Microwave assisted synthesis of some new thiazolopyrimidine and pyrimidothiazolopyrimidopyrimidine derivatives with potential antimicrobial activity

Background and objective A series of thiazolopyrimidine derivatives have been synthesized via multicomponent reaction and tested for biological activities. This research aims to develop a new synthetic method of poly fused pyrimidines under microwave irradiation. 6-Amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles reacted with bromomalono-nitrile to give 3,7-diamino-5-aryl-5H-thiazolo[3,2-a]pyrimidine-2,6-dicarbonitrile more willingly than the isomeric 7H-thiazolo[3,2-a]pyrimidines. Thiazolopyrimidine derivatives reacted with carbon disulphide to produce 11-aryl-11H-1,2,3,4,7,8,9,10-octahydropyrimido[4″,5″:4′,5′]thiazolo[3′,2′-a]pyrimido[4,5-d]pyrimidine-2,4,8,10-tetrathione. The above mentioned reactions were established by using both conventional methods and microwave-assisted irradiation. Conclusion This work provides a new method for preparing poly fused pyrimidines. The microwave-assisted technique is preferable due to the yield enhancements attained, time saving, and environmental safety reactions. The newly prepared compounds were verified for their antimicrobial activities. Also, the absorption and emission of some of the prepared compounds were studied.


Background
Pyrimidine derivatives are found to have a wide range of chemotherapeutic effects including angiogenic [1], enzyme inhibitory effects [2,3] and anti-leshiminal activity [4]. They have also been used as analgesics and anti-parkinsonian agents [5,6], as modulators of TRPV1 (Transient Receptor Potential Vanilloid Receptor 1) [7], as anticancer agents [8][9][10], as pesticides [11], as phosphate inhibitors [12,13], for treatment of circulatory system diseases [14]. They are also known to have antimicrobial [15][16][17], anti-inflammatory [18], and anti-insecticidal [19] properties in addition to acetyl cholinesterase inhibitory activity [20]. Thiazolopyrimidine and thiazolo-pyrimidopyrimidine compounds have attracted our interest due to the wide range of biological activities they exhibit. For instance, thiazolopyrimidines are known to exhibit hypoglycemic, hypolipidemic, antidiabetic [21] and antibacterial and anti-tubercular activities [22]. The microwave technique has many benefits over conventional synthetic methods. Reduction of reaction times, minimization energy consumption, management of analytical waste, improving yields and increasing safety for the operator were the main benefits of this technique [23][24][25][26][27][28]. The use of microwave depends on the ability of the reacting molecules to efficiently absorb microwave energy taking advantage of microwave dielectric heating phenomena such as dipolar polarization or ionic conduction mechanisms. This Open Access *Correspondence: amsyoussef@yahoo.co.uk 2 Chemistry Department, Faculty of Science, Fayoum University, Fayoum, Egypt Full list of author information is available at the end of the article leads to rapid internal heating (in-core volumetric heating) by direct interaction of electromagnetic radiation with the reacting molecules. Even though diverse types of microwave reactors and processing options are available currently, most of the microwave synthetic protocols have been reported in sealed reactors [29]. The rapid heating and high temperatures resulting in microwave chemistry makes it obvious based on the application of the Arrhenius equation, [k = A exp(− E a / RT)] that transformations that reach completion in hours under conventional heating in a solvent, would be completed in only minutes using superheated solvents under microwave conditions using a autoclave type sealed reactor. In addition the rapid heating generally produced in microwave chemistry may sometimes lead to altered product distributions as compared to reactions conducted under conventional heating if the product distribution is determined by complex temperature dependent kinetics [29,30]. This may be the reason why in many instances reactions performed under microwave irradiation at an optimized reaction temperature lead to lesser side products in comparison to reactions performed under conventional heating where the reaction temperature is often non-optimal [29][30][31]. Encouraged by the findings of the previously reported work [34][35][36] we herein report the use of microwave-assisted technique for preparing new derivatives of a series of thiazolopyrimidine and thiazolopyrimidothiazolopyrimidine for evaluation of their antimicrobial activity. The absorption and fluorescence emission of some of the prepared compounds were studied in dioxane, revealing that the substituents altered both the absorption and fluorescence emission maxima.

