Design, Synthesis, and Antimicrobial Evaluation of New Annelated Pyrimido[2,1-c][1,2,4]triazolo[3,4-f][1,2,4]triazines

A series of 34 new pyrimido[2,1-c][1,2,4]triazine-3,4-diones were synthesized and fully characterized using IR, NMR, MS, and microanalytical analysis. In vitro investigation of 12 compounds of this series revealed promising antimicrobial activity of the conjugates 15a and 15f–j that were tagged with electron-withdrawing groups, with sensitivities ranging from 77% to as high as 100% of the positive control. The investigation of antimicrobial activity included Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6535, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 8739 (EC), and fungal strains Candida albicans ATCC 10231 and Aspergillus brasiliensis ATCC 16404.


Introduction
Genetic mutations are major contributors to the prognosis of drug-resistant microbial strains [1]. These strains are, in most cases, able to detoxify drugs using mutant digestive enzymes like β-lactamases [2]. In addition, they are able to prevent the intracellular build-up of drugs to microbially nontoxic levels using mutant drug efflux proteins [3]. Spontaneous, error-prone replication bypass, errors introduced during DNA repair, and induced mutations are the four main modes of mutation encountered in nature. Induced mutations, in particular, emerge after a gene has come into contact with a mutagen or environmental inducer [4].
Therefore, seeking alternatives to commercially available drugs that will, sooner or later, no longer be effective remains a pharmacological challenge. The discovery of new antibiotics and innovative pharmacophore architectures for synthesis in particular, those based on computer-aided drug design (CADD) programs [5], in addition to molecular library approaches provide opportunities to develop new drug candidates [6].

Chemistry
As a part of our ongoing research toward the synthesis of a variety of nitrogen bridgehead heterocycles, we report the utility of the hydrazine derivative 1 [25,26] to construct fused pyrimidotriazine 2, (Scheme 1). Treatment of the cyclic 1,2-bioxygen analogue 2 with thiosemicarbazide produced the thioureido analogue 3, as mainly indicated by mass and/or NMR analyses. The presence of the thiocarbonyl group was deduced by 13 C-NMR as a singlet at 181.2 ppm and its IR absorption band at 1290 cm −1 , whereas the presence of the amino group was confirmed by 1 H-NMR as a singlet at 8.56 ppm. The recorded mass at m/z 397.08 corresponds to the formula C17H15N7OS2. All these data support the formation of the thiosemicarbazone derivative 3 via simple condensation of one amino group to afford the thioureido analogue 3 without further cyclocondensation.

Scheme 1. Reagents and conditions for synthesis of compounds (2 and 3).
The thioureido analogue 3 was subject to a sequence of treatments to investigate the reactivity of its thioureido moiety in an attempt to attain target thiazole and/or thiazine architectures. Thus, the treatment of compound 3 with benzylidenemalononitrile in boiling dioxane led to 1,3-thiazine-5carbonitrile derivative 5 via the carbonitrile intermediate 4. This intermediate undergoes intramolecular cyclization via the nucleophilic addition of NH2 to the nitrile group, affording the 1,3thiazine analogue 5 with a 75% yield (Scheme 2). Strong absorption bands at 3325 and 2219 cm −1 were observed for the NH2 and C≡N groups, respectively. Upon treatment of the thioureido 3 with 3chloropentane-2,4-dione in refluxing EtOH, the 2-substituted 4-methyl-5-acetylthiazole derivative 6

Chemistry
As a part of our ongoing research toward the synthesis of a variety of nitrogen bridgehead heterocycles, we report the utility of the hydrazine derivative 1 [25,26] to construct fused pyrimidotriazine 2, (Scheme 1). Treatment of the cyclic 1,2-bioxygen analogue 2 with thiosemicarbazide produced the thioureido analogue 3, as mainly indicated by mass and/or NMR analyses. The presence of the thiocarbonyl group was deduced by 13 C-NMR as a singlet at 181.2 ppm and its IR absorption band at 1290 cm −1 , whereas the presence of the amino group was confirmed by 1 H-NMR as a singlet at 8.56 ppm. The recorded mass at m/z 397.08 corresponds to the formula C 17 H 15 N 7 OS 2 . All these data support the formation of the thiosemicarbazone derivative 3 via simple condensation of one amino group to afford the thioureido analogue 3 without further cyclocondensation.

