
==== Front
J Enzyme Inhib Med Chem
J Enzyme Inhib Med Chem
Journal of Enzyme Inhibition and Medicinal Chemistry
1475-6366
1475-6374
Taylor & Francis

39258667
10.1080/14756366.2024.2386668
2386668
Version of Record
Research Article
Research Article
Challenging the Biginelli scaffold to surpass the first line antitubercular drugs: Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) inhibition activity and molecular modelling studies
M. S. el-Shoukrofy et al.
https://orcid.org/0000-0002-3016-5055
El-Shoukrofy Mai S. a
Atta Amal a
Fahmy Salwa a
Sriram Dharmarajan b
https://orcid.org/0000-0002-0418-456X
Shehat Michael G. c
https://orcid.org/0000-0001-7660-3287
Labouta Ibrahim M. a#
https://orcid.org/0000-0002-3456-941X
Mahran Mona A. a
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
b Medicinal Chemistry Research Laboratory, Pharmacy Group, Birla Institute of Technology and Science, Pilani, India
c Department of Microbiology and Immunology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
# This work is dedicated to the memory of Professor Ibrahim Labouta.

Supplemental data for this article can be accessed online at https://doi.org/10.1080/14756366.2024.2386668.

CONTACT Mai S. El-Shoukrofy mai.elshoukrofy@alexu.edu.eg Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, 21521, Egypt
11 9 2024
2024
11 9 2024
39 1 23866686 5 2024
6 7 2024
15 7 2024
KnowledgeWorks Global Ltd.10 9 2024
published online in a building issue10 9 2024
© 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
2024
The Author(s)
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. The terms on which this article has been published allow the posting of the Accepted Manuscript in a repository by the author(s) or with their consent.

Abstract

New Biginelli adducts were rationalised, via the introduction of selected anti-tubercular (TB) pharmacophores into the dihydropyrimidine (DHPM) ring of deoxythymidine monophosphate (dTMP), the natural substrate of Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt). Repurposing was one of the design rationale strategies for some selected mimics of the designed compounds. The anti-TB activity was screened against the Mtb H37Rv strain where 11a was superior to ethambutol (EMB), and was 9-fold more potent than pyrazinamide (PZA). Additionally, compounds 11b, 4a, 4b, 13a, 13b and 14a elicited higher anti-TB activity than PZA, showing better safety profiles than EMB against RAW 264.7 cells’ growth. The in vitro TMPKmt inhibition assay released compounds 11a, 11b and 13b as the most potent inhibitors. Docking studies presumed the binding modes and molecular dynamics (MD) simulation revealed the dynamic stability of 11a-TMPKmt complex over 100 ns. In silico prediction of the chemo-informatics properties of the most active compounds was conducted.

GRAPHICAL ABSTRACT

Keywords

Tetrahydropyrimidine
Biginelli
antitubercular activity
Mycobacterium tuberculosis thymidine monophosphate kinase
molecular dynamics simulation
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
==== Body
pmcIntroduction

Mycobacterium tuberculosis (Mtb) is the causative organism of tuberculosis (TB)1. TB is an airborne contagious disease that spreads via the respiratory route, causing pulmonary or extrapulmonary TB1,2. TB continues to rank among the top ten global killers2 infecting approximately 33% of the world’s population1,3. According to the World Health Organisation (WHO) Global TB Report 2022, 1.4 million people died from TB in 2021 where TB was ranked as the second cause of mortality from a single infection after COVID-19 in 2020, and 20214. Typically, the infection may not always result in active TB1 as Mtb may remain in a dormant state inside the macrophages1,5. Reactivation of such dormant form to a virulent form occurs as a result of weakness of the host’s immune system due to infection, use of certain drugs or aging1,5. The recommended primary treatment for TB is a combination of four antibiotics: isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (EMB)5,6. However, the long treatment duration, and patient non-compliance have led to the emergence of multidrug-resistant tuberculosis (MDR-TB), and extensively drug-resistant tuberculosis (XDR-TB)5,6. The WHO estimates that by 2050, drug-resistant tuberculosis strains will be responsible for nearly 25% of the total deaths attributed to the drug resistance3. The antibiotic-resistance and the ability of the pathogen to remain dormant hinder TB control3,5,7. Consequently, the urgent task for medicinal chemists is to develop new safe and efficient anti-TB agents that address novel targets and are less likely to develop resistance.

Nucleosides, and nucleotides are involved in protein biosynthesis as they are fundamental building blocks of DNA, and RNA3. Mycobacterium tuberculosis thymidine monophosphate kinase (TMPKmt) is a crucial enzyme in the synthesis of nucleotides1. Besides, it has low sequence similarity with the human isozyme (TMPKh) (22%)1. Thus, it was selected as a promising target to selectively inhibit mycobacterial growth. TMPK (TK) is a phosphotransferase that phosphorylates deoxythymidine (dT) into its monophosphorylated compound which is further converted to deoxythymidine triphosphate (dTTP), essential for DNA replication1,7. Hence, inhibiting TMPKmt causes dTTP pool imbalances, preventing Mtb replication leading to its death1. TMPKmt active site (PDB ID: 1G3U) was identified based on the coordinates of its complex with the natural substrate deoxythymidine momophosphate (dTMP) (I)8 where the thymine ring is recognised as the principal binding motif1. Structurally, the reported TMPKmt inhibitors are either thymine-containing or non-thymine-containing2. Thymine-containing inhibitors are further classified as thymine nucleoside, and thymine non-nucleoside derivatives2. Thymine is a pyrimidine nucleobase that is a promising core scaffold endowed with antitubercular (anti-TB) activities2,6,9,10. The pyrimidine-containing compounds SPR720 (II) TBA-7371 (III) are currently being evaluated in clinical trials as anti-TB agents2 (Figure 1). Several series of thymidine-nucleosides, with various modifications of the thymine ring, the ribose sugar, and the phosphate moieties were reported as potent, and selective TMPKmt inhibitors1–3 of which compounds IVa,b elicited potent TMPKmt inhibition11,12 (Figure 1). Moreover, several thymine-based non-nucleoside derivatives V-VIII exerted potent TMPKmt inhibition6,13–18 (Figure 1). These inhibitors share common features, including the thymine ring with variable substituents at position 5 (Moiety A), the aryl fragment at position 1 in some inhibitors (Moiety B), different chemical groups at position 4 (Moiety C), and the oxo or thioxo moieties at C2 (Moiety D) in other inhibitors (Figure 1).

Figure 1. Structures of dTMP (I); Pyrimidines (II, III) that are under clinical evaluation as anti-TB agents; Thymidine-nucleosides exerting TMPKmt inhibition (IV), and Thymine-based non-nucleosides exerting TMPKmt inhibition (V-VIII).

As part of our ongoing program to discover potent, and selective thymine based non-nucleoside TMPKmt inhibitors18, we further modify the TMPKmt natural substrate, dTMP (I), scaffold via multiple chemical modifications including variations at moieties A, B, C, and D. We decided to preserve the basic thymine ring, represented by the tetrahydropyrimidine (THPM) ring, and to attach different well known anti-TB pharmacophores at variable positions of the ring. Such optimizations could produce more bioactive, and effective hybrid molecules that are expected to have better anti-TB and TMPKmt inhibition activities6,18. Among these pharmacophores, the 4-fluorophenyl moiety that was included in several potent anti-TB agents19, where the activity was attributed to the similarity between fluorine, and hydrogen in steric properties20, and to the fluorine contribution in increasing lipid solubility, and subsequently penetration through the mycobacterial cell membrane21. In addition, propargyl22, pyrazolyl23,24, oxindolinyl19,25, quinoxalinyl26 and benzene sulfonamide6,27 moieties were proved to improve lipophilicity and to enhance the anti-TB activity. The 5-nitro-2-furanyl group was also essential for excellent pharmacokinetic properties, and for enhanced anti-TB activity of several agents owing to the presence of the furan ring, and to the polarity and electron-withdrawing properties of the nitro group28,29. These pharmacophores were included in several reported potent anti-TB agents IX-XV (Figure 2).

Figure 2. Design of the target THPM derivatives.

Our rational strategy first aimed to modify C4 of the THPM-based Biginelli adduct (moiety C) by introducing lipophilic anti-TB pharmacophores, including 4-fluorophenyl, propargyloxyphenyl, 1,3-diphenylpyrazole, and 5-nitro-2-furanyl, to study the impact of variation of lipophilicity on the anti-TB, and TMPKmt inhibition activities (Figure 2). Second, because carboxamide-containing compounds have considerably improved the anti-TB activity30, the variation of moiety A via the introduction of ester or different amide groups (CONH2 or CONHC6H5) at C5 of the THPM ring was suggested to compare the variable activities upon such modifications. Thirdly, the acetylation of position 1 of the THPM ring (moiety B) in some of the designed compounds was proposed. Fourth, the substitution at THPM-C2 (moiety D) with substituents of variable lipophilicity (=O, =S, =N-NH2 or = N-NR3) was also planned (Figure 2). The incorporation of oxindolinyl, quinoxalinyl and phenyl sulphonyl moieties into THPM-C2 (moiety D), via a two-atom spacer, was also within our consideration. Such substituents might improve the hydrophilic-lipophilic balance, and consequently the pharmacokinetic profile as well as the drug-likeness of the designed compounds.

Literature survey revealed that mimics of our compounds (XVI) showed heterogeneous biological activities31–39 (Figure 2). Hence, our design rationale was extended to explore their potential anti-TB and TMPKmt inhibition activities. Such repurposing strategy is an effective method utilised in several drug discovery disciplines40.

All the designed compounds were synthesised, and evaluated for their anti-TB activity against the Mtb H37Rv strain. Structure Activity Relationship (SAR) studies were discussed to compare the effect of such optimizations of Biginelli scaffold on the anti-TB activity. The most active anti-TB compounds were further investigated for their cytotoxicity against the growth of RAW 264.7 cells and also for their possible in vitro inhibition of TMPKmt. The mode of inhibition of TMPKmt by the most active compound was also investigated. Molecular modelling studies, including docking, molecular dynamics (MD) simulation and in silico prediction of the chemo-informatics properties, were conducted for the most active compounds.

Results and discussions

Chemistry

The designed compounds were prepared according to the steps shown in Schemes 1 and 2. Scheme 1 shows the application of the classical Biginelli reaction to obtain various 1-, 4-, and 5-substituted THPM compounds. The intermediates 1a,b, 2a,b, and 3a,b were prepared by refluxing ethyl acetoacetate with urea or thiourea, and the appropriate aldehyde in ethanol containing few drops of conc. HCl. The reaction of 5-nitro-2-furfural, urea or thiourea with ethyl acetoacetate using AlCl3 as a catalyst, furnished 5-nitro-2-furanyl derivatives 4a, 4b. The N-acetylated derivatives 5a, 5b, 6a, 6b, 7a, and 7b were prepared by refluxing the corresponding THPM derivatives 1a, 1b, 2a, 2b, 3a, and 3b in acetic anhydride, while trials to prepare the N-acetylated derivatives of compounds 4a, and 4b went in vein.1H-NMR spectra of compounds 5a, 5b, 6a, 6b, 7a, and 7b revealed a new upfield singlet at 2.25–2.65 ppm, assigned for the acetyl methyl protons while the 13C-NMR spectra of these compounds showed the acetyl C = O signals at 173.6–178.1 ppm.

Scheme 1. Synthesis of the target compounds 1–11.

Reagents and conditions: (i) Ethyl acetoacetate, urea or thiourea, EtOH/conc.HCl, reflux for 3–5 h, (1a,b: 69–72%; 2a-b: 64–80%, 3a-b: 65–67%); (ii) Ethyl acetoacetate, urea or thiourea. Polyethylene glycol, AlCl3, stir at 45°, 3 h, (4a-b: 41–45%); (iii) Ac2O, reflux for 1–2 h, (5a-b: 84–85%; 6a-b: 70–72%, 7a-b: 61–64%); (iv) Acetoacetanilide, urea or thiourea, acetonitrile/conc.HCl, reflux for 16h, (8a-b: 48–62%; 9a-b: 72–76%, 10a-b: 62%); (v) acetoacetamide, urea or thiourea, polyethylene glycol, AlCl3, stir at 45°, 3 h, 11a-b: 67–68%.

The N-phenyl carboxamide derivatives 9a, and 9b were obtained upon refluxing urea or thiourea with 2-(prop-2-yn-1-yloxy)benzaldehyde41 using acetoacetanilide instead of ethyl acetoacetate while the reaction of 5-nitro-2-furfural, urea or thiourea with acetoacetamide, using AlCl3 as a catalyst, furnished the carboxamide derivatives 11a, and 11b, respectively. An upfield D2O-exchangeable singlet assigned for NH2 protons was detected at 3.52 ppm in the 1H-NMR spectra of compounds 11a, and 11b while the amide NH resonated as a downfield D2O-exchangeable singlet at 9.66 ppm in the 1H-NMR spectra of compounds 9a,b. Moreover, signals for the amide C = O were shown at 164.9–174.7 ppm in the 13C spectra of compounds 9a, 9b, and 11a.