Chemical characterization
The above discussed medicinal and biological properties of fused pyrimidine derivatives, prompted us to carry out the synthesis of a series of new thiazolopyrimidine and thiazolodipyrimidine derivatives using microwave chemistry in conjunction with conventional chemical synthesis. The reaction of bifunctional reagents with 6-amino-4-aryl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile derivatives 1a-d, afforded a simple and efficient approach for the synthesis of the target molecules. The synthesized target molecules were evaluated for their antimicrobial activity. The starting materials 1ad were obtained by the one pot reaction of aromatic aldehydes, malononitrile and thiourea in an alcoholic sodium ethoxide solution (Scheme 1). Compounds 1a-d were characterized using elemental analysis as well as spectroscopic data. Compounds 1a, b were prepared according to literature procedures [33,40].
The IR (ʋ, cm −1 ) spectra of 1a-d showed absorption bands at 3350, 3270 and 3180 (NH, NH 2 ), 3050, 2980 (CH), 2217 (CN). 1  Each of 1a-d reacted with equimolar amount of monobromomalononitrile (2), in ethanolic potassium hydroxide solution, yielded in each case a single product which could be formulated to be either 5H-thiazolo[3,2-a]pyrimidine structure 3 or its isomeric structure 7H-thiazolo[3,2-a]pyrimidine 4 (Scheme 2). Preferring structure 3 over 4 was based on the comparison of the 1 H-NMR spectral data for compounds 1 and 3. Thus, the 1 H-NMR spectrum of 3b as an example revealed, in addition to the methoxy group, aromatic and NH 2 proton signals, a singlet (1H) at δ 6.41 assigned to the pyrimidine H-5. The downfield for the pyrimidine H-5 in 3b compared with the pyrimidine H-4 in 1b, which appeared at δ = 5.12 ppm, indicates that the moiety nearby H-5 in 3b differs from that of H-4 in 1b. Therefore, structure 3 could be initially assigned for the reaction products.
We have recently been attentive in carrying out synthesis of some heterocyclic compounds, with expected biological activity, under environmentally friendly, time saving microwave-assisted conditions [34][35][36][37][38][39]. Accordingly, we resynthesized the previously described compounds 1a-d, 3a-d, 5a, b and 6a, b under microwave conditions, aiming to increase reaction yields and reduce the reaction times, the difference in the outcome of the MW-assisted and thermal reactions are shown in Table 1. The outcomes of these preparations indicated that reaction yields were improved by 17-23% compared to the conventional methods. Also reaction times were considerably reduced. Figure 1 summarizes the outcome of using microwave technique for the preparation of the abovementioned compounds.

Biological evaluation Antimicrobial evaluation
The newly prepared compounds were verified for their antimicrobial action against different microorganisms Scheme 5 Synthesis of 6b in step wise sequence Scheme 6 Formation of 2-arylmethylene-7-amino-5-(4-chloropenyl)-3-oxo-2,3-dihydro-5-H-thiazolo[3,2-a]pyrimidine-6-carbonitrile such as: Escherichia coli, Pseudomonas putida, Bacillus subtilis, Streptococcus lactis, Aspergillus niger, Penicillium sp. and Candida albicans. The initial screening of the investigated compounds was achieved using the filter paper disc-diffusion method. Compounds 1a, b, 3a, b, 5a, 6a, 8, 9a and 10 showed moderate to slight inhibitory action towards the microorganisms. Other compounds showed slight to no sensitivity at all to the mentioned organisms, the results are listed in Table 2.

Fluorescence and absorption spectra
The UV-Vis absorption spectra of all compounds as well as the fluorescence spectra of the compounds exhibiting fluorescence in solution were measured in 1,4-dioxane. It is clear from Fig. 1 that the prepared compounds exhibit UV-Vis absorption spectra in the region of 250-500 nm with a maximum absorption at 326 nm. The difference in the intensity of the prepared compounds depends on the difference of their chemical structures. The probabilities of compounds towards excitation from the ground state to the singlet excited state (absorption cross-section σ a ) by absorbing photons at wavelength of 326 nm were calculated using Eq. (1) as follows [40]: σ a = 0.385 × 10 −20 ε where: the molar absorptivity ε was calculated from Beer-Lambert law Eq. (2): where: A: absorbance, I 0 and I: intensities of incident and emerged light from the sample, C: molar concentration of compounds and L is the light path (1 cm). The absorption and emission spectral maxima are listed in Table 3. The fluorescence properties of the compounds depend on the presence of electron-with donating and electron-withdrawing substituents on the acceptor part. The acceptor part of 2-carboxymethylthio derivative 10 contains carboxyl group when compared   with other compounds. Hence, due to the less positive inductive effect of 10, the donating tendency becomes less and compound 10 exhibits high quantum yield φ f of 0.73, much higher than other compounds.
No fluorescence was detected in solution for all studied compounds except 10, 3a and 5a (Table 3; Figs. 1, 2). Compounds 3a and 5a exhibited intense fluorescence while compounds 10 exhibited high quantum yield φ f of 0.68 and 0.63 and 0.73 respectively, which may be due to the presence a polycyclic compounds with tetrathione moiety and electron-withdrawing substituents, enabling extended conjugation (Table 3). Simultaneously, it was observed that only compound 10 showed fluorescence in both solution and solid phase, and the fluorescence maximum in solid phase was shifted bathochromically by about 50 nm compared with the maximum in solution. Conversely, compounds 3a and 5a exhibited fluorescence only in solution.

