Design, Synthesis, Pharmacodynamic and In Silico Pharmacokinetic Evaluation of Some Novel Biginelli-Derived Pyrimidines and Fused Pyrimidines as Calcium Channel Blockers

Abstract

Some new pyrimidine derivatives comprising arylsulfonylhydrazino, ethoxycarbonylhydrazino, thiocarbamoylhydrazino and substituted hydrazone and thiosemicarbazide functionalities were prepared from Biginelli-derived pyrimidine precursors. Heterocyclic ring systems such as pyrazole, pyrazolidinedione, thiazoline and thiazolidinone ring systems were also incorporated into the designed pyrimidine core. Furthermore, fused triazolopyrimidine and pyrimidotriazine ring systems were prepared. The synthesized compounds were evaluated for their calcium channel blocking activity as potential hypotensive agents. Compounds 2, 3a, 3b, 4, 11 and 13 showed the highest ex vivo calcium channel blocking activities compared with the reference drug nifedipine. Compounds 2 and 11 were selected for further biological evaluation. They revealed good hypotensive activities following intravenous administration in dogs. Furthermore, 2 and 11 displayed drug-like in silico ADME parameters. A ligand-based pharmacophore model was developed to provide adequate information about the binding mode of the newly synthesized active compounds 2, 3a, 3b, 4, 11 and 13. This may also serve as a reliable basis for designing new active pyrimidine-based calcium channel blockers.

1. Introduction

The American Heart Association reported an average of one death every 40 s due to cardiovascular diseases (CVDs) [1]. The WHO Global Atlas on Cardiovascular Disease Prevention and Control confirmed that CVDs are the leading cause of mortality worldwide [2]. In response to the burden posed by CVDs, the European Society of Cardiology (ESC) and the European Society of Hypertension (ESH) published their guidelines and recommended several cardiovascular agents in the clinic. However, in many cases, their clinical use is limited by their side effects [3]. Therefore, there is a continuous need for developing novel efficient cardiovascular agents.

Based on their chemical structures, many cardiovascular agents are pyrimidine derivatives (Figure 1), such as darusentan, a selective endothelin receptor antagonist [4]; minoxidil, a direct vasodilator [5]; rosuvastatin, a competitive inhibitor of HMG-CoA reductase [6]; and trapidil, a fused pyrimidine vasodilator [7]. Extensive exploration of the pyrimidine ring system led to the synthesis of novel orally active angiotensin II antagonists [8] and efficient calcium channel blockers (CCBs) which gained considerable interest [9]. Such a large representation of this heterocyclic nucleus in cardiovascular agents suggests that this heterocyclic moiety, if properly decorated with substituents, could lead to novel potential cardiovascular agents.

Here we report the synthesis of novel pyrimidine derivatives (Figure 2) designed by taking advantage of the high diversity initially generated on the pyrimidine core through Biginelli multicomponent reaction [10]. Inspired by the Biginelli-derived CCBs that are considered aza-analogs of dihydropyridine (DHP) CCBs [11,12,13,14,15] (Figure 2), the newly synthesized derivatives were rationalized as potential CCBs. This hypothesis was also supported by the representation of the pyrimidine ring in efficient CCBs [9]. Accordingly, all the target compounds were evaluated for their in vitro calcium channel blocking activity relative to the prototype CCB nifedipine. The most active derivatives were then evaluated for possible hypotensive activities in dogs, then subjected to molecular modeling studies. The substitution pattern was rationalized to keep the basic pharmacophoric core while modifying the C² position. In this regard, various alkyl and aryl moieties were introduced at the core’s C² via different functionalized linkers (hydrazones, thiosemicarbazides, etc.) following the SAR of previously reported CCBs [12,13,14,15]. Heterocyclic ring systems such as pyrazole, pyrazolidinedione, thiazoline and thiazolidinone were also incorporated. Furthermore, it was also aimed to synthesize fused pyrimidine ring systems such as triazolopyrimidine and pyrimidotriazine rings to extend the deduced structure–activity relationship study. It is worth mentioning that these nitrogenous heterocycles are obviously represented in various lead CCBs [12,13,14,15,16,17,18].

The synthesized pyrimidine and fused pyrimidine derivatives were evaluated for their potential calcium channel blocking activity as hypotensive agents. The compounds showing promising ex vivo calcium channel blocking activities were then tested for their hypotensive activity following intravenous administration in dogs. Nifedipine was selected as a reference as it is the prototype DHP CCB and the lead for Biginelli-derived dihydropyrimidine (DHPM) CCBs and pyrimidine-based CCBs. Additionally, a ligand-based pharmacophore model was developed to provide adequate information about the binding mode of the newly synthesized active compounds. This may also serve as a reliable basis for designing new active pyrimidine-based CCBs.

2. Results and Discussion

2.1. Chemistry

The synthetic strategies adopted for the preparation of the intermediate and final compounds are depicted in Scheme 1 and Scheme 2. As shown in Scheme 1, the starting compound ethyl 6-methyl-2-methylsulfonyl-4-phenylpyrimidine-5-carboxylate 1 [19] was conveniently converted to ethyl 2-hydrazino-6-methyl-4-phenylpyrimidine-5-carboxylate 2 by reaction with 99% hydrazine hydrate in ethanol. ¹H-NMR showed the absence of the methyl singlet and the appearance of the D₂O exchangeable signals at 4.33 and 8.62 ppm assigned to NH₂ and NH, respectively. Condensing equimolar amounts of hydrazine derivative 2 with aromatic aldehydes and acetophenone as representative ketone in refluxing EtOH following a conventional method [20] afforded the corresponding hydrazones 3a,b and 4, respectively. The IR spectra of these compounds lacked stretching absorption bands due to NH₂ and showed stretching absorption bands due to NH and C=N, while ¹H-NMR lacked the upfield D₂O-exchangeable singlet assigned for hydrazine NH₂ protons and showed a downfield D₂O-exchangeable singlet assigned for hydrazone NH proton. The structure of compound 4 was further verified by ¹³C-NMR spectral data. The new thiosemicarbazides 5a,b were prepared by reaction of the key intermediate 2 with representative aryl- and alkyl-substituted isothiocyanates at room temperature. Reaction of 5b with ethyl bromoacetate in boiling EtOH containing anhydrous sodium acetate [21] afforded the corresponding thiazolidinone derivative 6b. ¹H-NMR showed a highly deshielded D₂O-exchangeable singlet assigned for NH proton. In addition, a multiplet integrated for two protons was assigned for thiazolidinone C⁵ protons, while the ¹³C-NMR spectrum provided further confirmation of the structure. Moreover, condensing the thiosemicarbazides 5a with bromophenacyl bromide in presence of sodium acetate in absolute EtOH [21] afforded the thiazoline derivatives 7 in acceptable yields. Its ¹H-NMR spectrum showed a deshielded singlet, integrated for one proton assigned for thiazoline C⁵ proton. Compounds 8a,b were synthesized by stirring equimolecular amounts of the hydrazine 2 with the aryl sulfonyl chlorides in dry pyridine following a previously reported procedure [22]. Products were identified by IR, ¹H-NMR and ¹³C-NMR in addition to MS spectrum of 8b which showed a molecular ion peak at m/z 426 (20%) that matched its molecular weight.

