Synthesis of highly functionalized barbituric acids and study of their interactions with p-glycoprotein and Mg2+ - Potential candidates for multi drug resistance modulation

Article abstract of DOI:10.1016/j.ejmech.2009.12.033

A number of barbituric acids with appropriate substituent at C-5 position were synthesized and investigated for their interactions with p-gp and Mg²⁺. Compounds 5, 6, 8-10, 12-14 and 16 increased the basal activity of p-gp by more than 50% at 0

Full text of DOI:10.1016/j.ejmech.2009.12.033

European Journal of Medicinal Chemistry 45 (2010) 1256–1262
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis of highly functionalized barbituric acids and study of their interactions
with p-glycoprotein and Mg2þ – Potential candidates for multi drug
resistance modulation
*
Palwinder Singh , Jatinder Kaur, Atul Bhardwaj
Department of Chemistry, Guru Nanak Dev University, Amritsar-143005. India
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of barbituric acids with appropriate substituent at C-5 position were synthesized and inves-
tigated for their interactions with p-gp and Mg2þ. Compounds 5, 6, 8–10, 12–14 and 16 increased the
basal activity of p-gp by more than 50% at 0.05 mM concentration. Molecular docking indicate a number
of H-bond interactions between these molecules and the amino acid residues of ATP binding site of p-gp.
These molecules also showed appreciable interactions with Mg2þ, an important component of efflux
pump. All the results of these investigations favor the suitability of barbituric acids toward MDR
modulation.
Received 14 October 2009
Received in revised form
4 December 2009
Accepted 14 December 2009
Available online 22 December 2009
Keywords:
Ó 2009 Elsevier Masson SAS. All rights reserved.
P-Glycoprotein
Multi drug resistance
Barbituric acid
Docking
1. Introduction
and/or drug binding site), to work on the second approach for MDR
modulation, chelation of Mg2þ from the ATP hydrolysis site could
Amongst the transporter proteins (ABC class of transporters)
[1–3], P-glycoprotein (p-gp) [4] plays a significant role in the
development of multi drug resistance (MDR) [5,6] which becomes
a hurdle in the successful practice of chemotherapy of various
diseases (like cancer, AIDS, tuberculosis and malaria). Two or more
binding sites of p-gp (ATP binding site and drug binding site) and
its flip-flop type of mechanism for the drug transportation cause
a variety of substances to be transported by this protein. To get rid
of drug transportation by p-gp, use of combination of chemother-
apeutic agents (drug þ p-gp inhibitor/substrate) seems to be the
best option. P-gp inhibitors/substrates (MDR modulators [7–13])
through their interactions with this protein, either at ATP binding
site or drug binding site or both the sites, avoid the efflux of drug
molecules out of the cell thereby maintaining an optimum
concentration of drug inside the cells. Irrespective of the untiring
efforts of the scientific community, till date no MDR modulator is
successfully employed for the clinical use. Since drug trans-
portation by p-gp is supported by energy taken from ATP hydro-
lysis, another probable approach for MDR modulation is to cut off
the energy supply to p-gp by blocking ATP hydrolysis. In addition to
study the interactions of compounds directly with p-gp (at ATP
be a good option because of the pivotal role of Mg2þ in ATP
hydrolysis [14–16]. In continuation with our programme for
developing MDR modulators [17], here, compounds with a number
of H-donor/acceptor sites (it is assumed that more the number of
interacting sites, more is the inhibitory potency) and Mg2þ
chelating properties are selected for investigations. Since 5-fluo-
rouracil (an anti-cancer agent) is a substrate of p-gp, and the
usefulness of barbituric acids as anti convulsant [18], anti-cancer
[19] and sedative-hypnotic [20,21] agents, compounds 1–16 were
studied for their interactions with p-gp as well as Mg2þ
.
