH), 150.7(C), 139(C), 132.3(C), 130(CH),
126.4(CH), 122.9(CH), 119.1(CH), 93(C); MS (EI) m/z 275(M+ 100%), 258(9), 230(20),
188(99), 144(29), 65(18).
4-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic acid (3)
°
1
Yield 91%, off-white solid, m.p. 350-360 C (decompose); H NMR (d6-DMSO, δ, ppm);
12.92 (1H, s, COOH), 11.9 (1H, d, J 13.5, =CHNH-), 11.06 (1H, s, CONHCO), 10.91
(1H, s, CONHCO), 8.59 (1H, d, J 13.5, =CH-N), 7.95 (2H, d, J 8.7,ArH), 7.61 (2H, d, J
8.4, ArH); 13C NMR (d6-DMSO, δ, ppm): 166.5(C), 166(C), 163.4(C), 150.9(CH),
150.6(C), 142.1(C), 131(CH), 127.6(C), 118.1(CH), 93.7(C); MS (EI) m/z 275(M+ 83%),
188(63), 144(29), 89(30), 65(100), 53(76).
16

2-hydroxy-4-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (4)
°
1
Yield 75%, brown solid, m.p. 324-330 C (decompose); H NMR (d6-DMSO, δ,
ppm);13.13 (1H, s, COOH), 11.77 (1H, d, J 13.6, =CHNH-),11.05 (1H, s, CONHCO),
10.90(1H, s, CONHCO), 9.76 (1H, s, OH), 8.53 (1H, d, J 13.6, =CH-N), 7.79 (1H, d, J8.4,
ArH), 7.08 (1H, s, ArH), 7.03 (1H, d, J 8.8, ArH); 13C NMR (d6-DMSO, δ, ppm):
171.3(C), 166(C), 163.3(C), 162.4(C), 150.7(CH), 150.6(C), 144.3(C), 132(CH), 110.1(C),
109.1(CH), 105.8(CH), 93.9(C); MS (EI) m/z 291(M+ 54%), 273(21), 247(50), 159(36),
119(17), 103(20), 78(29), 53(100).
2-hydroxy-5-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (5)
°
1
Yield 74%, off-white solid, m.p. 252-260 C (decompose); H NMR (d6-DMSO, δ, ppm);
13.41(1H, s, COOH),11.79 (1H, d, J 13.6, =CHNH-), 10.89 (1H, s, CONHCO), 10.75
(1H, s, CONHCO), 9.86 (1H, s, OH), 8.39 (1H, d, J 12.4, =CH-N),7.77 (1H, s, ArH), 7.58
13
(1H, dd, J 8.8, ArH), 6.96 (1H, d, J 8.8, ArH); C NMR (d6-DMSO, δ, ppm): 171.5(C),
170.9(C), 165.9(C), 163.5(C), 159.6(C), 151.4(CH), 129.8(C), 124.2(CH), 120(CH),
117.2(CH), 113.8(C), 91.7(C); MS (EI) m/z 291(M+ 48%), 273(94), 174(20), 146(43),
103(29), 79(41), 53(100).
4-nitro-2-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (6)
°
1
Yield 90%, light yellow solid, m.p. 338-346 C (decompose); H NMR (d6-DMSO, δ,
ppm); 14.44 (1H, s, COOH), 13.36 (1H, d, J 13.6, =CHNH-), 11.04 (1H, s, CONHCO),
10.95 (1H, s, CONHCO), 8.66 (1H, d, J 13.2, =CH-N), 8.51 (1H, s, ArH), 8.2 (1H, d, J
13
8.4, ArH), 8.03 (1H, dd, J 8.4 ArH); C NMR (d6-DMSO, δ, ppm): 166.4(C), 165.1(C),
163.5(C), 150.6(C), 150.6(C), 150.4(CH), 141(C), 133.2(CH), 123.6(C), 118.5(CH),
112.4(CH), 95.3(C); MS (EI) m/z 320(M+ 29%), 302(15), 274(27), 188(27), 91(13),
53(100).