Referring to Scheme 2, the key intermediate 2 was heated with ethyl chloroformate in dry dioxane in accordance with conventional procedure [23] affording ethyl 2-[2-(ethoxycarbonyl)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylate 9. The ¹H-NMR spectrum showed an extra triplet and quartet characteristic of the ethyl moiety, while its MS spectrum revealed a molecular ion peak at m/z 344 (14%) which matched its molecular weight. Condensation of hydrazino derivative 2 with ethyl acetoacetate yielded the corresponding hydrazone 10. The chemical structure of 10 was confirmed by IR, ¹H-NMR, ¹³C-NMR and MS spectral data. The pyrazolyl derivatives 11 and 12 were successfully produced by heating 2 with acetylacetone and diethylmalonate, respectively, in ethanol/glacial acetic acid. ¹H-NMR spectra of compounds 11 and 12 were characterized by pyrazolyl C⁴ protons. ¹³C-NMR spectra for these compounds revealed a signal at 110.80 ppm due to pyrazolyl C⁴. Moreover, the MS spectrum of 11 showed a molecular ion peak at m/z 336 (100%) which is in accordance with its molecular formula.

Heating compound 2 with formic acid [24] gave the corresponding ethyl 5-methyl-7-phenyl-1,2,4-triazolo[4,3-a]pyrimidine-6-carbxylate 13. Its ¹H-NMR spectrum lacked the two D₂O-exchangeable singlets assigned for NHNH₂ protons and showed a new downfield singlet assigned for triazole C₃-H proton, confirming cyclization. Its ¹³C-NMR spectrum revealed two signals at 148.48 and 160.86 ppm corresponding to triazolopyrimidine C³ and C^8a, respectively. Additionally, its MS spectrum showed a molecular ion peak at m/z 282 (54%) which is in accordance with its molecular formula. Cyclization regioselectivity of 13 was unequivocally established by HMBC showing a correlation between C⁵-CH₃ at 2.9 ppm and the C³ at 148.48 ppm (Figure 3a), confirming cyclization at N¹ rather than N³ of the pyrimidine core. On the other hand, compound 2 was cyclized with the appropriate phenacyl bromides in boiling absolute ethanol [25] to give the pyrimido[2,1-c]-1,2,4-triazine derivatives 14a,b. ¹H-NMR spectra of these compounds showed the presence of singlets at 5.54–5.56 ppm assigned to the triazino C⁴ protons. The ¹³C-NMR spectrum of compound 14b revealed signals due to pyrimidotriazine C³ and C⁴ at their expected chemical shifts. Moreover, the MS spectrum of 14a showed a molecular ion peak at m/z 372 (65%) which matched its molecular weight. Similarly, cyclization regioselectivity of 14b was unequivocally established by HMBC showing a correlation between C⁶-CH₃ carbon at 17.47 ppm and the C⁴-H at 5.54 ppm (Figure 3b), confirming cyclization at N¹ rather than N³ of the pyrimidine core.

2.2. Biological Evaluation

All the newly synthesized derivatives were screened for calcium channel blocking activity by determining their ability to antagonize KCl-induced contractions of isolated rabbit jejunum and rat colon at a concentration of 10⁻⁵ M [26] (

Table 1. Preliminary screening of calcium channel blocking activity of the tested compounds at a concentration of 10⁻⁵ M in DMSO on isolated rat colon and rabbit jejunum (n = 4) ^a.

Cpd No.	Rat Colon	Rabbit Jejunum	Cpd No.	Rat Colon	Rabbit Jejunum
2	+	+	8b	-	-
3a	+	+	9	-	-
3b	+	+	10	-	-
4	+	+	11	+	+
5a	-	-	12	-	-
5b	-	-	13	+	+
6	-	-	14a	-	-
7	-	-	14b	-	-
8a	-	-	Nifedipine	+	+

^a refers to the number of observations used. (+) refers to compounds inhibiting KCl-induced contractions. (-) refers to inactive compounds.

). Results of the preliminary screening revealed that six compounds (2, 3a, 3b, 4, 11 and 13) showed inhibition of KCl-induced contractions, whereas other compounds failed to initiate any detectable activity. Candidate compounds were then evaluated at increasing doses (2 × 10⁻⁵, 4 × 10⁻⁵ and 6 × 10⁻⁵ M) (

Table 2. Quantitative assessment of active compounds expressed as % inhibition of KCl-induced contractions on isolated rabbit jejunum at different concentrations (n = 4) ^a.

Compound No.	% Inhibition of KCl-Induced Contractions
	2 × 10−5 M	4 × 10−5 M	6 × 10−5 M
2	44.45 ± 26.06	66.67 ± 33.33	100 ± 0
3a	25.00 ± 14.43	62.50 ± 23.94	72.33 ± 14.68
3b	17.00 ± 9.82	89.00 ± 11.00	100 ± 0
4	11.33 ± 9.81	33.45 ± 16.78	55.56 ± 24.22
11	15.55 ± 4.45	50.00 ± 30.00	70.00 ± 15.28
13	25.09 ± 16.03	55.09 ± 17.94	12.50 ± 7.98
Nifedipine	100 ± 0

^a refers to the number of experiments.

). Active compounds were less potent than nifedipine. However, they showed dose-dependent inhibition of KCl-induced contractions. The highest calcium channel blockade was exhibited by the hydrazine derivative 2 and the p-nitrophenylhydrazone 3b. They were equipotent, showing 100% inhibition of KCl-induced contractions at a concentration of 6 × 10⁻⁵ M. The hydrazones 3a and 4 lacking the nitro group showed lower activity at the same concentration. Moderate activity was exhibited when the hydrazine functionality was encaged in a planar heterocyclic pyrazole ring to furnish compound 11. The lowest detected activity was elicited when a triazole ring was fused to the pyrimidine ring in compound 13. For further quantitative assessment, IC₅₀ and pIC₅₀ were statistically calculated (

Table 3. Quantitative assessment of active compounds expressed as IC₅₀ and PIC₅₀ on isolated rabbit jejunum. (n = 3–4) ^a.

Compound No.	IC50 (µM)	pIC50
2	0.96	6.017
3a	1.089	5.962
3b	2.82	5.549
4	1.889	5.723
11	2.594	5.586
13	3.199	5.494
Nifedipine	6.279 (nM)	8.202

^a refers to the number of experiments used. pIC₅₀ scale = −log IC₅₀ (higher values indicate exponentially greater potency).

). Results showed that the lead compound 2 was the most potent. The hydrazones 3a, 3b and 4 as well as the pyrazole derivative 11 showed moderate activities. The fused triazolopyrimidine derivative 13 showed the least calcium channel blocking activity.

In addition, compounds 2 and 11 were evaluated for hypotensive activity (mg/kg, i.v.) in normotensive anesthetized dogs at different doses [27] (

Table 4. Hypotensive activity of selected test compounds (mg/kg, i.v.) in normotensive anesthetized dogs represented by change in MAP (mmHg) as mean ± SE (n = 3–5) ^a.