2. Result and discussion
2.1. Chemistry
1,3-Dimethyl-5-formyl barbituric acid was prepared by the
treatment of 1,3-dimethylbarbituric acid with CHCl3/KOH in ethanol/
H2O [22]. 1,3-Dimethyl-5-benzoyl/cinnamoyl barbituric acids
were prepared by microwave irradiations of 1,3-dimethylbarbituric
acid and benzoic anhydride/cinnamoyl chloride [23]. Stirring of
a solution of 1,3-dimethyl-5-formyl/benzoyl/cinnamoyl barbituric
acid and o-phenylene diamine/o-anisidine/anthranilic acid/o-nitro-
aniline/o-hydroxyaniline/2-aminoterephthalic acid in methanol at
35 ꢀC provided the desirable compounds 1–8 (Scheme 1) [24]. Under
* Corresponding author. Tel.: þ91 183 2258802 09x3495; fax: þ91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.12.033

P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
1257
O
R
O
H3C
O
N
NHNH
R1
R2
N
CH3
13-14
13: R = H, R1 = H, R2 = H
14: R = H, R1 =R2 = NO2
R1
R2
H2NHN
MeOH, stir
R2
R1
H2N
O
R
O
R
O
R2
H3C
H2N
R1
O
R
O
R1
N
N
O
H3C
O
H3C
O
( )n
N
N
H
N
N
O
N
OH
H
MeOH, stir
MeOH, stir
N
R1
N
CH3
N
CH3
CH3
1-8
15-16
( )n
H2N
NH2
15: R = H, R1 = NH2, n = 0
16: R = H, R1 = H, n = 1
1: R = H, R1 = NH2, R2 = H
MeOH, stir
2: R = CH=CH-Ph, R1 = NH2,R2 = H
3: R = Ph, R1 = NH2, R2 = H
4: R = H, R1 = COOH, R2 = H
5: R = H, R1 = OCH3, R2 = H
6: R = H, R1 = NO2, R2 = H
O
N
R
O
R
O
( )
nN
H
H3C
O
CH3
N
N
N
H
9: R = H, n = 2
10: R = CH=CH-Ph, n = 2
7: R = H, R1 = OH, R2 = H
O
N
O
8: R = H, R1 = COOH, R2 = COOH
11: R = CH=CH-Ph, n =0
12: R = Ph, n = 0
CH3
CH3
9-12
Scheme 1.
the same reaction conditions, 1,3-dimethyl-5-formyl/benzoyl/cin-
namoyl barbituric acid react with hydrazine/ethylene diamine to
provide compounds 9–12. Similar reactions of 1,3-dimethyl-5-
formyl barbituric acid with phenyl hydrazine, 2,4-dinitrophenyl
hydrazine and 2,6-diaminopyridine, 2-aminomethyl pyridine gave
compounds 13,14 and 15,16 respectively. Interestingly, the reactions
of hydrazine and ethylene diamine with 1,3-dimethyl-5-formyl/
benzoyl/cinnamoyl barbituric acids gave dimer type of products 9–
12 but similar reactions of these barbituric acids with o-phenylene
diamine and 2,6-diamino pyridine gave mono-substituted products
1–3 and 15. Probably, this is due to the low reactivity of aromatic
amines which further get decreased when one amino group gets
attached to the barbituric acid moiety. The structures of all the
compounds were elucidated with 1H, 13C NMR spectra, mass spectra
and CHN analysis. Characteristically, all these compounds exist in
enamine form (1H NMR spectra). Therefore, following a simple
synthetic procedure, a series of small organic molecules having
a number of polar interacting sites were prepared.
p-gp interactions are better gets increased which was manifested
as increase in the basal activity of p-gp. Compounds were tested for
their interactions with p-gp at 0.05
trations in triplicate.
mM, 0.5 mM and 5 mM concen-
2.2.1. Interactions of compounds 1–16 with P-gp
As per the manufacturer’s specifications for the ‘drug-p-glyco-
protein interactions’ assay kit, on addition of a compound, a 30%
change in the basal activity of p-gp implies that the compound is
interacting with p-gp. All the compounds under present investi-
gations changed the basal activity of p-gp by >30% at the three
concentrations (Table 1). Amongst compounds 1–8, compound 8
with two carboxyl groups present on phenyl ring exhibited best
relative activity and highest increase in the basal activity (57%) of
Table 1
Relative activity and percentage increase of basal activity of p-gp by compounds
1–16.
compound Relative activity of compd (% increase of basal activity of p-gp)
2.2. Biology
5
m
M
0.5
m
M
0.05 mM
The interactions of compounds 1–16 with p-gp were studied
using ‘Drug-P-glycoprotein Interaction’ assay kit which contains
the p-gp vesicles prepared from highly resistant MDR cells, the DC-
3F/ADX line. The interactions of compounds with p-gp were
assessed in terms of modulation of basal activity (MgATP hydrolysis
activity in the absence of drug) of p-gp measured by spectropho-
tometric method by continuous monitoring of ADP formation in the
vesicle suspension medium. The interactions of added compound
(test compound) in the ATP binding site of p-gp result in the
inhibition of ATPase activity of p-gp – slowing down of conversion
of phosphoenolpyruvate to pyruvate and slow formation of lactate.