17

4-hydroxy-3-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (7)
°
1
Yield 74%, off-white solid, m.p. 326-334 C (decompose); H NMR (d6-DMSO, δ, ppm);
12.81 (1H, s, COOH), 12.01 (1H, d, J 14, =CHNH-),11.40 (1H, s, OH), 10.97 (1H, s,
CONHCO), 10.84 (1H, s, CONHCO), 8.59 (1H, d, J 14, =CH-N), 7.99 (1H, s, ArH), 7.67
13
(1H, dd, J 8.4, ArH), 7.03 (1H, d, J 8.4 ArH); C NMR (d6-DMSO, δ, ppm): 166.5(C),
166.2(C), 163.3(C), 151C), 150.6(C), 149.9(CH), 127.8(CH), 126(C), 122.4(C), 117(CH),
115.4(CH), 92.7(C); MS (EI) m/z 291(M+ 87%), 181(21), 163(86), 153(58), 146(100),
118(22), 63(37).
2-nitro-5-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (8)
°
1
Yield 76%, yellow solid, m.p. 198-200 C (decompose); H NMR (d6-DMSO, δ, ppm);
13.95 (1H, s, COOH), 11.89 (1H, d, J 13.6, =CHNH-), 11.08 (1H, s, CONHCO), 10.95
(1H, s, CONHCO), 8.6 (1H, d, J 13.6, =CH-N), 8.04 (1H, d, J 8.8 ArH), 7.91 (1H, s,
ArH), 7.86 (1H, d, J 8.8 ArH); 13C NMR (d6-DMSO, δ, ppm): 168(C), 167.6(C), 165.3(C),
154.7(C), 151.5(C), 151(C), 148.4(CH), 133.8(C), 127.1(CH), 112.7(CH), 111.2(CH),
100.2(C); MS (EI) m/z 320(M+ 70%), 128(100), 100(7), 85(30), 69(20).
4-chloro-3-{[(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (9)
°
1
Yield 88%, off-white solid, m.p. 330-335 C (decompose); H NMR (d6-DMSO, δ, ppm);
13.42 (1H, s, COOH), 12.33 (1H, d, J 13.2, =CHNH-), 11.16 (1H, s, CONHCO), 10.99
(1H, s, CONHCO), 8.65 (1H, d, J 13.2, =CH-N), 8.18 (1H, s, ArH), 7.75 (1H, d, J 8.4,
ArH), 7.71 (1H, d, J 8.4 ArH); 13C NMR (d6-DMSO, δ, ppm): 166.6(C), 166(C), 163.1(C),
151.1(CH), 150.5(C), 135.4(C), 131.2(C), 130.3(CH), 127.2(C), 126.8(CH), 118.3(CH),
94.2(C);MS (EI) m/z 309(M+ 55%), 274(100), 231(70), 99(68), 53(78).
18

2-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (10)
°
1
Yield 89%, dark yellow solid, m.p. 340-344 C (decompose); H NMR (d6-DMSO, δ,
ppm); 13.84 (1H, s, COOH), 13.5 (1H, d, J 14, =CHNH-), 12.11 (1H, s, CONHCS), 12.02
(1H, s, CSNHCO), 8.65 (1H, d, J 14, =CH-N), 8.02 (1H, d, J8, ArH), 7.84 (1H, d, J 8,
ArH), 7.69 (1H, t, J 8 ArH), 7.34 (1H, t, J 8ArH); 13C NMR (d6-DMSO, δ, ppm): 178(C),
167.5(C), 162.9(C), 161.7(C), 150.9(CH), 139.5(C), 134.5(CH), 131.6(CH), 125.4(CH),
118.9(C), 117.2(CH), 95.4(C); MS (EI) m/z 291(M+ 73%), 170(32), 143(43), 115(43),
77(64), 53(100).
3-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (11)
°
1
Yield 91%, light yellow solid, m.p. 332-338 C (decompose); H NMR (d6-DMSO, δ,
ppm); 13.2 (1H, s, COOH), 12.16 (1H, d, J 14, =CHNH-), 12.04 (1H, s, CONHCS), 12
(1H, s, CSNHCO), 8.58 (1H, d, J 14, =CH-N), 7.99 (1H, s, ArH), 7.82 (1H, d, J 8, ArH),
13
7.8 (1H, d, J 8, ArH), 7.55 (1H, t, J 8,ArH); C NMR (d6-DMSO, δ, ppm): 177.9(C),
166.5(C), 163.7(C), 161.6(C), 152.5(CH), 138.8(C), 132.2(C), 130(CH), 126.9(CH),
123.3(CH), 119.6(CH), 94.3(C);MS (EI) m/z 291(M+ 100%), 188(57), 137(39), 116(38),
89(26), 65(83), 53(65).