Compound No.	Decrease in MAP (mmHg) as Mean ± SE
	6 mg/kg	12 mg/kg	24 mg/kg
Control 2	3 ± 1.29 9.6 ± 0.81	2.75 ± 1.60 15.4 ± 2.39	12.6 ± 3.57 35.4 ± 1.60
11	24.4 ± 2.56	28.75 ± 6.01	34 ± 4.16

Nifedipine (0.125 mg/kg) caused 50 mmHg drop in arterial blood pressure. ^a refers to the number of experiments. Results were significant at p < 0.05 according to Mann–Whitney test.

). Results are represented by the change in mean arterial blood pressure (MAP) (mmHg). The data indicated a poor correlation between in vitro calcium channel blocking activity and hypotensive activity in normotensive anesthetized dogs following i.v. administration of compounds 2 and 11 at doses up to 12 mg/kg. Additional studies were performed at higher doses, where both compounds exhibited approximately the same potency at 24 mg/kg i.v. dose.

2.3. Molecular Modeling

2.3.1. Pharmacophore Modeling

In the present investigation, a ligand-based pharmacophore model was developed for representative DHP CCBs, including the prototype nifedipine and its lead aza-analogs; DHPM CCBs; and pyrimidine-based CCBs [9,28,29,30] as a training set (Supplementary File Figure S29) in order to map common structural features of highly active CCBs (Figure 4).

In absence of the 3D structure of LCC, this hypothesis was employed as a valuable tool to provide adequate information about the binding mode of the newly synthesized active compounds. This may also provide a reliable basis for the design of new potentially active molecules of the pyrimidine type. All structures were built using MOE Builder in the Molecular Operating Environment program (MOE) [31]. The selected 3D-pharmacophore model (pharmacophore query) showed 100% accuracy and 7.8 overlap and was composed of five main features (Figure 4a):

• Hydrophobic center (green sphere) involving C⁴ phenyl ring (F1: Hyd).
• Hydrophobic center (green sphere) involving C⁶ methyl group (F2: Hyd).
• Hydrogen bond acceptor function (cyan sphere) involving C⁵ carbonyl group (F3: Acc).
• Hydrogen bond acceptor/donor function (pink sphere) involving ring N (F4: Acc/Don).
• Hydrophobic center with H-bond acceptor or donor function (pink sphere) involving C² substitutions (F5: Hyd/Acc/Don).

The 3D spatial relationship between these key features, identified by pharmacophore analysis, was reported as linear distances in angstroms (Figure 4b).

The selected pharmacophore model was validated for its predictive efficacy as a calcium channel model utilizing representative derivatives of DHP, DHPM [29] and pyrimidine [9] CCBs as a validation set (Supplementary File Figures S30 and S31). Biologically active compounds were subjected to conformational search and energy minimization and were superimposed onto the pharmacophore hypothesis. The most suitable alignment for each compound (lowest RMSD) was selected (

Table 5. RMSD values of hit compounds.

Compound No.	2	3a	3b	4	11
RMSD(Å)	0.5694	0.8392	0.7738	0.5678	0.6054

).

Results (Figure 5a–e) indicated that all compounds showing in vitro calcium channel blocking activity except compound 13 were able to satisfy pharmacophoric features of the model with RMSD values in the range 0.5678–0.8392, suggesting that they may share the same binding site on the receptor. Although compound 13 is active, it failed to fit the model, and this non-agreement might suggest a different binding mode.

2.3.2. In Silico Physicochemical Properties, Drug-Likeness and ADME

Recent drug discovery programs utilize in silico prediction of physicochemical and ADME parameters as useful lead identification tools. In this study, the physicochemical parameters formulating Lipinski’s rule [32] were computed for the most active compounds utilizing Molinspiration software [33] (

Table 6. In silico physicochemical properties, drug-likeness and ADME data of the most active compounds.

Cpd. No.	LogP a	M.Wt b	HBA c	HBD d	Lipinski’s Violation	TPSA e	%ABS f	Volumes (A)3	S g (mg/L)	Drug-Likeness Model Score	CaCo2 h	MDCK i	HIA j	BBB k	PPB l	CYP3A4 Inhibition	CYP2D6 Inhibition
2	1.82	272.31	6	3	0	90.14	77.90	248.72	77.22	0.16	20.44	77.38	92.60	0.67	69.58	Non	Non
11	3.17	336.39	6	0	0	69.92	84.87	310.77	3.06	0.06	33.73	18.32	98.72	1.58	88.16	inhibitor	Non

^aLog P: logarithm of compound partition coefficient between n-octanol and water. ^b M.Wt: molecular weight. ^c HBA: number of hydrogen bond acceptors. ^d HBD: number of hydrogen bond donors. ^e TPSA: polar surface area. ^f %ABS: percentage of absorption. ^g S: aqueous solubility. ^h CaCo2: permeability through cells derived from human colon adenocarcinoma. ^I MDCK: permeability through Madin–Darby canine kidney cells. ^j HIA: human intestinal absorption. ^k BBB: blood–brain barrier penetration. ^l PPB: plasma protein binding.

). Interestingly, the selected compounds 2 and 11 were in full accordance with Lipinski’s parameters. Molinspiration [33] was also employed to calculate topological polar surface area (TPSA), which is utilized to calculate the estimated absorption percentage [34] as an additional bioavailability descriptor [35]. Herein, compounds 2 and 11 displayed drug-like TPSA values (<140–150 A²) [36,37] and reasonable absorption percentages (77–84%), predicting promising oral bioavailability. Aqueous solubility of 2 and 11 and their drug-likeness scores (Table 6) were predicted utilizing Molsoft software [38]. Both compounds recorded excellent drug-like predicted solubility and drug-likeness model scores.

Furthermore, Pre-ADMET software [39] was used for ADME prediction of the selected compounds. Accordingly, CaCo2 and MDCK cell permeability coefficients, human intestinal absorption (HIA), blood–brain barrier penetration (BBB), plasma protein binding (PPB) and inhibition of cytochromes P450 2D6 (CYP2D6) and P450 3A4 (CYP3A4) were computed and listed in Table 6. Both 2 and 11 displayed acceptable CaCo2 cell model permeability values (20.44 and 33.73 nm/s, respectively) and MDCK cell model permeability values (77.38 and 18.32 nm/s, respectively). Their HIA (92–98%) and BBB (0.67 and 1.58, respectively) values demonstrated excellent predicted intestinal absorption and acceptable CNS bioavailability of both compounds. Additionally, 2 was predicted to be devoid of the undesirable CYP3A4 and CYP2D6 inhibition activities, and hence potential drug–drug interactions are most probably excluded.