This decreased the conversion of NADH to NADþ and hence higher
absorption at 340 nm (due to NADH). Therefore, the absorption
of NADH at 340 nm, in the wells (96 well plate) where compound –
1
2
3
4
5
6
7
8
2.66 (166)
2.33(133)
2.35 (135)
2.00 (100)
2.23 (123)
2.41 (141)
1.95 (95)
2.46 (146)
2.34 (134)
1.66 (66)
3.00 (200)
2.66 (166)
2.68 (168)
2.59 (159)
1.92 (92)
1.98 (98)
2.00 (102)
2.12 (112)
2.44 (144)
1.83 (83)
1.95 (95)
1.99 (99)
2.14 (114)
1.80 (80)
1.71 (71)
2.66 (166)
1.69 (69)
2.00 (100)
1.97 (97)
2.33 (133)
1.81 (81)
1.75 (75)
1.33 (33)
1.44 (44)
1.42 (42)
1.35 (35)
1.53 (53)
1.51 (51)
1.42 (42)
1.57 (57)
1.66 (66)
2.00 (100)
1.33 (33)
1.66 (66)
1.57(57)
2.00 (100)
1.44 (44)
1.63 (63)
9
10
11
12
13
14
15
16

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P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
p-gp at 0.05 mM concentration. From this series of compounds, 5
and 6 also showed more than 50% increase in the basal activity of
p-gp. In the second category of compounds i.e., 9–12, compound 10
was showing maximum relative activity and highest increase in
basal activity (100%) of p-gp at 0.05 mM. From compounds 13–14,
compound 14 with 2,4-dinitro phenyl hydrazine appendage at
barbituric acid exhibited better relative activity as well as increase
in basal activity (100%) at 0.05 mM in comparison to its analogue 13
indicating the additional role of nitro groups towards p-gp inter-
actions. Compounds 15 and 16 were taken to look into any addi-
tional advantage of pyridine moiety towards MDR modulation.
Although both these compounds showed appreciable increase in
the basal activity of p-gp but there was no improvement over their
analogues (compare 1 with 15). Overall, compounds 5, 6, 8–10,
12–14 and 16 showed more than 50% increase in the basal activity
of p-gp at 0.05 mM concentration.
Therefore, it seems as these barbituric acids interact in the ATP
binding site of p-gp, thereby inhibiting its ATPase activity and
hence the potential candidates for p-gp mediated MDR modulation.
The interactions of the compounds under present investigation in
the ATP and drug binding sites of p-gp, has also been explored with
molecular docking studies which indicate that these molecules
show more interactions in the ATP binding site of p-gp.
Fig. 1. Validation of docking programme. Space filled binding site of p-gp shows the
presence of two ADP molecules in the pocket. ADP1 (ADP docked in the ATP binding
site of p-gp, pink) is present in the same binding site pocket where the native ADP
molecule (present in the crystal structure of the protein, green) was crystallized. Hs’
are suppressed for clarity.
2.3. Docking studies
In order to elucidate the binding modes of these molecules with
p-gp, their dockings in the ATP binding site and drug binding site of
this protein were performed. Compounds were built using the
builder toolkit of the software package ArgusLab 4.0.1 [25] and
energy minimized using semi-empirical quantum mechanical
method PM3. The crystal coordinates of the p-glycoprotein (pdb ID
1MV5, pdb ID 3G60) [4] were downloaded from protein data bank.
Since the drug transportation by p-gp is linked with ATP hydrolysis,
blocking of ATP binding site or chelation of Mg2þ from this site
carbonyl oxygens of pyrimidine with S1383 and S1378; oxygens of
carboxyl group with S2355 and G1379; enaminic N with D2353.
One carbonyl oxygen and enaminic N of compound 4 are situated at
´
´
a distance of 2:96 Å and 4:01 Å from Mg2þ (Table S1). Therefore, as
per our design of the molecules and the observations from the
experimental results (interactions in the ATP binding site of p-gp),
docking studies also indicate the interactions of these compounds
´
with the ATP binding site amino acids of p-gp as well as Mg2þ
.
could alter the function of p-gp. We have taken 15 Å33 of ATP
´
Compounds 2 and 3 also show similar interactions with amino acid
binding site in case of 1MV5 crystal coordinates and 15 Å around
residues of p-gp but the nearest distance of any part of these
hexapeptide in case of 3G60 crystal coordinates, for the docking of
compounds 1–16. Mg2þ is also visible in the ATP binding site of
p-gp. Validation of the docking programme was checked by dock-
ing ADP in the binding site of p-gp (Fig. 1) in which the docked ADP
molecule stayed in the same binding site pocket where native ADP
molecule was present. The molecule to be docked in the active site
of the protein was pasted in the work space carrying the structure
of the protein. The docking programme implements an efficient
grid based docking algorithm which approximates an exhaustive
search within the free volume of the binding site cavity. The
conformational space was explored by the geometry optimization
of the flexible ligand (rings are treated as rigid) in combination with
the incremental construction of the ligand torsions. Thus, docking
occurs between the flexible ligand parts of the compound and
enzyme. The ligand orientation was determined by a shape scoring
function based on Ascore and the final positions were ranked by
lowest interaction energy values. H-bond and hydrophobic inter-
actions between the compound and enzyme were explored.