4-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}benzoic
acid (12)
°
1
Yield 86%, yellow solid, m.p. 335-340 C (decompose); H NMR (d6-DMSO, δ, ppm);
12.95 (1H, s, COOH), 12.21 (1H, d, J 14, =CHNH-), 12.07 (1H, s, CONHCS), 12.03 (1H,
s, CSNHCO), 8.64 (1H, d, J 14, =CH-N), 7.96 (2H, d, J 8.8,ArH), 7.62 (2H, d, J 8.8,
ArH); 13C NMR (d6-DMSO, δ, ppm): 177.9(C), 166.5(C), 163.7(C), 161.4(C), 151.9(CH),
141.8(C), 130.9(CH), 128(C), 118.5(CH), 95.00(C); MS (EI) m/z 291(M+ 100%), 188(49),
144(46), 116(39), 89(29), 65(87), 53(61).
19

4-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-2-hydroxy-
benzoic acid (13)
°
1
Yield 76%, green solid, m.p. 300-306 C (decompose); H NMR (d6-DMSO, δ, ppm);
13.17 (1H, s, COOH), 12.21 (1H, s, CONHCS),12.08 (1H, s, CSNHCO), 11.92 (1H, d, J
14, =CHNH-), 9.79 (1H, s, OH), 8.59 (1H, d, J 13.6, =CH-N), 7.81 (1H, d, J8.8, ArH),
13
7.15 (1H, s, ArH), 7.09 (1H, dd, J 8.8, ArH); C NMR (d6-DMSO, δ, ppm): 177.9(C),
171.1(C), 163.6(C), 162.2(C), 161.3(C), 151.7(CH), 144(C), 131.9(CH), 110.5(C),
109.4(CH), 106.3(CH), 95.1(C); MS (EI) m/z 307(M+ 53%), 263(27), 188(23), 103(32),
65(62), 53(100).
5-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-2-hydroxy-
benzoic acid (14)
°
1
Yield 77%, green solid, m.p. 300-305 C (decompose); H NMR (d6-DMSO, δ, ppm);
13.46 (1H, s, COOH),12.08 (1H, s, CONHCS), 11.95 (1H, s, CSNHCO),11.95 (1H, d, J
14, =CHNH-), 9.90 (1H, s, OH), 8.44 (1H, d, J 14, =CH-N),7.86 (1H, s, ArH), 7.72 (1H,
dd, J 9.2, ArH), 7.00 (1H, d, J 9.2, ArH); 13C NMR (d6-DMSO, δ, ppm): 177.7(C),
170.8(C), 163.5(C), 161.5(C), 159.3(C), 152.4(CH), 130.2(C), 126.6(CH), 120.6(CH),
118.2(CH), 113.8(C), 93.5(C); MS (EI) m/z 307(M+ 55%), 263(26), 188(25), 103(34),
65(63), 53(100).
2-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-4-
nitrobenzoic acid (15)
°
1
Yield 90%, light yellow solid, m.p. 320-326 C (decompose); H NMR (d6-DMSO, δ,
ppm); 14.5 (1H, s, COOH), 13.5 (1H, d, J 13.8, =CHNH-), 12.22 (1H, s, CONHCS), 12.13
(1H, s, CSNHCO), 8.74 (1H, d, J 13.5, =CH-N), 8.59 (1H, s, ArH), 8.23 (1H, d, J 8.7,
ArH), 8.08 (1H, dd, J 8.7 ArH); 13C NMR (d6-DMSO, δ, ppm): 178.1(C), 166.3(C),
163(C), 161.6(C), 151.8(CH), 150.6(C), 140.7(C), 133.2(CH), 124(C), 119.1(CH),
113(CH), 96.3(C); MS (EI) m/z 336(M+ 43%), 290(3), 216(17), 188(5), 114(19), 90(14),
69(37), 53(100).
20

3-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-4-
hydroxybenzoic acid (16)
°
1
Yield 76%, yellow solid, m.p. 323-330 C (decompose); H NMR (d6-DMSO, δ, ppm);
12.77 (1H, s, COOH), 12.2 (1H, d, J 14.4, =CHNH-), 12.16 (1H, s, CONHCS), 12.03 (1H,
s, CSNHCO), 11.5 (1H, s, OH), 8.66 (1H, d, J 14.4, =CH-N), 8.05 (1H, s, ArH), 7.7 (1H,
dd, J 8.4, ArH), 7.03 (1H, d, J 8.4 ArH); 13C NMR (d6-DMSO, δ, ppm): 177.8(C),
166.6(C), 164.1(C), 161.5(C), 151.3(C), 150.9(CH), 128.4(CH), 125.7(C), 122.4(C),
117.6(CH), 115.5(CH), 94.1(C); MS (EI) m/z 307(M+ 11%), 263(100), 188(23), 144(21),
120(77), 77(23), 53(53).