Chemistry
As a part of our ongoing research toward the synthesis of a variety of nitrogen bridgehead heterocycles, we report the utility of the hydrazine derivative 1 [25,26] to construct fused pyrimidotriazine 2, (Scheme 1). Treatment of the cyclic 1,2-bioxygen analogue 2 with thiosemicarbazide produced the thioureido analogue 3, as mainly indicated by mass and/or NMR analyses. The presence of the thiocarbonyl group was deduced by 13 C-NMR as a singlet at 181.2 ppm and its IR absorption band at 1290 cm −1 , whereas the presence of the amino group was confirmed by 1 H-NMR as a singlet at 8.56 ppm. The recorded mass at m/z 397.08 corresponds to the formula C17H15N7OS2. All these data support the formation of the thiosemicarbazone derivative 3 via simple condensation of one amino group to afford the thioureido analogue 3 without further cyclocondensation.

Scheme 1. Reagents and conditions for synthesis of compounds (2 and 3).
The thioureido analogue 3 was subject to a sequence of treatments to investigate the reactivity of its thioureido moiety in an attempt to attain target thiazole and/or thiazine architectures. Thus, the treatment of compound 3 with benzylidenemalononitrile in boiling dioxane led to 1,3-thiazine-5carbonitrile derivative 5 via the carbonitrile intermediate 4. This intermediate undergoes intramolecular cyclization via the nucleophilic addition of NH2 to the nitrile group, affording the 1,3thiazine analogue 5 with a 75% yield (Scheme 2). Strong absorption bands at 3325 and 2219 cm −1 were observed for the NH2 and C≡N groups, respectively. Upon treatment of the thioureido 3 with 3chloropentane-2,4-dione in refluxing EtOH, the 2-substituted 4-methyl-5-acetylthiazole derivative 6 Scheme 1. Reagents and conditions for synthesis of compounds (2 and 3).
The thioureido analogue 3 was subject to a sequence of treatments to investigate the reactivity of its thioureido moiety in an attempt to attain target thiazole and/or thiazine architectures. Thus, the treatment of compound 3 with benzylidenemalononitrile in boiling dioxane led to 1,3-thiazine-5-carbonitrile derivative 5 via the carbonitrile intermediate 4. This intermediate undergoes intramolecular cyclization via the nucleophilic addition of NH 2 to the nitrile group, affording the 1,3-thiazine analogue 5 with a 75% yield (Scheme 2). Strong absorption bands at 3325 and 2219 cm −1 were observed for the NH 2 and C≡N groups, respectively. Upon treatment of the thioureido 3 with 3-chloropentane-2,4-dione in refluxing EtOH, the 2-substituted 4-methyl-5-acetylthiazole derivative 6 was obtained. The most characteristic signal of compound 6 ( 1 H-NMR), due to the thiazole exchangeable (N-H) proton at 10.83 ppm, in addition to two new singlets, were observed at 2.51 and 2.72 ppm, attributed to the methyl and acetyl protons, respectively. Taken together, these data confirmed the structure of compound 6.
Similarly, compound 3 was cyclized with dimethyl but-2-ynedioate in refluxing dioxane to annulate the thiazole analogue 8 with an 80% yield (Scheme 2). Formation of compound 8 can be explained on the basis of an initial Michael-type addition of the thiol function in the thioureido moiety to the activated triple bond in dimethyl but-2-ynedioate to afford the non-isolable intermediate 7, which undergoes intramolecular cyclization via loss of another MeOH molecule (route a) to yield the thiazole derivative 8. The carbothioamide absorption bands originally observed in 3 at 1290 and 3230 cm −1 disappeared after this reaction.