Compound 1 on docking in the ATP binding site of p-gp
exhibited a number of interactions with the amino acid residues.
Two carbonyl oxygens of pyrimidine moiety of compound 1 show
H-bond interactions with E473 and Y2393 residues. Compound 1
also interacted through its Ns0 (enamine nitrogen and amino
´
compounds from Mg2þ is 4:0 Å.
In comparison to compounds 1–4, presence of two nitrogens (in
the form of hydrazine moiety) between pyrimidine and phenyl
rings and an additional substituent on the phenyl ring in compound
14 makes this molecule to approach T1384 of chain B where it
interacts with this residue through an H-bonding. Docking of
´
nitrogen) with D468 and D2353 from a distance of 2:95 Å and
´
2:99 Å respectively. Remarkably, these two nitrogens, situated at
´
´
a distance of 2:99 Å and 3:17 Å from Mg2þ, could also interact
with this metal ion (Fig. 2). Docking of compound 4 (COOH group
on the phenyl ring) indicate H-bond interactions between two
Fig. 2. Compound 1 docked in the active site of p-gp. Distances of various groups of
compound 1 from the amino acid residues (within the range of H-bond interactions
´
and electrostatic interactions) are given in Å. Hs’ are omitted for clarity.

P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
1259
Fig. 5. Compound 11 docked in the binding site pocket of p-gp. Carbonyl oxygens of
pyrimidine moiety interact through H-bonds with Y1352.
Fig. 3. Compound 14 docked in the active site of p-gp. Distances of various groups of
compound 14 from the amino acid residues (within the range of H-bond interactions
´
and electrostatic interactions) are given in Å. Hs’ are ommitted for clarity.
In order to make comparison between the interactions of bar-
bituric acids in the ATP and drug binding site of p-gp, docking of
these molecules were also performed in the drug binding site of
p-gp using crystal coordinates of p-gp with pdb ID 3G60. These
investigations indicated a single H-bond interaction of some of
these molecules with the drug binding site amino acids (Table S1).
As a representative, the docking of compound 1 in the drug binding
site of p-gp has been shown in Fig. 6. Compound 1 showed an H-
bond interaction between its carbonyl oxygen (C2]O) and S975.
Therefore, a comparison between the interactions of these barbi-
turic acids in the ATP binding site and drug binding site of p-gp
(Table S1) clearly indicated better interactions of these molecules in
the ATP binding site and hence support the experimental results
where a decrease in the ATPase activity of p-gp was observed.
compound 14 in the active site of p-gp indicates its interactions
with E473, Y2393, D468 and T1384 amino acids present in the
active site of p-gp. One of the two oxygens of C-2 nitro group
´
approached Mg2þ at a distance of 3:09 Å (Fig. 3).
Symmetrical molecule 9 with an ethylene diamine moiety
between two pyrimidines beautifully surrounds the Mg2þ where
carbonyl oxygen of each of the two pyrimidines is present at
´
´
a distance of 2:63 Å and 3:10 Å from Mg2þ (Fig. 4).
However, the replacement of methine Hs’ of compound 9 with
styryl moieties in compound 10 does not allow the molecule to
approach Mg2þ. Docking of compound 10 in the active site of p-gp
indicates its interactions with amino acid residues only. Removal of
ethylene unit from compound 10, as in compound 11, decreases the
overall volume of the molecule and docking of compound 11 (Fig. 5)
2.4. Interactions of compounds 1–16 with Mg2þ
´
in the binding site of p-gp shows its proximity to Mg2þ (1:33 Å) but
the H-bond interactions with amino acid residues of p-gp are less in
comparison to those observed for compound 10. Compound 11
exhibits interactions with Y1352 through two carbonyl oxygens of
pyrimidine moiety. Therefore, the overall volume of the molecule
and the presence of interacting sites are the critical parameters in
deciding its acceptability in the active site of the protein. Hence, the
docking studies indicate excellent interactions of all these
compounds in the ATP binding site of p-gp (Table S1).
The docking studies were indicating the possibility of interac-
tions of barbituric acids with Mg2þ and also the fact that
Fig. 4. Compound 9 docked in the binding site of p-gp. Chelation of Mg2þ by two
Fig. 6. Compound 1 docked in the drug binding site of p-gp. Hs’ are ommitted for
oxygens of carbonyl groups is visible.
clarity.