5-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-2-
nitrobenzoic acid (17)
°
Yield 72%, yellow solid, m.p.247- 250 C (decompose); 1H NMR (d6-DMSO, δ, ppm); 14
(1H, s, COOH), 12.04 (1H, d, J 13.6, =CHNH-), 12.25 (1H, s, CONHCS), 12.12 (1H, s,
CSNHCO), 8.66 (1H, d, J 13.6, =CH-N), 8.08 (1H, d, J 8.8 ArH), 8 (1H, s, ArH), 7.89
(1H, dd, J 9.2 ArH); 13C NMR (d6-DMSO, δ, ppm): 178(C), 165.7(C), 163.5(C), 161.3(C),
154.8(C), 152.3(CH), 142.6(C), 130.3(C), 125.8(CH), 119.3(CH), 111.2(CH), 95.9(C); MS
(EI) m/z 336(M+ 100%), 292(32), 155(49), 116(42), 103(40), 63(48), 53(81).
4-chloro-3-{[(4,6-dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)methyl]amino}-
benzoic acid (18)
°
1
Yield 86%, light yellow solid, m.p. 316-320 C (decompose); H NMR (d6-DMSO, δ,
ppm); 13.44 (1H, s, COOH), 12.45 (1H, d, J 13.2, =CHNH-), 12.31 (1H, s, CONHCS),
12.15 (1H, s, CSNHCO), 8.72 (1H, d, J 13.2, =CH-N), 8.23 (1H, s, ArH), 7.77 (1H, dd, J
8.4, ArH), 7.72 (1H, dd, J 8.4 ArH); 13C NMR (d6-DMSO, δ, ppm): 178(C), 165.9(C),
164.4(C), 161.2(C), 152.3(CH), 135.2(C), 131.3(C), 130.3(CH), 127.6(C), 127.3(CH),
118.8(CH), 95.5(C); MS (EI) m/z 325(M+ 67%), 290(46), 222(44), 182(27), 171(26),
98.9(36), 53(100).
5-({[2-(trifluoromethyl)phenyl]amino}methylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (19)
21

°
1
Yield 93%, white solid, m.p. 300-307 C (decompose); H NMR (d6-DMSO, δ, ppm);
12.47 (1H, d, J 12.8, =CHNH-), 11.15 (1H, s, CONHCO), 10.96 (1H, s, CONHCO), 8.61
(1H, d, J 12.8, =CH-N), 7.90 (1H, d, J 8 ArH), 7.78 (1H, d, J 8.4, ArH), 7.74 (1H, t, J 8,
ArH), 7.44 (1H, t, J 7.6, ArH); 13C NMR (d6-DMSO, δ, ppm): 166.7 (C), 163.1 (C), 152.3
(CH), 150.5 (C), 136.1 (C), 134.5(CH), 126.7 (CH), 126 (CH), 125 (C), 122.3(C), 119.8
(CH), 94.4 (C); MS (EI) m/z 299.1(M+ 100%), 254(5.1), 212(45.9), 154(7.6), 126(6.4),
68(6.6).
5-({[3-(trifluoromethyl)phenyl]amino}methylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (20)
°
1
Yield 80%, White solid, m.p. 301-306 C (decompose); H NMR (d6-DMSO, δ, ppm):
11.89 (1H, d, J 13.6, =CHNH-), 11.02 (1H, s, CONHCO), 10.88 (1H, s, CONHCO), 8.6
(1H, d, J 13.6, =CH-N), 7.98 (1H, s, ArH), 7.82 (1H, d, J 8, ArH), 7.61 (1H, t, J 8, ArH),
7.56 (1H, d, J 7.6, ArH); 13C NMR (d6-DMSO, δ, ppm): 165.9 (C), 163.4 (C), 151.8 (CH),
150.6 (C), 139.6 (C), 130.7 (CH), 130.3 (C), 125.1 (C), 122.5 (CH), 122 (CH), 115.7 (CH),
93.3 (C); MS (EI) m/z 299.0(M+ 100%), 212(75.7), 172(7), 144(3.3), 95(7.5).69(3.5).
