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Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies
Journal Pre-proofs Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies Mahmood Biglar, Roghieh Mirzazadeh, Mehdi Asadi, Saghi Sepehri, Yosef Valizadeh, Yaghoub Sarrafi, Massoud Amanlou, Bagher Larijani, Maryam Mohammadi-Khanaposhtani, Mohammad Mahdavi PII: DOI: Reference:
S0045-2068(19)31278-7 https://doi.org/10.1016/j.bioorg.2019.103529 YBIOO 103529
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
5 August 2019 16 December 2019 19 December 2019
Please cite this article as: M. Biglar, R. Mirzazadeh, M. Asadi, S. Sepehri, Y. Valizadeh, Y. Sarrafi, M. Amanlou, B. Larijani, M. Mohammadi-Khanaposhtani, M. Mahdavi, Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies, Bioorganic Chemistry (2019), doi: https:// doi.org/10.1016/j.bioorg.2019.103529
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Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies Mahmood Biglara, Roghieh Mirzazadehb, Mehdi Asadic, Saghi Sepehrid, Yosef Valizadeha, Yaghoub Sarrafie, Massoud Amanlouc, Bagher Larijania, Maryam Mohammadi-Khanaposhtanif,*, Mohammad Mahdavia,* a
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b Department
c
of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran d
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of
Medical Sciences, Ardabil, Iran e
f
Faculty of Chemistry, University of Mazandaran, Babolsar, Iran Cellular and Molecular Biology Research Center, Health Research Institute, Babol University
of Medical Sciences, Babol, Iran * Corresponding authors: E-mail
addresses:
[email protected]
[email protected] (M. Mahdavi)
(M.
Mohammadi-Khanaposhtani)
and
Abstract A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as Helicobacter pylori urease inhibitors. All the synthesized compounds (IC50 = 10.37 ± 1.0–77.52 ± 2.7 μM) were more potent than standard inhibitor hydroxyurea against urease (IC50 = 100.00 ± 0.2 μM). Furthermore, comparison of IC50 values of the synthesized compounds with the second standard inhibitor thiourea (IC50 = 22.0 ± 0.03 µM) revealed that compounds 7a-b and 7f-h were more potent than thiourea. Molecular modeling study of the most potent compounds 7a, 7b, 7f, and 7g was also conducted. Additionally, the drug-likeness properties of the synthesized compounds, based on Lipinski rule and other filters, were evaluated. Keywords: Urease inhibitor; N,N-dimethylbarbituric; Molecular docking; Barbituric acid; Helicobacter pylori 1. Introduction The urease (urea amidohydrolase EC 3.5.1.5) is a nickel-containing enzyme that found in various plants, bacteria, algae, fungi, and some invertebrates. Urease is responsible for use of urea as nitrogen source for growth of numerous microorganisms. This enzyme catalyzes the transformation of urea to ammonia and carbon dioxide [1]. High concentration of ammonia arising of the later transformation and increase in pH level help to survival of Helicobacter pylori (H. pylori). This bacteria is a successful human bacterial parasites, which infects up to 50% of the world’s human population [2]. Colonization of H. pylori leads to numerous gastroduodenal disorders in humans such as infection stones, peptic ulcer, gastric cancer, duodenal ulcers [3]. Therefore, H. pylori urease inhibition can be useful in the treatment of urease-relating diseases [4-
6]. Urease inhibitors are divided into four group based on the chemical structures: phosphorodiamidates, hydroxamic acid derivatives, nickel chelators, and thiolic compounds [7]. Barbituric acid was found in the many biological active compounds with anticancer, antifungal, antibacterial, and antidiabetic activities [8-11]. Furthermore, urease inhibitory activities of barbituric acid derivatives have been well documented [12-15]. Recently, several derivatives of N,N-dimethylbarbituric acid with inhibitory activities against urease have been reported [16,17]. Therefore, by using of the N,N-dimethylbarbituric scaffold and in continuation of our efforts for the development of new urease inhibitors, herein, we present for the first time a new series of N,Ndimethylbarbituric-pyridinium derivatives 7a-n as potent urease inhibitors [18-20]. All the synthesized derivatives were evaluated for their in vitro urease inhibitory activities. Furthermore, in silico study was also performed for prediction of the binding modes of these compounds in the active site of urease. 2. Results and discussion 2.1. Chemistry The synthesis of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was carried out by the synthetic route outlined in Scheme 1. This pathway was started of reaction between N,Ndimethylbarbituric acid 1 and trimethoxymethane 2 in propanol. Then, the reaction mixture allowed to stand overnight at room temperature to produce compound 3. Next step, the latter compound and pyridin-3-ylmethanamine 4 were refluxed in ethanol to produce compound 5. Finally, compound 5 was reacted with diverse benzyl bromide derivatives 6a-n in dry acetonitrile to afford the target compounds 7a-n. All the synthesized compounds were characterized by FTIR, 1H NMR, and 13C NMR spectroscopic techniques.
O N
N
O
NH2
O +
O
O
(a) O
O
O 1
N
O +
N
N
3
Br N
5
O
N
H N
R
N O
4
(c)
N
H N
N
O
2
O
(b)
Br O
+ R 6a-n
O
N O
7a-n
Scheme 1. Outline for the synthesis of N,N-dimethylbarbituric-pyridinium derivatives 7a-n. Reagents and Conditions: (a) propanol, reflux, 3 h; (b) EtOH, reflux, 4 h; (c) dry acetonitrile, Reflux, 2–3 h. 2.2. In vitro inhibition of urease All N,N-dimethylbarbituric-pyridiniums 7a-n were screened against H. pylori urease (Table 1). The obtained results revealed that the title compounds (IC50 = 10.37 ± 1.0-77.52 ± 2.7 µM) were more potent than the standard drug hydroxyurea (IC50 = 100.00 ± 0.2 µM). In addition, among the synthesized compounds, compounds 7a, 7b, 7f, 7g, and 7h with inhibitory activity in the range of 10.37 ± 1.0–20.04 ± 0.61 µM were more than potent than standard drug thiourea (IC50 = 22.0 ± 0.03 µM). Moreover, compound 7i with IC50 = 22.31 ± 0.2 µM was approximately as potent as thiourea against urease. Table 1. The urease inhibitory activity of the synthesized compounds 7a-n. O N R
N
H N
O
N O
Compound
R
IC50 (µM) a
7a
H
11.25 ± 0.011
7b
2-CH3
10.37 ± 1.0
7c
4-CH3
46.01 ± 1.3
7d
3-Fluoro
49.16 ± 2.02
7e
4- Fluoro
64.27 ± 2.5
7f
2-Chloro
11.02 ± 0.05
7g
3-Chloro
19.29 ± 1.6
7h
2,3-Dichloro
20.04 ± 0.61
7i
3,4-Dichloro
22.31 ± 0.2
7j
4-Bromo
66.10 ± 0.04
7k
2-Nitro
63.09 ± 2.30
7l
4-Nitro
68.36 ± 0.25
7m
2-Fluoro-6-nitro
77.52 ± 2.7
7n
4-Cyano
72.81 ± 0.8
Thiourea
-
22.0 ± 0.03
Hydroxyurea
-
100.00 ± 0.2
a Values
are the mean ± SEM. All experiments were performed at least three times.
Un-substituted compound 7a (IC50 = 11.25 ± 0.1) showed high inhibitory activity against urease. The introduction of a methyl group on 2-potision of pendant phenyl ring, as in compound 7b (IC50 = 10.37 ± 1.0), slightly improved anti-urease potency. Changing the position of the methyl group from C-2 to C-4, producing 7c (IC50 = 46.01 ± 1.3), dramatically diminished the activity. On the other hand, replacement 2-methyl group of compound 7b with 2-chloro substituent, as in compound 7f (IC50 = 11.02 ± 0.05), led to slightly decrease in the inhibitory activity. Movement of chloro substituent of compound 7f into 3-position caused decrease of potency as observed in compound 7g. Adding another chlorine atom to 2 or 4-position of pendant phenyl ring of
compound 7g, didn’t improve anti-urease potency as observed in the compounds 7h and 7i. The synthesized compounds 7d-e and 7j-n with 3-fluoro, 4-fluoro, 4-bromo, 2-nitro, 4-nitro, 2-fluoro6-nitro, and 4-cyano substituents demonstrated moderate activity against urease in comparison with standard drug thiourea. 2.3. Docking study To study the interaction modes of the synthesized N,N-dimethylbarbituric-pyridiniums in the active site of H. pylori urease and their related inhibitory activities, in silico studies were performed using Auto Dock Tools (version 1.5.6). For this purpose, the most potent compounds 7a, 7b, 7f, and 7g were selected and the crystal structure of H. pylori urease with PDB structure of 1E9Y was retrieved of the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). The superposed structure of the later compounds in the active site of urease is shown in Fig. 1.
Fig. 1. Superimposition structure of the most potent compounds 7a (pink), 7b (green), 7f (yellow), and 7g (cyan) in the H. pylori urease active site ( = Nickel).
