Design, Synthesis and Pharmacological Evaluation of Novel Annelated Pyrimido[2,1-c][1,2,4]triazolo[3,4-f][1,2,4]Triazines as Antimicrobial Agents

In our tactic to construct bio-active molecules, a series of novel pyrimido[2,1c][1,2,4]triazine-3,4-diones based heterocycles, were synthesized and evaluated for their in vitro antimicrobial impacts. The exploratory bioassay results declared that, the majority of the evaluated compounds exhibited considerable anti-microbial activity comparable to the reference drugs. Conjugates 15j, 15f, 15i, 15h, 15g and 15a were found to be the most potent antibacterial, indicating that conjugates bearing electron-attracting substituents exhibited higher potency than these with electron-releasing substituents.


Introduction
The vast use of antibiotics in the 1940s, puts a limit of the women's death from post-childbirth infections, and diseases such as tuberculosis were rife, using of antibiotics provide us the ability to avoid these tragedies.But so, micro-organisms become resistant to antimicrobial treatments, including antibiotics, there is a very real possibility that the drugs we have come to rely upon may become obsolete.A potential solution to counteract antibiotic resistance is to design and explore innovative heterocyclic scaffolds with novel modes of action.Thus, vast attention is given by medicinal chemists to 1,2,4-triaoles, pyrimidines and triazines motifs for developing of many drugs with various biological activities.Due to their ability to bind easily with enzymes and receptors in organisms, via H-bonding and other weak interactions, azoles and/or azines have been attracting increasing interest in pharmaceutical chemistry [1,2].Potent pharmacological activity of azoles and/or azines can attributed to the presence of toxophoric azomethine moiety (-C=N-), that used in forming H-bonding with nucleophilic centers of various cellular constituents, thus upsetting the normal metabolic operation of these organisms [3].
Finally, as third motif in this preface, pyrimidines have a long and eminent history extending from the days of their discovery as important constituents of nucleic acids to their current use in the chemotherapy of AIDS.Alloxan is known with its diabetogenic action in a number of animals [18].Uracil, thymine and cytosine (Figure 1), are the three important constituents of nucleic acids, pyrimidine derivatives.In addition, the pyrimidine ring is found in vitamins like barbitone [18], the first barbiturate hypnotic sedative and anticonvulsant is a pyrimidine derivative [18].5-Fluorouracil is a pyrimidine derivative, that is the early metabolites prepared [19], also, 5-Thiouracil derivatives exhibits some useful antineoplastic activities [20].On the basis of the above considerations about the chemistry as well as the biological significance of triazole, pyrimidine and triazine analogues, and to continue our previously initiated program [20,39], aiming at merge of multi pharmacophores in single architecture, to study and develop a novel innovative route for synthesis of various heterocyclic systems of promising biological activities.Herein, we describe the synthesis and the utility of 8phenyl-6-(thiophen-2-yl)-6,7-dihydro-2H-pyrimido[2,1-c][1,2,4]triazine-3,4-dione (2), as reactive precursor to attain a series of the target compounds.
Signals of the hydrazinyl protons, originally observed in 7 ( 1 H NMR) at 4.82 and 8.32 ppm, were disappeared in each case.The mass spectrum of compound 12 showed a peak at m/z 396.06, corresponding to its molecular formula C18H13ClN6OS.As a chemical confirmation the triazolotriazine compound 10, was obtained in another design via treating the hydrazinyl 7 with dimethylformamidedimethyl acetal (DMF-DMA) in refluxing dry xylene, but in low yield 60% (Scheme 2).
On the other hand, the reactivity of the endocyclic imino group in compound 8 was investigated.
Thus, hydroxymethylation of 8 via refluxing with MeOH/CH2O solution, afforded 2-(hydroxymethyl)- (9) in 75% yield (Scheme 2).The structure of compound 9 was deduced based on its spectral data, where, its mass spectrum recorded molecular ion peak (C25H20N6O2S) at m/z 468.15, while the IR spectrum showed characteristic absorption bands at 3315 cm -1 due to stretching of the O-H group.The 1 H NMR spectrum displayed D2O exchangeable broad singlet at 5.19 ppm for the O-H group, besides singlet signal at 4.32 ppm attributed to methylene protons of hydroxy methyl moiety, (Tables 1 and 2).Scheme 2. Reagent, conditions and synthesis of compounds (6)(7)(8)(9)(10)(11)(12) On the other hand, aiming at annulation a new Mannich analogues to investigate their biological impact, the thiol tautomer 14 was synthesized, then the reactivity of its thiol tag was investigated via subjection to one-pot three component reaction with various substituted anilines and excess of HCHO solution, in EtOH to give the new N-Mannich derivatives (15a-j), in good to excellent yields (70-80%), (Scheme 3).Formation of the compounds (15a-j), can be demonstrated on the basis of initial Mannich reaction which proceeds via two steps, first the reaction between HCHO and the amine leads to the formation of the non-isolable iminium ion intermediate, which lose H2O molecule in situ.