5-({[4-(trifluoromethyl)phenyl]amino}methylidene)pyrimidine-2,4,6(1H,3H,5H)-
trione (21)
°
1
Yield 79%, white solid, m.p. 270-274 C (decompose); H NMR (d6-DMSO, δ, ppm):
11.96 (1H, d, J 13.8, =CHNH-), 11.10 (1H, s, CONHCO), 10.95 (1H, s, CONHCO), 8.65
13
(1H, d, J 14.1, =CH-N), 8.06 (2H, d, J 8.7, ArH), 7.74 (2H, d, J 9, ArH); C NMR (d6-
DMSO, δ, ppm): 166 (C), 163.4 (C), 151.2 (CH), 150.8(C), 131 (C), 129.5 (2CH),
125.8(C), 123.7 (C), 120.1 (2CH), 93.5 (C); MS (EI) m/z 401.2(MH+ 100%), 299.1(10.7),
212.1(10.7), 172.1(6.6), 144.1 (3.9), 95(6.7), 69(5).
5-({[5-nitro-2-(trifluoromethyl)phenyl]amino}methylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (22)
°
Yield 78%, yellow solid, m.p. 333-338 C (decompose) ; 1H NMR (d6-DMSO, δ, ppm);
12.62 (1H, d, J 12, =CHNH-), 11.29 (1H, s, CONHCO), 11.11 (1H, s, CONHCO), 8.76
(1H, d, J 12, =CH-N), 8.51 (1H, d, J 9.2 ArH), 8.42 (1H, s, ArH), 8.20 (1H, d, J 9.2, ArH),
22

13C NMR (d6-DMSO, δ, ppm): 166.8 (C), 162.7 (C), 151.3 (CH), 150.9 (C), 150.4(C),
143.38(C), 141.4 (C), 129.3 (CH), 122.8(CH), 120.7 (C), 120 (CH), 96.7 (C); MS (EI) m/z
334.2(M+ 100%), 314.2(6.8), 273.2(9.7), 171.1(4.6), 154.1(17.3), 144.1(13.2), 75.1(8.2),
69.1(10.7).
5-{[(3,5-dichlorophenyl)amino]methylidene}pyrimidine-2,4,6(1H,3H,5H)-trione (23)
°
1
Yield 94%, off white solid, m.p. 315-320 C (decompose); H NMR (d6-DMSO, δ, ppm):
11.76 (1H, d, J 13.5, =CHNH-), 11.05 (1H, s, CONHCO), 10.90 (1H, s, CONHCO), 8.52
13
(1H, d J 13.5, =CH-N), 7.71 (2H, s, ArH), 7.40 (1H, s, ArH); C NMR (d6-DMSO, δ,
ppm): 165.8 (C), 163.3(C), 151.7 (CH), 150.6 (C), 141.2 (C), 134.9 (C), 124.8 (CH), 117.6
(CH), 93.9(C); MS (EI) m/z 298.9(M+ 100%), 171.9(6.1), 154(11.7), 144.9(18.8), 69(3.8).
5-{[(2,4,5-trichlorophenyl)amino]methylidene}pyrimidine-2,4,6(1H,3H,5H)-trione
(24)
°
1
Yield 73%, light yellow solid, m.p. 312-318 C (decompose); ); H NMR (d6-DMSO, δ,
ppm): 12.38 (1H, d, J 13.2, =CHNH-), 11.20 (1H, s, CONHCO), 11.06 (1H, s,
CONHCO), 8.76 (1H, d, J 13.2, =CH-N), 8.34 (1H, s, ArH), 8.00 (1H, s, ArH); 13C NMR
(d6-DMSO, δ, ppm): 166.7(C), 162.9 (C), 151.3 (CH), 150.5 (C), 135.4 (C), 131.5 (C),
130.7 (CH), 127.6 (C), 122.1 (C), 119.4 (CH), 94.8 (C); MS (EI) m/z 346(M+ 2%),
298(100), 144.9(4.8), 69(3.1).
5-{[(4-iodophenyl)amino]methylidene}pyrimidine-2,4,6(1H,3H,5H)-trione (25)
o
1
Yield 90%, white solid, m.p. 306-310 C (decompose); H NMR (d6-DMSO, δ, ppm):
11.78 (1H, d, J 13.6, =CHNH-), 11.01 (1H, s, CONHCO), 10.87 (1H, s, CONHCO), 8.49
(1H, d, J 13.6, =CH-N), 7.74 (2H, d, J 8.8, ArH), 7.35 (2H, d, J 8.8, ArH); 13C NMR (d6-
DMSO, δ, ppm): 166 (C), 163.4 (C), 151 (CH), 150.6 (C), 138.4 (C), 138.2(2CH), 129.7
(C), 120.7 (2CH), 92.9 (C).