In the case of compound 7a, the barbituric moiety interacted with active site residues Ala169, Asp223, His221, His248, Gly279, and Asp362 (Fig. 2a). NH unit of this compound established a hydrogen bond with carbonyl unit of Asn168. Furthermore, pyridinium ring and pendant phenyl ring formed hydrophobic interactions with Ala365 and Arg368, respectively. Introduction of a methyl substituent on 2-potision of pendant phenyl ring led to a slightly increase in the inhibitory activity and minor changes in interaction mode as observed in Table 1 and Figs. 2a and 2b. As can be seen in the latter figures, the barbituric moiety of the most potent compound 7b formed additional interactions with Ala365 and His322 in comparison to compound 7a. Interestingly, compound 7a with un-substituted phenyl ring and compound 7f with 2-chlorophenyl ring that exhibited approximately similar activity against urease also have same interaction modes in the active site of this enzyme (Figs. 2a vs. 2c). Movement of chloro substituent of the compound 7f into 3-position, as in compound 7g, caused small decrease of potency presumably because pyridinium ring of the compound 7g formed no interaction with active site in comparison to this ring of the compound 7f (Fig. 2c vs. 2d). Furthermore, barbituric moiety of the compound 7f established two interactions with His248 while this moiety in the compound 7g formed only one interaction with His248. On the other hand, 3chlorophenyl group of the compound 7g interacted with residues Arg368 and Pro302 via 3-chloro substituent while 2-chlorophenyl group of compound 7f interacted only with Arg368 via phenyl ring. (a)
(b)
(c)
(d)
Fig. 2. The predicted binding modes of compounds (a) 7a, (b) 7b, (c) 7f, and (d) 7g in the active site pocket ( = Nickel). 2.4. Screening of pharmacokinetic properties To predict of the pharmacokinetic properties of N,N-dimethylbarbituric-pyridiniums 7a-n, physicochemical properties such as a number of H-bond acceptors (HBA), a number of H-bond donors (HBD), octanol/water partition coefficients (log P), and topological factors such as polar surface area (PSA) and number of rotatable bonds (RBC) of all the synthesized compounds were calculated and shown in Table 2. As can be seen in the Table 2, the title compounds 7a-n can be a
likely orally active drug in humans because they follow of Lipinski rule of 5 (MW ≤ 500, HBA ≤ 10, HBD ≤ 5, and log P ≤ 5) [21, 22]. Furthermore, theoretically, all the synthesized compounds except compounds 7k-m able to permeate cells because showed optimal amounts of PSA (< 89 A2) [23]. The RBC is a descriptor for prediction of oral bioavailability of drugs and prior research data showed that compounds with RBC <10 have good oral bioavailability in rat [24]. As can be seen in Table 2, all the synthesized compounds have RBC = 5, so it is expected that our compounds will have high oral bioavailability. Table 2. Molecular descriptors a of the synthesized compounds 7a-n. Compoun
MW
HBA
HBD
LogP
PSA
RBC
7a
365.16
4
0
1.26
56.74
5
7b
379.18
4
0
1.54
56.74
5
7c
379.18
4
0
1.66
56.74
5
7d
383.15
4
0
1.53
56.74
5
7e
383.15
4
0
1.53
56.74
5
7f
399.12
4
0
1.85
56.74
5
7g
399.12
4
0
1.97
56.74
5
7h
433.08
4
0
2.44
56.74
5
7i
433.08
4
0
2.56
56.74
5
7j
443.07
4
0
2.11
56.74
5
7k
410.15
6
0
0.81
89.83
5
7l
410.15
6
0
0.93
90.13
5
d
7m
428.14
6
0
0.95
89.83
5
7n
390.16
5
0
1.18
73.80
5
a
MW: Molecular Weight, HBA: a number of H-bond acceptors, HBD: a number of H-bond donors, log P: the
octanol-water partition coefficient, PSA: the polar surface area, RBC: a number of rotatable bonds.
3. Conclusion In conclusion, a new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and screened as anti-urease agents. These compounds showed good to excellent inhibitory activity against H. pylori urease in comparison to the standard inhibitors (hydroxyurea and thiourea). The obtained results revealed that all the title compounds 7a-n were more potent than hydroxyurea and compounds 7a, 7b, 7f, 7g, and 7h were more potent than thiourea against urease. The molecular docking results of the most potent compounds 7a, 7b, 7f, and 7g showed that these compounds interacted with important residues in the active site of urease. 4. Methods and Materials Melting points of the N,N-dimethylbarbituric-pyridinium derivatives 7a-n were measured on a Kofler hot stage apparatus and were uncorrected. 1H and 13C NMR spectra of the title compounds were recorded on a Bruker FT-500, using TMS as an internal standard. FT-IR spectra of these compounds were obtained on a Nicolet Magna FTIR 550 spectrophotometer (KBr disks). Mass spectroscopy was done by an Agilent Technology (HP) mass spectrometer performing at ionization potential = 70 eV. The elemental analysis of the compounds 7a-n for C, H, and N was carried out with an Elementar Analysen system GmbH VarioEL. 4.1. General procedure for the synthesis of 5-(ethoxymethylene)pyrimidine-2,4,6(1H,3H,5H)trione 3
A mixture of barbituric acid 1 (1 mmol) and trimethoxymethane 2 (3 mmol) in propanol (5ml) was stirred at reflux for 3 h. Then, the obtained mixture was allowed to stand overnight at room temperature for formation pure compound 3. 4.2. General procedure for the synthesis of 5-(((pyridin-4-ylmethyl)amino)methylene)pyrimidine2,4,6(1H,3H,5H)-trione 5 A mixture of compound 3 (1 mmol) and pyridin-3-ylmethanamine 4 (1 mmol) in ethanol (5ml) was stirred at reflux for 4 h. Then, the reaction mixture was allowed to cool at room temperature and poured into crushed ices, and the pure white precipitate 5 were filtered off. 4.3.
General
procedure
for
the
synthesis
of
1-aryl-4-((((1,3-dimethyl-2,4,6-
trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)pyridin-1-ium bromides 7a-n A solution of the compound 5 (1 mmol) and benzyl bromide derivatives 6a-n (1.4 mmol) in dry acetonitrile (10 ml) was heated under reflux condition for 2-3 h. The reaction progress was monitored by TLC. The obtained precipitate was filtered off and washed with dry acetonitrile (2 ml) and recrystallized in ethyl acetate to give pure compounds 7a-n. The synthesized compounds 7b, 7e, 7i-j, and 7m-n were obtained as a mixture of E and Z isomers and the percentage of E and Z isomers was calculated by NMR. The remaining compounds were obtained as E isomer. 4.3.1.
(E)-1-benzyl-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7a Yield 83%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3325, 2925, 2850, 1685, 1610. 1H NMR (500 MHz, DMSO-d6): δ = 10.55 (br. s, 1H, NH), 9.29 (s, 1H, H2 pyridine), 9.22 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.60 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.44 (m, 1H, =CH), 8.20 (m, 1H, H5 pyridine), 7.55 (dd, 2H, H2,6 phenyl, J1 = 7.2 Hz, J2 = 1.2 Hz ), 5.91 (s, 2H, CH2N+), 4.95 (s, 2H,
CH2N), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.9 (C=O), 162.6 (C=O), 160.6 (C=O), 152.0 (C), 145.5 (CH), 144.6 (CH), 144.4 (CH), 139.5 (C), 134.6 (C), 129.8 (CH), 129.6 (CH), 129.3 (CH), 128.6 (CH), 91.2 (C), 63.8 (CH2), 50.1 (CH2), 27.9 (CH3), 27.2 (CH3). MS: m/z = 445 [M+]. Anal. Calcd for C20H21BrN4O3: C, 53.94; H, 4.75; N, 12.58. Found: C, 53.81; H, 7.87; N, 12.41. 4.3.2. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(2-methylbenzyl)pyridin-1-ium bromide 7b Yield 88% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3356, 2985, 1695, 1680, 1605. NMR data for the major isomer: 1H NMR (500 MHz, DMSO-d6): δ = 10.56 (br. s, 1H, NH), 9.09 (s, 1H, H2 pyridine), 9.07 (m, 1H, H6 pyridine), 8.64 (d, 1H, H4 pyridine, J = 7.6 Hz), 8.43 (d, 1H, =CH, J = 14.0 Hz), 8.22 (t, 1H, H5 pyridine, , J = 7.2 Hz), 7.277.35 (m, 2H, H3,5 phenyl), 7.24 (d, 1H, H6 phenyl, J = 7.4 Hz), 7.16 (m, 1H, H4 phenyl), 5.98 (s, 2H, CH2N+), 4.96 (s, 2H, CH2N, J = 6.0 Hz), 3.13 (s, 6H, 2NMe), 2.31 (s, 3H, Me). 13C NMR (125 MHz, DMSO-d6): δ = 163.9 (C=O), 162.6 (C=O), 160.6 (C=O), 152.0 (C),145.6 (CH), 144.6 (CH), 144.5 (CH), 138.5 (CH), 132.5 (C), 131.8 (C), 129.6 (CH), 129.3(CH), 128.6 (C), 90.5 (C), 62.9 (CH2), 51.0 (CH2), 27.9 (CH3), 27.2 (CH3), 23.3 (CH3). MS: m/z = 459 [M+]. Anal. Calcd for C21H23BrN4O3: C, 54.91; H, 5.05; N, 12.20. Found: C, 55.06; H, 4.93; N, 12.32. 4.3.3.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(4-methylbenzyl)pyridin-1-ium bromide 7c Yield 84%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3358, 2981, 1698, 1683, 1604. 1H NMR (500 MHz, DMSO-d6): δ = 10.15 (m, 1H, NH), 9.11 (s, 1H, H2 pyridine), 9.08 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.72 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.38 (d, 1H, =CH, J = 14.5 Hz), 8.04
(m, 1H, H5 pyridine), 7.91 (d, 1H, H6 pyridine, J = 9.0 Hz), 7.16 (dd, 2H, Hα phenyl, J1 = 8.0 Hz, J2 = 2.5 Hz), 6.86 (d, 2H, Hβ phenyl, J = 8.0 Hz), 6.11 (s, 2H, CH2N+), 4.56 (m, 2H, CH2N), 3.12 (s, 6H, 2NMe), 2.33 (s, 3H, Me). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 161.4 (C=O), 160.6 (C=O), 156.0 (CH), 140.8 (C), 137.9 (CH), 135.9 (CH), 132.5 (CH), 129.2(CH), 127.6(CH), 124.0 (CH), 123.8 (CH), 123.5 (CH), 116.5 (CH), 116.3 (CH), 92.2 (C), 57.6 (CH2), 48.3 (CH2), 27.8 (CH3), 26.4 (CH3), 22.9 (CH3). Anal. Calcd for C21H23BrN4O3: C, 54.91; H, 5.05; N, 12.20. Found: C, 54.85; H, 4.97; N, 12.11. 4.3.4.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(3-fluorobenzyl)pyridin-1-ium bromide 7d Yield 83%; yellow solid; mp > 250 ºC; IR (KBr, cm-1): ν = 3340, 2925, 1705, 1687, 1612. 1H NMR (500 MHz, DMSO-d6): δ = 10.57 (br. s, 1H, NH), 9.24 (s, 1H, H2 pyridine), 9.21 (br.s, 1H, H6 pyridine, J = 5.5 Hz), 8.16 (br. s, 1H, H4 pyridine), 8.45 (d, 1H, =CH, J = 13.0 Hz), 8.20 (s, 1H, H5 pyridine), 7.51-7.28 (m, 4H, H2,4,5,6 phenyl), 5.91 (s, 2H, CH2N+), 4.94 (s, 2H, CH2N), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.2 (C=O), 151.5 (C), 145.3 (CH), 144.4 (CH), 143.8 (CH), 139.1 (C), 136.4 (C), 131.4 (CH), 128.2(CH), 125.1 (CH), 116.1 (CH), 115.6 (CH), 90.7 (C), 62.5 (CH2), 49.7 (CH2), 27.3 (CH3), 26.7 (CH3). MS: m/z = 463 [M+]. Anal. Calcd for C20H20BrFN4O3: C, 51.85; H, 4.35; N, 12.09. Found: C, 51.74; H, 4.29; N, 12.16. 4.3.5. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(4-fluorobenzyl)pyridin-1-ium bromide 7e Yield 91% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3341, 2927, 1702, 1685, 1614. NMR data for the major isomer: 1H NMR (500 MHz, DMSO-d6):
δ = 10.58-10.55 (m, 1H, NH), 9.27 (s, 1H, H2 pyridine), 9.24 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.61 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.20 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.67 (t, 1H, Hα phenyl, J = 6.0 Hz), 7.31 (t,1H, Hβphenyl, J = 8.5 Hz), 5.90 (s, 2H, CH2N+), 4.94 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 162.1 (C=O), 160.1 (C=O), 160.0 (C), 151.5 (C=O), 145.1 (CH), 144.2 (CH), 144.0 (CH), 143.8 (CH), 139.0 (C), 136.0 (C), 131.5 (CH), 130.3 (CH), 128.2 (CH), 128.1 (CH), 116.0 (CH), 90.7 (C), 62.4 (CH2), 49.6 (CH2), 27.3 (CH3), 26.7 (CH3). Anal. Calcd for C20H20BrFN4O3: C, 51.85; H, 4.35; N, 12.09. Found: C, 51.93; H, 4.41; N, 11.97. 4.3.6.