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P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
Table 2
Association constants for Mg2þ binding with compounds 1–16 in HEPES buffer
(Mꢁ1).
Compd
Ka
Compd
Ka
1
2
3
4
5
6
7
8
7.06 ꢂ 105
2.7 ꢂ 104
2.41 ꢂ 104
3.6 ꢂ 106
5.79 ꢂ 105
1.40 ꢂ 105
5.76 ꢂ 105
7.46 ꢂ 105
9
8.68 ꢂ 105
5.84 ꢂ 105
4.82 ꢂ 105
1.11 ꢂ 106
1.53 ꢂ 107
1.2 ꢂ 106
10
11
12
13
14
15
16
2.57 ꢂ 105
3.20 ꢂ 105
apparent from the values of association constants that all the
compounds show appreciable bindings with Mg2þ
.
Therefore, the interactions of these compounds with Mg2þ
could also be responsible for inhibition of ATPase activity which in
consonance with the results of interactions of compounds with
p-gp resulted in the modulation of p-gp activity.
Fig. 7. Absorption spectra of compound 4 in presence of increasing concentration of
Mg2þ(0–0.1 ꢂ 10ꢁ4 M). Arrows denote change in absorption with increasing concen-
tration of Mg2þ
.
sequestering of Mg2þ results in slowing down of ATP hydrolysis and
hence the supply of energy to p-gp during drug effluxing, the
barbituric acids under present investigations were checked for
their interactions with Mg2þ with the help of UV-vis studies.
Solutions of compounds 1–16 were prepared at 10ꢁ4 M concen-
trations in HEPES buffer (10ꢁ2 M) at pH 7.2 and titrated with Mg2þ
solutions (0–0.1 ꢂ 10ꢁ4 M). All these compounds exhibit hyper-
chromicity in the region 230–250 nm and hypochromicity in the
region 280–380 nm on addition of Mg2þ solution with a shift of 5
and 9 nm in case of compounds 4 and 13. Absorption spectrum of
compound 4 showing two peaks at 234 nm and 352 nm upon
addition of Mg2þ exhibited a hypsochromic shift of 5 nm in the
peak at 234 nm. Moreover, with continuous addition of Mg2þ, an
increase in absorbance at 229 nm and a decrease in absorbance at
352 nm was observed (Fig. 7). Absorption spectra of compound 13
showing two absorption peaks at 231 nm and 300 nm, upon
titration with Mg2þ, exhibited a continuous increase in absorbance
at 231 nm with a concomitant decrease in absorbance at 300 nm
(Fig. 8). There was also a hypsochromic shift of 9 nm in the 300 nm
peak which gets shifted to 291 nm (Fig. 8).
3. Conclusions
The barbituric acid based compounds carrying a number of H-
donor/acceptor sites were synthesized and using ‘Drug–p-gp
interaction assay kit’, they were found to inhibit the ATPase activity
of p-gp. The interactions of these molecules with Mg2þ also indicate
the inhibition of ATPase activity in presence of these compounds.
Molecular docking of these molecules in the ATP and drug binding
site of p-gp showed their significantly better interactions in the ATP
binding site which support the experimental results of decrease in
ATPase activity by these compounds.
4. Experimental
4.1. Chemistry
Melting points were determined in capillaries and are uncor-
rected. 1H and 13C NMR spectra were run on JEOL 300 MHz and
75 MHz NMR spectrometer respectively using CDCl3 as solvent.
Chemical shifts are given in ppm with TMS as an internal reference.
J values are given in Hertz. IR and UV spectral data were recorded
on FTIR 8400S Shimadzu and BioTek PowerWave XS instruments
respectively. Chromatography was performed with silica 100–200
mesh and reactions were monitored by thin layer chromatography
(TLC) with silica plates coated with silica gel HF-254. In 13C NMR
spectral data, þve, ꢁve terms correspond to CH3, CH, CH2 signals in
DEPT-135 NMR spectra.
The change in absorbance was plotted against mole fraction of
metal ion concentration (Job plot for compound 4 and 13 is given in
figure S1) to obtain the stoichiometry of Mg2þ - compound
complexes. In all the cases, the stoichiometry of the complexes is
1:1. The association constants of Mg2þ-compound complexes were
calculated using Benesi–Hildebrand equation [26] (Table 2). It is
4.2. General procedure for synthesis of compounds 1–16
The solution of 5-substituted-6-hydroxy-1,3-dimethylpyr-
imidin-2,4-dione and the appropriate amine (phenylene diamine/
ethylene
diamine/hydrazine/phenyl
hydrazine/2,4-dini-
trophenylhydrazine/anthranilic acid/o-anisidine/2,6-diaminopyr-
idine/2-aminomethylpyridine/2-nitroaniline/2-aminophenol/2-
aminoterephthalic acid) (1.2 equiv for compounds 1–8, 13–16 and
0.5 equiv for compounds 9–12) in methanol was stirred at 35 ꢀC for
3–5 h. The solid suspension on filtration and washing with diethyl
ether provided the pure compounds 1–16. Spectral data of
compounds 1–3 and 9–12 has been already reported [24].