5-{[(2-hydroxyphenyl)amino]methylidene}pyrimidine-2,4,6(1H,3H,5H)-trione (26)
o
1
Yield 97%, yellow solid, m.p. 323-330 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.11 (1H, d, J 14.4, =CHNH-), 10.94 (1H, s, CONHCO), 10.81 (1H, s, CONHCO), 10.53
23

(1H, s, OH), 8.60 (1H, d, J 14.4, =CH-N), 7.58 (1H, d, J 8, ArH), 7.05 (1H, t, J 7.6, ArH),
13
6.95 (1H, d, J 7.6, ArH), 6.87 (1H , t, J 7.6, ArH); C NMR (d6-DMSO, δ, ppm): 166.3
(C), 163.5 (C), 150.7 (C), 149.6(CH), 147 (C), 126.4 (CH), 126 (C), 120 (CH), 115.9
(CH), 115.7 (CH), 92.3 (C); MS (EI) m/z 231(M+ 1.5%), 159(22.6), 120(100%), 69(3.6).
5-{[(2-sulfanylphenyl)amino]methylidene}pyrimidine-2,4,6(1H,3H,5H)-trione (27)
o
1
Yield 70%, light yellow solid, m.p. 239-243 C (decompose); H NMR (d6-DMSO, δ,
ppm): 12.53 (1H, d, J 8.8, =CHNH-), 11.10 (1H, s, CONHCO), 11.03 (1H, s, CONHCO),
10.90 (1H, s, SH), 8.52 (1H, d, J 13.6, =CH-N), 7.73 (1H, d, J 8, ArH), 7.32 (1H, d, J 7.2,
13
ArH), 7.11 (1H, m, ArH), 7.08 (1H, m, ArH); C NMR (d6-DMSO, δ, ppm): 166.2 (C),
163.2 (C), 150.6 (C), 150.3 (CH), 140.2 (C), 126.1 (CH), 124.2(C), 117.2 (CH), 115.8
(CH), 114.9 (CH), 93.8 (C); MS (EI) m/z 125(M+ 100%), 95.0(3.2),69.0(12.0).
2-thioxo-5-({[2-(trifluoromethyl)phenyl]amino}methylidene)dihydropyrimidine-4,6
(1H,5H)-dione (28)
°
1
Yield 73%, yellow solid, m.p. 316-321 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.59 (1H, d, J 12.6, =CHNH-), 12.31 (1H, s, CONHCS), 12.15 (1H, s, CSNHCO),
8.68(1H, d, J 12.6, =CH-N), 7.94 (1H, d, J 7.5, ArH), 7.80 (1H, d, J 6.9, ArH), 7.78 (1H, t,
13
J 6.9, ArH), 7.48 (1H, t, J 6.9, ArH); C NMR (d6-DMSO, δ, ppm): 178 (C), 164.6 (C),
161.1 (C), 153.6 (CH), 135.8 (C), 134.5 (CH), 126.8 (CH), 126.6 (CH), 125.4 (C), 121.8
(C), 120.3 (CH), 95.6 (C).
2-thioxo-5-({[3-(trifluoromethyl)phenyl]amino}methylidene)dihydropyrimidine-
4,6(1H,5H)-dione (29)
°
1
Yield 83%, light greenish, m.p. 312-318 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.19 (1H, s, CONHCS), 12.06 (1H, d, J 13.6, =CHNH-), 12.05 (1H, s, CSNHCO), 8.66
(1H, d, J 14, =CH-N), 8.04 (1H, s, ArH), 7.86 (1H, d, J 8, ArH), 7.66 (1H, t, J 7.6, ArH),
13
7.59 (1H, d, J 7.6, ArH); C NMR (d6-DMSO, δ, ppm); 177.9 (C), 163.6 (C), 161.5 (C),
152.9 (CH), 139.3 (C), 130.7 (CH), 130.3 (C), 125.1 (C), 122.9 (CH), 122.5(CH), 116.2
(CH), 94.7 (C); MS (EI) m/z 315(M+ 100%), 172.1(21.4), 155.0(9.3), 144(5), 75.1(3.5),
69.0(4.9).