(E)-1-(2-chlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7f Yield 76%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3338, 2968, 2865, 1712, 1685, 1598. 1H NMR (500 MHz, DMSO-d6): δ = 10.61-10.55 (m, 1H, NH), 9.13 (br. s, 1H, H2 pyridine), 9.07 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.67 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.77 (d, 1H, H6 phenyl, J = 8.0 Hz), 7.50 (t, 1H, H5 phenyl, J =7.5 Hz), 7.43 (t, 1H, H4 phenyl, J = 7.5 Hz), 7.36 (d, 1H, H3 phenyl, J = 7.0 Hz), 6.00 (s, 2H, CH2N+), 4.95 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSOd6): δ = 162.1 (C=O), 160.1 (C=O), 151.5 (C=O), 145.7 (CH), 145.7 (CH), 145.5 (CH), 144.6 (CH), 144.3 (CH), 139.0 (C), 136.0 (C), 133.3 (CH), 132.9 (C), 131.6 (CH), 128.5(CH), 128.1 (CH), 123.4 (CH), 90.8 (C), 63.4 (CH2), 49.5 (CH2), 27.4 (CH3), 26.7 (CH3). Anal. Calcd for C20H20BrClN4O3: C, 50.07; H, 4.20; N, 11.68. Found: C, 50.14; H, 4.31; N, 11.75.
4.3.7.
(E)-1-(3-chlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7g Yield 83%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3345, 2978, 2898, 1680. 1H NMR (500 MHz, DMSO-d6): δ = 10.58-10.54 (m, 1H, NH), 9.22 (s, 1H, H2 pyridine), 9.18 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.60 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.20 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.69 (s, 1H, H2 phenyl), 7.50-7.47 (m, 3H, H4,5,6 phenyl), 5.90 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J =6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 162.2 (C=O), 160.2 (C=O), 151.5 (C=O), 145.3 (CH), 144.4 (CH), 144.2 (CH), 143.8 (CH), 139.1 (C), 136.2 (C), 133.6 (CH), 130.9 (C), 129.5 (CH), 128.8(CH), 128.3 (CH), 127.5 (CH), 90.7 (C), 62.5 (CH2), 49.7 (CH2), 27.4 (CH3), 26.8 (CH3). Anal. Calcd for C20H20BrClN4O3: C, 50.07; H, 4.20; N, 11.68. Found: C, 49.94; H, 4.13; N, 11.53. 4.3.8.
(E)-1-(2,3-dichlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7h Yield 78%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3340, 2966, 2905, 1688, 1608. 1H NMR (500 MHz, DMSO-d6): δ = 10.59-10.55 (m, 1H, NH), 9.12 (br. s, 2H, H2, H6 pyridine), 8.67 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.77 (d, 1H, H6 phenyl, J = 7.5 Hz), 7.50 (t, 1H, H5 phenyl, J = 7.5 Hz), 7.41 (d,1H, H4 phenyl, J = 7.5 Hz), 6.08 (s, 2H, CH2N+), 4.95 (d, 2H, CH2N, J = 6.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 145.7 (CH), 144.6 (CH), 144.3 (CH), 139.0 (C), 133.8 (C), 132.6 (CH), 131.6 (C), 130.1 (C), 129.8 (CH), 128.9 (CH), 128.1 (CH), 90.7 (C), 61.6 (CH2), 49.6 (CH2), 27.3 (CH3), 26.8 (CH3). Anal. Calcd for C20H19BrCl2N4O3: C, 46.72; H, 3.72; N, 10.90. Found: C, 46.85; H, 3.83; N, 10.98.
4.3.9.
1-(3,4-dichlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7i Yield 82% (E isomer: 65% and Z isomer: 35%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3335, 2925, 1710, 1670, 1605. 1H NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.57-10.53 (m, 1H, NH), 9.22 (br. s, 2H, H2, H6 pyridine), 8.61 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.44 (d, 1H, =CH, J = 14.5 Hz), 8.19 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.93 (s, 1H, H3 phenyl), 7.74 (d, 1H, H6 phenyl, J = 8.0 Hz), 7.58 (d,1H, H5 phenyl, J = 8.0 Hz), 5.90 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 145.3 (CH), 144.2 (CH), 143.9 (CH), 139.0 (C), 134.6 (C), 132.3 (C), 131.2 (C), 129.4 (CH), 128.2 (CH), 128.1 (CH), 90.7 (C), 72.7 (CH2), 49.6 (CH2), 27.3 (CH3), 26.7 (CH3). Anal. Calcd for C20H19BrCl2N4O3: C, 46.72; H, 3.72; N, 10.90. Found: C, 46.61; H, 3.86; N, 11.04. 4.3.10.
1-(4-bromobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7j Yield 88% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3350, 2958, 2888, 1700, 1675, 1610. NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.55 (br. s, 1H, NH), 9.18 (s, 1H, H2 pyridine), 9.16 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.60 (br. s, 1H, H4 pyridine), 8.44 (d, 1H, =CH, J = 14.0 Hz), 8.19 (br.s, 1H, H5 pyridine), 7.66 (br. s, 2H, Hα phenyl), 7.51 (br. s, 2H, Hβ phenyl), 5.86 (s, 2H, CH2N+), 4.92 (s, 2H, CH2N, J = 6.0 Hz), 3.16 (s, 6H, 2NMe).
13C
NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.1
(C=O), 145.1 (CH), 144.2 (CH), 144.1 (CH), 143.8 (CH), 139.1 (C), 136.0 (C), 133.3 (CH), 131.2 (CH), 131.0 (CH), 128.1(CH), 122.9 (C), 90.7 (C), 62.6 (CH2), 49.6 (CH2), 26.9 (CH3), 26.8 (CH3). Anal. Calcd for C20H20Br2N4O3: C, 45.82; H, 3.85; N, 10.69. Found: C, 45.76; H, 3.71; N, 10.53.
4.3.11.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(2-nitrobenzyl)pyridin-1-ium bromide 7k Yield 75%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3352, 3008, 1715, 1685, 1622, 1580. 1H NMR (500 MHz, DMSO-d6): δ = 10.59-10.57 (m, 1H, NH), 9.11 (s, 1H, H2 pyridine), 9.09 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.69 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.27-8.25 (m, 2H, H6 phenyl, H5 pyridine), 7.85 (t, 1H, H4 phenyl, J = 7.0 Hz), 7.76 (t, 1H, H5 phenyl, J = 7.0 Hz), 7.25 (d, 1H, H3 phenyl, J = 7.5 Hz), 6.27 (s, 2H, CH2N+), 4.96 (d, 2H, CH2N, J = 5.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 147.6 (C), 145.5 (CH), 144.7 (CH), 144.4 (CH), 139.0 (C), 134.9 (C), 130.9 (C), 128.7 (CH), 128.1 (CH), 125.6 (CH), 125.5 (CH), 90.7 (C), 60.6 (CH2), 49.6 (CH2), 27.5 (CH3), 26.7 (CH3). MS: m/z = 490 [M+]. Anal. Calcd for C20H20BrN5O5: C, 48.99; H, 4.11; N, 14.28. Found: C, 48.88; H, 4.21; N, 14.34. 4.3.12.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(4-nitrobenzyl)pyridin-1-ium bromide 7l Yield 95%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3338, 3025, 1690, 1610, 1590. 1H NMR (500 MHz, DMSO-d6): δ = 10.12 (m, 1H, NH), 9.17 (d, 1H, H6 pyridine, J = 6.0 Hz), 9.11 (s, 1H, H2 pyridine), 8.66 (m, 1H, H4 pyridine), 8.42 (d, 1H, =CH, J = 14.5 Hz), 8.11 (d, 2H, Hα phenyl, J = 8.0 Hz), 7.93 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.51 (d, 2H, Hβ phenyl, J = 8.0 Hz), 6.07 (s, 2H, CH2N+), 5.03 (s, 2H, CH2N), 3.12 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.7 (C=O), 161.2 (C=O), 160.2 (C=O), 157.3 (CH), 148.1 (C), 140.7 (C), 136.4 (CH), 132.2 (CH), 132.0 (CH), 129.7 (CH), 127.0 (CH), 128.3 (CH), 124.6 (CH), 124.5 (CH), 123.6 (CH), 123.5 (CH), 121.1 (CH), 120.6 (CH), 90.0 (C), 60.5 (CH2), 49.9 (CH2), 27.7 (CH3), 26.6 (CH3). Anal. Calcd for C20H20BrN5O5: C, 48.99; H, 4.11; N, 14.28. Found: C, 49.06; H, 4.03; N, 14.36.