Scheme 2 illustrates the variation of the substituents at position-2 (moiety D) of the THPM ring, with 5-ethyl ester (moiety A), unsubstituted NH (moiety B), and either 4-fluorophenyl or 4–(1,3-diphenyl-1H-pyrazol-4-yl) scaffolds (moiety C). The 2-hydrazono derivatives 12a, and 12b were the key intermediates used to prepare the designed derivatives. Compounds 12a, and 12b were prepared by reacting the corresponding 2-thioxo THPM derivatives 1b, and 3b with hydrazine hydrate 99% in 1,4-dioxane, using a catalytic amount of conc. sulphuric acid. Structures of the 2-hydrazono derivatives 12a, and 12b were confirmed by the appearance of characteristic stretching absorption bands for the NH2 group at 3244–3325, and 3175–3179 cm-1 in their IR spectra. Moreover, the 1H-NMR spectra of compounds 12a, and 12b revealed a characteristic upfield D2O-exchangeable singlet assigned for NH2 protons at 3.34–3.58 ppm in addition to two downfield D2O-exchangeable singlets assigned for two NH protons at 9.66–9.74, and 10.28–10.36 ppm.

Scheme 2. Synthesis of the designed compounds 12–15.

Reagents and conditions: (i) NH2 NH2.H2O (99%), dioxane/C.H2SO4, reflux, 4 h (67–69%); (ii) isatin or 5-methylisatin, abs. EtOH, reflux, 6 h (70–71%); (iii) quinoxaline-2,3(1H,4H)-dione, abs.EtOH, reflux, 6 h (68–70%); (iv) benzenesulfonyl chloride, dioxane/(C2H5)3N, reflux, 3 h (63–66%).

The oxindolinyl 13a-c, and the quinoxalinyl 14a,b derivatives were obtained upon reacting the 2-hydrazono derivatives 12a, and 12b with equimolar amounts of isatin, 5-methylisatin or quinoxaline-2,3(1H,4H)-dione in absolute ethanol. Furthermore, reacting compounds 12a, and 12b with benzenesulfonyl chloride in dry 1,4-dioxane containing triethyl amine afforded phenylsulfonyl derivatives 15a, and 15b.

The 1H-NMR spectra of 13a-c, 14a, 14b, 15a, and 15b lacked the upfield D2O-exchangeable singlet for NH2 protons present in their precursors 12a, and 12b, and showed three downfield D2O-exchangeable singlets assigned for three NH protons at 9.50–9.68, 10.16–10.37, and 10.94–11.87 ppm while the fourth NH of 14a, and 14b was included within the aromatic multiplet. An upfield singlet, assigned for the C5-methyl protons, was shown at 2.51 ppm in the 1H-NMR spectrum of 13b. The oxindolinyl protons of 13a were shown as two triplets, and two doublets, while those of 13b resonated as a doublet, and a multiplet, whereas the oxindolinyl, and quinoxalinyl protons of 13c, 14a, and 14b were included within the aromatic multiplet in the corresponding 1H-NMR spectra.

The EI-MS of the designed compounds showed both molecular ion, and base peaks at their expected values. The detailed spectral data of the newly designed compounds are provided in the experimental section, and the spectra are presented in the supplementary information (SI).

In vitro biological evaluation

In vitro anti-TB screening

All the synthesised compounds were evaluated for their in vitro anti-TB activity against the Mtb H37Rv strain using the Microplate Alamar Blue Assay (MABA)42. EMB, and PZA were used as references. The results, presented in Table 1, indicated that the 2-oxo derivative 11a was the most active anti-TB agent (MIC = 5.86 µM), being more potent than EMB, and 9-fold more potent than PZA. Moreover, nine compounds, in the order 11b > 4b > 4a > 14a > 13b > 13a > 14b > 15b > 13c, elicited higher anti-TB activity than PZA (Supplementary Figure 1). In this context, compound 11b (MIC = 11.07 µM) was 4.5-fold more potent than PZA, while the ester derivatives 4a, and 4b exhibited about 2.5-fold the activity of PZA with MIC values of 20.07, and 21.17 µM, respectively. Compounds 13a, 13b and 14a elicited approximately twice the activity of PZA with MIC values of 29.66, 28.70, and 28.64 µM, respectively. Compounds 13c, 14b, and 15b were slightly more potent than PZA showing MIC values in the range 44.59–45.82 µM. Other compounds revealed moderate to weak anti-TB activity. It is to be noted that PZA is a prodrug that is converted to its active form (pyrazinoic acid) by pyrazinamidase enzyme under acidic conditions43. Thus, the activity of PZA is pH-dependent where it elicited MIC values of 6.25–12.5 µg/mL (50.76–101.53 μM) in acidic media while MIC values get higher at neutral conditions44.

Table 1. Results of in vitro anti-TB screening (MIC, µM) against the Mtb H37Rv strain using MABA.

Compound ID	MIC (µM)	
1a	89.83	
1b	84.93	
2a	79.53	
2b	75.66	
3a	62.11	
3b	59.73	
4a	21.17	
4b	20.07	
5a	78.04	
5b	74.32	
6a	70.15	
6b	67.12	
7a	56.24	
7b	54.28	
8a	76.84	
8b	73.22	
9a	69.17	
9b	66.63	
10a	55.61	
10b	53.69	
11a	5.86	
11b	11.07	
12a	85.52	
12b	60.02	
13a	29.66	
13b	28.70	
13c	45.82	
14a	28.64	
14b	44.59	
15a	57.80	
15b	44.91	
PZA	50.77	
EMB	7.64	

Structure-Activity Relationship (SAR) studies (Figure 3) revealed that modification of moiety C via the introduction of a 5-nitro-2-furanyl moiety to the THPM-C4 furnished the most potent anti-TB agents (compounds 4a, 4b, 11a, and 11b), with the carboxamide derivatives 11a, and 11b being more potent than the ester analogues 4a, and 4b. Moreover, the 2-oxo derivative 11a was the most active compound in this study, declaring superior anti-TB activity than its 2-thioxo analogue 11b.

Figure 3. SAR study of the anti-TB activity of the designed compounds against the MtbH37Rv strain.

Although the acetylated derivatives 5a,b, 6a,b, and 7a,b were less potent than PZA, they elicited enhanced anti-TB activity than their precursors 1a,b, 2a,b, and 3a,b, indicating the impact of modification of moiety B through acetylation on improving the anti-TB activity. Studying the impact of variation of moiety A, revealed that the carboxamide derivatives 8a,b, 9a,b, and 10a,b were more potent than their ester counterparts 1a,b, 2a,b, and 3a,b, although being less potent than PZA. It is to be noted that the thioxo derivatives 1b-10b exerted slightly more potent activity than their oxo analogues 1a-10a.

Modification of moiety D via the incorporation of oxindolinyl, quinoxalinyl or phenylsulfonyl moieties through (=N-N=) spacer, significantly enhanced the anti-TB activity where compounds 13a-c, 14a,b, and 15a,b elicited higher anti-TB activity than their precursors 12a,b. Comparing the effect of modification of moiety C within compounds 13a-c, 14a,b, revealed that the incorporation of 4-fluorophenyl moiety at THPM-C4 ring was of better impact on the anti-TB activity than introducing 1,3-diphenylpyrazolyl moiety at the same position. In this context, compounds 13a, 13b, and 14a followed the nitrofuranyl derivatives 11a,b, and 4a,b in the decreasing order of the anti-TB activity, eliciting approximately twice the activity of PZA. Additionally, compounds 13a, 13b, and 14a showed higher anti-TB activity than their pyrazolyl analogues 13c, and 14b that elicited slightly more potent activity than PZA. The 5-methyloxindolinyl derivative 13b was the most potent within the oxindolinyl series exhibiting MIC value of 28.70 µM. This is consistent with the reported data illustrating the role of properly positioned lipophilic group (as methyl group) in improving the physicochemical properties, and hence enhancing the activity45.

Surprisingly, the activity was reversed within the phenylsulfonyl series where the incorporation of the 1,3-diphenylpyrazolyl moiety into THPM-C4 was better for the anti-TB activity where compound 15b was a more potent anti-TB agent than its 4-fluorophenyl counterpart 15a. Again, compound 15b showed slightly more potent activity than PZA, with MIC value of 44.91 µM.

These SAR findings confirmed our rationale for optimising the Biginelli scaffold, where all the final compounds were of more potent anti-TB activity than their precursors. Additionally, the incorporation of oxindolinyl, quinoxalinyl or phenylsulfonyl moieties through (=N-N=) spacer significantly enhanced the anti-TB activity.

In vitro cytotoxicity assay

Compounds 4a, 4b, 11a, 11b, 13a, 13b, and 14a, exhibiting the highest activity in the in vitro anti-TB screening, were evaluated for their cytotoxicity against RAW 264.7 cells’ growth at a concentration of 25 μg/mL using the MTT assay46,47. Results, presented in Figure 4(a) demonstrated that the most active compounds were safer, and less cytotoxic to RAW 264.7 cells than EMB eliciting % inhibition in the range 17.09 − 19.87% compared to 23.57% for EMB. Moreover, compound 11a, showing the least % inhibition, demonstrated very low cytotoxicity against RAW 264.7 cells with IC50 value of 932.7 µM and a selectivity index (SI)17 of 159. Thus, compound 11a was predicted as a promising and safe anti-TB candidate.

Figure 4. (a) In vitro cytotoxicity assay against RAW 264.7 cells (% inhibition) of the most active compounds, and EMB, (b) In vitro TMPKmt inhibition activity (IC50, µM) of the most active compounds, and dTMP.

In vitro TMPKmt inhibitory activity

Compounds 4a, 4b, 11a, 11b, 13a, 13b, and 14a were further screened for their in vitro inhibition of TMPKmt according to the reported assay method48,49 using dTMP as a reference. Results presented in Figure 4(b) revealed that the 2-oxo derivative 11a exhibited inhibitory activity comparable to that of dTMP, with an IC50 value of 0.08 µM. Furthermore, 5-methyl-2-oxindolinyl derivative 13b, and 2-thioxo analogue 11b were slightly less active than dTMP, with IC50 values of 0.10, and 0.19 µM, respectively.

Kinetic analysis of the inhibition of TMPKmt

The most potent compound 11a was investigated for its mode of inhibition of TMPKmt. The potential of 11a to inhibit the free enzyme and enzyme-substrate complex was expressed in terms of enzyme inhibitor (EI) and enzyme-substrate-inhibitor (ESI) constants, Ki and Ki′, respectively50. The Lineweaver–Burk plot of 1/V versus 1/[S] in the presence of different concentrations of 11a showed a series of straight lines that intersected within the second quadrant (Figure 5(a)). The analysis revealed that Michaelis-Menten constant (Km) increased to new values and then became constant with decreasing values of maximum velocity (Vmax) in the presence of increasing concentrations of 11a (Table 2). This behaviour indicated that compound 11a acts as a mixed inhibitor that inhibits TMPKmt competitively, forming an EI complex, and also interrupts the ESI complex in a non-competitive manner. Ki, and Ki′ were obtained from the secondary plots (Figure 5(b,c)) where a lower value of Ki than Ki′ indicated stronger binding between 11a and TMPKmt suggesting preferred competitive over non-competitive manners.

Figure 5. Kinetic study of TMPKmt inhibition by compound 11a. (a) Lineweaver-Burk plot in the absence and presence of different concentrations of 11a. (b) The secondary plot between the slopes versus various concentrations of 11a. (c) The secondary plot between the vertical intercepts versus various concentrations of 11a.

Table 2. Kinetic constants and inhibition constants of compound 11a on TMPKmt.

Code	Concentration (µM)	Vmax
(ΔA /min)	Km (µg/mL)	Inhibition type	Ki
(µM)	Ki′
(µM)	
11a	0.1	340118	48.33	Mixed inhibitor	6.552	15.571	
 	1	239455	80.10	 	 	 	
 	10	193204	80.20	 	 	 	

Molecular modelling studies

Docking into TMPKmt crystal structure

Compounds 11a, 11b, and 13b, which exhibited the highest in vitro TMPKmt inhibitory activity, were docked into the active site of TMPKmt (PDB ID: 1G3U)8 to analyse their probable binding modes. The docking study was performed using MOE, version 2019.01 software51. The binding energies, and interactions are listed in Supplementary Table 1. The 2D and 3D binding interactions are illustrated in Figure 6. The overlay of the modelled 3D binding poses of 11a, 11b, and 13b with the modelled 3D binding pose of dTMP was shown in Figure 7. The overlay of the modelled 2D binding poses of 11a, 11b, and 13b with the modelled 2D binding pose of dTMP was shown in Supplementary Figure 2.

Figure 6. The docked poses of dTMP, 11a, 11b, and 13b into TMPKmt active site (PDB ID: 1G3U). (a), (c), (e), (g) The 2D binding interactions of dTMP, 11a, 11b, and 13b, respectively with the amino acid residues. (b), (d), (f), (h) The 3D binding interactions of dTMP, 11a, 11b, and 13b, respectively with the amino acid residues.

Figure 7. The overlay of the modelled 3D docking poses of dTMP, 11a, 11b, and 13b into TMPKmt active site (PDB ID: 1G3U). (a) Comparison of the modelled 3D binding mode of the co-crystallised ligand dTMP (colored with red) with its superimposed docking conformation (colored with green). (b), (c), (d) Comparison of the modelled 3D binding modes of compounds 11a (colored with blue), 11b (colored with cyan), and 13b (colored with yellow) superimposed with the co-crystallised ligand dTMP (colored with green), respectively.

The redocked dTMP exhibited a binding pattern similar to that reported by I. Li de la Sierra et al.8, where several hydrogen bonds were observed with Asn100, Arg74, Asp9, Tyr39, and Arg95 residues. Additionally, Tyr165, Asp163, Arg160, Lys13, Phe36, and Pro37 residues were involved in hydrogen bonds through the water molecules W1002, W1009, W1050, W1014, W1026, and W1024, respectively. Moreover, a hydrophobic interaction was observed between the DHPM ring of dTMP, and Phe70 residue while both W1009, and W1050 coordinate Mg2+ ion in metallic bonds.