24

2-thioxo-5-({[4-(trifluoromethyl)phenyl]amino}methylidene)dihydropyrimidine-4,6
(1H,5H)-dione (30)
°
1
Yield 76%, light yellow, m.p. 279-281 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.22 (1H, s, CONHCS), 12.09 (1H, s, CSNHCO), 12.00 (1H, d, J 14.4, =CHNH-), 8.69
13
(1H, d J 13.6, =CH-N), 8.00 (2H, m, ArH), 7.74 (2H, m, ArH); C NMR (d6-DMSO, δ,
ppm): 178.2(C), 164.1(C), 161.8 (C), 152.2 (CH), 141.4 (C), 129.8 (2CH), 126.2 (C),
120.4 (2CH), 118.7 (C), 96.2 (C); MS (EI) m/z 315(M+ 33.1%), 172(7.4), 144(4.0).
5-({[5-nitro-2-(trifluoromethyl)phenyl]amino}methylidene)-2-thioxodihydropyri-
midine-4,6(1H,5H)-dione (31)
1
Yield 97%, light yellow solid, m.p. 308-312oC (decompose); H NMR (d6-DMSO, δ,
ppm): 12.77 (1H, d, J 12, =CHNH-), 12.45 (1H, s, CONHCS), 12.28 (1H, s, CSNHCO),
8.81 (1H, d, J 12, =CH-N), 8.54 (1H, d, J 9.2, ArH), 8.48 (1H, s, ArH), 8.25 (IH, d, J 9.2,
ArH); 13C NMR (d6-DMSO, δ, ppm): 178.2 (C), 164.7 (C), 160.8 (C), 152.6 (CH),
143.7(C), 141 (C), 129.2 (CH), 123.9(C), 122.8 (CH), 121.2 (C), 120.6 (CH), 97.7 (C); MS
(EI) m/z 360.1(M+ 100%), 172.1(5), 155(30.5), 144.1(19.5), 116(11.1), 76.1(5.5),
69.1(9.8).
5-{[(3,5-dichlorophenyl)amino]methylidene}-2-thioxodihydropyrimidine-4,6(1H,5H)-
dione (32)
1
Yield 79%, light yellow solid, m.p. 335-341°C (decompose); H NMR (d6-DMSO, δ,
ppm): 12.20 (1H, s, CONHCS), 12.07 (1H, s, CSNHCO), 11.90 (1H, d, J 13.8, =CHNH-),
8.58 (1H, d, J 13.8, =CH-N), 7.75 (2H, s, ArH), 7.43 (1H, s, ArH); 13C NMR (d6-DMSO,
δ, ppm): 178 (C), 163.6 (C), 161.4 (C), 152.7 (CH), 140.9 (C), 134.8 (C), 125.3 (CH),
118(CH), 95.1 (C); MS (EI) m/z 315(M+ 100%), 172(23.4), 155(21.1), 145(18.2),
76.1(3.3), 69.1(6.6).
2-thioxo-5-{[(2,4,5-trichlorophenyl)amino]methylidene}dihydropyrimidine-
4,6(1H,5H)-dione (33)
25

°
1
Yield 78%, white solid, m.p. 350-360 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.51 (1H, d, J 13.2, =CHNH-), 12.35 (1H, s, CONHCS), 12.17 (1H, s, CSNHCO), 8.82
13
(1H, d, J 13.2, =CH-N), 8.39 (1H, s, ArH), 8.03 (1H, s, ArH); C NMR (d6-DMSO, δ,
ppm): 178 (C), 164.6 (C), 161 (C), 152.5 (CH), 135.1 (C), 131.5 (C), 130.8(CH), 128.2
(C), 122.4 (C), 119.8 (CH), 96 (C); MS (EI) m/z 351 (M+ 100%), 315(16.2), 172(3.4),
155(13.2), 69(11.3).
5-{[(4-iodophenyl)amino]methylidene}-2-thioxodihydropyrimidine-4,6(1H,5H)-dione
(34)
°
1
Yield 90%, white solid, m.p. 329-335 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.17 (1H, s, CONHCS), 12.04 (1H, s, CSNHCO), 11.94 (1H, d, J 14, =CHNH-), 8.55
13
(1H, d, J 14, =CH-N), 7.76 (2H, d, J 8.8, ArH), 7.39 (2H, d, J 8.8, ArH); C NMR (d6-
DMSO, δ, ppm): 177.8 (C), 163.7 (C), 161.5 (C), 152 (CH), 138.2 (2CH), 138.1 (C),
129.7 (C), 121.1 (2CH), 94.3 (C); MS (EI) m/z 373.1(M+ 100%), 315.1(6.0), 155.0(3.8),
141.1(8.9), 76.0(14.2), 69.0(3.2).