Experimental
General A Gallenkamp melting point apparatus was used to determine melting points and IR spectra (KBr discs) were recorded on a Shimadzu FTIR-8201PC Spectrophotometer. 1 H-NMR and 13 C-NMR spectra were verified on a Varian Mercury 300 MHz and a Varian Gemini 200 MHz spectrometers using TMS as an internal standard and DMSO-d6 as a solvent and the chemical shifts were expressed as δ (ppm) units. Shimadzu GCMS-QP1000EX instrument were used to record Mass spectra using an inlet type sample injection at 70 eV. The Microanalytical Center of Cairo University performed the microanalyses. Microwave reactions were performed with a Millstone Organic Synthesis Unit (Micro SYNTH with touch control terminal) with a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. A calibrated infrared temperature control was used to monitor the temperature of the reaction mixture under the reaction vessel with a pressure sensor connected to the septum of the vessel to control the pressure. Ultraviolet-visible absorption spectra were measured on a PerkinElmer Lambda 35 Spectrophotometer at room temperature. Steady-state fluorescence spectra were measured on a PerkinElmer LS 55 spectrophotometer. The prepared compounds were dissolved in precleaned amber glass vials (1-cm cell) containing dioxane as solvent in concentration of 1 × 10 −5 M (King Khalid University).

11-Aryl
Method B Each of compounds 3a, b (3a, 1.09 g, 3b, 1.08 g; 0.03 mol) in 6 mL carbon disulphide were heated in microwave oven at 500 W and 140 °C for 8 min. The reaction mixture was treated in a similar manner to method A to yield compounds 5a, b.

11-Aryl
Method B Each of compounds 3a, b (3a, 1.09 g; 3b, 1.08 g; 0.03 mol) in 5 mL formic acid (80%) were heated in microwave oven at 500 W and 140 °C for 8 min. compounds 6a, b was obtained by treating the reaction mixture in a similar manner to method A.
Method B A solution of 11 (2.73 g, 0.01 mol) in a mixture of glacial acetic acid (20 mL)/acetic anhydride (8 mL) containing 1 g fused sodium acetate was heated with p-methoxybenzaldehyde (1.49 g, 0.01 mol) under reflux for 1 h. The obtained solid was found to be identical in all aspects (IR, m.p., mixed m.p.) with compound 9b.

Reaction of 1a with chloroacetic acid: formation of 10
A solution of 1a (2.64 g; 0.01 mol) in glacial acetic acid (40 mL) containing 1.0 g fused sodium acetate was heated under reflux for 3 h with 0.95 g (0.01 mol) of chloroacetic acid. The reaction mixture was then poured onto water and the precipitated solid was filtered off, dried and recrystallized from dioxane as yellow crystals, yield, 43%, 1.
Method B A solution of 10 (3.22 g, 0.01 mol) in glacial acetic acid (20 mL)/acetic anhydride (8 mL) was refluxed in water bath for 3 h. The reaction mixture was then cooled and the precipitated solid was filtered off and dried. The obtained solid was found to be identical in all aspects (IR, m.p., mixed m.p.) with compound 11. Yield, 61%, 1.83 g, m.p. 223-225 °C. 1

Antimicrobial screening
The newly prepared compounds were verified for their antimicrobial activity against: (a) Gram-negative: Escherichia coli and Pseudomonas putida; (b) Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) fungi: Aspergillus niger and Penicillium sp.; (d) yeast: Candida albicans.
For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min. Procedure (Filter Paper Diffusion Method) [42]. Suitable concentrations of microbial suspensions were prepared from (1 for bacteria to 3 for yeast and fungi)-day-old liquid stock cultures in cubated on a rotary shaker (100 rpm). In the case of fungi, 5 sterile glass beads were added to each culture flask. The mycelia were then subdivided by mechanical stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a spectrophotometer at 350 nm to give an optical density of 1.0. Appropriate agar plates were aseptically surface inoculated uniformly by a standard volume (ca. 1 mL) of the microbial broth culture of the tested microorganism, namely E. coli, P. putida, B. subtilis, S. lactis, A. niger, Penicillium sp. and C. albicans. Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at 121 °C. Test compounds were dissolved in 80% ethyl alcohol (final concentrations are ~ 70% ethanol, ~ 5% methanol and ~ 5% isopropanol. Contains ~ 20% water) to give final concentration of 5 mg/mL. The sterile discs were impregnated with the test compounds (50 μg/disc). After the impregnated discs have been air dried, they were placed on the agar surface previously seeded with the organism to be tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs were arranged per dish, suitably spaced apart, i.e., the discs should be separated by a distance that is equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone. Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at 30 °C for 72 h for yeast and fungi [32].

Conclusions
New polycyclic fused pyrimidines have been synthesized using both conventional methods and microwave assisted conditions. The latter methods proved very efficient in reducing reaction times, minimization of energy consumption, management of analytical waste and increased safety for the operator as well as better reaction yields. All prepared compounds were verified for their antimicrobial activities. Some compounds showed moderate or weak antimicrobial activity. The absorption and fluorescence emission of some of the prepared compounds were studied in dioxane.