The overlay of the modelled 3D docking poses (Figure 7) revealed that the THPM rings of 11a, and 11b overlaid the DHPM ring of dTMP. Moreover, the furan ring of 11a overlapped with the tetrahydrofuran of dTMP, and the nitro groups of 11a, and 11b superimposed with the phosphate moiety of dTMP. Furthermore, the oxindolinyl moiety of 13b partially overlapped both the DHPM, and tetrahydrofuran rings of dTMP, while the two-atom spacer overlaid the phosphate moiety of dTMP. This superimposition had an impact on the binding modes of 11a, 11b, and 13b where they showed several interactions with TMPKmt, similar to those observed with the natural substrate, dTMP.

As shown in Figure 6, the THPM ring of 11a, and 11b formed two hydrogen bonds with Asn100 residue, while the 2-oxo-O and 2-thioxo-S atoms were involved in two hydrogen bonds with Tyr165 residue either directly or through the water bridge (W1002). Moreover, the 2-thioxo-S of 11b showed an extra hydrogen bond with Phe70 residue at a distance of 3.86 Å. Furthermore, Arg95, and Tyr39 residues were involved in three hydrogen bonds (as shown in the 3D docking pose) with the NO2-O atoms of 11a, and 11b. In addition, the NO2-O atom of 11b was engaged in a hydrogen bond with Arg160 residue through the water molecule (W1050) that was also involved in a metallic bond with the Mg+2 ion. Additionally, a metallic bond was formed between NO2-O atom, and the Mg+2 ion in both compounds 11a, and 11b. On the other hand, the carboxamide C = O-O, and NH2-N of 11a showed two additional hydrogen bonds with Pro37, and Phe36 residues at distances of 3.09, and 3.08 Å, respectively. Furthermore, hydrophobic interactions were shown between the furan rings of 11a, and 11b with both Phe70, and Pro37 residues.

The binding mode of 13b was quite different. In this context, Tyr39 residue was involved in hydrogen bonds with THPM-N3 and one of the hydrazono-N atoms, while the second hydrazono-N atom was involved in hydrogen bonds with Phe36, and Pro37 residues through the water molecules W1026, and W1026, W1024, respectively. Furthermore, the 2-oxindolinyl-O was involved in a hydrogen bond with the Arg95 residue, and in a metallic bond with the Mg+2 ion. Additionally, Tyr103, and Tyr39, were engaged in hydrophobic interactions with indolinyl-C7, and 4-fluorophenyl-C2, respectively.

These similar binding interactions of compounds 11a, 11b, and 13b with that of the natural substrate dTMP was reflected on their in vitro TMPKmt inhibition activity where compound 11a was nearly equipotent to dTMP with an IC50 value of 0.08 µM (compared to IC50 = 0.07 µM for dTMP), while compounds 11b, and 13b showed IC50 values of 0.19, 0.01 µM, respectively. The improved activity of 11a over 11b could be attributed to the extra hydrogen bonds formed via its carboxamide moiety.

Figure 8 illustrates the cartoon representation of the molecular surface of TMPKmt (PDB ID: 1G3U) demonstrating the orientation of compounds 11a, 11b, 13b, and the natural substrate dTMP in the binding pocket, and confirming that all compounds are within the same binding pocket. Figure 8 also illustrates the comparison of the modelled 3D binding modes of compounds 11a, 11b, 13b overlaying the modelled 3D binding mode of dTMP where all compounds are well overlapped with dTMP.

Figure 8. The binding mode of compounds 11a, 11b, 13b, and dTMP in TMPKmt (PDB ID: 1G3U). (a) The cartoon representation of the molecular surface of TMPKmt (displayed on a gray surface) showing the binding mode of compounds 11a (colored with blue), 11b (colored with cyan), 13b (colored with yellow), and dTMP (colored with green) in the same binding pocket. (b) Zoomed-in view of TMPKmt active site, shown in the same orientation as in (a). (c) Comparison of the modelled 3D binding mode of compounds 11a (colored with blue), 11b (colored with cyan), and 13b (colored with yellow) superimposed with the co-crystallised ligand dTMP (colored with green).

The contact preference and electrostatic potential surface maps of compounds 11a, 11b, 13b, and dTMP were generated using MOE, version 2019.01 software51 and illustrated in Figure 9. Contact preference maps demonstrate the preferred locations of hydrophobic, and hydrophilic ligand atoms in green, and purple, respectively52. The contact preference data showed that the DHPM of dTMP, the THPM, furan (of 11a, and 11b), the indolinyl, and 4-fluorophenyl moieties (of 13b) represent hydrophobic sites. Moreover, the phosphate, the ribose 3′-OH of dTMP in addition to the oxygen atoms of the nitro, carboxamide, oxindolinyl, and 5-ester moieties were hydrophilic sites. Electrostatic potential surface maps determine the charge distribution around molecules, where the red sites indicate the highly electronegative regions that accept hydrogen bonds, and are prone to electrophilic reactions, while the blue sites are the electron-deficient regions53,54. The negative electrostatic potential (red shades) was located mainly around oxygen, sulphur and fluorine atoms while the OH, NHs, and NH2 groups were the main contributors to the positive electrostatic potential (blue shades).

Figure 9. (a) Contact preference maps for compounds 11a, 11b, 13b, and dTMP in the TMPKmt (PDB ID: 1G3U) binding pocket showing the hydrophobic and hydrophilic moieties (colored with green, and purple, respectively), (b) Electrostatic potential surface maps for compounds 11a, 11b, 13b, and dTMP in the TMPKmt (PDB ID: 1G3U) binding pocket showing the negative, and positive electrostatic potential sites (colored with red, and blue, respectively).

Molecular dynamics simulation of 11a-TMPKmt

A post-docking Molecular Dynamics (MD) simulation was employed for the best pose of 11a-TMPKmt docking complex, for 100 ns at room temperature, to further evaluate its dynamic stability. MD simulation was performed using the open-source software, GROMACS55,56. Figure 10(a) shows the Root mean square deviation (RMSD) for 11a-TMPKmt complex, TMPKmt backbone, and the ligand 11a. The complex and backbone RMSD showed great stability and very low values after 10 ns of the simulation time, while the ligand RMSD was stable after 20 ns up to the end of the simulation. The average RMSD values were 2.250 ± 0.151, 1.453 ± 0.112 and 1.538 ± 0.317 Å for the complex, backbone and ligand, respectively. Furthermore, the Root mean square fluctuation (RMSF) (Figure 10(b)) was calculated for the protein complex based on “C-alpha” atoms where the fluctuation intensity remains below 2.0 Å through most of the simulation with an average RMSF value of 0.0834 Å. The radius of gyration analysis (rGyr) (Figure 10(c)) was also consistent with RMSD, and RMSF results for 11a-TMPKmt complex, showing stability and very little fluctuations (< 0.7 Å). The recorded rGyr values varied from 16.3 to 16.9 Å throughout the simulation time. Figure 10(d) illustrates the total number of hydrogen bonds where 11a displayed an average of three hydrogen bonds with TMPKmt with a maximum of seven hydrogen bonds throughout 100 ns simulation time. The average centre of mass (COM) distance between 11a and TMPKmt (Figure 10(e)) showed a very stable pattern with little to no fluctuations, indicating the stability of the ligand in the TMPKmt binding site. The COM distance varied between 6.0 to 7.4 Å only from the beginning to the end of the simulation with an average of 6.706 ± 0.231 Å. Furthermore, the average computed solvent accessible surface area (SASA) of the simulated 11a-TMPKmt complex was 1060.817 ± 18.849 Å2 with nearly constant plot over the simulation time (Figure 10(f)).

Figure 10. Predicted molecular dynamics simulation criteria for 11a-TMPKmt complex over 100 ns. (a) RMSD of 11a-TMPKmt complex, TMPKmt backbone and 11a. (b) RMSF, (c) rGyr, (d) hydrogen bonding analysis, (e) COM and (f) SASA analysis of 11a-TMPKmt complex over 100 ns.

To further evaluate the binding between TMPKmt and 11a, trajectory visualisation and analysis of detailed hydrogen bonding information and contact frequency (CF) during simulation was performed using the visual molecular dynamics (VMD)57. Figure 11(a) showed the results of the CF analysis where a contact is defined as the amino acid being within a distance cut-off of 4 Å. The residues eliciting the highest CF% were Arg74, Phe70, Tyr103, Asn100, Arg95, Ser99, and Tyr165 with CF% ranging from 97.30–100%. Ligand interactions network was then mapped (Figure 11(b)). Multiple hydrogen bonds were shown with Arg107, Arg74, Arg95, Ser99, Pro37, and Tyr103 residues. The ligand’s core was also stabilised by an ionic bond with Arg95 residue. Several hydrophobic interactions were noticed with Arg95, Tyr103, Tyr165, Ala67, Phe70, and Met66 residues. Thus, post-simulation visual analysis of the trajectory indicated that 11a remained bound to TMPKmt and did not separate from it. These results collectively confirmed the reliability of the docking pose of 11a and provided additional information concerning the dynamic stability of the 11a-TMPKmt simulated complex.

Figure 11. (a) CF analysis of residues within a distance cut-off of 4 Å from 11a. (b) ligand interactions network of 11a-TMPKmt complex during 100 ns MD simulation. Dotted lines represent conventional hydrogen bond (green), carbon hydrogen bond (yellow) or π-donor hydrogen bond (cyan), attractive charge (orange), π-π stacked (purple), π-alkyl (violet), and alkyl interactions (pink).

Molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) calculations

The Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) force field-based method58,59 was utilised to calculate the ligand-receptor binding free energies using g-mmpbsa software. Results presented in Table 3 showed that the computed binding free energy was −124.808 ± 39.367 kJ/mol highlighting that 11a could remain bound within TMPKmt in a stable form during simulation. Obviously, electrostatic and Van der Waal energies were significant contributors to the binding affinity recording −167.189 ± 44.204 and −141.087 ± 13.451 kJ/mol, respectively. These results are consistent with the promising TMPKmt inhibitory activity of 11a.

Table 3. Calculated binding free energies of 11a-TMPKmt complex (kJ/mol).

Type of energy	E (kJ/mol)	
ΔG	−124.808 ± 39.367	
Van der Waal energy	−141.087 ± 13.451	
Electrostatic energy	−167.189 ± 44.204	
Polar solvation energy	197.618 ± 41.740	
SASA energy	−14.150 ± 0.591	

In silico prediction of chemo-informatics properties

The SwissADME online server60 was used to predict the physicochemical, and pharmacokinetic properties of the most active compounds 11a, 11b, 13b as well as the reference drugs PZA, and EMB, as shown in Supplementary Table 2. Orally bioavailable drug candidates should have ilogP value ≤ 5, and TPSA ≤ 160 Å² 61. Compounds 11a, 11b, and 13b showed suitable parameters for oral bioavailability with ilogP and TPSA values within the acceptable ranges. These results were confirmed by the bioavailability radar charts62 (Supplementary Figure 3) where compounds 11a, and 11b were included in the pink area with two slight deviations of saturation, and polarity, while 13b was completely visible inside the pink area with a very slight deviation of saturation. As for the pharmacokinetic properties, the predicted BOILED-Egg model (Supplementary Figure 4) revealed that only 13b, PZA, and EMB, fell in the white area of the model, displaying high gastrointestinal (GI) absorption63,64. Moreover, none of our compounds were found in the yolk, indicating no blood brain barrier (BBB) penetration, highlighting their safety regarding CNS side effects63,64. All compounds, except 13b, are presented as red dots, indicating that they are non-substrates for P-glycoprotein (PGP-), which is a probable drug resistance mechanism64. Moreover, compounds 11a, 11b, and 13b complied with Lipinski’s “rule of five” parameters indicating promising drug-likeness properties.

Conclusions

New Biginelli adducts were designed and optimised through various modifications of the DHPM ring of dTMP, the natural substrate of TMPKmt. The anti-TB screening against the Mtb H37Rv strain revealed that 11a was superior to EMB, and was 9-fold more potent than PZA, while 11b was 4.5-times more potent than PZA. The ester derivatives 4a, and 4b were 2.5-fold more potent than PZA whereas the oxindolinyl and quinoxalinyl derivatives 13a, 13b, and 14a elicited approximately twice the activity of PZA. The most active compounds exhibited better safety profiles than EMB against RAW 264.7 cells’ growth with the most active compound 11a eliciting IC50 value of 932.7 µM and a selectivity index of 159. The in vitro TMPKmt inhibition assay showed that the most potent inhibitors were in order 11a, 13b and 11b with equipotent or comparable IC50 values to dTMP. SAR findings confirmed our rationale for optimising the Biginelli scaffold, where all the final compounds were of more potent anti-TB activity than their precursors. Additionally, the incorporation of oxindolinyl or quinoxalinyl moieties through (=N-N=) spacer significantly enhanced the anti-TB activity. The THPM-based Biginelli scaffold possessing 5-carboxamide, 4–(5-nitro-2-furanyl), and 2-oxo groups (11a), exerted the best anti-TB, and TMPKmt inhibition activities. Kinetic analysis of TMPKmt inhibition revealed that 11a inhibits TMPKmt by the mixed type of inhibition with preferred competitive over non-competitive manners (Ki = 6.552 µM, Ki′ = 15.571 µM). Docking studies into TMPKmt active site (PDB ID: 1G3U) showed that compounds 11a, 11b, and 13b occupied the same binding pocket as dTMP, showing similar binding interactions. Molecular Dynamics simulation revealed the dynamic stability of 11a-TMPKmt simulated complex over 100 ns. Moreover, compounds 11a, 11b and 13b showed good oral bioavailability and promising drug-likeness properties. Thus, compounds 11a, 11b, and 13b (Figure 12) are effective anti-TB agents, and TMPKmt inhibitors possessing high safety profiles, and acceptable chemo-informatics properties with compound 11a being the most promising.