5-{[(2-hydroxyphenyl)amino]methylidene}-2-thioxodihydropyrimidine-4,6(1H,5H)-
dione (35)
°
1
Yield 82%, light yellow solid, m.p. 323-330 C (decompose); H NMR (d6-DMSO, δ,
ppm): 12.30 (1H, d, J 14, =CHNH-), 12.13 (1H, s, CONHCS), 12.00 (1H, s, CSNHCO),
10.63 (1H, s, OH), 8.66 (1H, d, J 14.4, =CH-N), 7.62 (1H, d, J 8, ArH), 7.08 (1H, t, J 7.6
13
), 6.95 (1H, d, J 8, ArH), 6.87 (1H, t, J 7.6, ArH); C NMR (d6-DMSO, δ, ppm): 177.6
(C), 164.1 (C), 161.5 (C), 150.3 (CH), 147.3 (C), 126.9 (CH), 125.7 (C), 120(CH),
116.3(CH), 115.8 (CH), 93.8 (C); MS (EI) m/z 263(M+ 100%), 188(19.1), 159(13.8),
65(5.3).
5-{[(2-sulfanylphenyl)amino]methylidene}-2-thioxodihydropyrimidine-4,6(1H,5H)-
dione (36)
°
1
Yield 80%, white solid, m.p. 225-228 C (decompose); H NMR (d6-DMSO, δ, ppm):
12.66 (1H, d, J 14, =CHNH-), 12.17 (1H, s, CONHCS), 12.10 (1H, s, CSNHCO), 10.90
(1H, s, SH), 8.59 (1H, d, J 14, =CH-N), 7.79 (1H, d, J 8, ArH), 7.33 (1H, d, J 7.6, ArH),
26

7.14 (1H, m, ArH), 7.12 (1H, m, ArH); 13C NMR (d6-DMSO, δ, ppm): 177.8 (C), 164 (C),
161.5 (C), 152.2(CH), 140.1 (C), 126.1 (CH), 125.4 (C), 123(CH), 122.5 (CH), 117.7
(CH), 81.9 (C); MS (CI) m/z 264(MH+ 1%), 175(11.8), 156(10.4), 144(34.8), 76(4.4),
69(36.2).
4.3. Urease inhibition assay
Reaction mixtures comprising 25µL of enzyme (jack bean urease) solution and 55 µL of
buffers containing 100 mM urea were incubated with 5 µL of test compounds (0.5 mM
concentration) at 30 °C for 15 min in 96-well plates. Urease activity was determined by
measuring ammonia production using the indophenol method as described by
Weatherburn. Briefly, 45 µL each phenol reagent (1% w/v phenol and 0.005% w/v sodium
nitroprussside) and 70 µL of alkali reagent(0.5% w/v NaOH and 0.1% active chloride
NaOCl) were added to each well. The increasing absorbance at 630 nm was measured after
50min, m using a micro-plate reader (Molecular Device, USA). All reactions were
performed in triplicate in a final volume of 200 µL. The results (change in absorbance per
min) were processed by using soft Max Pro software (molecular Device, USA). The entire
assays were performed at pH 6.8. Percentage inhibitions were calculated from the formula
100-(ODtestwell/ODcontrol)×100. Thiourea was used as standard inhibitor of urease.
4.4 Molecular modelling studies
The three-dimensional structures of the compounds were built with Corina on-line tool [63].
Protonation states were predicted at pH of reaction mixture by Marvin online plugin [64]. The atom
and bond types were checked and Gasteiger-Marsili charges were assigned by Sybyl-X 1.1 [65].
Finally, ligand structures were saved in the mol2 format.
The structure of Jack bean (Canavalia ensiformis) urease of resolution 1.49 Å was downloaded
from Protein Data Bank (PDB code: 4GY7) [66]. Then, all histidine residues except His407,
His409 and His545 (Nδ protonation) were protonated at Nε atom, ligand and water molecules were
removed, and missing hydrogens were added using Hermes 1.5 [67]. The default coordination
states were assigned to both nickel cations, and the unusual amino acids were checked. The binding
site was defined as all amino acid residues within 12 Å from nickel ions. Dockings were performed
with Gold 5.1 software [68] using genetic algorithm with default settings. GoldScore function and
27

analysis of binding mode were applied to find final ligand poses. Results were visualized by
PyMOL 0.99rc2 [69]. Thiourea was used as a reference compound in the docking studies.
Acknowledgments: We acknowledge Higher Education Commission (HEC) Pakistan for
financial support.
References and notes
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29