4.3.13. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(2-fluoro-6-nitrobenzyl)pyridin-1-ium bromide 7m Yield 86% (E isomer: 62% and Z isomer: 38%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3348, 3028, 1705, 1680, 1618, 1589. NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.57-10.55 (m, 1H, NH), 9.12 (d, 1H, H6 pyridine, J = 6.0 Hz), 9.03 (d, 1H, H6 pyridine), 8.67 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.42-8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 8.16 (dd, 1H, H5 phenyl, J = 7.5 Hz), 7.90-7.93 (m, 2H, H3,4 phenyl), 6.17 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.0 Hz), 3.13 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.9 (C=O), 163.2 (C=O), 162.6 (C=O), 161.5 (JC-F =145 Hz), 152.1 (C), 149.9 (CH), 149.2 (CH), 146.1(CH), 144.46(CH), 144.2 (CH), 139.2 (C), 133.8 (CH), 128.4 (CH), 123.0 (CH), 122.8 (CH), 122.5 (CH), 122.4 (CH), 115.4 (CH), 115.2 (C), 91.2 (C), 54.4 (CH2), 49.6 (CH2), 27.5 (CH3), 26.8 (CH3). Anal. Calcd for C20H19BrFN5O5: C, 47.26; H, 3.77; N, 13.78. Found: C, 47.20; H, 3.82; N, 13.65. 4.3.14.
1-(4-cyanobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7n Yield 86% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3341, 3018, 2965, 17055, 1688, 1625, 1590. NMR data for the major isomer: 1H NMR (500 MHz, DMSO- d6): δ = 10.58-10.55 (m, 1H, NH), 9.23 (s, 1H, H2 pyridine), 9.19 (d, 1H, H6 pyridine, J = 6.0 Hz ), 8.63 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.96 (d, 2H, Hα phenyl, J = 7.5 Hz), 7.71 (d, 2H, Hβ phenyl, J = 7.5 Hz), 6.00 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.2 (C=O), 151.5 (C), 145.4 (CH), 144.4 (CH), 144.0 (CH), 139.1 (C), 133.0 (CH), 129.6 (CH), 128.3 (CH), 128.2 (CH), 118.2 (CN), 112.0 (C),
90.7 (C), 62.5 (CH2), 49.6 (CH2), 27.5 (CH3), 26.8 (CH3). Anal. Calcd for C21H20BrN5O3: C, 53.63; H, 4.29; N, 14.89. Found: C, 53.57; H, 4.17; N, 14.76. 4.4. Urease Inhibitory Activity Urease inhibitory activity of the synthesized compounds 7a-n was evaluated exactly according to our pervious paper [20]. Results are expressed as mean ± SEM (n = 3 experiments). 4.5. Molecular docking study The three-dimensional (3D) protein structure of H. pylori urease (PDB ID: 1E9Y), determined by X-ray
crystallography,
was
retrieved
from
the
RCSB
Protein
Databank
(http://www.rcsb.org/pdb/home/home.do.). Then, extra molecules of the enzyme were removed using Accelrys discovery studio visualizer 4.0 (DSVisualizer, Accelrys Software Inc., San Diego, CA, USA, 2014.) and polar hydrogens and charges to the enzyme were added by Auto Dock Tools version 1.5.6. Protonation step corrects the ionization and tautomeric states of residues and modifies the total Kollman charges on the protein structure. 3D structures of the selected ligands were drawn by MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com). The resulting protein and ligand structures were converted to pdbqt coordinate using Auto dock Tools and these pdbqt files used as input files for the AutoGrid program. AutoGrid determined precalculated atomic affinity grid maps for each atom type in the selected ligands, plus an electrostatics map and a separate desolvation map presented. All maps were provided with 0.375 Å spacing between grid points and grid box was set at 60 × 60 × 60 Å. Each docking was performed by 50 runs of the AutoDock search by the Lamarckian genetic algorithm. Lastly, the conformations with the lowest predicted binding free energy were selected for the each test compound. Graphic manipulations were visualized using Accelrys discovery studio visualizer 4.0 software.
4.6. Prediction of pharmacokinetic properties Prediction of the molecular properties of the synthesized compounds 7a-n was performed using the
online
servers
as
Molinspiration
(http://www.molinspiration.com/),
and
Molsoft
(http://www.molsoft.com/). References [1] E. Mentese, H. Bektas, B.B. Sokmen, M. Emirik, D. Çakır, B. Kahveci, Synthesis and molecular docking study of some 5, 6-dichloro-2-cyclopropyl-1H-benzimidazole derivatives bearing triazole, oxadiazole, and imine functionalities as potent inhibitors of urease, Bioorg. Med. Chem. Lett. 27 (2017) 3014-3018. [2] B.E. Lacy, J. Rosemore, Helicobacter pylori: ulcers and more: the beginning of an era, J. Nutr. 131 (2001) 2789S-2793S. [3] B. Ali, K.M. Khan, U.S. Kanwal, S. Hussain, M. Ashraf, M. Riaz, A. Wadood, M. Taha, S. Perveen, 1-[(4′-Chlorophenyl) carbonyl-4-(aryl) thiosemicarbazide derivatives as potent urease inhibitors: Synthesis, in vitro and in silico studies, Bioorg. Chem. 79 (2018) 363-371. [4] E. Menteşe, G. Akyuz, M. Emirik, N. Baltaş, Synthesis, in vitro urease inhibition and molecular docking studies of some novel quinazolin-4 (3H)-one derivatives containing triazole, thiadiazole and thiosemicarbazide functionalities, Bioorg. Chem. 83 (2019) 289-296. [5] M. Alomari, M. Taha, S. Imran, W. Jamil, M. Selvaraj, N. Uddin, F. Rahim, Design, synthesis, in vitro evaluation, molecular docking and ADME properties studies of hybrid bis-coumarin with thiadiazole as a new inhibitor of Urease, Bioorg. Chem. 92 (2019) 103235. [6] F. Rahim, M. Taha, H. Ullah, A.Wadood, M. Selvaraj, A. Rab, M. Sajid, S.A.A. Shah, N. Uddin, M. Gollapalli, Synthesis of new arylhydrazide bearing Schiff bases/thiazolidinone: αamylase, urease activities and their molecular docking studies, Bioorg. Chem. 91 (2019) 103112.
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Graphical abstract
Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies Mahmood Biglar, Roghieh Mirzazadeh, Mehdi Asadi, Saghi Sepehri, Yosef Valizadeh, Yaghoub Sarrafi, Massoud Amanlou, Bagher Larijani, Maryam Mohammadi-Khanaposhtani*, Mohammad Mahdavi*
N+ R
O
N
H N
O N
Urease inhibitory assay
CH3 N+
O
O Compounds 7a-n
N
H N Compound 7b
O N
O
Docking study
A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated for their urease inhibitory activity. All the synthesized compounds (IC50 = 10.37 ± 1.0– 77.52 ± 2.7 μM) exhibited better inhibitory activity against urease when compared with standard inhibitor hydroxyurea (IC50 = 100.00 ± 0.2 μM).
Highlights
A novel series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as new urease inhibitors. These compounds showed urease inhibition superior to standard drug Hydroxyurea. Compounds 7a-7b and 7f-h were more potent than standard drug thiourea. Compound 7b was the most active compound. The most potent compounds interacted with important residues of urease active site.
Conflict of interest The authors have declared no conflict of interest.
S0045-2068(19)31278-7 https://doi.org/10.1016/j.bioorg.2019.103529 YBIOO 103529
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
5 August 2019 16 December 2019 19 December 2019
Please cite this article as: M. Biglar, R. Mirzazadeh, M. Asadi, S. Sepehri, Y. Valizadeh, Y. Sarrafi, M. Amanlou, B. Larijani, M. Mohammadi-Khanaposhtani, M. Mahdavi, Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies, Bioorganic Chemistry (2019), doi: https:// doi.org/10.1016/j.bioorg.2019.103529
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Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies Mahmood Biglara, Roghieh Mirzazadehb, Mehdi Asadic, Saghi Sepehrid, Yosef Valizadeha, Yaghoub Sarrafie, Massoud Amanlouc, Bagher Larijania, Maryam Mohammadi-Khanaposhtanif,*, Mohammad Mahdavia,* a
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b Department
c
of Biochemistry, Pasteur Institute of Iran, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Tehran University of Medical Sciences, Tehran, Iran d
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of
Medical Sciences, Ardabil, Iran e
f
Faculty of Chemistry, University of Mazandaran, Babolsar, Iran Cellular and Molecular Biology Research Center, Health Research Institute, Babol University
of Medical Sciences, Babol, Iran * Corresponding authors: E-mail
addresses:
[email protected]
[email protected] (M. Mahdavi)
(M.