Figure 12. Structures of the most active compounds 11a, 11b and 13b.

Experimental

Chemistry

Solvents and reagents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA), El Gomhoureya for Drugs Trade & Medical Supplies (Alexandria, Egypt), and EL-Nasr Pharmaceutical Chemicals Company (Qalyubia, Egypt). Melting points were determined on a Stuart capillary melting point apparatus (Stuart scientific Stone, Model SMP1, UK) in open glass capillaries, and were uncorrected. Infra-red (IR) spectra were recorded on Perkin-Elmer 1430, Beaconsfield, UK spectrophotometer in Alexandria, Egypt using the KBr plate technique, (ύ, cm-1). Nuclear magnetic resonance (1H, and 13C-NMR) were scanned on BrukerAvance III apparatus 400 MHz, and 100.63 MHz spectrophotometer (Cairo, and BeniSuef, Egypt), and on Jeol (ECA-500 II) apparatus 500.17 MHz, and 125.77 MHz spectrophotometer (Mansoura, Egypt) using deuterated Dimethylsulphoxide (DMSO-d6) or chloroform (CDCl3) as solvents. Electron impact mass spectra (EI-MS) were carried out on Gas chromatograph/mass spectrometer Shimadzu GCMS-Qp2010 plus, single quad (70 eV) (Al-Azhar University, Egypt). Elemental Microanalyses were performed at the microanalytical unit (Al-Azhar University, Egypt). Values were within ± 0.4% of the theoretical values. Compounds 1a,b65, 2a66, 3a,b23,67, 4a68, 8a69, 8b39, and 10a,b23 were reported. Structures of the prepared reported compounds were confirmed by comparing their experimental spectral data with the reported ones. The obtained spectral data of the reported compounds are presented in the Supplementary Information.

Ethyl 6-methyl-4–(2-(prop-2-yn-1-yloxy)phenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2b)

A mixture of thiourea (1 mmol), 2-(prop-2-yn-1-yloxy)benzaldehyde41 (1 mmol), and ethyl acetoacetate (0.195 g, 0.19 ml, 1.5 mmol) in EtOH (10 ml) containing drops of conc. HCl was heated under reflux for 5h. The reaction mixture was cooled to RT, and the separated product was filtered, washed with cold EtOH, dried, and crystallised from EtOH. Yield 0.21 g (64%); mp 180–182oC; IR ύ (KBr, cm-1): 3143, 3100 (NH), 2117 (C≡C), 1733 (C = O ester), 1566, 1476 (C = C Ar), 1252, 1023 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.05 (t, 3H, CH2CH3, J = 8.0 Hz); 2.30 (s, 3H, THPM-C6-CH3); 3.37 (s, 1H,-C≡CH); 4.00 (q, 2H, CH2CH3, J = 8.0 Hz); 4.82 (s, 2H, CH2-C≡CH); 5.52 (s, 1H, THPM C4-H); 6.95 (t, 1H, phenyl C5-H, J = 8.0 Hz); 7.07–7.11 (m, 2H, phenyl C3,6-Hs); 7.28 (t, 1H, phenyl C4-H, J = 8.0 Hz); 9.28, 10.24 (2s, each 1H, 2NH, D2O exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.0, 17.1, 49.2, 55.8, 59.4, 78.2, 79.4, 99.4, 112.7, 121.0, 128.0, 129.0, 131.2, 145.3, 154.6, 165.1 (ester C = O), 174.1 (C = S); Anal. Calcd for C17H18N2O3S (330.40): C, 61.80; H, 5.49; N, 8.48. Found: C, 61.79; H, 5.46; N, 8.39.

(R,S) Ethyl 6-methyl-4–(5-nitro-2-furanyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (4b)

A mixture of 5-nitro-2-furfural (0.14 g, 1 mmol), ethyl acetoacetate (0.13 g, 0.13 ml, 1 mmol), thiourea (1.2 mmol), and AlCl3 (0.3 mmol) in polyethylene glycol (5 ml) was stirred at 45 °C for 3h. The reaction mixture was cooled, and distilled H2O (4 ml) was added. The formed precipitate was filtered, washed with EtOH, and dried. Yield 0.14 g (45%); mp 190–192oC; IR ύ (KBr, cm-1): 3332 (NH), 1701 (C = O ester), 1592, 1507 (C = C Ar), 1534, 1368, (υas, and υs NO2), 1240, 1011 (υas, and υs C-O-C); 1H-NMR (400 MHz, CDCl3, δ ppm): 1.37, 1.43 (2t, 3H, CH2CH3, J = 7.1 Hz, 2 isomers); 1.68 (s, 1H, NH, D2O-exchangeable); 2.44, 2.58 (2s, 3H, THPM-C6-CH3, 2 isomers); 3.69, 3.80 (2s, 1H, THPM C4-H, 2 isomers); 4.35, 4.55 (2q, 2H, CH2CH3, J = 7.1 Hz, 2 isomers); 6.91, 6.94 (2d, 1H, 5-nitro-2-furanyl C3-H, J = 3.8 Hz, 2 isomers); 7.34, 7.37 (2d, 1H, 5-nitro-2-furanyl C4-H, J = 3.8 Hz, 2 isomers); 13C-NMR (100.63 MHz, DMSO-d6, δ ppm): 14.5, 17.6, 53.9, 60.1, 101.1, 115.8 (5-nitro-2-furanyl C3,4), 128.9 (5-nitro-2-furanyl C2,5), 145.7, 165.5 (C = O), 174.6 (C = S); EI-MS m/z (%): 311 [M+•] (4), 77 (100); Anal. Calcd for C12H13N3O5S (311.31): C, 46.30; H, 4.21; N, 13.50. Found: C, 46.30; H, 4.21; N, 13.50.

General procedure for preparation of ethyl 1-acetyl-4-substituted-6-methyl-2-oxo (or thioxo)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a,b, 6a,b,7a,b)

A solution of appropriate THPM derivatives 1a,b, 2a,b, 3a,b (1 mmol) in Ac2O (5 ml) was heated under reflux for 1–2 h. The reaction mixture was cooled, and poured onto ice-cold H2O. The formed crystals were filtered, washed with H2O, dried, and crystallised from EtOH.

Ethyl 1-acetyl-4–(4-fluorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5a)

Yield 0.27 g (84%); mp 185–187oC; IR ύ (KBr, cm-1): 3119 (NH), 1708 (ester C = O), 1655 (C = O), 1584, 1507 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.19 (t, 3H, CH2CH3, J = 7.0 Hz); 2.36 (s, 3H, THPM-C6-CH3); 2.65 (s, 3H, COCH3); 4.15 (q, 2H, CH2CH3, J = 7.0 Hz); 6.40 (s, 1H, THPM C4-H); 7.16–7.25 (m, 4H, 4-fluorophenyl-Hs); 11.69 (s, 1H, NH, D2O-exchangeable); 13C-NMR (100.63 MHz, DMSO-d6, δ ppm): 14.6, 16.9, 27.6, 52.6, 60.8, 107.6, 115.8 (2 C), 128.7 (2 C), 135.5, 145.7, 163.3, 165.2 (C = O, ester C = O), 173.6 (acetyl C = O); EI-MS m/z (%): 320 [M+•] (20), 97 (100); Anal. Calcd for C16H17FN2O4 (320.32): C, 59.99; H, 5.35; N, 8.75. Found: C, 59.97; H, 5.27; N, 8.78.

Ethyl 1-acetyl-4–(4-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5b)

Yield 0.29 g (85%); mp 189–191oC; IR ύ (KBr, cm-1): 3159 (NH), 1682 (C = O ester), 1639 (COCH3), 1546, 1510 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.20 (t, 3H, CH2CH3, J = 7.0 Hz); 2.36 (s, 3H, THPM-C6-CH3); 2.65 (s, 3H, COCH3); 4.15 (q, 2H, CH2CH3, J = 7.0 Hz); 6.41 (s, 1H, THPM C4-H); 7.05–7.38 (m, 4H, 4-fluorophenyl-Hs); 11.70 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.1, 16.5, 27.2, 52.1, 60.3, 107.1, 115.4 (2 C), 128.5 (2 C), 135.1, 145.2, 160.7, 164.7 (ester C = O), 173.1 (C = S), 177.8 (acetyl C = O); EI-MS m/z (%): 336 [M+•] (5), 293 (100); Anal. Calcd for C16H17FN2O3S (336.38): C, 57.13; H, 5.09; N, 8.33. Found: C, 57.16; H, 5.12; N, 8.25.

Ethyl 1-acetyl-6-methyl-2-oxo-4–(2-(prop-2-yn-1-yloxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6a)

Yield 0.26 g (72%); mp 180–182oC; IR ύ (KBr, cm-1): 3280 (NH), 2123 (C≡C), 1690 (ester C = O), 1659 (C = O), 1229, 1085 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.20 (t, 3H, CH2CH3, J = 7.0 Hz); 2.25 (s, 3H, THPM-C6-CH3); 2.63 (s, 3H, COCH3); 3.54 (s, 1H, -C≡CH); 4.12 (q, 2H, CH2CH3, J = 7.0 Hz); 4.75 (s, 2H, CH2-C≡CH); 6.58 (s, 1H, THPM C4-H); 6.92 (t, 1H, phenyl C5-H, J = 7.4 Hz); 7.08 (d, 1H, phenyl C3-H, J = 8.0 Hz); 7.16 (d, 1H, phenyl C6-H, J = 7.1 Hz); 7.28 (t, 1H, phenyl C4-H, J = 7.2 Hz); 11.53 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.1, 16.1, 27.5, 50.7, 55.8, 60.1, 78.2, 79.4, 107.3, 112.9, 120.7, 127.2, 127.7, 129.1, 142.6, 154.6, 165.1 (C = O), 172.3 (ester C = O), 178.1 (acetyl C = O); EI-MS m/z (%): 356 [M+•] (13); 307 (100%); Anal. Calcd for C19H20N2O5 (356.37): C, 64.04; H, 5.66; N, 7.86. Found: C, 63.99; H, 5.70; N, 7.89.

Ethyl 1-acetyl-6-methyl-2-thioxo-4–(2-(prop-2-yn-1-yloxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6b)

Yield 0.26 g (70%); mp 175–177oC; IR ύ (KBr, cm-1): 3120 (NH), 2124 (C≡C), 1708 (C = O ester), 1655 (COCH3), 1584, 1508 (C = C Ar), 1256, 1073 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.14 (t, 3H, CH2CH3, J = 7.0 Hz); 2.25 (s, 3H, THPM-C6-CH3); 2.59 (s, 3H, COCH3); 3.55 (s, 1H, -C≡CH); 4.11 (q, 2H, CH2CH3, J = 7.0 Hz); 4.75 (s, 2H, CH2-C≡CH); 6.59 (s, 1H, THPM C4-H); 6.93 (t, 1H, phenyl C5-H, J = 7.4 Hz); 7.07 (d, 1H, phenyl C3-H, J = 8.0 Hz); 7.17 (d, 1H, phenyl C6-H, J = 7.1 Hz); 7.28 (t, 1H, phenyl C4-H, J = 7.2 Hz); 11.43 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 13.9, 16.1, 27.5, 50.7, 55.5, 60.1, 78.2, 79.0, 107.3, 112.9, 120.7, 127.2, 127.7, 129.1, 142.6, 154.7, 164.8 (ester C = O), 172.3 (C = S), 178.1 (acetyl C = O); EI-MS m/z (%): 372 [M+•] (54), 323 (100); Anal. Calcd for C19H20N2O4S (372.44): C, 61.27; H, 5.41; N, 7.52. Found: C, 61.55; H, 5.39; N, 7.58.

Ethyl 1-acetyl-4–(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (7a)

Yield 0.28 g (64%); mp 195–197oC; IR ύ (KBr, cm-1): 3373 (NH), 1694 (ester C = O), 1634 (C = O), 1592 (C = N mixed with C = C Ar), 1515 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 0.97 (t, 3H, CH2CH3, J = 7.0 Hz); 2.09 (s, 3H, THPM-C6-CH3); 2.25 (s, 3H, COCH3); 3.91 (q, 2H, CH2CH3, J = 7.0 Hz); 5.58 (s, 1H, THPM C4-H); 7.40–7.53 (m, 6H, C-phenyl C3,4,5-Hs, N-phenyl C3,4,5-Hs); 7.90 (d, 2H, N-phenyl C2,6-Hs, J = 7.0 Hz); 8.10 (s, 1H, pyrazole C5-H); 8.16 (d, 2H, C-phenyl C2,6-Hs, J = 7.0 Hz); 9.68 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 13.9 (2 C), 18.0, 55.9, 60.0, 105.1, 112.8, 121.3, 127.3 (2 C), 128.2 (2 C), 128.6 (2 C), 129.1 (2 C), 130.3 (2 C), 138.4, 145.4 (2 C), 151.9, 154.4 (C = O), 165.4 (ester C = O), 177.4 (acetyl C = O); EI-MS m/z (%): 444 [M+•] (16), 43 (100); Anal. Calcd for C25H24N4O4 (444.48): C, 67.55; H, 5.44; N, 12.60. Found: C, 67.87; H, 5.43; N, 12.30.