Secondly, the thiocarbonyl compound undergoes tautomerization to give its thiol tautomer, which proceeds to execute an attack on the iminium ion, this attack finally yields the target β-amino-carbonyl compounds (15a-j) as shown in Scheme 4. The IR spectra of the isolated compounds (15a-j) displayed the common characteristic absorption bands around the region 3165-3281 cm 1 due to secondary amine groups.Further, evidence for compounds (15a-j) is based on their 1 H NMR spectra; the secondary amine proton appeared at  4.80 ppm as a singlet (D2O exchangeable), while the methylene singlet ( 1 H NMR) of their phenylaminomethyl moiety were observed at  5.40 ppm.The IR spectrum of 15j showed characteristic absorption bands at 1390 and 1520 cm -1 due to NO2str (Asy.& Sy.), respectively.
Its mass spectrum displayed an ion peak at m/z 530.09 (M + , 30%) corresponding to the expected molecular formula C24H18N8O3S2, (Tables 1 and 2).Scheme 3. Reagent, conditions and synthesis of compounds (14 and15) Scheme 4. The proposed mechanism for the formation of compounds 15.
The reaction of hydrazinyl derivative 7 with KSCN was assumed to proceed via formation of the nonisolable intermediates which, undergo further intramolecular cyclization via loss of H2S molecule to afford the final product 5, as shown in the considered mechanism (Scheme 6).The spectral data together with elemental analyses of the isolated compounds (5,(16)(17)(18)(19), were in covenant with the suggested structures.The 1 H NMR spectra of these compounds showed the lack of the signals of the hydrazinyl protons, originally observed in 7 ( 1 H NMR) at 4.82 and 8.32 ppm.A recorded m/z value of 387.08, supported the formation of compound 16, while, its IR spectrum declared strong absorption bands at 1686 and 2218 cm -1 due to C=O and C≡N groups, respectively.Scheme 5. Reagent, conditions and synthesis of compounds (5,(16)(17)(18)(19), i = KSCN /AcOH; ii = CNCH2CO2Et; iii = Ac2O; iv = PhCOCl; v = SOCl2/ EtOH Scheme 6.The proposed mechanism for formation of compound 5.
Furthermore, the reactivity of the endocyclic imino moiety in compound 2, was explored aiming at annulation of pendent N-pyrimido [c] On the other hand, the thioureido analogue 3 was incorporated in a sequence of manipulations, aiming at investigation the reactivity of its thioureido moiety to attain the target thiazole and/or thiazine architectures.Thus, compound 3 was treated with benzylidenemalononitrile in boiling dioxane, to           (CA), and Aspergillus brasiliensis ATCC 16404 (AB).The reference drug for the evaluation of antibacterial activity against Gram positive bacteria was Ampicillin, and Gentamicin was the reference in assessing the activity against Gram negative bacteria, while Amphotericin B was taken as the stander for the anti-fungal impact.The inhibitory effects of the synthesized compounds versus these organisms, are depicted in Table 3.The parent precursors 7 and 14, were the lowest potent anti the evaluated species, as compared to the title compounds 15a-j.The anti-microbial data presented in Table 3 declared that, the majority of the evaluated compounds exhibited proficient inhibitory effects on the growth of the tested microorganisms.In general, most of the screened compounds revealed better potency contra Gram-positive bacteria than Gram-negative ones [39,40].This result is supported by the fact that, Gram-positive bacteria have a thick cell wall consisting multilayered peptidoglycan, while, in Gram-negative bacteria the cell wall is very thin, consisting of very few layers of peptidoglycan along with a second layer of lipopolysaccharides and lipoproteins.This outer membrane is selectively permeable and thus, control access to the underlying structures.This renders the Gram-negative bacteria generally less susceptible to tested compounds than the Gram-positive bacteria.In addition, the outcome of the anti-microbial investigation declared that, majority of the tested compounds showed less potency against fungal species comparable to bacterial species.As evident from Table 3, compounds (15j, 15f, 15i, 15h and 15g), showed the proficient potency with MIC values ≤ 14.15 and 17.13 µg/ mL in hindering the growth of (SA) and (BS) respectively.
Compound 15j was the most powerful with MIC values 11.32 and 11.34 µg/ mL, to be nearly as effective as the positive control Ampicillin in hindering the growth of both (SA) and (BS), respectively.
On the other hand, for the antifungal potency of the tested compounds the trend alters ( showed the lowest antifungal activity against the tested organisms comparable to Amphotericin B with MIC values > 30.05 µg/ mL.
Table 3: Antimicrobial evaluation of some synthesized compounds using the disc-diffusion method.
a Values are the average of three readings.
* Ampicillin in the case of Gram-positive bacteria, Gentamicin in the case of Gram-negative bacteria, and Amphotericin B in the case of fungi.