Mohammadi-Khanaposhtani)
and
Abstract A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as Helicobacter pylori urease inhibitors. All the synthesized compounds (IC50 = 10.37 ± 1.0–77.52 ± 2.7 μM) were more potent than standard inhibitor hydroxyurea against urease (IC50 = 100.00 ± 0.2 μM). Furthermore, comparison of IC50 values of the synthesized compounds with the second standard inhibitor thiourea (IC50 = 22.0 ± 0.03 µM) revealed that compounds 7a-b and 7f-h were more potent than thiourea. Molecular modeling study of the most potent compounds 7a, 7b, 7f, and 7g was also conducted. Additionally, the drug-likeness properties of the synthesized compounds, based on Lipinski rule and other filters, were evaluated. Keywords: Urease inhibitor; N,N-dimethylbarbituric; Molecular docking; Barbituric acid; Helicobacter pylori 1. Introduction The urease (urea amidohydrolase EC 3.5.1.5) is a nickel-containing enzyme that found in various plants, bacteria, algae, fungi, and some invertebrates. Urease is responsible for use of urea as nitrogen source for growth of numerous microorganisms. This enzyme catalyzes the transformation of urea to ammonia and carbon dioxide [1]. High concentration of ammonia arising of the later transformation and increase in pH level help to survival of Helicobacter pylori (H. pylori). This bacteria is a successful human bacterial parasites, which infects up to 50% of the world’s human population [2]. Colonization of H. pylori leads to numerous gastroduodenal disorders in humans such as infection stones, peptic ulcer, gastric cancer, duodenal ulcers [3]. Therefore, H. pylori urease inhibition can be useful in the treatment of urease-relating diseases [4-
6]. Urease inhibitors are divided into four group based on the chemical structures: phosphorodiamidates, hydroxamic acid derivatives, nickel chelators, and thiolic compounds [7]. Barbituric acid was found in the many biological active compounds with anticancer, antifungal, antibacterial, and antidiabetic activities [8-11]. Furthermore, urease inhibitory activities of barbituric acid derivatives have been well documented [12-15]. Recently, several derivatives of N,N-dimethylbarbituric acid with inhibitory activities against urease have been reported [16,17]. Therefore, by using of the N,N-dimethylbarbituric scaffold and in continuation of our efforts for the development of new urease inhibitors, herein, we present for the first time a new series of N,Ndimethylbarbituric-pyridinium derivatives 7a-n as potent urease inhibitors [18-20]. All the synthesized derivatives were evaluated for their in vitro urease inhibitory activities. Furthermore, in silico study was also performed for prediction of the binding modes of these compounds in the active site of urease. 2. Results and discussion 2.1. Chemistry The synthesis of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was carried out by the synthetic route outlined in Scheme 1. This pathway was started of reaction between N,Ndimethylbarbituric acid 1 and trimethoxymethane 2 in propanol. Then, the reaction mixture allowed to stand overnight at room temperature to produce compound 3. Next step, the latter compound and pyridin-3-ylmethanamine 4 were refluxed in ethanol to produce compound 5. Finally, compound 5 was reacted with diverse benzyl bromide derivatives 6a-n in dry acetonitrile to afford the target compounds 7a-n. All the synthesized compounds were characterized by FTIR, 1H NMR, and 13C NMR spectroscopic techniques.
O N
N
O
NH2
O +
O
O
(a) O
O
O 1
N
O +
N
N
3
Br N
5
O
N
H N
R
N O
4
(c)
N
H N
N
O
2
O
(b)
Br O
+ R 6a-n
O
N O
7a-n
Scheme 1. Outline for the synthesis of N,N-dimethylbarbituric-pyridinium derivatives 7a-n. Reagents and Conditions: (a) propanol, reflux, 3 h; (b) EtOH, reflux, 4 h; (c) dry acetonitrile, Reflux, 2–3 h. 2.2. In vitro inhibition of urease All N,N-dimethylbarbituric-pyridiniums 7a-n were screened against H. pylori urease (Table 1). The obtained results revealed that the title compounds (IC50 = 10.37 ± 1.0-77.52 ± 2.7 µM) were more potent than the standard drug hydroxyurea (IC50 = 100.00 ± 0.2 µM). In addition, among the synthesized compounds, compounds 7a, 7b, 7f, 7g, and 7h with inhibitory activity in the range of 10.37 ± 1.0–20.04 ± 0.61 µM were more than potent than standard drug thiourea (IC50 = 22.0 ± 0.03 µM). Moreover, compound 7i with IC50 = 22.31 ± 0.2 µM was approximately as potent as thiourea against urease. Table 1. The urease inhibitory activity of the synthesized compounds 7a-n. O N R
N
H N
O
N O
Compound
R
IC50 (µM) a
7a
H
11.25 ± 0.011
7b
2-CH3
10.37 ± 1.0
7c
4-CH3
46.01 ± 1.3
7d
3-Fluoro
49.16 ± 2.02
7e
4- Fluoro
64.27 ± 2.5
7f
2-Chloro
11.02 ± 0.05
7g
3-Chloro
19.29 ± 1.6
7h
2,3-Dichloro
20.04 ± 0.61
7i
3,4-Dichloro
22.31 ± 0.2
7j
4-Bromo
66.10 ± 0.04
7k
2-Nitro
63.09 ± 2.30
7l
4-Nitro
68.36 ± 0.25
7m
2-Fluoro-6-nitro
77.52 ± 2.7
7n
4-Cyano
72.81 ± 0.8
Thiourea
-
22.0 ± 0.03
Hydroxyurea
-
100.00 ± 0.2
a Values
are the mean ± SEM. All experiments were performed at least three times.
Un-substituted compound 7a (IC50 = 11.25 ± 0.1) showed high inhibitory activity against urease. The introduction of a methyl group on 2-potision of pendant phenyl ring, as in compound 7b (IC50 = 10.37 ± 1.0), slightly improved anti-urease potency. Changing the position of the methyl group from C-2 to C-4, producing 7c (IC50 = 46.01 ± 1.3), dramatically diminished the activity. On the other hand, replacement 2-methyl group of compound 7b with 2-chloro substituent, as in compound 7f (IC50 = 11.02 ± 0.05), led to slightly decrease in the inhibitory activity. Movement of chloro substituent of compound 7f into 3-position caused decrease of potency as observed in compound 7g. Adding another chlorine atom to 2 or 4-position of pendant phenyl ring of
compound 7g, didn’t improve anti-urease potency as observed in the compounds 7h and 7i. The synthesized compounds 7d-e and 7j-n with 3-fluoro, 4-fluoro, 4-bromo, 2-nitro, 4-nitro, 2-fluoro6-nitro, and 4-cyano substituents demonstrated moderate activity against urease in comparison with standard drug thiourea. 2.3. Docking study To study the interaction modes of the synthesized N,N-dimethylbarbituric-pyridiniums in the active site of H. pylori urease and their related inhibitory activities, in silico studies were performed using Auto Dock Tools (version 1.5.6). For this purpose, the most potent compounds 7a, 7b, 7f, and 7g were selected and the crystal structure of H. pylori urease with PDB structure of 1E9Y was retrieved of the RCSB protein data bank (http://www.rcsb.org/pdb/home/home.do). The superposed structure of the later compounds in the active site of urease is shown in Fig. 1.
Fig. 1. Superimposition structure of the most potent compounds 7a (pink), 7b (green), 7f (yellow), and 7g (cyan) in the H. pylori urease active site ( = Nickel).
In the case of compound 7a, the barbituric moiety interacted with active site residues Ala169, Asp223, His221, His248, Gly279, and Asp362 (Fig. 2a). NH unit of this compound established a hydrogen bond with carbonyl unit of Asn168. Furthermore, pyridinium ring and pendant phenyl ring formed hydrophobic interactions with Ala365 and Arg368, respectively. Introduction of a methyl substituent on 2-potision of pendant phenyl ring led to a slightly increase in the inhibitory activity and minor changes in interaction mode as observed in Table 1 and Figs. 2a and 2b. As can be seen in the latter figures, the barbituric moiety of the most potent compound 7b formed additional interactions with Ala365 and His322 in comparison to compound 7a. Interestingly, compound 7a with un-substituted phenyl ring and compound 7f with 2-chlorophenyl ring that exhibited approximately similar activity against urease also have same interaction modes in the active site of this enzyme (Figs. 2a vs. 2c). Movement of chloro substituent of the compound 7f into 3-position, as in compound 7g, caused small decrease of potency presumably because pyridinium ring of the compound 7g formed no interaction with active site in comparison to this ring of the compound 7f (Fig. 2c vs. 2d). Furthermore, barbituric moiety of the compound 7f established two interactions with His248 while this moiety in the compound 7g formed only one interaction with His248. On the other hand, 3chlorophenyl group of the compound 7g interacted with residues Arg368 and Pro302 via 3-chloro substituent while 2-chlorophenyl group of compound 7f interacted only with Arg368 via phenyl ring. (a)
(b)
(c)
(d)
Fig. 2. The predicted binding modes of compounds (a) 7a, (b) 7b, (c) 7f, and (d) 7g in the active site pocket ( = Nickel). 2.4. Screening of pharmacokinetic properties To predict of the pharmacokinetic properties of N,N-dimethylbarbituric-pyridiniums 7a-n, physicochemical properties such as a number of H-bond acceptors (HBA), a number of H-bond donors (HBD), octanol/water partition coefficients (log P), and topological factors such as polar surface area (PSA) and number of rotatable bonds (RBC) of all the synthesized compounds were calculated and shown in Table 2. As can be seen in the Table 2, the title compounds 7a-n can be a
likely orally active drug in humans because they follow of Lipinski rule of 5 (MW ≤ 500, HBA ≤ 10, HBD ≤ 5, and log P ≤ 5) [21, 22]. Furthermore, theoretically, all the synthesized compounds except compounds 7k-m able to permeate cells because showed optimal amounts of PSA (< 89 A2) [23]. The RBC is a descriptor for prediction of oral bioavailability of drugs and prior research data showed that compounds with RBC <10 have good oral bioavailability in rat [24]. As can be seen in Table 2, all the synthesized compounds have RBC = 5, so it is expected that our compounds will have high oral bioavailability. Table 2. Molecular descriptors a of the synthesized compounds 7a-n. Compoun
MW
HBA
HBD
LogP
PSA
RBC
7a
365.16
4
0
1.26
56.74
5
7b
379.18
4
0
1.54
56.74
5
7c
379.18
4
0
1.66
56.74
5
7d
383.15
4
0
1.53
56.74
5
7e
383.15
4
0
1.53
56.74
5
7f
399.12
4
0
1.85
56.74
5
7g
399.12
4
0
1.97
56.74
5
7h
433.08
4
0
2.44
56.74
5
7i
433.08
4
0
2.56
56.74
5
7j
443.07
4
0
2.11
56.74
5
7k
410.15
6
0
0.81
89.83
5
7l
410.15
6
0
0.93
90.13
5
d
7m
428.14
6
0
0.95
89.83
5
7n
390.16
5
0
1.18
73.80
5
a
MW: Molecular Weight, HBA: a number of H-bond acceptors, HBD: a number of H-bond donors, log P: the
octanol-water partition coefficient, PSA: the polar surface area, RBC: a number of rotatable bonds.