Ethyl 1-acetyl-4–(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (7b)

Yield 0.28 g (61%); mp 190–192oC; IR ύ (KBr, cm-1): 3159 (NH), 1682 (C = O ester), 1638 (COCH3), 1585, 1510 (C = C Ar), 1256, 1073 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 0.98 (t, 3H, CH2CH3, J = 7.0 Hz); 2.09 (s, 3H, THPM-C6-CH3); 2.25 (s, 3H, COCH3); 3.92 (q, 2H, CH2CH3, J = 7.0 Hz); 5.64 (s, 1H, THPM C4-H); 7.29–7.53 (m, 6H, C-phenyl C3,4,5-Hs, N-phenyl C3,4,5-Hs); 7.90 (d, 2H, N-phenyl C2,6-Hs, J = 7.0 Hz); 8.10 (s, 1H, pyrazole C5-H); 8.16 (d, 2H, C-phenyl C2,6-Hs, J = 7.0 Hz); 9.66 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 13.9 (2 C), 18.0, 55.9, 60.0, 105.0, 112.8, 121.3, 127.3 (2 C), 128.2, 128.6 (2 C), 129.1, 130.3 (2 C), 132.1 (2 C), 137.3, 140.4, 145.4, 154.4, 165.4 (ester C = O), 177.4 (acetyl C = O, C = S); EI-MS m/z (%): 460 [M+•] (18), 279 (100); Anal. Calcd for C25H24N4O3S (460.55): C, 65.20; H, 5.25; N, 12.17. Found: C, 65.14; H, 5.11; N, 11.88.

General procedure for preparation of 6-methyl-2-oxo(or thioxo)-N-phenyl-4–(2-(prop-2-yn-1-yloxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (9a,b)

A mixture of urea or thiourea (1 mmol), 2-(prop-2-yn-1-yloxy)benzaldehyde (1 mmol), and acetoacetanilide (0.18 g, 1 mmol), in acetonitrile (10 ml) containing drops of conc. HCl was heated under reflux for 16h. The reaction mixture was concentrated, cooled to RT, and the separated product was filtered, washed with acetonitrile, dried, and crystallised from EtOH.

6-Methyl-2-oxo-N-phenyl-4–(2-(prop-2-yn-1-yloxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (9a)

Yield 0.26 g (72%); mp 190–192oC; IR ύ (KBr, cm-1): 3385, 3177 (NH), 2100 (C≡C), 1667 (C = O), 1564, 1464 (C = C Ar), 1372, 1062 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.00 (s, 3H, THPM-C6-CH3); 3.61 (s, 2H, -C≡CH, NH, D2O exchangeable); 4.88 (s, 2H, CH2-C≡CH); 5.64 (s, 1H, THPM C4-H); 7.07 (t, 1H, N-phenyl C4-H, J = 7.4 Hz); 7.17–7.21 (m, 3H, N-phenyl C3,5-Hs, phenyl-O-propargyl C5-H); 7.32–7.47 (m, 5H, N-phenyl C2,6-Hs, phenyl-O-propargyl C3,4,6-Hs); 9.66 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 16.2, 49.9, 55.8, 78.4, 79.2, 107.1, 112.6, 119.5 (2 C), 121.2, 123.1, 127.5, 128.5, 128.8 (2 C), 131.0, 135.0, 139.2, 154.0, 165.0 (C = O), 174.7 (CONH); EI-MS m/z (%): 361 [M+•] (24), 77 (100); Anal. Calcd for C21H19N3O3 (361.39): C, 69.79; H, 5.30; N, 11.63. Found: C, 69.66; H, 5.22; N, 11.61.

6-Methyl-2-thioxo-N-phenyl-4–(2-(prop-2-yn-1-yloxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (9b)

Yield 0.29 g (76%); mp 195–197oC; IR ύ (KBr, cm-1): 3328, 3176 (NH), 2100 (C≡C), 1682 (CONH), 1574, 1466 (C = C Ar), 1335, 1118 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.00 (s, 3H, THPM-C6-CH3); 3.46 (s, 1H, -C≡CH); 4.88 (s, 2H, CH2-C≡CH); 5.64 (s, 1H, THPM C4-H); 7.07 (t, 1H, N-phenyl C4-H, J = 7.4 Hz); 7.17–7.21 (m, 2H, N-phenyl C3,5-Hs); 7.32–7.46 (m, 6H, N-phenyl C2,6-Hs, phenyl-O-propargyl C3,4,5,6-Hs); 9.66 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 16.3, 49.9, 55.8, 78.4, 79.2, 107.1, 112.6, 119.5 (2 C), 121.2, 123.1, 127.5, 128.5, 128.8 (2 C), 131.0, 135.0, 139.2, 154.0, 164.9 (CONH), 174.7 (C = S); EI-MS m/z (%): 377 [M+•] (57), 81 (100); Anal. Calcd for C21H19N3O2S (377.46): C, 66.82; H, 5.07; N, 11.13. Found: C, 66.78; H, 5.02; N, 10.97.

General procedure for preparation of 6-methyl-4–(5-nitro-2-furanyl)-2-oxo (or thioxo)-1,2,3,4-tetrahydropyrimidine-5-carboxamide (11a,b)

A mixture of 5-nitro-2-furfural (0.14 g, 1 mmol), acetoacetamide (0.10 g, 1 mmol), urea or thiourea (1.2 mmol), and AlCl3 (0.3 mmol) in polyethylene glycol (5 ml) was stirred at 45 °C for 3h. The reaction mixture was cooled, and distilled H2O (4 ml) was added. The formed precipitate was filtered, washed with EtOH, and dried in air.

6-Methyl-4–(5-nitro-2-furanyl)-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (11a)

Yield 0.18 g (67%); mp 170–172oC; IR ύ (KBr, cm-1): 3325, 3175, 3105 (NH2, NH), 1686 (CONH), 1654 (C = O), 1574, 1369 (υas, and υs NO2), 1284, 1118 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.16 (s, 3H, THPM-C6-CH3); 3.52 (s, 2H, NH2, D2O exchangeable); 6.01 (s, 1H, THPM C4-H); 7.45 (s, br, 1H, 5-nitro-2-furanyl C3-H); 7.68 (s, br, 1H, 5-nitro-2-furanyl C4-H); 11.43 (s, 1H, NH, D2O-exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 16.9, 60.2, 100.5, 115.8 (2 C), 121.6 (2 C), 150.0, 162.1 (C = O), 168.4 (CONH); EI-MS m/z (%): 266 [M+•] (33), 96 (100); Anal. Calcd for C10H10N4O5 (266.21): C, 45.12; H, 3.79; N, 21.05. Found: C, 45.02; H, 3.75; N, 20.97.

6-Methyl-4–(5-nitro-2-furanyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (11b)

Yield 0.19 g (68%); mp 180–182o:C; IR ύ (KBr, cm-1): 3325, 3175, 3108 (NH2, NH), 1685 (CONH), 1573, 1368 (υas, and υs NO2), 1257, 1118 (υas, and υs C-O-C); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 2.16 (s, 3H, THPM-C6-CH3); 3.52 (s, 2H, NH2, D2O exchangeable); 6.05 (s, 1H, THPM C4-H); 7.51 (s, br, 1H, 5-nitro-2-furanyl C3-H); 7.67 (s, br, 1H, 5-nitro-2-furanyl C4-H); 11.43 (s, 1H, NH, D2O-exchangeable); EI-MS m/z (%): 282 [M+•] (16), 211 (100); Anal. Calcd for C10H10N4O4S (282.28): C, 42.55; H, 3.57; N, 19.85. Found: C, 42.46; H, 3.46; N, 19.83.

General procedure for preparation of ethyl 4-substituted-2-hydrazono-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12a,b)

To a solution of THPM derivatives (1b or 3b) (1 mmol) in 1,4-dioxane (20 ml), hydrazine hydrate (99%) (3 mmol) was added dropwise followed by a catalytic amount of conc. H2SO4. The reaction mixture was refluxed with stirring for 4h. The crude mass was allowed to cool, and poured onto crushed ice. The separated product was filtered, washed with H2O, dried, and crystallised from EtOH.

Ethyl 4–(4-fluorophenyl)-2-hydrazono-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12a)

Yield 0.20 g (69%); mp 180–182o:C; IR ύ (KBr, cm-1): 3325, 3175, 3105 (NH2, NH), 1728 (C = O), 1612 (C = N), 1573, 1462 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, CH2CH3, J = 7.0 Hz); 2.30 (s, 3H, THPM-C6-CH3); 3.34 (s, 2H, NH2, D2O exchangeable); 4.01 (q, 2H, CH2CH3, J = 7.0 Hz); 5.18 (s, 1H, THPM C4-H); 7.17–7.27 (m, 4H, 4-fluorophenyl-Hs); 9.66, 10.36 (2s, each 1H, 2NH, D2O exchangeable); 13C-NMR (100.63 MHz, DMSO-d6, δ ppm): 17.0, 19.0, 54.9, 56.5, 107.3, 120.2 (2 C), 129.1 (2 C), 132.8, 139.4, 142.4, 165.3 (C = N-NH2, C = O); EI-MS m/z (%): 292 [M+•] (15), 100 (100); Anal. Calcd for C14H17FN4O2 (292.31): C, 57.52; H, 5.86; N, 19.17. Found: C, 57.58; H, 5.79; N, 19.22.

Ethyl 4–(1,3-diphenyl-1H-pyrazol-4-yl)-2-hydrazono-6-methyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (12b)

Yield 0.28 g (67%); mp 185–187o:C; IR ύ (KBr, cm-1): 3244, 3179, 3117 (NH2, NH), 1725 (C = O), 1632 (C = N), 1577, 1450 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 0.81 (t, 3H, CH2CH3, J = 7.0 Hz); 2.28 (s, 3H, THPM-C6-CH3); 3.58 (s, 2H, NH2, D2O exchangeable); 3.80 (q, 2H, CH2CH3, J = 7.0 Hz); 5.41 (s, 1H, THPM C4-H); 7.32–7.53 (m, 6H, pyrazole C-phenyl C3,4,5-Hs, pyrazole N-phenyl C3,4,5-Hs); 7.79 (d, 2H, pyrazole N-phenyl C2,6-Hs, J = 7.1 Hz); 7.89 (d, 2H, pyrazole C-phenyl C2,6-Hs, J = 7.9 Hz); 8.36 (s, 1H, pyrazole C5-H); 9.74, 10.28 (2s, each 1H, 2NH, D2O exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 13.6, 17.2, 59.3, 66.3, 101.2, 118.4, 125.2, 126.5 (2 C), 127.8, 128.0 (2 C), 128.3, 128.4 (2 C), 129.5 (2 C), 132.9, 139.3, 144.8, 150.4, 164.9 (C = N-NH2), 173.8 (C = O); Anal. Calcd for C23H24N6O2 (416.48): C, 66.33; H, 5.81; N, 20.18. Found: C, 66.35; H, 5.90; N, 19.88.

General procedure for preparation of ethyl 4-substituted-6-methyl-2-((5-substituted-2-oxindolin-3-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13a-c)

A mixture of THPM derivatives 12a or 12b (1 mmol), and isatin or 5-methylisatin (1 mmol) in absolute EtOH (10 ml) was heated under reflux for 6h, then allowed to cool. The separated product was filtered, washed with EtOH, dried, and crystallised from EtOH.

Ethyl 4–(4-fluorophenyl)-6-methyl-2-((2-oxindolin-3-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13a)

Yield 0.30 g (71%); mp 190–192o:C; IR ύ (KBr, cm-1): 3325, 3175, 3105 (NH), 1728 (ester C = O), 1682 (CONH), 1612 (C = N), 1574, 1504 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, CH2CH3, J = 7.0 Hz); 2.30 (s, 3H, THPM-C6-CH3); 4.03 (q, 2H, CH2CH3, J = 7.0 Hz); 5.18 (s, 1H, THPM C4-H); 6.92 (d, 1H, oxindolinyl C7-H, J = 7.8 Hz); 7.06 (t, 1H, oxindolinyl C5-H, J = 7.4 Hz); 7.17–7.27 (m, 4H, 4-fluorophenyl-Hs); 7.50 (d, 1H, oxindolinyl C4-H, J = 7.4 Hz,); 7.58 (t, 1H, oxindolinyl C6-H, J = 6.5 Hz); 9.65, 10.36, 11.04 (3s, each 1H, 3NH, D2O exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.0, 17.2, 53.4, 59.6, 100.6, 112.2, 115.4 (2 C), 117.8, 122.8, 124.7 (2 C), 128.5 (2 C), 138.4, 139.8, 145.2, 150.7, 159.4, 160.6, 165.0 (ester C = O), 174.2 (oxindolinyl C = O); EI-MS m/z (%): 421 [M+•] (43), 62 (100); Anal. Calcd for C22H20FN5O3 (421.42): C, 62.70; H, 4.78; N, 16.62. Found: C, 62.66; H, 4.79; N, 16.55.