2.4. Structure–Activity Relationship

The preliminary calcium channel blockade screening (Table 2) revealed that the designed 2-substituted Biginelli-derived scaffolds (Figure 2), when appropriately substituted, conserved the intrinsic calcium channel blocking activities of their DHP mimics [40], DHPM precursors [11,12,13,14,15,20] and pyrimidine-based leads [9]. It is worth mentioning this observation echoes previous structure–activity relation (SAR) studies showing that nifedipine [40] and DHPM CCBs [15,28,29,41] tolerate various C² substituents. The promising group (Figure 6) included the 2-hydrazino derivative 2, its hydrazones 3a,b and 4, the dimethyl-1H-pyrazol-1-yl derivative 11 and the triazolopyrimidine derivative 13. Quantitative assessment of active compounds (Table 3) showed that most of the derivatives, namely 2, 3a, 3b and 4, that can display (donate) hydrogen bond(s) within the vicinity of the heterocyclic core showed notable calcium channel blockade activities. This correlation is consistent with previous SAR studies highlighting the critical hydrogen bonding interaction offered by the heterocyclic core of various DHP and DHPM CCBs. [42,43]. Herein, this hypothesis was supported by the elucidated pharmacophore model (Figure 4). However, it seems that the C² substituent’s size and the number of possible hydrogen bond donors tuned the compounds’ potency (Table 3). The hydrazino moiety in compound 2 conferred the highest potency (IC₅₀ = 0.96 µM) to the Biginelli-derived scaffold, followed by 2-benzylidenehydrazino (IC₅₀ = 1.089 µM) and 2-(phenylethylidene)hydrazino (IC₅₀ = 1.889 µM) groups in thee hydrazones 3a and 4, respectively. Notably, the introduction of the p-nitro group to the 2-benzylidenehydrazino motif in 3b critically decreased the potency (IC₅₀ = 2.82 µM) by approximately 2.5-fold relative to the unsubstituted derivative 3a. Obviously, thiocarbamoylation, sulfonation, acylation and condensation with ethyl acetoacetate afforded the inactive derivatives 5a,b, 8a,b, 9 and 10, respectively. These results point to the unfavorable effect of introducing electron-withdrawing and/or bulky groups to the hydrazino group on calcium channel antagonism. Another correlation between C² flexibility and the calcium channel blockade could be deduced from monitoring the activity of the pyrazolyl 11 and the triazolopyrimidine 13 derivatives, where the free hydrazino group was encaged in isolated or fused ring systems, respectively. Results (Table 3) showed that the pyrazolyl derivative 11 (IC₅₀ = 2.594 µM) was 2.7-fold less potent than the hydrazino derivative 2, whereas the triazolopyrimidine derivative 13 was the least potent (IC₅₀ = 3.199 µM) among the group (3-fold less potent than 2). These findings clarified the influence of C² substituent flexibility on calcium channel antagonism. Again, introducing electron-withdrawing moieties to the ring systems was detrimental to activity, as evidenced by loss of activity in the case of the dioxopyrazolidin-1-yl derivative 12 and the pyrimidotriazine derivatives 14a,b. Collectively, it could be concluded that the designed Biginelli-derived scaffold was optimized as the 2-hydrazino derivative 2. It may tolerate hydrazones or isolated heterocycles of suitable size and electronic environment. On the other hand, it may be deduced that derivatizing the C² hydrazino group into various electron-withdrawing functionalities either flexible (thiosemicarbzides 5a,b, arylsulfonylhydrazines 8a,b, ethoxycarbonylhydrazine 9 and ethoxyoxobutan-3-ylidene hydrazine 10), in ring systems (thiazolidinone 6, thiazoline 7 and pyrazolidinedione 12) or fused with the heterocyclic core (pyrimidotriazines 14a,b) was detrimental to activity.

In other words, the hydrazino group may be utilized as a spacer to introduce aromatic moieties taking into consideration keeping the linker flexible while avoiding electron-withdrawing groups.

Further evaluation of selected CCBs (2 and 11) for their hypotensive activities (mg/kg, i.v.) in normotensive anesthetized dogs at different doses (Table 4) revealed that the pyrazolyl derivative 11 exhibited superior in vivo hypotensive activity relative to the hydrazino derivative 2, at doses up to 12 mg/kg, despite being less active as a CCB according to in vitro studies. This poor correlation between the in vitro calcium channel blockade and in vivo hypotensive activities when prioritizing the evaluated derivatives refers to the influence of a secondary hypotensive mechanism that might have contributed to the in vivo potency of the pyrazolyl derivative 11. Interestingly, higher doses (at 24 mg/kg i.v.) of both compounds exhibited approximately the same potency.

3. Materials and Methods

3.1. Chemistry

Melting points were determined in open-glass capillaries using a Griffin melting point apparatus and are uncorrected. IR spectra (KBr) were recorded using a Bruker Vector 22 spectrophotometer at the Microanalytical Center, Faculty of Science, Cairo University. ¹H NMR spectra were scanned on a Mercury spectrometer (300 MHz) at the Faculty of Science, Cairo University. ¹³C NMR, distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple bond coherence (HMBC) spectra were recorded on Jeol spectrometer (500 MHz) at the National Research Centre, Dokki, Cairo, using tetramethylsilane (TMS) as internal standard and DMSO-d₆ as the solvent (chemical shifts are given in δ ppm). Splitting patterns were designated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, dist = distorted. Mass spectra were recorded using a Shimadzu GCMS-Qp2010 plus (70 ev) at the Faculty of Science, Cairo University. Microanalyses were performed at the Microanalytical Unit, Faculty of Science, Cairo University. Results of the microanalyses were within ±0.4% of the calculated values. Follow-up of the reactions and checking the purity of the compounds were performed by thin-layer chromatography (TLC) on aluminum sheets precoated with silica gel (Type 60 GF254; Merck, Germany) and the spots were detected by exposure to UV lamp at 254 nm for a few seconds. Compound 1 was synthesized as described in [19].

Ethyl 2-hydrazino-6-methyl-4-phenylpyrimidine-5-carboxylate (2): Hydrazine hydrate 99% (3.75 g, 75 mmol) was slowly added to a solution of the methylsulfonyl derivative 1 (8 g, 25 mmol) in absolute EtOH (15 mL). The reaction mixture was stirred at RT for 30 min, during which complete dissolution and reprecipitation occurred. The obtained product was filtered, washed thoroughly with H₂O, dried and crystallized from EtOH/H₂O (5:1). Yield: (quantitative), m.p: 94–96 °C. IR (KBr, cm⁻¹): 3254, 3221, 3060 (NH₂, NH), 1710 (C=O), 1553 (C=N and C=C Ar), 1436 (C=C Ar), 1260, 1082 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.91 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.39 (s, 3H, C⁶-CH₃), 4.01 (q, J = 7.2 Hz, 2H, CH₃CH₂), 4.33 (s, br, 2H, NH₂, D₂O-exchangeable), 7.40–7.55 (m, 5H, Ar-Hs), 8.62 (s, 1H, NH, D₂O-exchangeable). EI-MS m/z (relative intensity): 272 ([M⁺], 100). Anal. Calcd. for C₁₄H₁₆N₄O₂ (272.3): C 61.75, H 5.92, N 20.58. Found: C 62.02, H 5.88, N 21.00.

Ethyl 2-(2-arylidenehydrazino)-6-methyl-4-phenylpyrimidine-5-carboxylates (3a,b): A solution of hydrazine derivative 2 (0.27 g, 1 mmol) in absolute EtOH (5 mL) was treated with a solution of an equimolar amount of the appropriate aromatic aldehyde in absolute EtOH. The reaction mixture was heated under reflux for 15 h and cooled. The separated product was filtered, washed with petroleum ether (40–60°), dried and crystallized from EtOH.