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 17 was obtained. The most characteristic signal of compound 6 ( 1 H-NMR), due to the thiazole exchangeable (N-H) proton at 10.83 ppm, in addition to two new singlets, were observed at 2.51 and 2.72 ppm, attributed to the methyl and acetyl protons, respectively. Taken together, these data confirmed the structure of compound 6. Similarly, compound 3 was cyclized with dimethyl but-2-ynedioate in refluxing dioxane to annulate the thiazole analogue 8 with an 80% yield (Scheme 2). Formation of compound 8 can be explained on the basis of an initial Michael-type addition of the thiol function in the thioureido moiety to the activated triple bond in dimethyl but-2-ynedioate to afford the non-isolable intermediate 7, which undergoes intramolecular cyclization via loss of another MeOH molecule (route a) to yield the thiazole derivative 8. The carbothioamide absorption bands originally observed in 3 at 1290 and 3230 cm −1 disappeared after this reaction. Chlorination of the 1,2-dioxo compound 2 with POCl3 afforded dielectrophile 3-chloro-8-phenyl-6-(thiophen-2-yl)-6,7-dihydro-4H-pyrimido [2,1-c] [1,2,4]triazin-4-one (9) with a 75% yield (Scheme 3). The N-H stretching band and its 1 H-NMR signal for compound 2 disappeared after this step. Hydrazinolysis of compound 9 with an excess of N2HNH2.H2O afforded the hydrazinyltriazine 10, which undergoes further cyclization due to the reactivity of its hydrazinyl tag, which can be exploited in developing triazolotriazine derivatives. Thus, cyclocondensation of intermediate 10 with phenacyl bromide, triethyl orthoformate, ethyl chloroformate, chloroacetyl chloride, and, finally, dimethylformamide dimethyl acetal (DMF-DMA) afforded the series of compounds 11, 13, 14, and 15 displayed in Scheme 2 under the given conditions. Further hydroxymethylation of compound 11 Scheme 2. Reagents and conditions for the synthesis of compounds 5, 6, and 8.
Chlorination of the 1,2-dioxo compound 2 with POCl 3 afforded dielectrophile 3-chloro-8-phenyl-6-(thiophen-2-yl)-6,7-dihydro-4H-pyrimido [2,1-c] [1,2,4]triazin-4-one (9) with a 75% yield (Scheme 3). The N-H stretching band and its 1 H-NMR signal for compound 2 disappeared after this step. Hydrazinolysis of compound 9 with an excess of N 2 HNH 2 .H 2 O afforded the hydrazinyltriazine 10, which undergoes further cyclization due to the reactivity of its hydrazinyl tag, which can be exploited in developing triazolotriazine derivatives. Thus, cyclocondensation of intermediate 10 with phenacyl bromide, triethyl orthoformate, ethyl chloroformate, chloroacetyl chloride, and, finally, dimethylformamide dimethyl acetal (DMF-DMA) afforded the series of compounds 11, 13, 14, and 15 displayed in Scheme 2 under the given conditions. Further hydroxymethylation of compound 11 afforded derivative 12 with a 75% yield. The structure of compound 12 was deduced based on its spectral data, where its mass spectrum recorded a molecular ion peak (C 25 H 20 N 6 O 2 S) at m/z 468.15, whereas the IR spectrum showed characteristic absorption bands at 3315 cm −1 due to stretching of the Intermediate 10 was cyclocondensed with carbon disulfide to produce the Mannich base precursor 17, which upon a classical one-pot three-component reaction, produced a set of Mannich bases (18a-j) with high yields (Scheme 4). The presence of these bases on different pharmacophores have unique potential for medical research [31]. The formation of compounds 18a-j are demonstrated on the basis of the initial Mannich reaction, which proceeds in two steps: First, the reaction between HCHO and the amine leads to the formation of the non-isolable iminium ion intermediate, which loses a H2O molecule in situ. Secondly, the thiocarbonyl compound undergoes tautomerization to produce its thiol tautomer, which proceeds to attack the iminium ion, which finally yields the target β-amino-thiocarbonyl compounds (18a-j) [30]. The IR spectra of the isolated compounds (18a-j) displayed common characteristic absorption bands around the region 3165-3281 cm −1 due to the secondary amine groups. This was further evidenced by their 1 H-NMR broad singlets at ~4.80 ppm (D2O exchangeable), whereas the methylene singlet ( 1 H-NMR) of their phenylaminomethyl moiety was observed at ~5.40 ppm. The presence of the nitro group in 18j was