Ethyl 4–(4-fluorophenyl)-6-methyl-2-((5-methyl-2-oxindolin-3-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13b)

Yield 0.31 g (70%); mp 189–191o:C; IR ύ (KBr, cm-1): 3325, 3175, 3105 (NH), 1686 (C = O), 1654 (CONH), 1574 (C = N mixed with C = C Ar), 1508 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, CH2CH3, J = 7.0 Hz); 2.26, 2.51 (2s, each 3H, THPM-C6-CH3, oxindolinyl C5-CH3); 4.02 (q, 2H, CH2CH3, J = 7.0 Hz); 5.18 (s, 1H, THPM C4-H); 6.81 (d, 1H, oxindolinyl C7-H, J = 8.0 Hz); 7.16–7.42 (m, 6H, oxindolinyl C4,6-Hs,4-fluorophenyl-Hs); 9.66, 10.36, 10.94 (3s, each 1H, 3NH, D2O exchangeable); Anal. Calcd for C23H22FN5O3 (435.45): C, 63.44; H, 5.09; N, 16.08. Found: C, 63.48; H, 5.12; N, 16.44.

Ethyl 4–(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-2-((2-oxindolin-3-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (13c)

Yield 0.39 g (71%); mp 198–200o:C; IR ύ (KBr, cm-1): 3325, 3174, 3059 (NH), 1728 (C = O), 1681 (CONH), 1612 (C = N), 1573, 1504 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.10 (t, 3H, CH2CH3, J = 7.0 Hz); 2.29 (s, 3H, THPM-C6-CH3); 4.01 (q, 2H, CH2CH3, J = 7.0 Hz); 5.18 (s, 1H, THPM C4-H); 7.07–7.27 (m, 15H, N-phenyl-Hs, 3-phenyl-Hs, pyrazole C5-H, oxindolinyl C4,5,6,7-Hs); 9.66, 10.37, 11.87 (3s, each 1H, 3NH, D2O exchangeable); EI-MS m/z (%): 545 [M+•] (38), 156 (100); Anal. Calcd for C31H27N7O3 (545.59): C, 68.24; H, 4.99; N, 17.97. Found: C, 68.18; H, 5.02; N, 17.66.

General procedure for preparation of ethyl 4-substituted-6-methyl-2-((3-oxo-3,4-dihydroquinoxalin-2(1H)-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (14a,b)

A mixture of THPM derivatives 12a or 12b (1 mmol), and quinoxaline-2,3(1H,4H)-dione (0.16 g, 1 mmol) in absolute EtOH (10 ml) was heated under reflux for 6h then allowed to cool. The separated product was filtered, washed with EtOH, dried, and crystallised from EtOH.

Ethyl 4–(4-fluorophenyl)-6-methyl-2-((3-oxo-3,4-dihydroquinoxalin-2(1H)-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (14a)

Yield 0.31 g (70%); mp 192–194o:C; IR ύ (KBr, cm-1): 3325, 3175, 3105 (NH), 1728 (C = O), 1682 (CONH), 1612 (C = N), 1574, 1504 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.00 (t, 3H, CH2CH3, J = 7.2 Hz); 2.26 (s, 3H, THPM-C6-CH3); 3.92 (q, 2H, CH2CH3, J = 7.2 Hz); 5.59 (s, 1H, THPM C4-H); 6.97–7.29, 7.68–8.25 (2 m, 9H, quinoxaline C5,6,7,8-Hs + 4-fluorophenyl-Hs + NH, D2O exchangeable); 9.50, 10.16, 11.10 (3s, each 1H, 3NH, D2O exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.0, 17.2, 53.4, 59.6, 100.6, 115.1 (2 C), 115.3, 123.0 (2 C), 125.6 (2 C), 128.4 (2 C), 139.8, 145.2 (2 C), 155.2, 160.6, 162.5 (C = O), 165.0, 174.2 (ester C = O); EI-MS m/z (%): 436 [M+•] (61), 265 (100); Anal. Calcd for C22H21FN6O3 (436.44): C, 60.54; H, 4.85; N, 19.26. Found: C, 60.38; H, 5.14; N, 19.16.

Ethyl 4–(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-2-((3-oxo-3,4-dihydroquinoxalin-2(1H)-ylidene)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (14b)

Yield 0.38 g (68%); mp 196–198o:C; IR ύ (KBr, cm-1): 3402, 3325, 3171 (NH), 1670 (C = O), 1654 (CONH), 1597 (C = N mixed with C = C Ar), 1516 (C = C Ar); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 0.98 (t, 3H, CH2CH3, J = 7.0 Hz); 2.26 (s, 3H, THPM-C6-CH3); 3.92 (q, 2H, CH2CH3, J = 7.0 Hz); 5.59 (s, 1H, THPM C4-H); 7.28–8.18 (m, 16H, pyrazole-N-phenyl-Hs, pyrazole-C-phenyl-Hs, pyrazole C5-H, quinoxaline C5,6,7,8-Hs + NH, D2O exchangeable); 9.68, 10.36, 11.43 (3s, each 1H, 3NH, D2O exchangeable); 13C-NMR (125.77 MHz, DMSO-d6, δ ppm): 14.7, 17.4, 50.5, 62.0, 101.0, 105.4, 105.5, 113.1, 121.3, 126.7 (2 C), 128.6 (2 C), 128.8 (2 C), 129.6 (3 C), 129.9 (2 C), 131.20 (2 C), 134.2, 138.2, 142.1, 145.2, 145.5, 147.4, 165.6 (C = O), 168.8, 174.2 (ester C = O); EI-MS m/z (%): 560 [M+•] (28), 45 (100); Anal. Calcd for C31H28N8O3 (560.61): C, 66.42; H, 5.03; N, 19.99. Found: C, 66.78; H, 4.99; N, 20.12.

General procedure for preparation of ethyl 4-substituted-6-methyl-2–(2-(phenylsulfonyl)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15a,b)

A mixture of appropriate THPM derivatives 12a or 12b (1 mmol), and benzenesulfonyl chloride (0.18 g, 0.13 ml, 1 mmol) in dry 1,4-dioxane (10 ml) containing triethyl amine (1 ml) was heated under reflux for 3h. After cooling, the reaction mixture was poured onto ice-water, and the solid formed was filtered off, dried, and crystallised from EtOH.

Ethyl 4–(4-fluorophenyl)-6-methyl-2–(2-(phenylsulfonyl)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15a)

Yield 0.27 g (63%); mp 193–195oC; IR ύ (KBr, cm-1): 3391, 3180 (NH), 1727 (C = O), 1616 (C = N), 1564, 1462 (C = C Ar), 1272,1125 (υas, and υs SO2); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.02 (t, 3H, CH2CH3, J = 7.2 Hz); 2.25 (s, 3H, THPM-C6-CH3); 3.91 (q, 2H, CH2CH3, J = 7.2 Hz); 5.58 (s, 1H, THPM C4-H); 6.92–7.30 (m, 4H, 4-fluorophenyl-Hs); 7.83–8.26 (m, 5H, phenyl-Hs), 9.66, 10.17, 11.69 (3s, each 1H, 3NH, D2O exchangeable); EI-MS m/z (%): 432 [M+•] (49), 414 (100); Anal. Calcd for C20H21FN4O4S (432.47): C, 55.54; H, 4.89; N, 12.96. Found: C, 55.46; H, 4.88; N, 12.87.

Ethyl 4–(1,3-diphenyl-1H-pyrazol-4-yl)-6-methyl-2–(2-(phenylsulfonyl)hydrazono)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (15b)

Yield 0.37 g (66%); mp 192–194oC; IR ύ (KBr, cm-1): 3429, 3332, 3214 (NH), 1701 (C = O), 1617 (C = N), 1240, 1172 (υas, and υs SO2); 1H-NMR (400 MHz, DMSO-d6, δ ppm): 1.20 (t, 3H, CH2CH3, J = 7.0 Hz); 2.30 (s, 3H, THPM-C6-CH3); 4.03 (q, 2H, CH2CH3, J = 7.0 Hz); 5.18 (s, 1H, THPM C4-H); 7.07–7.27 (m, 16H, pyrazole-N-phenyl-Hs, pyrazole-C-phenyl-Hs, pyrazole C5-H, phenyl-Hs); 9.66, 10.36, 11.69 (3s, each 1H, 3NH, D2O exchangeable); EI-MS m/z (%): 556 [M+•] (29), 214 (100); Anal. Calcd for C29H28N6O4S (556.64): C, 62.57; H, 5.07; N, 15.10. Found: C, 62.44; H, 4.97; N, 14.88.

In vitro anti-TB screening

The microplate alamar blue assay (MABA) was performed to screen the anti-TB activity of the newly synthesised compounds against the Mtb H37Rv strain using EMB, and PZA as references42. The Mtb H37Rv strain was obtained from the National Institute of Research in Tuberculosis, Chennai, India (Indian Council of Medical Research Laboratory). Mycobacterium growth occurred in Lowenstein-Jensen medium (LJ medium), and the Mycobacterium cells were resuspended in the 7H9-S medium supplemented with oleic acid, albumin, dextrose, and catalase (OADC). Following that, the previously prepared medium was adjusted to approximately 3 × 108 CFU/mL in McFarland tube number 1, and diluted 1:20, where 100 μL was employed as inoculum. Stock solutions of test compounds, and references were defrosted, and diluted at four-fold the final highest concentration tested in 7H9-S medium. Next, a sterile 96-well microtiter plate was used to prepare two-fold serial dilutions of each test compound, and reference drugs using 100 μl of 7H9-S, and sterile controls were prepared for each plate. Subsequently, sterile deionised water was added to all outer perimeter wells of the plate, and the plate was covered, and incubated for seven days at 37 °C (normal atmosphere). After incubation, 30 μL of alamar blue solution was added to each well, and the plate was re-incubated overnight. Visual examination signifies bacterial growth by a colour change from blue (oxidized state) to pink (reduced state), where blue indicates no growth while pink indicates bacterial growth. The minimal inhibitory concentration (MIC) is the lowest concentration that prevents colour change from blue to pink42.

In vitro cytotoxicity assay

The MTT assay was used to evaluate the in vitro cytotoxicity of the most active anti-TB agents against growth inhibition of RAW 264.7 cells at a concentration of 25 μg/mL46. RAW 264.7 cells were procured from National Centre for Cell Science, Pune, India. RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS), and 100 μg/mL penicillin/streptomycin. The medium was incubated in a 5% CO2 atmosphere at 37 °C, and sub-cultured every 3 days. Following the growth of adherent cells, they were transferred to a sterile centrifuge tube, centrifuged at 2500 rpm for 5 min for pellet formation, and a haemocytometer was used to mix the formed pellet with fresh media. A 96-well plate was used to add 100 µL of media with RAW 264.7 cells (ranging from 5000–6000 cells per well), and the plate was incubated in 5% CO2 for 24 h at 37 °C to allow cell adherence. For determination of % inhibition: The cells were then treated with the test compounds, and the reference at 25 µM. Zero-hour reading was determined for untreated cells, and for the control cells treated with 1% DMSO. For IC50 determination, the cells were then treated with 11a at 5-fold serial dilution47. Then, the plate was incubated for 48 h at 37 °C with humidified 5% CO2, followed by the addition of MTT dissolved in Phosphate-buffered saline (PBS) to each well to obtain a final cell concentration of 5 mg/mL. The plate was kept for 4 h at 37 °C with 5% CO2 to confirm the cleavage of MTT in viable cells, producing purple formazan crystals that were then dissolved in 100 µL of DMSO. The viability was measured at 540 nm using a multimode reader (Spectra max). The concentration of purple formazan crystals was directly proportional to the number of viable cells, and inversely proportional to the degree of cytotoxicity. The tests were repeated in triplicate for each concentration. The % inhibition was calculated46. The (IC50), representing the compound concentration needed to produce a 50% inhibition of cell growth compared to untreated controls after 48 h of incubation, was determined from the exponential curve of viability, calculated as a percentage of the Control, versus concentration47.

In vitro TMPKmt inhibition activity

Recombinant TMPKmt, expressed, and purified (in two steps) as described by Munier-Lehmann et al.48, was used for the enzymatic assay. Two E. coli strains, namely NM554 (Agilent technologies) and BLi5 (derived from the BL21 (DE3)70, Novagen, Sigma-Aldrich) were utilised for purification, sequencing of DNA, and for production of recombinant TMPKmt expressed by the plasmid pHL50, respectively. 2YT medium supplemented with ampicillin and chloramphenicol (100, and 30 µg/mL, respectively) was used for cultures growth at 37 °C. Production of recombinant TMPKmt was prompted with 1 mM isopropyl-1-thio-β-D-thiogalactoside (when the culture attained an absorbance of 1.5 at 600 nm) followed by harvesting via centrifugation after 3h of production. The first purification step involved disruption of cells (suspended in Tris-HCl pH 7.4, 50 mM) by sonication followed by centrifugation (at 14000 rpm for 30 min) and loading the obtained bacterial lysate onto a Blue-Sepharose column pre-equilibrated with the same buffer at a concentration of 10 mg of protein/mL of swollen gel. Washing of the column was performed using 10 volumes of 50 mM Tris-HCl pH 7.4 followed by elution of TMPKmt with 4 volumes of 1 M NaCl in 50 mM Tris-HCl pH 7.4. A second elution of TMPKmt was carried out using 4 volumes of elution buffer containing 2 M NaCl in 50 mM Tris-HCl pH 7.4. The second purification step was performed via pooling and concentration of the fractions containing TMPKmt followed by loading of such fractions onto a 1 × 110 cm Ultrogel AcA54 column equilibrated with 50 mM Tris-HCl pH 7.4 where fractions of 1.8 ml were collected at a flow rate of 9 ml/h. The most active compounds were dissolved in DMSO and evaluated for their TMPKmt activities at different concentrations (from 0.006 to 0.12 mM) and at fixed concentrations of both dTMP (0.05 mM) and ATP (0.5 mM). The coupled spectrophotometric assay was performed at 30 °C, as described by Blondin et al.49, where the reaction medium consists of 50 mM Tris–HCl (pH 7.4), KCl (50 mM), MgCl2 (2 mM), NADH (0.2 mM), Phosphoenolpyruvate (PEP) (1 mM), and the coupling enzymes, Lactate dehydrogenase (LDH) (2 U), Pyruvate kinase (PK) (2 U), and nucleoside diphosphate kinase (NDP-kinase) (2 U). The absorbance was measured spectrophotometrically at 334 nm on an Eppendorf ECOM 6122 photometer, and IC50 values were calculated using Kaleidagraph.