4.2.1. 2-((Tetrahydro-1,3-dimethyl-2,4,6-trioxopyrimidin-5(6H)-
ylidene)methylamino) benzoic acid (4)
Fig. 8. Absorption spectra of compound 13 in presence of increasing concentration of
Creamish white solid, 78% yield, mp 210 ꢀC; nmax (KBr): 1632
Mg2þ (0–0.1
ꢂ
10ꢁ4 M). Arrows denote change in absorption with increasing
concentration of Mg2þ
.
(C]O), 1675 (C]O), 3425 (NH), 3480 (COOH); UV lmax (3)

P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
1261
(DMSO þ HEPES buffer) 234 (16470) and 352 (32040) nm; 1H NMR
4.2.7. 5-(N0-(2,4-Dinitrophenyl)-hydrazinomethylene)-1,3-
dimethylpyrimidine-2,4,6(1H,3H, 5H)-trione (14)
Brownish solid, 75% yield, mp 225 ꢀC; nmax (KBr): 1635 (C]O),
(CDCl3 þ TFA)
d
3.39 (s, 6H, CH3), 7.40–7.45 (t, J ¼ 7.5 Hz, 1H, ArH),
7.68–7.70 (d, J ¼ 8.1 Hz, 1H, ArH), 7.76–7.81 (t, J ¼ 7.8 Hz, 1H, ArH),
8.22–8.25 (dd, J2 ¼ 8.1 Hz, J3 ¼ 1.5 Hz, 1H, ArH), 8.91–8.96 (d,
J ¼ 13.8 Hz, 1H, ]CH), 13.69 (broad doublet, J ¼ 13.2 Hz, 1H, NH);
1655 (C]O), 3390 (NH); lmax
(
3
) UV (DMSO þ HEPES buffer) 236
(6250) and 373 (11850) nm; 1H NMR (CDCl3 þ TFA):
d 3.25 (s, 6H,
13C NMR (normal/DEPT-135) (CDCl3 þ TFA):
d
27.90 (þve, CH3),
CH3), 7.36–7.38 (d, J ¼ 6.6 Hz, 1H, ArH), 8.40–8.42 (d, J ¼ 7.5 Hz, 1H,
ArH), 8.50–8.52 (d, J ¼ 5.7 Hz,1H, ArH), 8.61–8.65 (d, J ¼ 10.8 Hz,1H,
]CH), 9.18 (broad singlet, 1H, NH), 10.23 (broad singlet, 1H, NH);
28.72 (þve, CH3), 108.07 (C5), 112.48 (þve, ArCH), 116.25 (þve,
ArCH), 117.12 (þve, ArCH), 120.03 (absent, ArC), 126.77 (þve, ArCH),
136.29 (absent, ArC), 150.90 (C2), 152.64 (þve, CH), 160.14 (C6/C4),
160.71 (C6/C4). FAB mass m/z 304 (Mþ); (Found: C 55.50, H 4.39, N
13.91; C14H13N3O5 requires C 55.45, H 4.32, N 13.86)
13C NMR (normal/DEPT-135) (CDCl3 þ TFA):
d
28.25 (þve, CH3),
28.98 (þve, CH3), 108.74 (C5), 112.51 (þve, ArCH), 114.90 (þve,
ArCH), 116.27 (þve, ArCH), 123.88 (absent, ArC), 131.07 (absent,
ArC), 140.21 (absent, ArC), 146.44 (C2), 161.61 (þve, CH), 162.76
(C6/C4), 163.04 (C6/C4). FAB mass m/z 365 (Mþ); (Found C 42.89, H
3.38, N 23.10; C14H13N3O5 requires C 42.86, H 3.32, N 23.07).