elucidated based on the IR spectrum, which showed two characteristic absorption bands at 1390 and 1520 cm −1 due to NO2str (as and sym), respectively. The mass spectrum of 18j displayed an ion peak at m/z 530.09 (M + , 30%) corresponding to the expected molecular formula C24H18N8O3S2. Intermediate 10 was cyclocondensed with carbon disulfide to produce the Mannich base precursor 17, which upon a classical one-pot three-component reaction, produced a set of Mannich bases (18a-j) with high yields (Scheme 4). The presence of these bases on different pharmacophores have unique potential for medical research [31]. The formation of compounds 18a-j are demonstrated on the basis of the initial Mannich reaction, which proceeds in two steps: First, the reaction between HCHO and the amine leads to the formation of the non-isolable iminium ion intermediate, which loses a H 2 O molecule in situ. Secondly, the thiocarbonyl compound undergoes tautomerization to produce its thiol tautomer, which proceeds to attack the iminium ion, which finally yields the target β-amino-thiocarbonyl compounds (18a-j) [30]. The IR spectra of the isolated compounds (18a-j) displayed common characteristic absorption bands around the region 3165-3281 cm −1 due to the secondary amine groups. This was further evidenced by their 1 H-NMR broad singlets at~4.80 ppm (D 2 O exchangeable), whereas the methylene singlet ( 1 H-NMR) of their phenylaminomethyl moiety was observed at~5.40 ppm. The presence of the nitro group in 18j was elucidated based on the IR spectrum, which showed two characteristic absorption bands at 1390 and 1520 cm −1 due to NO 2str (as and sym), respectively. The mass spectrum of 18j displayed an ion peak at m/z 530.09 (M + , 30%) corresponding to the expected molecular formula C 24 H 18 N 8 O 3 S 2. Upon smooth cyclocondensation of compound 10 with KSCN, ethyl cyanoacetate, acetic anhydride, benzoyl chloride, and thionyl chloride, a series of pyrimido-[1,2,4]triazolo-[1,2,4]triazine derivatives (19)(20)(21)(22)(23) were obtained (Scheme 5). The 1 H-NMR spectra of these compounds showed the lack of signals corresponding to the hydrazinyl protons originally observed in 10 ( 1 H-NMR) at 4.82 and 8.32 ppm. The formation of compound 20 was confirmed through its mass spectrum, which showed a m/z value at 387.08 corresponding to the expected molecular formula C19H13N7OS, whereas its IR spectrum indicated strong absorption bands at 1686 and 2218 cm −1 attributed to the C=O and C≡N groups, respectively.  Upon smooth cyclocondensation of compound 10 with KSCN, ethyl cyanoacetate, acetic anhydride, benzoyl chloride, and thionyl chloride, a series of pyrimido-[1,2,4]triazolo- [1,2,4]triazine derivatives (19)(20)(21)(22)(23) were obtained (Scheme 5). The 1 H-NMR spectra of these compounds showed the lack of signals corresponding to the hydrazinyl protons originally observed in 10 ( 1 H-NMR) at 4.82 and 8.32 ppm. The formation of compound 20 was confirmed through its mass spectrum, which showed a m/z value at 387.08 corresponding to the expected molecular formula C 19 H 13 N 7 OS, whereas its IR spectrum indicated strong absorption bands at 1686 and 2218 cm −1 attributed to the C=O and C≡N groups, respectively. Upon smooth cyclocondensation of compound 10 with KSCN, ethyl cyanoacetate, acetic anhydride, benzoyl chloride, and thionyl chloride, a series of pyrimido- [1,2,4]triazolo- [1,2,4]triazine derivatives (19)(20)(21)(22)(23) were obtained (Scheme 5). The 1 H-NMR spectra of these compounds showed the lack of signals corresponding to the hydrazinyl protons originally observed in 10 ( 1 H-NMR) at 4.82 and 8.32 ppm. The formation of compound 20 was confirmed through its mass spectrum, which showed a m/z value at 387.08 corresponding to the expected molecular formula C19H13N7OS, whereas its IR spectrum indicated strong absorption bands at 1686 and 2218 cm −1 attributed to the C=O and C≡N groups, respectively.