Kinetic analysis of the inhibition of TMPKmt

A series of experiments were performed to determine the inhibition kinetics of TMPKmt by 11a50. In this analysis the concentrations of inhibitor 11a (0.1, 1, and 10 µM), and the substrate concentrations (31.25, 62.5, 125, 250, 500, and 1000 µM) were used. Maximal initial velocity was determined from initial linear portion of absorbance up to 15 min after addition of the enzyme at per minute’s interval. The inhibition type on the enzyme was assayed by Lineweaver–Burk plots of inverse of velocities (1/V) versus inverse of substrate concentration 1/[S] mM−1. Enzyme inhibition (EI) dissociation constant (Ki) was determined by the secondary plot of slope versus various concentrations of 11a while Enzyme-substrate-inhibitor (ESI) dissociation constant (Ki′) was determined by plotting the intercept versus various concentrations of 11a.

Docking into TMPKmt crystal structure

MOE (Chemical Computing Group Inc. 2019.01 software51, Montreal, QC, Canada), installed on a 2.5 G Core (TM) i5, was used to build the 3D structures of compounds 11a, 11b, and 13b. The crystal structures of the natural substrate dTMP bound to TMPKmt (PDB ID: 1G3U)8 were obtained from the PDB. Protein structures were prepared for docking using the QuickPrep wizard in MOE 2019.01, with default options, and without deletion of water. The default Triangle Matcher method was used to generate poses that were scored, and ranked by the London dG, and GBVI/WSA dG scoring functions. The final five poses were ranked, and visualised using MOE. The protocol was validated by redocking the co-crystallised ligand into TMPKmt crystal structure with a RMSD value of 0.1664 Å, and applying the same protocol for docking of compounds 11a, 11b, and 13b. The binding free energy values of the redocked dTMP, 11a, 11b, and 13b were calculated using the best docking poses. The lowest energy conformer was subjected to surface mapping.

Molecular dynamics simulation

Molecular dynamics (MD) calculations were performed on the best docking pose of 11a to get further insights about its dynamic stability within TMPKmt binding site. CHARMM-GUI solution builder was used to generate the input files for MD calculations using CHARMM force field parameters for TMPKmt71,72. CHARMM General Force Field73 was used to generate the topologies of 11a through CgenFF server. The coordinates of 11a–TMPKmt complex were first read by CHARMM-GUI solution builder followed by solvation of 11a–TMPKmt complex after addition of 3 Cl- ions for neutralisation of the system and determination of its size and shape. Next, Periodic Boundary Conditions (PBC) were determined where the simulation occurs only for the atoms located inside the PBC box. The complex had a 72 Å, 72 Å and 72 Å dimensions for the PBC box in x, y and z, respectively. At this step, running short minimisation was used for elimination of bad contacts. Then, equilibration of the system through NVT and NPT ensembles took place to ensure that the system has reached the required temperature and pressure. Following that, the equilibration and production input files were downloaded where the necessary changes, as number of steps of MD run, frequency of trajectories saving, and calculation of energy, were made. Non-bonded interactions were treated with a 12 Å cut-off distance and the Verlet cut-off-scheme was used to buffer the neighbour searching list while the particle mesh Ewald (PME) method was used for the treatment of the long-range electrostatic interactions. Energy minimisation, using steepest descent algorithm (5000 steps), was performed before the production simulation. Then, the complex was equilibrated, by subjecting it to NVT and NPT ensemble, to ensure stability of its temperature and pressure and simulation occurred for 125 ps at 300.15 K using 400 and 40 kJ mol−1 nm−2 positional restraints on the backbone and side chains, respectively. At the end, production simulation run was performed for 100 ns in NPT ensemble at 300.15 K and 1 bar. Nose-Hoover thermostat and Parrinello-Rahman barostat were used for maintaining the temperature and the pressure, respectively. Hydrogen bonds were constrained using the LINCS algorithm and the inputs provided by CHARMM-GUI. The V-rescale thermostat at 300 K with a coupling constant of 1 ps was used. The trajectories were saved every 2 ps. Simulation of 100 ns in NPT assembly was carried out for the production stage employing GROMACS software55,56. GROMACS utilities including gmx_rms, gmx_rmsf, gmx_gyrate, gmx_hbond, gmx_distance subprograms were used for the calculation of RMSD, RMSF, rGyr, number of hydrogen bonds (inside the protein–ligand interface), and COM between TMPKmt and 11a, respectively during the simulation. Trajectory visualisation and contact frequency (CF) analysis was performed using the VMD molecular graphics program53 while the GROMACS tool, g_mmpbsa was used for both performing MM-PBSA calculations and estimation of the energy contribution per residue to the binding energy. The binary run input file (.tpr) needed for MM-PBSA calculation through the g_mmpbsa was regenerated by GROMACS 5.1.4 while the necessary files to generate the binary run input file, such as molecular structure (.gro), topology (.top), and MD-parameter (.mdp) files, were obtained from the MD process.

The binding free energy of the protein with ligand in solvent can be expressed as: ΔGBinding=ΔGComplex−(ΔGProtein−ΔGLignad)

where ΔGComplex,ΔGProtein,ΔGLignad are the total free energies of the protein–ligand complex, the isolated protein and ligand in solvent, respectively. To know the share of each residue to the binding energy, ΔEMM, ΔGpolar and ΔGnon−polar were separately calculated for each residue and were then summed up to obtain the contribution.

In silico prediction of chemo-informatics properties

SwissADME online server (http://swissadme.ch/index.php,)60 was used to predict the chemo-informatics properties of compounds 11a, 11b, 13b, and the references PZA, and EMB.

Supplementary Material

Supplemental Material

Authors’ contributions

Conception and design: M.E., S.F., I.L., M.M.; methodology: M.E., A.A., D.S., M.S., I.L., M.M.; analysis and interpretation of the data: M.E., A.A., S.F., D.S., M.S., I.L., M.M.; original draft preparation: M.E., A.A., S.F., I.L., M.M.; revising for intellectual content: M.E., A.A., S.F., D.S., I.L., M.M.; writing-review and editing: M.E., A.A., S.F., D.S., M.S., I.L., M.M; Final approval of the version to be published: M.E., A.A., S.F., D.S., M.S., M.M; accountability for all aspects of the work: M.E., A.A., S.F., D.S., M.S., I.L., and M.M.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article or its supplementary materials. Any other required data are available from the corresponding author upon request.
==== Refs
References