Ethyl 2-(2-benzylidenehydrazino)-6-methyl-4-phenylpyrimidine-5-carboxylate (3a): Yield: 63%, m.p: 133–135 °C. IR (KBr, cm⁻¹): 3199 (NH), 1719 (C=O), 1578, 1541 (C=N occasionally mixed with C=C Ar), 1443 (C=C Ar), 1268, 1081 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.94 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.47 (s, 3H, C⁶-CH₃), 4.06 (q, J = 7.2 Hz, 2H, CH₃CH₂), 7.34–7.71 (m, 10H, Ar-Hs), 8.20 (s, 1H, N=CH), 11.64 (s, 1H, NH, D₂O-exchangeable). Anal. Calcd. for C₂₁H₂₀N₄O₂ (360.41): C 69.98, H 5.59, N 15.55. Found: C 70.08, H 6.00, N 15.50.

Ethyl 6-methyl-2-(2-(4-nitrobenzylidene)hydrazino)-4-phenylpyrimidine-5-carboxylate (3b): Yield: 69%, m.p: 220–222 °C. IR (KBr, Cm-1): 3325 (NH), 1706 (C=O), 1544 (C=N occasionally mixed with C=C Ar), 1434 (C=C Ar), 1257, 1082 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.96 (t, dist, J = 7.2 Hz, 3H, CH₃CH₂), 2.50 (s, 3H, C⁶-CH₃), 4.09 (q, dist, J = 7.5 Hz, 2H, CH₃CH₂), 7.50–7.58 (m, 5H, Ar-Hs), 7.93 (d, dist, J = 8.7 Hz, 2H, Ha, C^2′,6′-Hs), 8.25 (s, 1H, N=CH), 8.27 (d, J = 9 Hz, 2H, Hb, C^3′,5′-Hs), 11.95 (s, 1H, NH, D₂O-exchangeable). EI-MS m/z (relative intensity): 405 ([M⁺], 14), 228 (100). Anal. Calcd. for C₂₁H₁₉N₅O₄ (405.41): C 62.22, H 4.72, N 17.27. Found: C 62.09, H 4.83, N 16.91.

Ethyl 2-[2-(1-phenylethylidene)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylate (4): A solution of hydrazine 2 (0.27 g, 1 mmol) in absolute EtOH (5 mL) was treated with a solution of an equimolar amount of acetophenone in absolute EtOH. The reaction mixture was heated under reflux for 19 h and cooled to RT. The separated product was filtered, washed with petroleum ether (40–60°), dried and crystallized from EtOH unless otherwise stated. Yield: 75%, m.p: 162–164 °C. IR (KBr, cm⁻¹): 3223 (NH), 1717 (C=O), 1551 (C=N occasionally mixed with C=C Ar), 1438 (C=C Ar), 1259, 1082 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.94 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.35 (s, 3H, N=C-CH₃(Ar)), 2.49 (s, 3H, C⁶-CH₃ overlapping with DMSO), 4.06 (q, J = 7.5 Hz, 2H, CH₃CH₂), 7.36–7.83 (m, 10H, Ar-Hs), 10.50 (s, 1H, NH, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 14.0, 14.4, 23.1, 61.5, 117.5, 126.6, 128.4, 128.9, 129.2, 130.2, 138.8, 139.1, 149.4, 160.0, 165.7, 167.1, 168.4; EI-MS m/z (relative intensity): 374 ([M⁺], 64), 373 (100). Anal. Calcd. for C₂₂H₂₂N₄O₂ (374.44): C 70.57, H 5.92, N 14.96. Found: C 70.79, H 6.02, N 15.05.

Ethyl 6-methyl-4-phenyl-2-[2-(substituted thiocarbamoyl)hydrazino]pyrimidine-5-carboxylates (5a,b): The appropriate isothiocyanate derivative (0.01 mol) was added to a well-stirred suspension of hydrazine derivative 2 (2.7 g, 0.01 mol) in absolute EtOH (20 mL). The reaction mixture was stirred at RT for 2 h, during which dissolution and reprecipitation occurred. The obtained precipitate was filtered, washed with petroleum ether (40–60°), dried and crystallized from EtOH.

Ethyl 6-methyl-4-phenyl-2-[2-(phenylthiocarbamoyl)hydrazino]pyrimidine-5-carboxylates (5a): Yield: 90%, mp: 140–142 °C. IR (KBr, cm⁻¹): 3274, 3151 (NH), 1715 (C=O), 1557 (C=N mixed with C=C Ar), 1438 (C=C Ar), 1526, 1358, 1219, 1016 (N-C=S amide I, II, III, IV bands), 1252, 1089 (υ_as and υ_s C-O-C).¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.97 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.44 (s, 3H, C⁶-CH₃), 4.08 (q, J = 7.2 Hz, 2H, CH₃CH₂), 7.13–7.56 (m, 10H, Ar-Hs), 9.52 (s, 1H, NHNHC=S, D₂O-exchangeable), 9.73 (s, 1H, NHC₆H₅, D₂O-exchangeable), 9.75 (s, 1H, C²-NH, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 14.0, 23.0, 61.7, 117.7, 125.4, 126.1, 128.4, 128.9, 130.4, 138.5, 139.9, 162.2, 165.0, 166.9, 168.5, 181.6; Anal. Calcd. for C₂₁H₂₁N₅O₂S.H₂O (425.50): C 59.28, H 5.45, N 16.46. Found: C 59.47, H 5.05, N 15.75.

Ethyl 2-[2-(butyl thiocarbamoyl)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylates (5b): Yield: 97%, m.p: 125–127 °C. IR (KBr, cm⁻¹): 3226 (NH), 1724 (C=O), 1565 (C=N mixed with C=C Ar), 1439 (C=C Ar), 1539, 1377, 1171, 1020 (N-C=S amide I, II, III, IV bands), 1253, 1088 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.83 (t, J = 7.2 Hz, 3H, C^dH₃), 0.96 (t, J = 7.2 Hz, 3H, ester CH₃), 1.24 (m, 2H, C^cH₂), 1.47 (m, 2H, C^bH₂), 2.41 (s, 3H, C⁶-CH₃), 3.44 (q, J = 6.6 Hz, 2H, C^aH₂, appearing as t after deuteration), 4.08 (q, J = 7.2 Hz, 2H, ester CH₂), 7.40–7.46 (m, 5H, C⁴-Ar-Hs), 8.02 (t, J = 6.9 Hz, br, 1H, NHC₄H₉, D₂O-exchangeable), 9.23 (s, 1H, NHC=S, D₂O-exchangeable), 9.30 (s, 1H, C²-NH, D₂O-exchangeable). Anal. Calcd. for C₁₉H₂₅N₅O₂S (387.5): C 58.89, H 6.50, N 18.07. Found: C 58.77, H 6.37, N 18.69.