3. Conclusion In conclusion, a new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and screened as anti-urease agents. These compounds showed good to excellent inhibitory activity against H. pylori urease in comparison to the standard inhibitors (hydroxyurea and thiourea). The obtained results revealed that all the title compounds 7a-n were more potent than hydroxyurea and compounds 7a, 7b, 7f, 7g, and 7h were more potent than thiourea against urease. The molecular docking results of the most potent compounds 7a, 7b, 7f, and 7g showed that these compounds interacted with important residues in the active site of urease. 4. Methods and Materials Melting points of the N,N-dimethylbarbituric-pyridinium derivatives 7a-n were measured on a Kofler hot stage apparatus and were uncorrected. 1H and 13C NMR spectra of the title compounds were recorded on a Bruker FT-500, using TMS as an internal standard. FT-IR spectra of these compounds were obtained on a Nicolet Magna FTIR 550 spectrophotometer (KBr disks). Mass spectroscopy was done by an Agilent Technology (HP) mass spectrometer performing at ionization potential = 70 eV. The elemental analysis of the compounds 7a-n for C, H, and N was carried out with an Elementar Analysen system GmbH VarioEL. 4.1. General procedure for the synthesis of 5-(ethoxymethylene)pyrimidine-2,4,6(1H,3H,5H)trione 3
A mixture of barbituric acid 1 (1 mmol) and trimethoxymethane 2 (3 mmol) in propanol (5ml) was stirred at reflux for 3 h. Then, the obtained mixture was allowed to stand overnight at room temperature for formation pure compound 3. 4.2. General procedure for the synthesis of 5-(((pyridin-4-ylmethyl)amino)methylene)pyrimidine2,4,6(1H,3H,5H)-trione 5 A mixture of compound 3 (1 mmol) and pyridin-3-ylmethanamine 4 (1 mmol) in ethanol (5ml) was stirred at reflux for 4 h. Then, the reaction mixture was allowed to cool at room temperature and poured into crushed ices, and the pure white precipitate 5 were filtered off. 4.3.
General
procedure
for
the
synthesis
of
1-aryl-4-((((1,3-dimethyl-2,4,6-
trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)pyridin-1-ium bromides 7a-n A solution of the compound 5 (1 mmol) and benzyl bromide derivatives 6a-n (1.4 mmol) in dry acetonitrile (10 ml) was heated under reflux condition for 2-3 h. The reaction progress was monitored by TLC. The obtained precipitate was filtered off and washed with dry acetonitrile (2 ml) and recrystallized in ethyl acetate to give pure compounds 7a-n. The synthesized compounds 7b, 7e, 7i-j, and 7m-n were obtained as a mixture of E and Z isomers and the percentage of E and Z isomers was calculated by NMR. The remaining compounds were obtained as E isomer. 4.3.1.
(E)-1-benzyl-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7a Yield 83%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3325, 2925, 2850, 1685, 1610. 1H NMR (500 MHz, DMSO-d6): δ = 10.55 (br. s, 1H, NH), 9.29 (s, 1H, H2 pyridine), 9.22 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.60 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.44 (m, 1H, =CH), 8.20 (m, 1H, H5 pyridine), 7.55 (dd, 2H, H2,6 phenyl, J1 = 7.2 Hz, J2 = 1.2 Hz ), 5.91 (s, 2H, CH2N+), 4.95 (s, 2H,
CH2N), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.9 (C=O), 162.6 (C=O), 160.6 (C=O), 152.0 (C), 145.5 (CH), 144.6 (CH), 144.4 (CH), 139.5 (C), 134.6 (C), 129.8 (CH), 129.6 (CH), 129.3 (CH), 128.6 (CH), 91.2 (C), 63.8 (CH2), 50.1 (CH2), 27.9 (CH3), 27.2 (CH3). MS: m/z = 445 [M+]. Anal. Calcd for C20H21BrN4O3: C, 53.94; H, 4.75; N, 12.58. Found: C, 53.81; H, 7.87; N, 12.41. 4.3.2. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(2-methylbenzyl)pyridin-1-ium bromide 7b Yield 88% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3356, 2985, 1695, 1680, 1605. NMR data for the major isomer: 1H NMR (500 MHz, DMSO-d6): δ = 10.56 (br. s, 1H, NH), 9.09 (s, 1H, H2 pyridine), 9.07 (m, 1H, H6 pyridine), 8.64 (d, 1H, H4 pyridine, J = 7.6 Hz), 8.43 (d, 1H, =CH, J = 14.0 Hz), 8.22 (t, 1H, H5 pyridine, , J = 7.2 Hz), 7.277.35 (m, 2H, H3,5 phenyl), 7.24 (d, 1H, H6 phenyl, J = 7.4 Hz), 7.16 (m, 1H, H4 phenyl), 5.98 (s, 2H, CH2N+), 4.96 (s, 2H, CH2N, J = 6.0 Hz), 3.13 (s, 6H, 2NMe), 2.31 (s, 3H, Me). 13C NMR (125 MHz, DMSO-d6): δ = 163.9 (C=O), 162.6 (C=O), 160.6 (C=O), 152.0 (C),145.6 (CH), 144.6 (CH), 144.5 (CH), 138.5 (CH), 132.5 (C), 131.8 (C), 129.6 (CH), 129.3(CH), 128.6 (C), 90.5 (C), 62.9 (CH2), 51.0 (CH2), 27.9 (CH3), 27.2 (CH3), 23.3 (CH3). MS: m/z = 459 [M+]. Anal. Calcd for C21H23BrN4O3: C, 54.91; H, 5.05; N, 12.20. Found: C, 55.06; H, 4.93; N, 12.32. 4.3.3.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(4-methylbenzyl)pyridin-1-ium bromide 7c Yield 84%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3358, 2981, 1698, 1683, 1604. 1H NMR (500 MHz, DMSO-d6): δ = 10.15 (m, 1H, NH), 9.11 (s, 1H, H2 pyridine), 9.08 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.72 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.38 (d, 1H, =CH, J = 14.5 Hz), 8.04
(m, 1H, H5 pyridine), 7.91 (d, 1H, H6 pyridine, J = 9.0 Hz), 7.16 (dd, 2H, Hα phenyl, J1 = 8.0 Hz, J2 = 2.5 Hz), 6.86 (d, 2H, Hβ phenyl, J = 8.0 Hz), 6.11 (s, 2H, CH2N+), 4.56 (m, 2H, CH2N), 3.12 (s, 6H, 2NMe), 2.33 (s, 3H, Me). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 161.4 (C=O), 160.6 (C=O), 156.0 (CH), 140.8 (C), 137.9 (CH), 135.9 (CH), 132.5 (CH), 129.2(CH), 127.6(CH), 124.0 (CH), 123.8 (CH), 123.5 (CH), 116.5 (CH), 116.3 (CH), 92.2 (C), 57.6 (CH2), 48.3 (CH2), 27.8 (CH3), 26.4 (CH3), 22.9 (CH3). Anal. Calcd for C21H23BrN4O3: C, 54.91; H, 5.05; N, 12.20. Found: C, 54.85; H, 4.97; N, 12.11. 4.3.4.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(3-fluorobenzyl)pyridin-1-ium bromide 7d Yield 83%; yellow solid; mp > 250 ºC; IR (KBr, cm-1): ν = 3340, 2925, 1705, 1687, 1612. 1H NMR (500 MHz, DMSO-d6): δ = 10.57 (br. s, 1H, NH), 9.24 (s, 1H, H2 pyridine), 9.21 (br.s, 1H, H6 pyridine, J = 5.5 Hz), 8.16 (br. s, 1H, H4 pyridine), 8.45 (d, 1H, =CH, J = 13.0 Hz), 8.20 (s, 1H, H5 pyridine), 7.51-7.28 (m, 4H, H2,4,5,6 phenyl), 5.91 (s, 2H, CH2N+), 4.94 (s, 2H, CH2N), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.2 (C=O), 151.5 (C), 145.3 (CH), 144.4 (CH), 143.8 (CH), 139.1 (C), 136.4 (C), 131.4 (CH), 128.2(CH), 125.1 (CH), 116.1 (CH), 115.6 (CH), 90.7 (C), 62.5 (CH2), 49.7 (CH2), 27.3 (CH3), 26.7 (CH3). MS: m/z = 463 [M+]. Anal. Calcd for C20H20BrFN4O3: C, 51.85; H, 4.35; N, 12.09. Found: C, 51.74; H, 4.29; N, 12.16. 4.3.5. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(4-fluorobenzyl)pyridin-1-ium bromide 7e Yield 91% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3341, 2927, 1702, 1685, 1614. NMR data for the major isomer: 1H NMR (500 MHz, DMSO-d6):
δ = 10.58-10.55 (m, 1H, NH), 9.27 (s, 1H, H2 pyridine), 9.24 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.61 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.20 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.67 (t, 1H, Hα phenyl, J = 6.0 Hz), 7.31 (t,1H, Hβphenyl, J = 8.5 Hz), 5.90 (s, 2H, CH2N+), 4.94 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 162.1 (C=O), 160.1 (C=O), 160.0 (C), 151.5 (C=O), 145.1 (CH), 144.2 (CH), 144.0 (CH), 143.8 (CH), 139.0 (C), 136.0 (C), 131.5 (CH), 130.3 (CH), 128.2 (CH), 128.1 (CH), 116.0 (CH), 90.7 (C), 62.4 (CH2), 49.6 (CH2), 27.3 (CH3), 26.7 (CH3). Anal. Calcd for C20H20BrFN4O3: C, 51.85; H, 4.35; N, 12.09. Found: C, 51.93; H, 4.41; N, 11.97. 4.3.6.
(E)-1-(2-chlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7f Yield 76%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3338, 2968, 2865, 1712, 1685, 1598. 1H NMR (500 MHz, DMSO-d6): δ = 10.61-10.55 (m, 1H, NH), 9.13 (br. s, 1H, H2 pyridine), 9.07 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.67 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.77 (d, 1H, H6 phenyl, J = 8.0 Hz), 7.50 (t, 1H, H5 phenyl, J =7.5 Hz), 7.43 (t, 1H, H4 phenyl, J = 7.5 Hz), 7.36 (d, 1H, H3 phenyl, J = 7.0 Hz), 6.00 (s, 2H, CH2N+), 4.95 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSOd6): δ = 162.1 (C=O), 160.1 (C=O), 151.5 (C=O), 145.7 (CH), 145.7 (CH), 145.5 (CH), 144.6 (CH), 144.3 (CH), 139.0 (C), 136.0 (C), 133.3 (CH), 132.9 (C), 131.6 (CH), 128.5(CH), 128.1 (CH), 123.4 (CH), 90.8 (C), 63.4 (CH2), 49.5 (CH2), 27.4 (CH3), 26.7 (CH3). Anal. Calcd for C20H20BrClN4O3: C, 50.07; H, 4.20; N, 11.68. Found: C, 50.14; H, 4.31; N, 11.75.