4.2.2. 5-((4-Methoxyphenylamino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)–trione (5)
Creamish white solid, 81% yield, mp 190 ꢀC; nmax (KBr): 1630
(C]O),1650 (C]O), 3220 (NH); UV lmax
(
3
) (DMSO þ HEPES buffer)
4.2.8. 5-(((6-Aminopyridine-2-yl)methylamino)methylene)-1,3-
dimethylpyrimidine-2,4,6(1H, 3H,5H)-trione (15)
231 (9090) and 349 (16120) nm; 1H NMR (CDCl3 þ TFA)
d 3.36
(s, 6H, CH3), 3.85 (s, 3H, OCH3), 6.95–6.98 (d, J ¼ 8.7 Hz, 2H, ArH),
7.19–7.28 (m, 2H, ArH), 8.59–8.64 (d, J ¼ 14.1 Hz, 1H, ]CH), 12.06–
12.10 (broad doublet, J ¼ 13.2 Hz, 1H, NH); 13C NMR (normal/DEPT-
Brownish solid, 72% yield, mp 240 ꢀC; nmax (KBr): 1632 (C]O),
1675 (C]O), 3258 (NH); UV lmax
(
3
) (DMSO þ HEPES buffer) 237
(10920), 280 (14040) and 362 (6050) nm; 1H NMR (CDCl3 þ TFA):
135) (CDCl3 þ TFA):
d
27.24 (þve, CH3), 27.96 (þve, CH3), 55.60 (þve,
d 3.38 (s, 6H, CH3), 4.00 (s, 2H, NH2), 6.10–6.19 (m, 2H, ArH), 7.28–
OCH3), 92.30 (C5), 115.21 (þve, ArCH), 119.48 (þve, ArCH), 131.35
(absent, ArC), 152.09 (þve, CH), 158.35 (absent, ArC), 162.77 (C2),
165.04 (C6/C4), FAB mass m/z 290 (Mþ); (Found: C 58.15, H 5.22, N
14.48; C14H13N3O5 requires C 58.13, H 5.23, N 14.53)
7.35 (m, 1H, ArH), 7.66–7.68 (d, J ¼ 13.9 Hz, 1H, ]CH), 12.25–12.29
(broad doublet, J ¼ 13.2 Hz, 1H, NH); FAB mass m/z 276 (Mþ);
(Found: C 52.32, H 4.75, N 25.48; C14H13N3O5 requires C 52.36, H
4.76, N 25.44).
4.2.3. 5-((2-Nitrophenylamino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)-trione (6)
4.2.9. 5-(((Pyridine-2-yl)methylamino)methylene)-1,3-dimethyl-
pyrimidine-2,4,6(1H,3H,5H)-trione (16)
Greenish solid, 73% yield, mp 235 ꢀC; nmax (KBr): 1635 (C]O),
Brownish solid, 70% yield, mp 170 ꢀC; nmax (KBr): 1645 (C]O),
1680 (C]O), 3250 (NH); UV lmax
(
3
) (DMSO þ HEPES buffer) 232
1687 (C]O), 3236 (NH); UV lmax
(
3
) (DMSO þ HEPES buffer) 238
(12900) and 360 (16150) nm; 1H NMR (CDCl3 þ TFA):
d
3.30 (s, 6H,
(11190), 282 (14310) and 360 (7030) nm; 1H NMR (CDCl3 þ TFA):
CH3), 6.83–6.96 (m, 2H, ArH), 7.19–7.28 (m, 2H, ArH), 8.59–8.64
(d, J ¼ 13.9 Hz, 1H, ]CH), 12.00–12.12 (broad doublet, J ¼ 13.2 Hz,
1H, NH); FAB mass m/z 305 (Mþ); (Found: 51.35, H 3.96, N 18.43;
C14H13N3O5 requires C 51.32, H 3.98, N 18.41).
d 3.38 (s, 6H, CH3), 4.12 (s, 2H, CH2), 6.90–6.98 (m, 2H, ArH), 7.85–
7.95 (m, 2H, ArH), 8.66–8.69 (d, J ¼ 13.9 Hz, 1H, ]CH), 12.35–12.38
(broad, 1H, NH); FAB mass m/z 275 (Mþ); (Found: C 56.92, H 5.17, N
20.45; C14H13N3O5 requires C 56.93, H 5.14, N 20.43).
4.2.4. 5-((2-hydroxyphenylamino)methylene)-1,3-dimethylpyrimi-
dine-2,4,6(1H,3H,5H)–trione (7)
4.2.10. p-gp interaction studies
Solutions of compounds 1–16 which are tested for their inter-
Dark brown solid, 70% yield, mp 250 ꢀC; nmax (KBr): 1642 (C]O),
actions with p-gp were primarily prepared at 10ꢁ2 M concentration
1665 (C]O), 3225 (NH); UV lmax
(
3
) (DMSO þ HEPES buffer) 233
and diluted to three concentrations 0.5
Further dilutions take place during the assay and the final
concentrations become 0.05 M, 0.5 M and 5 M. Each well of 96
well plate was dispensed with 80 L of enzymatic buffer, 20 L of
PK/LDH solution, 10 L of PEP solution and 10 L of NADH solution.