Ethyl 6-methyl-2-[2-(3-butyl-4-oxothiazolidin-2-ylidene)-hydrazino]-4 phenylpyrimidine-5-carboxylate (6): Ethyl bromoacetate (0.167 g, 1 mmol) was added to a suspension of thiosemicarbazide derivative 5b (1 mmol) in absolute EtOH (5 mL) containing anhydrous NaOAc (0.12 g, 1.5 mmol). The reaction mixture was heated under reflux for 3 h, concentrated to half its volume, allowed to attain RT and poured onto ice-cold H₂O (10 mL). The obtained precipitate was filtered, dried and crystallized from EtOH. Yield: 62%, m.p: 121–123 °C. IR (KBr, cm⁻¹): 3159 (NH), 1710 (C=O ester), 1624 (C=O amide), 1553 (C=N), 1470 (C=C Ar), 1262, 1092 (υ_as and υ_s C-O-C), 1168, 1018 (υ_as and υ_s C-S-C). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.9, 14.2, 20.0, 23.1, 29.2, 33.0, 42.8, 61.4, 116.4, 128.4, 128.8, 130.2, 138.9, 159.4, 161.1, 165.3, 166.9, 168.5, 172.1; Anal. Calcd. for C₂₁H₂₅N₅O₃S (427.54): C 59.00, H 5.89, N 16.38. Found: C 59.13, H 5.94, N 16.47

Ethyl 2-[2-(4-(4-bromophenyl)-3-phenylthiazol-2(3H)-ylidene)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylate (7): A solution of 4-bromophenacyl bromide (1 mmol) in absolute EtOH (5 mL) was gradually added to a suspension of the thiosemicarbazide derivative 5a (1 mmol) and the equimolar amount of anhydrous NaOAc (0.082 g, 1 mmol) in absolute EtOH (5 mL). The reaction mixture was heated under reflux for 3–4 h, concentrated to half its volume and left to attain RT. The obtained precipitate was filtered, washed with H₂O and crystallized from EtOH. Yield: 54%, m.p: 172–174 °C. IR (KBr, cm⁻¹): 3161 (NH), 1713 (C=O), 1627 (C=N), 1584, 1497 (C=C Ar), 1259, 1087 (υ_as and υ_s C-O-C), 1150, 1039 (υ_as and υ_s C-S-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.97 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.41 (s, 3H, C⁶-CH₃), 4.08 (q, J = 7.2 Hz, 2H, CH₃CH₂), 6.47 (s, 1H, thiazoline-H), 6.82–7.55 (m, 12 H, Ar-Hs), 7.64 (d, J = 8.4 Hz, 2H, Hb), 10.38, 10.43 (2 s, 1H, NH, D₂O exchangeable). Anal. Calcd. for C₂₉H₂₄BrN₅O₂S (586.5): C 59.39, H 4.12, N 11.94. Found: C 59.15, H 3.87, N 11.83.

2-[2-(Arylsulfonyl)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylates (8a,b): A mixture of hydrazine 2 (0.27 g, 1 mmol) and the appropriate arylsulfonyl chloride (1 mmol) in dry pyridine (5 mL) was stirred at RT for 24 h. The reaction mixture was diluted with crushed ice and neutralized with dilute HCl (10%). The obtained precipitate was filtered, dried and crystallized from benzene.

Ethyl 6-methyl-4-phenyl-2-[2-(phenylsulfonyl)hydrazino]-pyrimidine-5-carboxylate (8a): Yield: 66%, m.p: 98–100 °C. IR (KBr, Cm-1): 3218 (NH), 1719 (C=O), 1556 (C=N mixed with C=C Ar), 1438 (C=C Ar), 1342, 1170 (υ_as and υ_s SO₂), 1268, 1089 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.93 (t, J = 7.5 Hz, 3H, CH₃CH₂), 2.22 (s, br, 3H, C⁶-CH₃), 4.03 (q, J = 7.2 Hz, 2H, CH₃CH₂), 7.28–7.80 (m, 10H, Ar-Hs), 9.71 (s, 1H, C²-NH, D₂O-exchangeable), 9.85 (s, 1H, NHSO₂, D₂O-exchangeable). Anal. Calcd. for C₂₀H₂₀N₄O₄S (412.46): C 58.24, H 4.89, N 13.58. Found: C 58.51, H 5.20, N 15.32.

Ethyl 6-methyl-4-phenyl-2-[2-(4-tolylsulfonyl)hydrazino]-pyrimidine-5-carboxylate (8b): Yield: 64%, m.p: 127–129 °C. IR (KBr, cm⁻¹): 3211 (NH), 1721 (C=O), 1555 (C=N mixed with C=C Ar), 1438 (C=C Ar), 1345, 1165 (υ_as and υ_s SO₂), 1266, 1088 (υ_as and υ_s C-O-C).¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.92 (t, J = 7.5 Hz, 3H, CH₃CH₂), 2.22 (s, br, 6H, C⁶-CH₃ + C^4′’-CH₃), 4.02 (q, J = 7.5 Hz, 2H, CH₃CH₂), 7.33 (d, J = 7.8 Hz, dist, 2H, Ha), 7.40–7.51 (m, 5H, Ar-Hs), 7.60 (d, J = 7.8 Hz, 2H, Hb), 9.71 (s, 1H, C²-NH, D₂O-exchangeable), 9.80 (s, 1H, NHSO₂, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.9, 21.4, 22.6, 61.6, 116.9, 127.9, 128.2, 128.6, 129.5, 130.3, 137.2, 138.2, 143.4, 161.3, 164.6, 166.7, 168.3; EI-MS m/z (relative intensity): 426 ([M⁺], 20), 242 (100). Anal. Calcd. for C₂₁H₂₂N₄O₄S (426.49): C 59.14, H 5.20, N 13.14. Found: C 59.49, H 5.00, N 13.00.

Ethyl 2-[2-(ethoxycarbonyl)hydrazino]-6-methyl-4-phenylpyrimidine-5-carboxylate (9): Ethyl chloroformate (0.165 g, 1.5 mmol) was added to a suspension of the hydrazine 2 (0.27 g, 1 mmol) and anhydrous K₂CO₃ (0.27 g, 2 mmol) in dry dioxane (5 mL). The reaction mixture was heated under reflux while stirring for 5 h, left to cool to RT, diluted with ice-cold H₂O (30 mL) and refrigerated overnight. The separated product was filtered, dried and crystallized from CH₂Cl_2/petroleum ether (40–60°) (1:4). Yield: 70%, m.p: 112–114 °C. IR (KBr, cm⁻¹): 3332, 3307 (NH), 1710 (C=O), 1557 (C=N mixed with C=C Ar), 1444 (C=C Ar), 1256, 1089 (υ_as and υ_s C-O-C).¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.97 (t, J = 7.2 Hz, 3H, C⁵-ester CH₂CH₃), 1.21 (t, J = 7.2 Hz, 3H, carbamate CH₂CH₃), 2.49 (s, 3H, C⁶-CH₃), 4.07 (2 overlapping q, J = 7.2 Hz, 4H, 2 × CH₂), 7.40–7.55 (m, 5H, Ar-Hs), 8.76 and 9.13 (2s, br, 1H, C²-NH, D₂O-exchangeable), 9.33 (d, dist, 1H, NHCOOEt, D₂O-exchangeable). EI-MS m/z (relative intensity): 344 ([M⁺], 14), 242 (100). Anal. Calcd. for C₁₇H₂₀N₄O₄ (344.37): C 59.29, H 5.85, N 16.27. Found: C 59.00, H 5.60, N 16.50.