4.3.7.
(E)-1-(3-chlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7g Yield 83%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3345, 2978, 2898, 1680. 1H NMR (500 MHz, DMSO-d6): δ = 10.58-10.54 (m, 1H, NH), 9.22 (s, 1H, H2 pyridine), 9.18 (d, 1H, H6 pyridine, J = 5.5 Hz), 8.60 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.20 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.69 (s, 1H, H2 phenyl), 7.50-7.47 (m, 3H, H4,5,6 phenyl), 5.90 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J =6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 162.2 (C=O), 160.2 (C=O), 151.5 (C=O), 145.3 (CH), 144.4 (CH), 144.2 (CH), 143.8 (CH), 139.1 (C), 136.2 (C), 133.6 (CH), 130.9 (C), 129.5 (CH), 128.8(CH), 128.3 (CH), 127.5 (CH), 90.7 (C), 62.5 (CH2), 49.7 (CH2), 27.4 (CH3), 26.8 (CH3). Anal. Calcd for C20H20BrClN4O3: C, 50.07; H, 4.20; N, 11.68. Found: C, 49.94; H, 4.13; N, 11.53. 4.3.8.
(E)-1-(2,3-dichlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7h Yield 78%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3340, 2966, 2905, 1688, 1608. 1H NMR (500 MHz, DMSO-d6): δ = 10.59-10.55 (m, 1H, NH), 9.12 (br. s, 2H, H2, H6 pyridine), 8.67 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.77 (d, 1H, H6 phenyl, J = 7.5 Hz), 7.50 (t, 1H, H5 phenyl, J = 7.5 Hz), 7.41 (d,1H, H4 phenyl, J = 7.5 Hz), 6.08 (s, 2H, CH2N+), 4.95 (d, 2H, CH2N, J = 6.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 145.7 (CH), 144.6 (CH), 144.3 (CH), 139.0 (C), 133.8 (C), 132.6 (CH), 131.6 (C), 130.1 (C), 129.8 (CH), 128.9 (CH), 128.1 (CH), 90.7 (C), 61.6 (CH2), 49.6 (CH2), 27.3 (CH3), 26.8 (CH3). Anal. Calcd for C20H19BrCl2N4O3: C, 46.72; H, 3.72; N, 10.90. Found: C, 46.85; H, 3.83; N, 10.98.
4.3.9.
1-(3,4-dichlorobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7i Yield 82% (E isomer: 65% and Z isomer: 35%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3335, 2925, 1710, 1670, 1605. 1H NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.57-10.53 (m, 1H, NH), 9.22 (br. s, 2H, H2, H6 pyridine), 8.61 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.44 (d, 1H, =CH, J = 14.5 Hz), 8.19 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.93 (s, 1H, H3 phenyl), 7.74 (d, 1H, H6 phenyl, J = 8.0 Hz), 7.58 (d,1H, H5 phenyl, J = 8.0 Hz), 5.90 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 145.3 (CH), 144.2 (CH), 143.9 (CH), 139.0 (C), 134.6 (C), 132.3 (C), 131.2 (C), 129.4 (CH), 128.2 (CH), 128.1 (CH), 90.7 (C), 72.7 (CH2), 49.6 (CH2), 27.3 (CH3), 26.7 (CH3). Anal. Calcd for C20H19BrCl2N4O3: C, 46.72; H, 3.72; N, 10.90. Found: C, 46.61; H, 3.86; N, 11.04. 4.3.10.
1-(4-bromobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7j Yield 88% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3350, 2958, 2888, 1700, 1675, 1610. NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.55 (br. s, 1H, NH), 9.18 (s, 1H, H2 pyridine), 9.16 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.60 (br. s, 1H, H4 pyridine), 8.44 (d, 1H, =CH, J = 14.0 Hz), 8.19 (br.s, 1H, H5 pyridine), 7.66 (br. s, 2H, Hα phenyl), 7.51 (br. s, 2H, Hβ phenyl), 5.86 (s, 2H, CH2N+), 4.92 (s, 2H, CH2N, J = 6.0 Hz), 3.16 (s, 6H, 2NMe).
13C
NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.1
(C=O), 145.1 (CH), 144.2 (CH), 144.1 (CH), 143.8 (CH), 139.1 (C), 136.0 (C), 133.3 (CH), 131.2 (CH), 131.0 (CH), 128.1(CH), 122.9 (C), 90.7 (C), 62.6 (CH2), 49.6 (CH2), 26.9 (CH3), 26.8 (CH3). Anal. Calcd for C20H20Br2N4O3: C, 45.82; H, 3.85; N, 10.69. Found: C, 45.76; H, 3.71; N, 10.53.
4.3.11.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(2-nitrobenzyl)pyridin-1-ium bromide 7k Yield 75%; white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3352, 3008, 1715, 1685, 1622, 1580. 1H NMR (500 MHz, DMSO-d6): δ = 10.59-10.57 (m, 1H, NH), 9.11 (s, 1H, H2 pyridine), 9.09 (d, 1H, H6 pyridine, J = 6.0 Hz), 8.69 (d, 1H, H4 pyridine, J = 7.5 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.27-8.25 (m, 2H, H6 phenyl, H5 pyridine), 7.85 (t, 1H, H4 phenyl, J = 7.0 Hz), 7.76 (t, 1H, H5 phenyl, J = 7.0 Hz), 7.25 (d, 1H, H3 phenyl, J = 7.5 Hz), 6.27 (s, 2H, CH2N+), 4.96 (d, 2H, CH2N, J = 5.5 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.0 (C=O), 151.5 (CH), 147.6 (C), 145.5 (CH), 144.7 (CH), 144.4 (CH), 139.0 (C), 134.9 (C), 130.9 (C), 128.7 (CH), 128.1 (CH), 125.6 (CH), 125.5 (CH), 90.7 (C), 60.6 (CH2), 49.6 (CH2), 27.5 (CH3), 26.7 (CH3). MS: m/z = 490 [M+]. Anal. Calcd for C20H20BrN5O5: C, 48.99; H, 4.11; N, 14.28. Found: C, 48.88; H, 4.21; N, 14.34. 4.3.12.
(E)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)-1-(4-nitrobenzyl)pyridin-1-ium bromide 7l Yield 95%; yellow solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3338, 3025, 1690, 1610, 1590. 1H NMR (500 MHz, DMSO-d6): δ = 10.12 (m, 1H, NH), 9.17 (d, 1H, H6 pyridine, J = 6.0 Hz), 9.11 (s, 1H, H2 pyridine), 8.66 (m, 1H, H4 pyridine), 8.42 (d, 1H, =CH, J = 14.5 Hz), 8.11 (d, 2H, Hα phenyl, J = 8.0 Hz), 7.93 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.51 (d, 2H, Hβ phenyl, J = 8.0 Hz), 6.07 (s, 2H, CH2N+), 5.03 (s, 2H, CH2N), 3.12 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.7 (C=O), 161.2 (C=O), 160.2 (C=O), 157.3 (CH), 148.1 (C), 140.7 (C), 136.4 (CH), 132.2 (CH), 132.0 (CH), 129.7 (CH), 127.0 (CH), 128.3 (CH), 124.6 (CH), 124.5 (CH), 123.6 (CH), 123.5 (CH), 121.1 (CH), 120.6 (CH), 90.0 (C), 60.5 (CH2), 49.9 (CH2), 27.7 (CH3), 26.6 (CH3). Anal. Calcd for C20H20BrN5O5: C, 48.99; H, 4.11; N, 14.28. Found: C, 49.06; H, 4.03; N, 14.36.
4.3.13. 4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)methyl)amino)methyl)1-(2-fluoro-6-nitrobenzyl)pyridin-1-ium bromide 7m Yield 86% (E isomer: 62% and Z isomer: 38%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3348, 3028, 1705, 1680, 1618, 1589. NMR data for the major isomer: 1H NMR (500 MHz, DMSOd6): δ = 10.57-10.55 (m, 1H, NH), 9.12 (d, 1H, H6 pyridine, J = 6.0 Hz), 9.03 (d, 1H, H6 pyridine), 8.67 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.42-8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 8.16 (dd, 1H, H5 phenyl, J = 7.5 Hz), 7.90-7.93 (m, 2H, H3,4 phenyl), 6.17 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.0 Hz), 3.13 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO- d6): δ = 163.9 (C=O), 163.2 (C=O), 162.6 (C=O), 161.5 (JC-F =145 Hz), 152.1 (C), 149.9 (CH), 149.2 (CH), 146.1(CH), 144.46(CH), 144.2 (CH), 139.2 (C), 133.8 (CH), 128.4 (CH), 123.0 (CH), 122.8 (CH), 122.5 (CH), 122.4 (CH), 115.4 (CH), 115.2 (C), 91.2 (C), 54.4 (CH2), 49.6 (CH2), 27.5 (CH3), 26.8 (CH3). Anal. Calcd for C20H19BrFN5O5: C, 47.26; H, 3.77; N, 13.78. Found: C, 47.20; H, 3.82; N, 13.65. 4.3.14.