L of enzymatic buffer was added to the total
L of non-specific ATPase inhibitor solution and
mM, 5 mM and 50 mM.
(4920) and 348 (9760) nm; 1H NMR (CDCl3 þ TFA):
d 3.35 (s, 6H,
CH3), 6.89–6.98 (m, 2H, ArH), 7.25–7.37 (m, 2H, ArH), 8.66–8.69 (d,
J ¼ 13.9 Hz, 1H, ]CH), 12.45–12.47 (broad doublet, J ¼ 13.2 Hz, 1H,
NH); FAB mass m/z 276 (Mþ); (Found: C 56.78, H 4.78, N 15.29;
C14H13N3O5 requires C 56.72, H 4.76, N 15.27).
m
m
m
m
m
m
m
Additionally, 60
activity well; 30
m
m
4.2.5. 2-((Tetrahydro-1,3-dimethyl-2,4,6-trioxopyrimidin-5(6H)
ylidene)methylamino) benzene ꢁ1,4-dioic acid (8)
30
non-specific ATPase inhibitor solution, 30
non-specific activity well. Blank well contains 200
buffer. The plate was incubated for 30 min at 37 ꢀC. 10
enzymatic buffer was added to non-specific activity wells and 10
of membrane vesicles were added to all other wells except blank
well. Plate was incubated for 5 min at 37 ꢀC, dispensed 20
L of
compound at each concentration and again incubated for 5 min at
m
L enzymatic buffer was added to basal activity well and 30
L enzymatic buffer to
L of enzymatic
L of
mL
m
Creamish white solid, 75% yield, mp 255 ꢀC; nmax (KBr):
m
1624(C]O), 1655(C]O), 3220 (NH), 3450 (COOH); UV lmax
(3)
m
(DMSO þ HEPES buffer) 232 (12990) and 350 (21240) nm; 1H NMR
mL
(CDCl3 þ TFA):
d 3.32 (s, 6H, CH3), 6.90–6.97 (m, 2H, ArH), 7.75–7.80
(m, 1H, ArH), 8.66–8.69 (d, J ¼ 14.4 Hz, 1H, ]CH), 13.21 (broad
doublet, J ¼ 13.7 Hz, 1H, NH); FAB mass m/z 348 (Mþ); (Found: C
51.82, H 3.79, N 12.12; C14H13N3O5 requires C 51.88, H 3.77, N 12.10).
m
37 ꢀC. Finally, 10
mL of MgATP was added to each well except blank
well and plate was incubated for 20 min at 37 ꢀC. Plate was read at
340 nm followed by incubation and again reading (after 20 min).
4.2.6. Compound 13
White solid, 80% yield, mp 210 ꢀC; nmax (KBr): 1635 (C]O), 1680
(C]O), 3385 (NH); UV lmax
(
3
) (DMSO þ Hepes buffer) 231 (8980)
4.3. Mg2þ binding studies
and 300 (14780) nm; 1H NMR (CDCl3 þ TFA):
d 3.27 (s, 6H, CH3),
7.27–7.29 (d, J ¼ 6.5 Hz, 1H, ArH), 8.29–8.31 (d, J ¼ 7.6 Hz, 1H, ArH),
8.31–8.43 (d, J ¼ 5.5 Hz, 1H, ArH), 8.51–8.56 (m, 2H, ArH), 8.67–8.65
(d, J ¼ 10.7 Hz, 1H, ]CH), 9.25 (broad singlet, 1H, NH), 10.16 (broad
singlet, 1H, NH); FAB mass m/z 275 (Mþ); (Found: C 56.95, H 5.17, N
20.46; C14H13N3O5 requires C 56.93, H 5.14, N 20.43)
Stock solutions (10ꢁ2 M) of compounds 1–16 were prepared by
dissolving the compounds in 2–3 drops of DMSO and diluting with
HEPES buffer (10ꢁ2 M) at pH 7.2. The complex formation was
studied by incremental addition of metal ion to 10
ml of ligand
solution making final volume 1 ml. The binding constants of

1262
P. Singh et al. / European Journal of Medicinal Chemistry 45 (2010) 1256–1262
compounds 1–16 with Mg2þ were calculated using Benesi Hilde-
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Acknowledgements
Financial assistance from DST, New Delhi is gratefully
acknowledged. JK thanks UGC and AB thanks DST for fellowships.
Department of Applied Chemistry, GNDU, Amritsar is acknowl-
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Appendix. Supplementary data
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Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2009.12.033.