Ethyl 2-[2-(1-ethoxy-1-oxobutan-3-ylidene)hydrazino]-6-methyl-4- phenylpyrimidine-5-carboxylate (10): A mixture of hydrazine derivative 2 (0.27 g, 1 mmol) and ethyl acetoacetate (0.13 g, 1 mmol) in absolute EtOH (5 mL) was heated under reflux for 4 h. the reaction mixture was concentrated and cooled to RT. The obtained precipitate was filtered, washed with petroleum ether (40–60°), dried and crystallized from EtOH. Yield: 64%, m.p: 98–100 °C. IR (KBr, cm⁻¹): 3196 (NH), 1721 (C=O), 1577 (C=N mixed with C=C Ar), 1544 (C=C Ar), 1255, 1087 (υ_as and υ_s C-O-C).¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.93 (t, J = 7.2 Hz, 3H, C⁵-CO₂CH₂CH₃), 1.20 (t, J = 7.2 Hz, 3H, CH₂CO₂CH₂CH₃), 2.00 (s, 3H, CH₃-C=N), 2.44 (s, 3H, C⁶-CH₃), 3.36 (s, 2H, CH₂CO₂C₂H₅), 4.05 (q, J = 7.2 Hz, 2H, C⁵-CO₂CH₂CH₃), 4.11 (q, J = 7.2 Hz, 2H, CH₂CO₂CH₂CH₃), 7.49–7.51 (m, 5H, Ar-Hs), 10.17 (s, 1H, NH, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.9, 14.6, 17.1, 22.9, 40.2, 61.0, 61.5, 117.3, 128.3, 128.8, 130.2, 138.8, 148.5, 159.9, 165.7, 167.0, 168.3, 170.5; EI-MS m/z (relative intensity): 384 ([M⁺], 6), 297 (100). Anal. Calcd. for C₂₀H₂₄N₄O₄ (384.43): C 62.49 H 6.29, N 14.57. Found: C 62.56, H 6.38, N 15.00.

Ethyl 6-methyl-2-(3,5-dimethyl-1H-pyrazol-1-yl)-4-phenylpyrimidine-5-carboxylate (11): A mixture of hydrazine derivative 2 (0.27 g, 1 mmol) and acetylacetone (0.1 g, 1 mmol) in absolute EtOH (5 mL) was heated under reflux for 5 h. The reaction mixture was concentrated, diluted with ice-cold H₂O (20 mL) and refrigerated overnight. The obtained precipitate was filtered, dried and crystallized from petroleum ether (40–60°). Yield: 92%, m.p: 60–62 °C. IR υ (KBr, cm⁻¹): 1719 (C=O), 1550 (C=N mixed with C=C Ar), 1437 (C=C Ar), 1263, 1093 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 1.04 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.21 (s, 3H, C⁶-CH₃), 2.59 (s, 3H, C^3″-CH₃), 2.62 (s, 3H, C^5″-CH₃), 4.20 (q, J = 7.2 Hz, 2H, CH₃CH₂), 6.19 (s, 1H, C^4′’-H), 7.51–7.67 (m, 5H, Ar-Hs). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 14.0, 15.5, 23.0, 62.3, 110.8, 122.4, 128.7, 129.2, 131.0, 137.4, 143.2, 150.9, 156.2, 164.8, 167.6, 167.8; EI-MS m/z (relative intensity): 336 ([M⁺], 100). Anal. Calcd. for C₁₉H₂₀N₄O₂ (336.39): C 67.84, H 5.99, N 16.66. Found: C 67.79, H 6.13, N 16.54

Ethyl 6-methyl-2-(3,5-dioxopyrazolidin-1-yl)-4-phenylpyrimidine-5-carboxylate (12): A mixture of hydrazine 2 (0.27 g, 1 mmol) and diethyl malonate (0.32 g, 2 mmol) in absolute EtOH/glacial HOAc (4:1) (5 mL) was heated under reflux for 11 h, concentrated to a small volume and diluted with ice-cold H₂O. The separated product was filtered, dried and crystallized from CH₂Cl_2/petroleum ether (40–60°) (1:4). Yield: 42%, m.p: 166–168 °C. IR (KBr, cm⁻¹): 3327, 3276 (OH, NH), 1710 (C=O ester), 1668 (C=O amide), 1557 (C=N mixed with C=C Ar), 1440 (C=C Ar), 1263, 1091 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.90 (t, J = 6.85 Hz, 3H, CH₂CH₃), 1.87 (s, 3H, C⁶-CH₃), 4.01 (q, J = 6.75 Hz, 2H, CH₂CH₃), 4.13 (s, 1H, C^4′’-H), 7.44 (s, 5H, Ar-Hs), 9.25 (s, 1H, NH, D₂O-exchangeable), 9.83 (s, br, 1H, OH, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.91, 21.1, 61.5, 116.8, 128.3, 128.9, 130.3, 138.7, 162.1, 162.5, 165.2, 167.0, 168.5, 169.7; Anal. Calcd. for C₁₇H₁₆N₄O₄ (340.33): C 59.99, H 4.74, N 16.46. Found: C 59.54, H 5.10, N 16.68.

Ethyl 5-methyl-7-phenyl-1,2,4-triazolo[4,3-a]pyrimidine-6-carbxylate (13): A solution of hydrazine derivative 2 (0.27 g, 1 mmol) in formic acid (5 mL) was heated under reflux for 27 h. The reaction mixture was concentrated to a small volume and diluted with ice-cold H₂O. The obtained precipitate was filtered, washed with H₂O, dried and crystallized from EtOH/H₂O. Yield 34%, m.p: 104–106 °C. IR (KBr, cm⁻¹): 1724 (C=O), 1608 (C=N), 1520, 1442 (C=C Ar), 1285, 1087 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 0.96 (t, J = 7.5 Hz, 3H, CH₃CH₂), 2.90 (s, 3H, C⁵-CH₃), 4.15 (q, J = 7.5 Hz, 2H, CH₃CH₂), 7.50–7.63 (m, 5H, Ar-Hs), 8.80 (s, 1H, C³-H). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.4, 15.4, 62.2, 116.9, 128.1, 128.7, 130.2, 138.0, 148.5, 153.7, 157.0, 160.9, 165.5; EI-MS m/z (relative intensity): 282 ([M⁺], 54), 253 (100). Anal. Calcd. for C₁₅H₁₄N₄O₂ (282.3): C 63.82, H 5.00. N 19.85. Found: C 63.86, H 5.05, N 19.00

Ethyl 6-methyl-8-phenyl-3-(substituted phenyl)-4H-pyrimido[2,1-c]-1,2,4-triazine-7-carboxylates (14a,b): The appropriate 4-substituted phenacyl bromide (1 mmol) was added to a solution of the hydrazine derivative 2 (0.27 g, 1 mmol) in absolute EtOH (5 mL). The reaction mixture was heated under reflux for 2 h, concentrated to half its volume and then left to cool to RT. The obtained precipitate was filtered, washed with petroleum ether (40–60°), dried and crystallized from EtOH.

Ethyl 6-methyl-3,8-diphenyl-4H-pyrimido[2,1-c]-1,2,4-triazine-7-carboxylate (14a): Yield: 22%, m.p: 234–236 °C (charring). IR (KBr, cm⁻¹): 1724 (C=O), 1611 (C=N), 1553, 1446 (C=C Ar), 1257, 1049 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 300 MHz) δ ppm: 1.00 (t, J = 7.2 Hz, 3H, CH₃CH₂), 2.80 (s, 3H, C⁶-CH₃), 4.20 (q, J = 7.2 Hz, 2H, CH₃CH₂), 5.56 (s, 2H, C⁴-H₂), 7.57–7.96 (m, 10 H, Ar-Hs), 13.26 (s, 1H, NH, D₂O-exchangeable). EI-MS m/z (relative intensity): 372 ([M⁺], 65), 77 (100). Anal. Calcd. for C₂₂H₂₀N₄O₂ (372.42): C 70.95, H 5.41, N 15.04. Found: C 58.86, H 5.02, N 14.89.