Pharmacological Evaluation
Antimicrobial Impact According to the disc diffusion method [32], compounds 18a-j, 10, and 17 were screened for their in vitro antimicrobial activity. This series was proposed for antimicrobial screening as it represents the largest homologous series that is suitable for structure-activity relationship (SAR) considerations. The investigations included two Gram-positive strains, Bacillus subtilis ATCC 6633 (BS) and Staphylococcus aureus ATCC 6535 (SA); two Gram-negative strains, Pseudomonas aeruginosa

Pharmacological Evaluation
Antimicrobial Impact According to the disc diffusion method [32], compounds 18a-j, 10, and 17 were screened for their in vitro antimicrobial activity. This series was proposed for antimicrobial screening as it represents the largest homologous series that is suitable for structure-activity relationship (SAR) considerations. The investigations included two Gram-positive strains, Bacillus subtilis ATCC 6633 (BS) and Staphylococcus aureus ATCC 6535 (SA); two Gram-negative strains, Pseudomonas aeruginosa Scheme 7. Reagents and conditions for the synthesis of compounds 28 and 31.

Pharmacological Evaluation
Antimicrobial Impact According to the disc diffusion method [32], compounds 18a-j, 10, and 17 were screened for their in vitro antimicrobial activity. This series was proposed for antimicrobial screening as it represents the largest homologous series that is suitable for structure-activity relationship (SAR) considerations. The investigations included two Gram-positive strains, Bacillus subtilis ATCC 6633 (BS) and Staphylococcus aureus ATCC 6535 (SA); two Gram-negative strains, Pseudomonas aeruginosa ATCC 27853 (PA) and Escherichia coli ATCC 8739 (EC); and two fungal strains, Candida albicans ATCC 10231 (CA) and Aspergillus brasiliensis ATCC 16404 (AB). Positive controls included ampicillin and gentamicin for Gram-positive and Gram-negative bacteria, respectively, and amphotericin B for fungi, while DMSO was used as the negative control. The minimum inhibitory concentration (MIC) was determined according to the reported method [32].
The inhibitory effects of the synthesized compounds versus these organisms are presented in Table 1. The parent precursors 10 and 17 were less active compared with the triazole's N1-substituted series 18a-j. This finding agrees with the activity of most azole-based antifungal drugs; for instance, fluconazole, ravuconazole, and rufinamide.  Analyses of the MIC values and the inhibition zone diameters, as given in Table 1, show that the test organisms were generally sensitive to compounds 18a-j. The sensitivity ranged from 77% to as high as 100% of the positive controls.
In the case of bacteria, congeners tagged with electron-withdrawing groups 18f-j showed better activity than those modified with electron-donating groups. Electron-withdrawing substituents at the para position, as in compounds 18j and 18f, displayed higher activity than on the ortho or meta positions, as in compounds 18i, 18h, and 18g.
This trend deviated for fungi, where derivatives supported by electron-donating groups 18a-e displayed higher activity than compounds 18f-j. Compound 18d, with a p-methoxy tag, was the most potent among those in the tested series. The observed prominent antifungal profile of compounds 18a-j supports our hypothesis that new N1-substituted triazole architectures show potential as renewable antifungals. The latent antifungal activity of azoles is attributed to their ability to interfere with and disrupt fungal lanosterol biosynthesis [33], which is required for membrane permeability.
In conclusion, a series of new aza-heterocycles was prepared according to classical chemical methods. They are tripod and tetrapod pharmacophoric architectures that can enhance antimicrobial potency. The derivatives 18a-g displayed promising antimicrobial activities. Derivatives supported by electron withdrawing groups (EWGs) displayed excellent antibacterial activities, whereas those tagged with electron donating groups (EDGs) were better as antifungals. These results support the case for a second phase of biochemical research to elucidate their possible modes of action and determine whether or not these are in line with classical mechanisms.

General Information
Reagents were purchased from Sigma Aldrich (Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia) and used without further purification. The reaction progress was monitored by TLC on silica gel pre-coated F254 Merck plates (Merck, Darmstadt, Germany). Spots were visualized by ultraviolet irradiation. All melting points were determined on a digital Gallen-Kamp MFB-595 instrument (Gallenkamp, London, UK) using open capillary tubes and were uncorrected. IR spectra were recorded as potassium bromide discs using Bruker-Vector 22 FTIR spectrophotometer (Bruker, Manasquan, NJ, USA). The NMR spectra were recorded with a Varian Mercury VXR-300 NMR spectrometer (Bruker, Marietta, GA, USA) at 300 and 75 MHz for 1 H and 13 C NMR spectra, respectively, using DMSO-d 6 as the solvent. Mass spectra were recorded on a Hewlett Packard MS-5988 spectrometer (Hewlett Packard, Palo Alto, CA, USA) at 70 eV. Elemental analyses were conducted at the Micro-Analytical Center of Taif University, Taif, KSA.

Methodology
The antimicrobial activity of the new synthesized compounds was evaluated using the disc diffusion method [32]. Plates 90 mm in diameter containing either Müller-Hinton agar for the growth of bacteria or Sabouraud dextrose agar for the growth of fungi were prepared, and each plate was separately inoculated with different cultures of the test bacteria and fungi by aseptically swabbing onto the entire surface of the agar with cotton wool. A 6-mm-diameter filter paper disc was saturated with 200 µg/mL of the test compound in DMSO. The discs were air-dried and placed aseptically at the center of the plates. The plates were left in a refrigerator for 1 h before incubation to allow the extract to diffuse into the agar. Ampicillin and gentamicin were used as bacterial standards and amphotericin B as the fungal reference to evaluate the efficacy of the tested compounds, with DMSO used as a negative control. After incubation of the plate at a suitable temperature (37 • C for bacteria and 25 • C for fungi), the results were recorded for each tested compound as the average diameter (mm) of the inhibition zone (IZ) of bacterial or fungal growth around the discs. The minimum inhibitory concentration (MIC) was determined for compounds that exhibited significant growth inhibition zones of more than 15 mm using the two-fold serial dilution method [34]. The MIC (µM) and IZ values are listed in Table 1.