Structure activity relationship (SAR)
Therefore, structure activity relationship (SAR) studies were discussed to determine the effect of substituents on antimicrobial activities of the newly synthesized compounds.The obtained results declared implicitly some definite and interesting facts about the structure activity relationship (SAR), of synthesized compounds.The substitution pattern of 1,2,4-triazole analogue is affected by the electronic environment, and hence anti-microbial potency was also altered.and 15g), displayed significant anti-bacterial activity against the tested organisms, indicating that conjugates bearing electron-withdrawing (EWG) groups such as NO2, F and Cl exhibited higher potency than the rest of other compounds.While, electron donating (EDG) substituents, enhanced the antifungal potency.
It worth mentioned that, the highest antibacterial activity was observed when the electron attracting substituents were at para position (15j and 15f), rather than if they at ortho or meta positions (15i, 15h and 15g) in the phenyl ring.On the other hand, considering the antifungal activity, the trend altered and compound 15d was the most potent antifungal analogy, having (MeO) group as an electron releasing substituent at para position in its phenyl ring.

General Information
Reagents were purchased from Sigma Aldrich (Bayouni Trading Co. Ltd., Al-Khobar, Saudi Arabia) and used without further purification.Reaction progress was monitored by TLC on silica gel pre-coated F254Merck plates.Spots were visualized by ultraviolet irradiation.Melting points were determined on a Gallenkamp electro thermal melting point apparatus and are uncorrected.IR spectra were recorded as potassium bromide disks using Bruker-Vector 22 FTIR Spectrophotometer.The NMR spectra were recorded with a Varian Mercury VXR-300 NMR spectrometer at 300 and 75 MHz for 1 H and 13 C NMR spectra, respectively, using DMSO-d6 as solvents.Mass spectra were recorded on a Hewlett Packard MS-5988 spectrometer at 70 eV.Elemental analyses were carried out at the Microanalytical Center of Taif University, Taif, KSA.
Method B: A solution of 7 (0.33 g, 1 mmol) and DMF-DMA (0.22 mL, 2 mmol) in dry xylene (25 mL) was heated under reflux for 8 h.The solvent was then removed in vacuo and the solid so obtained was triturated with petroleum ether, collected by filtration and recrystallized from EtOH to yield 10 (60%).18).A mixture of compound 7 (0.33 g, 1 mmol), and benzoyl chloride (15 mL) was heated at reflux for 4 h, the reaction mixture was allowed to cool to rt, the resulted solid was filtered off, washed with cold MeOH, dried, and recrystallized using EtOH to yield 18 as orange crystals.pyridine (15 mL), malononitrile (0.12 g, 2 mmol) was added, the mixture was heated under reflux for 6 h. the reaction mixture was left to cool at rt.The precipitate that formed was collected and recrystallized from CHCl3 to yield 22 as yellow-orange powder.mmol) in dioxane (20 mL) was added dropwise.The mixture was stirred for further 2 h at rt., then the reaction mixture was heated at reflux for further, 8 h (the reaction was monitored by TLC analyses).

2-(5-Hydroxy
The solvent was evaporated under vacuum, the formed solid was filtered off and then recrystallized from MeOH-H2O to afford 35 as yellowish-brown powder.

Methodology
The antimicrobial activity of the new synthesized compounds was evaluated using the diskdiffusion method [41].A plate of 90 mm diameter containing the Muller-Hinton agar for the growth of bacteria and the 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 swabbing aseptically on the whole surface of the agar with cotton wool.A 6 mm diameter filter paper disc was saturated with 200 μg /mL of the tested compounds in DMSO.The discs were air dried and placed aseptically at the center of the plates.The plates were left in refrigerator for 1h, before incubation to allow the extract to diffuse into the agar.Ampicillin and gentamicin were used as bacterial standards, while, amphotericin B was used as fungal reference to evaluate the efficacy of the tested compounds, DMSO was used as negative control.After incubation of the plate at the suitable temperature (37 °C for bacteria and 25 °C for fungi), the results were recorded for each tested compound as the average diameter of inhibition zones (IZ) of bacterial or fungal growth around the disks in mm.The minimum inhibitory concentration (MIC) measurement was determined for compounds exhibited significant growth inhibition zones more than 15 mm using two-fold serial dilution method [42].The MIC (µg/ mL) and IZ diameters values are recorded in Table 3.

Conclusions
In conclusion, a novel series of annelated pyrimido

Table 1 .
Physical and analytical data of compounds 2 to 35