Ethyl 3-(4-bromophenyl)-6-methyl-8-phenyl-4H-pyrimido[2,1-c]-1,2,4-triazine-7-carboxylate (14b): Yield: 27%, m.p: 242–244 °C (charring). IR (KBr, cm⁻¹): 1725 (C=O), 1615 (C=N), 1548, 1446(C=C Ar), 1256, 1056 (υ_as and υ_s C-O-C). ¹H-NMR (DMSO-d₆, 500 MHz) δ ppm: 0.97 (t, J = 6.9 Hz, 3H, CH₃CH₂), 2.79 (s, 3H, C⁶-CH₃), 4.17 (q, J = 6.9 Hz, 2H, CH₃CH₂), 5.54 (s, 2H, C⁴-H₂), 7.54–7.64 (m, 3H, Ar-Hs), 7.64–7.70 (m, 2H, Ar-Hs), 7.74 (d, J = 8.4Hz, 2H, Ha), 7.88 (d, J = 8.4Hz, 2H, Hb), 13.28 (s, 1H, NH, D₂O-exchangeable). ¹³C-NMR (DMSO-d₆, 125 MHz) δ ppm: 13.3, 17.5, 39.7, 62.9, 120.1, 125.7, 128.5, 128.6, 129.7, 131.6, 132.4, 133.0, 135.6, 144.7, 147.7, 160.6, 164.9, 169.6; Anal. Calcd. for C₂₂H₁₉BrN₄O₂ (451.32): C 58.55, H 4.24, N 12.41. Found: C 53.11, H 4.29, N 12.20.

3.2. Biological Evaluation

3.2.1. Experimental Animals

Animals were obtained and housed in Moassat Hospital Animal House, Pharmacology Department, Faculty of Medicine, Alexandria University. Rats and rabbits were kept in cages with wide mesh wire bottoms under standard conditions of light and temperature and allowed food and H₂O ad libitum (dogs were kept at separate theatres). The experimental protocol was approved by the Animal Care and Use Committee, Faculty of Pharmacy, Alexandria University (ACUC project number 15).

3.2.2. Data Recoding

Intestinal responses were recorded using an isometric transducer (Model TRI 201, Panlab S.I.) connected to an amplifier (Model Iso 510, Panlab S.I.). Tissues were mounted in a Bioscience organ bath.

In normotensive anesthetized dog experiments, mean arterial blood pressure (MAP) was recorded on a Grass polygraph via a pressure transducer (Model TRA 021, Panlab S.I.) triggered by an amplifier (Model Iso 510, Panlab S.I.) and connected to a mercury manometer.

3.2.3. Statistical Analysis and Data Interpretation

Statistical analysis was conducted using GraphPad Prism version 3.02 software package [44] to calculate IC₅₀, mean, standard deviation and standard error of each mean and for comparison between different groups involved. One-way test was used for comparison between independent samples.

3.2.4. In Vitro Calcium Channel Blocking Activity

Rat Colon

Thirty Wistar albino rats (200–250 g) of either sex were starved with free access to H₂O for 24 h prior to experiments and sacrificed by cervical dislocation on the day of the experiment; the abdominal cavity was opened and the ascending colon was rapidly removed and immersed in Kreb’s solution of the following composition (mM): NaCl 118.4, KCl 4.7, MgSO₄.H₂O 1.2, KH₂PO₄.2H₂O 1.2, NaHCO₃ 25, CaCl₂ 1.25 and glucose 11.1.

Segments (1.5–2 cm) were mounted vertically under 1g tension in a 25 mL organ bath containing Kreb’s solution maintained at 37 °C and aerated with carbogen (95% O₂ and 5% CO₂). Preparations were allowed to equilibrate for about 30 min with regular washes.

Solutions of nifedipine and test compounds in DMSO, selected for in vitro calcium channel blocking activity [26], were freshly prepared, protected from light and added to the organ bath to give a final concentration of 10⁻⁵ M.

Tissues were contracted with 100 mM KCl and the maximum response was recorded. Tissues were then washed thoroughly with Kreb’s solution and, after reaching a steady state, were preincubated for 5 min with test compounds (10⁻⁵ M); again, KCl was added with the same final concentration and maximum contractions were recorded.

Rabbit Jejunum

Eight white New Zealand rabbits (1.5–2 kg) of either sex were starved with free access to H₂O for 24 h prior to experiments and then slaughtered; the abdomen was opened and the jejunal portion was immediately isolated and kept in Tyrode’s solution of the following composition (mM): KCl 2.68, NaCl 136.9, MgCl₂ 1.05, NaHCO₃ 11.90, NaH₂PO₄ 0.42, CaCl₂ 1.8 and glucose 5.55.

Segments (1.5–2 cm) were mounted vertically under 1g tension in a 25 mL organ bath containing Tyrode’s solution maintained at 37 °C and aerated with carbogen (95% O₂ and 5% CO₂). Preparations were allowed to equilibrate for about 30 min with regular washes. The same steps were followed as in rat colon for preliminary screening.

For quantitative studies, contractions produced by KCl (100 mM) were recorded in the absence and presence of different concentrations of active compounds. The percentage of inhibition of KCl-induced contractions was plotted against the concentration of the compounds for the determination of IC₅₀.

3.2.5. In Vivo Hypotensive Activity on Normotensive Anesthetized Dogs

Eight adult normotensive dogs (15–25 kg) of either sex were anesthetized with thiopental sodium (35 mg/kg, i.v.), and additional doses were administered when needed. A 5 cm incision was made in the skin of the groin and underlying muscles were cut. Both femoral vein and artery were exposed, and each was cannulated for drug administration and determination of arterial blood pressure, respectively. The arterial cannula was connected to the pressure transducer, and arterial blood pressure was then recorded on the manometer and changes were displayed on the polygraph. Normal saline (0.90% w/v NaCl) was infused slowly throughout the experiments.

Solutions of nifedipine and test compounds (0.7 M) in DMSO were injected i.v. [27], DMSO alone did not influence the dogs’ mean MAP in control experiments. At least 15 min was allowed between challenge doses and appropriate vehicle controls. Records for test compounds were compared to the corresponding control values.

4. Conclusions

The Biginelli-derived pyrimidines and fused pyrimidines 2, 3a, 3b, 4, 11 and 13 showed the highest ex vivo calcium channel blocking activities. It was noticed that the potency among the promising compounds could be a function of the number and size of possible hydrogen bond donors/acceptors at C². The substituent flexibility also critically contributed to the detected activity. Moreover, 2 and 11 revealed good hypotensive activities in dogs. A ligand-based pharmacophore model described the binding mode of the newly synthesized active compounds. This may also serve as a reliable basis for designing new active pyrimidine-based CCBs. Finally, the selected most active compounds 2 and 11 displayed drug-like in silico ADME parameters.