1 Van Calenbergh S, Pochet S, Munier-Lehmann H. Drug design and identification of potent leads against Mycobacterium tuberculosis thymidine monophosphate kinase. Curr Top Med Chem. 2012;12 (7 ):694–705.22283813
2 Finger V, Kufa M, Soukup O, Castagnolo D, Roh J, Korabecny J. Pyrimidine derivatives with antitubercular activity. Eur J Med Chem. 2023;246 :114946.36459759
3 Alexandrova LA, Khandazhinskaya AL, Matyugina ES, Makarov DA, Kochetkov SN. Analogues of pyrimidine nucleosides as mycobacteria growth inhibitors. Microorganisms. 2022;10 (7 ):1299.35889017
4 Global Tuberculosis Report. 2022. Available from: https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2022.
5 Perveen S, Sharma R. Screening approaches and therapeutic targets: the two driving wheels of tuberculosis drug discovery. Biochem Pharmacol. 2022;197 :114906.34990594
6 Dhameliya TM, Devani AA, Patel KA, Shah KC. Comprehensive coverage on anti‐mycobacterial endeavour reported in 2021. ChemistrySelect. 2022;7 (19 ):e202200921.
7 Capela R, Félix R, Clariano M, Nunes D, Perry MD, Lopes F. Target identification in anti-tuberculosis drug discovery. Int J Mol Sci. 2023;24 (13 ):10482.37445660
8 De La Sierra IL, Munier-Lehmann H, Gilles AM, Bârzu O, Delarue M. X-ray structure of TMP kinase from Mycobacterium tuberculosis complexed with TMP at 1.95 Å resolution. J Mol Biol. 2001;311 (1 ):87–100.11469859
9 Dhameliya TM, Vekariya DD, Patel HY, Patel JT. Comprehensive coverage on anti-mycobacterial endeavour reported during 2022. Eur J Med Chem. 2023;255 :115409.37120997
10 Dhameliya TM, Bhakhar KA, Gajjar ND, Patel KA, Devani AA, Hirani RV. Recent advancements and developments in search of anti-tuberculosis agents: a quinquennial update and future directions. J Mol Struct. 2022;1248 :131473.
11 Van Daele I, Munier-Lehmann H, Froeyen M, Balzarini J, Van Calenbergh S. Rational design of 5’-thiourea-substituted α-thymidine analogues as thymidine monophosphate kinase inhibitors capable of inhibiting mycobacterial growth. J Med Chem. 2007;50 (22 ):5281–5292.17910427
12 Van Poecke S, Munier-Lehmann H, Helynck O, Froeyen M, Van Calenbergh S. Synthesis and inhibitory activity of thymidine analogues targeting Mycobacterium tuberculosis thymidine monophosphate kinase. Bioorg Med Chem. 2011;19 (24 ):7603–7611.22061826
13 Gasse C, Douguet D, Huteau V, Marchal G, Munier-Lehmann H, Pochet S. Substituted benzyl-pyrimidines targeting thymidine monophosphate kinase of Mycobacterium tuberculosis: synthesis and in vitro anti-mycobacterial activity. Bioorg Med Chem. 2008;16 (11 ):6075–6085.18467107
14 Yan M, Xu L, Wang Y, Wan J, Liu T, Liu W, Wan Y, Zhang B, Wang R, Li Q. Opportunities and challenges of using five‐membered ring compounds as promising antitubercular agents. Drug Dev Res. 2020;81 (4 ):402–418.31904877
15 Song L, Merceron R, Hulpia F, Lucía A, Gracia B, Jian Y, Risseeuw MD, Verstraelen T, Cos P, Aínsa JA, et al. Structure-aided optimization of non-nucleoside M. tuberculosis thymidylate kinase inhibitors. Eur J Med Chem. 2021;225 :113784.34450493
16 Jian Y, Risseeuw MDP, Froeyen M, Song L, Cappoen D, Cos P, Munier-Lehmann H, van Calenbergh S. 1-(piperidin-3-yl) thymine amides as inhibitors of M. tuberculosis thymidylate kinase. J Enzyme Inhib Med Chem. 2019;34 (1 ):1730–1739.31822127
17 Jian Y, Hulpia F, D P Risseeuw M, Forbes HE, Caljon G, Munier-Lehmann H, I M Boshoff H, Van Calenbergh S. 1-(1-arylethylpiperidin-4-yl) thymine analogs as antimycobacterial TMPK inhibitors. Molecules. 2020;25 (12 ):2805.32560578
18 El-Shoukrofy MS, Atta A, Fahmy S, Sriram D, Mahran MA, Labouta IM. New tetrahydropyrimidine-1,2,3-triazole clubbed compounds: antitubercular activity and thymidine monophosphate kinase (TMPKmt) inhibition. Bioorg Chem. 2023;131 :106312.36528922
19 Kancharla SK, Birudaraju S, Pal A, Reddy LK, Reddy ER, Vagolu SK, Sriram D, Bonige KB, Korupolu RB. Synthesis and biological evaluation of isatin oxime ether-tethered aryl 1H-1,2,3-triazoles as inhibitors of Mycobacterium tuberculosis. New J Chem. 2022;46 (6 ):2863–2874.
20 Ramesh D, Sarkar D, Joji A, Singh M, Mohanty AK, Vijayakumar BG, Chatterjee M, Sriram D, Muthuvel SK, Kannan T. First‐in‐class pyrido[2,3‐d]pyrimidine‐2,4(1H, 3H)‐diones against leishmaniasis and tuberculosis: rationale, in vitro, ex vivo studies and mechanistic insights. Arch Pharm. 2022;355 :2100440.
21 Lagu SB, Yejella RP, Nissankararao S, Bhandare RR, Golla VS, Subrahmanya Lokesh BV, Rahman MM, Shaik AB. Antitubercular activity assessment of fluorinated chalcones, 2-aminopyridine-3-carbonitrile and 2-amino-4H-pyran-3-carbonitrile derivatives: in vitro, molecular docking and in-silico drug likeliness studies. Plos One. 2022;17 (6 ):e0265068.35709194
22 Khani-Meinagh H, Mostafavi H, Reiling N, Mahdavi M, Zarrini G. Design, synthesis and evaluation of biological activities of some novel anti-TB agents with bio-reducible functional group. Bioimpacts. 2019;9 (4 ):199–209.31799156
23 Yadlapalli RK, Chourasia OP, Vemuri K, Sritharan M, Perali RS. Synthesis and in vitro anticancer and antitubercular activity of diarylpyrazole ligated dihydropyrimidines possessing lipophilic carbamoyl group. Bioorg Med Chem Lett. 2012;22 (8 ):2708–2711.22437116
24 Soares de Melo C, Singh V, Myrick A, Simelane SB, Taylor D, Brunschwig C, Lawrence N, Schnappinger D, Engelhart CA, Kumar A, et al. Antitubercular 2-pyrazolylpyrimidinones: structure–activity relationship and mode-of-action studies. J Med Chem. 2021;64 (1 ):719–740.33395287
25 Karunanidhi S, Chandrasekaran B, Karpoormath R, Patel HM, Kayamba F, Merugu SR, Kumar V, Dhawan S, Kushwaha B, Mahlalela MC. Novel thiomorpholine tethered isatin hydrazones as potential inhibitors of resistant Mycobacterium tuberculosis. Bioorg Chem. 2021;115 :105133.34329993
26 Raphoko LA, Lekgau K, Lebepe CM, Leboho TC, Matsebatlela TM, Nxumalo W. Synthesis of novel quinoxaline-alkynyl derivatives and their anti-Mycobacterium tuberculosis activity. Bioorg Med Chem Lett. 2021;35 :127784.33422606
27 Kumar AS, Kudva J, Bharath BR, Ananda K, Sadashiva R, Kumar SM, Revanasiddappa BC, Kumar V, Rekha PD, Naral D. Synthesis, structural, biological and in silico studies of new 5-arylidene-4-thiazolidinone derivatives as possible anticancer, antimicrobial and antitubercular agents. New J Chem. 2019;43 (3 ):1597–1610.
28 Sanka BM, Tadesse DM, Bedada ET, Mengesha ET, Babu N. Design, synthesis, biological screening and molecular docking studies of novel multifunctional 1,4-di(aryl/heteroaryl) substituted piperazine derivatives as potential antitubercular and antimicrobial agents. Bioorg Chem. 2022;119 :105568.34968884
29 Sahoo SK, Maddipatla S, Gajula SN, Ahmad MN, Kaul G, Nanduri S, Sonti R, Dasgupta A, Chopra S, Yaddanapudi VM. Identification of nitrofuranylchalcone tethered benzoxazole-2-amines as potent inhibitors of drug resistant Mycobacterium tuberculosis demonstrating bactericidal efficacy. Bioorg Med Chem. 2022;64 :116777.35487101
30 Reddyrajula R, Dalimba U. The bioisosteric modification of pyrazinamide derivatives led to potent antitubercular agents: synthesis via click approach and molecular docking of pyrazine-1,2,3-triazoles. Bioorg Med Chem Lett. 2020;30 (2 ):126846.31839540
31 Braga TC, Silva TF, Maciel TM, da Silva EC, da Silva-Júnior EF, Modolo LV, Figueiredo IM, Santos JC, de Aquino TM, de Fátima Â. Ionic liquid-assisted synthesis of dihydropyrimidin (thi) one Biginelli adducts and investigation of their mechanism of urease inhibition. New J Chem. 2019;43 (38 ):15187–15200.
32 Bais J, Benedetti F, Berti F, Cerminara I, Drioli S, Funicello M, Regini G, Vidali M, Felluga F. One pot synthesis of micromolar BACE-1 inhibitors based on the dihydropyrimidinone scaffold and their thia and imino analogues. Molecules. 2020;25 (18 ):4152.32927879
33 da Silva DL, Reis FS, Muniz DR, Ruiz AL, de Carvalho JE, Sabino AA, Modolo LV, de Fátima Â. Free radical scavenging and antiproliferative properties of Biginelli adducts. Bioorg Med Chem. 2012;20 (8 ):2645–2650.22410248
34 de Azambuja GO, Svetaz L, Gonçalves IL, Corbelini PF, von Poser GL, Kawano DF, Zacchino S, Eifler-Lima VL. In vitro antifungal activity of dihydropyrimidinones/thiones against Candida albicans and Cryptococcus neoformans. CBC. 2019;15 (6 ):648–655.
35 Kumar P, Kumar A, Verma SS, Dwivedi N, Singh N, Siddiqi MI, Tripathi RP, Dube A, Singh N. Leishmania donovani pteridine reductase 1: biochemical properties and structure-modeling studies. Exp Parasitol. 2008;120 (1 ):73–79.18617167
36 Attaby FA, Ramla MM, Harukuni T. Synthesis and inhibitory activity against Epstein-Barr virus of some new 1,2,3,4-tetrahydropyrimidine-2-thiones. Phosphorus Sulfur Silicon. 2008;183 (12 ):2956–2967.
37 Ravendra Babu K, Nanda Kumar Y, Raghavendra A, Phanindra V, Madhava G, Ravi N, Bhaskar M, Raju NC. Design, synthesis, in silico and in vitro studies of substituted 1,2,3,4-tetrahydropyrimidine phosphorus derivatives. Comb Chem High Throughput Screen. 2015;18 :862.26004048
38 Malani K, Thakkar SS, Thakur MC, Ray A, Doshi H. Synthesis, characterization and in silico designing of diethyl-3-methyl-5-(6-methyl-2-thioxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxamido)thiophene-2,4-dicarboxylate derivative as anti-proliferative and anti-microbial agents. Bioorg Chem. 2016;68 :265–274.27616159
39 Buzmakova NA, Rudakova IP, Zamaraeva TM. Synthesis and anti-inflammatory activity of N,6-diaryl-4-methyl-2-thioxo-1,2,3,6-tetrahydropyrimidine-5-carboxamides. Pharm Chem J. 2021;55 (8 ):775–777.
40 Desai NC, Vaghani HV, Jethawa AM, Khedkar VM. In silico molecular docking studies of oxadiazole and pyrimidine bearing heterocyclic compounds as potential antimicrobial agents. Arch Pharm. 2021;354 :2100134.
41 Khoshkholgh MJ, Balalaie S, Gleiter R, Rominger F. Intramolecular hetero-Diels–Alder reaction of 1-oxa-1,3-butadienes with terminal acetylenes in aqueous media using CuI. Tetrahedron. 2008;64 (48 ):10924–10929.
42 Franzblau SG, Witzig RS, McLaughlin JC, Torres P, Madico G, Hernandez A, Degnan MT, Cook MB, Quenzer VK, Ferguson RM, et al. Rapid, low-technology MIC determination with clinical Mycobacterium tuberculosis isolates by using the microplate alamar blue assay. J Clin Microbiol. 1998;36 (2 ):362–366.9466742
43 Sriram D, Yogeeswari P, Reddy SP. Synthesis of pyrazinamide Mannich bases and its antitubercular properties. Bioorg Med Chem Lett. 2006;16 (8 ):2113–2116.16464574
44 Nawrot DE, Bouz G, Janďourek O, Konečná K, Paterová P, Bárta P, Novák M, Kučera R, Zemanová J, Forbak M, et al. Antimycobacterial pyridine carboxamides: from design to in vivo activity. Eur J Med Chem. 2023;258 :115617.37423128
45 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 1997;23 (1–3 ):3–25.
46 Van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. Cancer cell culture: methods and protocols. Totowa (NJ): Humana Press; 2011. p. 237.
47 Ismail RS, Abou-Seri SM, Eldehna WM, Ismail NS, Elgazwi SM, Ghabbour HA, Ahmed MS, Halaweish FT, Abou El Ella DA. Novel series of 6-(2-substituted acetamido)-4-anilinoquinazolines as EGFR-ERK signal transduction inhibitors in MCF-7 breast cancer cells. Eur J Med Chem. 2018;155 :782–796.30047410
48 Munier‐Lehmann HÉ, Chaffotte A, Pochet S, Labesse G. Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes. Protein Sci. 2001;10 (6 ):1195–1205.11369858
49 Blondin C, Serina L, Wiesmüller L, Gilles AM, Bârzu O. Improved spectrophotometric assay of nucleoside monophosphate kinase activity using the pyruvate kinase/lactate dehydrogenase coupling system. Anal Biochem. 1994;220 (1 ):219–221.7978251
50 Ashraf Z, Rafiq M, Seo SY, Babar MM, Zaidi NU. Design, synthesis and bioevaluation of novel umbelliferone analogues as potential mushroom tyrosinase inhibitors. J Enzyme Inhib Med Chem. 2015;30 (6 ):874–883.25643758
51 Molecular Operating Environment (MOE). 2019.01, Chemical Computing Group Inc.: Montreal, QC, Canada. Available from: http://www.chemcomp.com.
52 Nawareg NA, Mostafa AS, El-Messery SM, Nasr MN. New benzimidazole based hybrids: synthesis, molecular modeling study and anticancer evaluation as TopoII inhibitors. Bioorg Chem. 2022;127 :106038.35870412
53 Bhat AR, Dongre RS, Almalki FA, Berredjem M, Aissaoui M, Touzani R, Hadda TB, Akhter MS. Synthesis, biological activity and POM/DFT/docking analyses of annulated pyrano[2,3-d]pyrimidine derivatives: identification of antibacterial and antitumor pharmacophore sites. Bioorg Chem. 2021;106 :104480.33279245
54 Qi L, Li MC, Bai JC, Ren YH, Ma HX. In vitro antifungal activities, molecular docking, and DFT studies of 4-amine-3-hydrazino-5-mercapto-1,2,4-triazole derivatives. Bioorg Med Chem Lett. 2021;40 :127902.33684439
55 GROMACS: Fast, Flexible, Free. Available from: http://www.gromacs.org/.
56 Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2 :19–25.
57 Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14 (1 ):33–38.8744570
58 Massova I, Kollman PA. Combined molecular mechanical and continuum solvent approach (MM-PBSA/GBSA) to predict ligand binding. Perspect Drug Discovery Des. 2000;18 :113.
59 Kairys V, Baranauskiene L, Kazlauskiene M, Matulis D, Kazlauskas E. Binding affinity in drug design: experimental and computational techniques. Expert Opin Drug Discov. 2019;14 (8 ):755–768.31146609
60 SwissADME. Available from: http://swissadme.ch/index.php.
61 Othman DI, Hamdi A, Abdel-Aziz MM, Elfeky SM. Novel 2-arylthiazolidin-4-one-thiazole hybrids with potent activity against Mycobacterium tuberculosis. Bioorg Chem. 2022;124 :105809.35447406
62 Raslan RR, Ammar YA, Fouad SA, Hessein SA, Shmiess NA, Ragab A. Evaluation of the anti-proliferative activity of 2-oxo-pyridine and 1′H-spiro-pyridine derivatives as a new class of EGFRWt and VEGFR-2 inhibitors with apoptotic inducers. RSC Adv. 2023;13 (15 ):10440–10458.37020892
63 Sravika N, Priya S, Divya N, Jyotsna PM, Anusha P, Kudumula N, Bai SA. Swiss ADME properties screening of the phytochemical compounds present in Bauhinia acuminata. J Pharmacogn Phytochem. 2021;10 (4 ):411–419.
64 Şahin S, Dege N. (E)-N-(3-chlorophenyl)-1-(5-nitro-2-(piperidin-1-yl)phenyl) methanimine: X-ray, DFT, ADMET, boiled-egg model, druggability, bioavailabilty, and human cyclophilin D (CypD) inhibitory activity. J Mol Struct. 2022;1250 :131744.
65 Dandia A, Saha M, Taneja H. Synthesis of fluorinated ethyl 4-aryl-6-methyl-1,2,3,4-tetrahydropyrimidin-2-one/thione-5-carboxylates under microwave irradiation. J Fluor Chem. 1998;90 (1 ):17–21.
66 Vendrusculo V, de Souza VP, Fontoura LA, D’Oca MG, Banzato TP, Monteiro PA, Pilli RA, de Carvalho JE, Russowsky D. Synthesis of novel perillyl–dihydropyrimidinone hybrids designed for antiproliferative activity. Medchemcomm. 2018;9 (9 ):1553–1564.30288229
67 Bratenko MK, Chornous VA, Vovk MV. 4-Functionally substituted 3-heterylpyrazoles: XIII. 3-aryl (heteryl)-4-(4-pyrazolyl)-1,2,3,4-tetrahydropyrimidin-2-ones (thiones). Russ J Org Chem. 2005;41 :95.
68 Hirao I, Kato Y, Hujimoto T. Studies on synthesis of 2-keto-4-(5’-nitro-2’-furyI)-5-carbethoxy-6-methyl-1,2,3,4-tetrahydropyrimidine. Nippon Kagaku Zasshi. 1964;85 (1 ):52–54,A4.
69 Kurmach MN, Ryabitskiy AB, Britsun VN. 2-Acylthioacetamides in the Biginelli reaction. Chem Heterocycl Comp. 2014;49 (12 ):1770–1776.
70 Serina L, Blondin C, Krin E, Sismeiro O, Danchin A, Sakamoto H, Gilles AM, Bârzu O. Escherichia coli UMP kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochem. 1995; 34 (15 ):5066–5074.7711027
71 Brooks BR, Brooks III CL, Mackerell Jr AD, JrNilsson L, Petrella RJ, Roux B, Won Y, Archontis G, Bartels C, Boresch S, et al. CHARMM: the biomolecular simulation program. J Comput Chem. 2009;30 (10 ):1545–1614.19444816
72 Jo S, Kim T, Iyer VG, Im W. CHARMM‐GUI: a web‐based graphical user interface for CHARMM. J Comput Chem. 2008;29 (11 ):1859–1865.18351591
73 Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, Darian E, Guvench O, Lopes P, Vorobyov I, et al. CHARMM general force field: a force field for drug‐like molecules compatible with the CHARMM all‐atom additive biological force fields. J Comput Chem. 2010;31 (4 ):671–690.19575467