1-(4-cyanobenzyl)-4-((((1,3-dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
ylidene)methyl)amino)methyl)pyridin-1-ium bromide 7n Yield 86% (E isomer: 66% and Z isomer: 34%); white solid; mp > 250 ºC. IR (KBr, cm-1): ν = 3341, 3018, 2965, 17055, 1688, 1625, 1590. NMR data for the major isomer: 1H NMR (500 MHz, DMSO- d6): δ = 10.58-10.55 (m, 1H, NH), 9.23 (s, 1H, H2 pyridine), 9.19 (d, 1H, H6 pyridine, J = 6.0 Hz ), 8.63 (d, 1H, H4 pyridine, J = 8.0 Hz), 8.45 (d, 1H, =CH, J = 14.5 Hz), 8.22 (t, 1H, H5 pyridine, J = 7.0 Hz), 7.96 (d, 2H, Hα phenyl, J = 7.5 Hz), 7.71 (d, 2H, Hβ phenyl, J = 7.5 Hz), 6.00 (s, 2H, CH2N+), 4.93 (d, 2H, CH2N, J = 6.0 Hz), 3.15 (s, 6H, 2NMe). 13C NMR (125 MHz, DMSO-d6): δ = 163.5 (C=O), 162.1 (C=O), 160.2 (C=O), 151.5 (C), 145.4 (CH), 144.4 (CH), 144.0 (CH), 139.1 (C), 133.0 (CH), 129.6 (CH), 128.3 (CH), 128.2 (CH), 118.2 (CN), 112.0 (C),
90.7 (C), 62.5 (CH2), 49.6 (CH2), 27.5 (CH3), 26.8 (CH3). Anal. Calcd for C21H20BrN5O3: C, 53.63; H, 4.29; N, 14.89. Found: C, 53.57; H, 4.17; N, 14.76. 4.4. Urease Inhibitory Activity Urease inhibitory activity of the synthesized compounds 7a-n was evaluated exactly according to our pervious paper [20]. Results are expressed as mean ± SEM (n = 3 experiments). 4.5. Molecular docking study The three-dimensional (3D) protein structure of H. pylori urease (PDB ID: 1E9Y), determined by X-ray
crystallography,
was
retrieved
from
the
RCSB
Protein
Databank
(http://www.rcsb.org/pdb/home/home.do.). Then, extra molecules of the enzyme were removed using Accelrys discovery studio visualizer 4.0 (DSVisualizer, Accelrys Software Inc., San Diego, CA, USA, 2014.) and polar hydrogens and charges to the enzyme were added by Auto Dock Tools version 1.5.6. Protonation step corrects the ionization and tautomeric states of residues and modifies the total Kollman charges on the protein structure. 3D structures of the selected ligands were drawn by MarvineSketch 5.8.3, 2012, ChemAxon (http://www.chemaxon.com). The resulting protein and ligand structures were converted to pdbqt coordinate using Auto dock Tools and these pdbqt files used as input files for the AutoGrid program. AutoGrid determined precalculated atomic affinity grid maps for each atom type in the selected ligands, plus an electrostatics map and a separate desolvation map presented. All maps were provided with 0.375 Å spacing between grid points and grid box was set at 60 × 60 × 60 Å. Each docking was performed by 50 runs of the AutoDock search by the Lamarckian genetic algorithm. Lastly, the conformations with the lowest predicted binding free energy were selected for the each test compound. Graphic manipulations were visualized using Accelrys discovery studio visualizer 4.0 software.
4.6. Prediction of pharmacokinetic properties Prediction of the molecular properties of the synthesized compounds 7a-n was performed using the
online
servers
as
Molinspiration
(http://www.molinspiration.com/),
and
Molsoft
(http://www.molsoft.com/). References [1] E. Mentese, H. Bektas, B.B. Sokmen, M. Emirik, D. Çakır, B. Kahveci, Synthesis and molecular docking study of some 5, 6-dichloro-2-cyclopropyl-1H-benzimidazole derivatives bearing triazole, oxadiazole, and imine functionalities as potent inhibitors of urease, Bioorg. Med. Chem. Lett. 27 (2017) 3014-3018. [2] B.E. Lacy, J. Rosemore, Helicobacter pylori: ulcers and more: the beginning of an era, J. Nutr. 131 (2001) 2789S-2793S. [3] B. Ali, K.M. Khan, U.S. Kanwal, S. Hussain, M. Ashraf, M. Riaz, A. Wadood, M. Taha, S. Perveen, 1-[(4′-Chlorophenyl) carbonyl-4-(aryl) thiosemicarbazide derivatives as potent urease inhibitors: Synthesis, in vitro and in silico studies, Bioorg. Chem. 79 (2018) 363-371. [4] E. Menteşe, G. Akyuz, M. Emirik, N. Baltaş, Synthesis, in vitro urease inhibition and molecular docking studies of some novel quinazolin-4 (3H)-one derivatives containing triazole, thiadiazole and thiosemicarbazide functionalities, Bioorg. Chem. 83 (2019) 289-296. [5] M. Alomari, M. Taha, S. Imran, W. Jamil, M. Selvaraj, N. Uddin, F. Rahim, Design, synthesis, in vitro evaluation, molecular docking and ADME properties studies of hybrid bis-coumarin with thiadiazole as a new inhibitor of Urease, Bioorg. Chem. 92 (2019) 103235. [6] F. Rahim, M. Taha, H. Ullah, A.Wadood, M. Selvaraj, A. Rab, M. Sajid, S.A.A. Shah, N. Uddin, M. Gollapalli, Synthesis of new arylhydrazide bearing Schiff bases/thiazolidinone: αamylase, urease activities and their molecular docking studies, Bioorg. Chem. 91 (2019) 103112.
[7] W.K. Shi, R.C. Deng, P.F. Wang, Q.Q. Yue, Q. Liu, K.L. Ding, M.H. Yang, H.Y. Zhang, S.H. Gong, M. Deng, W.R. Liu, Q.J. Feng, Z.P. Xiao, H.L. Zhu, 3-Arylpropionylhydroxamic acid derivatives as Helicobacter pylori urease inhibitors: Synthesis, molecular docking and biological evaluation, Bioorg. Med. Chem. 24 (2016) 4519-4527. [8] P. Bhatt, M. Kumar, A. Jha, Design, Synthesis and Anticancer Evaluation of Oxa/Thiadiazolylhydrazones of Barbituric and Thiobarbituric Acid: A Collective In Vitro and In Silico Approach, Chem. Select. 3 (2018) 7060-7065. [9] B.D. Dhorajiya, R.G. Bhuva, B.Z. Dholakiya, Design, Synthesis and Comparative Study of Anti-Microbial Activities on Barbituric Acid and Thiobarbituric Acid based Chalcone Derivatives Bearing the Pyrimidine Nucleus, Chem. Sci. J. 7 (2016) 1000126. [10] Y.C. Jeong, M.G. Moloney, Antibacterial barbituric acid analogues inspired from natural 3acyltetramic acids; synthesis, tautomerism and structure and physicochemical propertyantibacterial activity relationships, Molecules 20 (2015) 3582-3627. [11] H.M. Faidallah, K.A. Khan, Synthesis and biological evaluation of new barbituric and thiobarbituric acid fluoro analogs of benzenesulfonamides as antidiabetic and antibacterial agents, J. Fluorine. Chem. 142 (2012) 96-104. [12] F. Rahim, M. Ali, S. Ullah, U. Rashid, H. Ullah, M. Taha, M.T. Javed, W. Rehman, A.A. Khan, O.U.R. Abid, M. Bilal, Development of bis-thiobarbiturates as successful urease inhibitors and their molecular modeling studies, Chin. Chem. Lett. 27 (2016) 693-697. [13] K.H. Khan, F. Rahim, A. Khan, M. Shabeer, S. Hussain, W. Rehman, M. Taha, M. Khan, S. Perveen, M.I. Choudhary, Synthesis and structure–activity relationship of thiobarbituric acid derivatives as potent inhibitors of urease, Bioorg. Med. Chem. 22 (2014) 4119-4123.
[14] A. Rauf, S. Shahzad, M. Bajda, M. Yar, F. Ahmed, N. Hussain, M.N. Akhtar, A. Khan, J. Jończyk, Design and synthesis of new barbituric-and thiobarbituric acid derivatives as potent urease inhibitors: Structure activity relationship and molecular modeling studies, Bioorg. Med. Chem. 23 (2015) 6049-6058. [15] B. Bano, K.M. Khan, A. Lodhi, U. Salar, F. Begum, M. Ali, M. Taha, S. Perveen, Synthesis, in vitro urease inhibitory activity, and molecular docking studies of thiourea and urea derivatives, Bioorg. Chem. 80 (2018) 129-144. [16] A. Barakat, H.A. Ghabbour, A.M. Al-Majid, R. Imad, K. Javaid, N.N. Shaikh, S. Yousuf, M.I. Choudhary, A. Wadood, Synthesis, X-ray crystal structures, biological evaluation, and molecular docking studies of a series of barbiturate derivatives, J. Chem. (2016) 1-11. [17] A. Barakat, A.M. Al-Majid, G. Lotfy, F. Arshad, S. Yousuf, M.I. Choudhary, S. Ashraf, Z. Ul-Haq, Synthesis and dynamics studies of barbituric acid derivatives as urease inhibitors, Chem. Cent. J. 9 (2015), 63-77. [18] H. Azizian, F. Nabati, A. Sharifi, F. Siavoshi, M. Mahdavi, M. Amanlou, Large-scale virtual screening for the identification of new Helicobacter pylori urease inhibitor scaffolds, J. Mol. Model. 18 (2012) 29172927. [19] M. Vosooghi, S. Farzipour, M. Saeedi, N.B. Shareh, M. Mahdavi, S. Mahernia, A. Foroumadi,
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Graphical abstract
Novel N,N-dimethylbarbituric-pyridinium derivatives as potent urease inhibitors: Synthesis, in vitro, and in silico studies Mahmood Biglar, Roghieh Mirzazadeh, Mehdi Asadi, Saghi Sepehri, Yosef Valizadeh, Yaghoub Sarrafi, Massoud Amanlou, Bagher Larijani, Maryam Mohammadi-Khanaposhtani*, Mohammad Mahdavi*
N+ R
O
N
H N
O N
Urease inhibitory assay
CH3 N+
O
O Compounds 7a-n
N
H N Compound 7b
O N
O
Docking study
A new series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated for their urease inhibitory activity. All the synthesized compounds (IC50 = 10.37 ± 1.0– 77.52 ± 2.7 μM) exhibited better inhibitory activity against urease when compared with standard inhibitor hydroxyurea (IC50 = 100.00 ± 0.2 μM).
Highlights
A novel series of N,N-dimethylbarbituric-pyridinium derivatives 7a-n was synthesized and evaluated as new urease inhibitors. These compounds showed urease inhibition superior to standard drug Hydroxyurea. Compounds 7a-7b and 7f-h were more potent than standard drug thiourea. Compound 7b was the most active compound. The most potent compounds interacted with important residues of urease active site.
Conflict of interest The authors have declared no conflict of interest.