Synthesis and biological screening of new thiadiazolopyrimidine-based polycyclic compounds

Novel tri-and tetra-cyclic compounds based on the thiadiazolopyrimidine ring system were synthesized, and their antimicrobial activity was estimated. The obtained results evidenced the substantial efficiencies of pyrano-thiadiazolopyrimidine compounds 8a–b and 9a–b toward the two strains of gram-positive bacteria (S. aureus and B. cereus). Besides, tetracyclic pyrazolopyrimido-thiadiazolopyrimidine derivatives 16a–b and 17a–b displayed prominent efficiencies toward the two strains of gram-negative bacteria (E. coli and P. aeruginosa). In addition, compounds 8a–b and 9a–b displayed good efficacy toward C. albicans. The activity of antiquorum sensing (anti-QS) inhibition of the newly synthesized thiadiazolopyrimidine-based compounds toward C. violaceum was tested, suggesting satisfactory activity for derivatives 16a–b, 17a–b, 8b, and 9a. The cytotoxic activity of these derivatives was screened toward various cancer cell lines (MCF-7, PC3, Hep-2, and HepG2) and standard normal fibroblast cells (WI38) by utilizing the MTT assay. The pyrazolopyrimido-thiadiazolopyrimidine derivatives 16a, 16b17a, and 17b showed potent cytotoxic efficacy against the MCF-7 cells with the IC50 values ranging from 5.69 to 9.36 µM. Also, the endorsed structural activity relationship (SAR) of the inspected thiadiazolopyrimidine derivatives provided a correlation between the chemical structure and anticancer efficiency. The in silico docking studies were implemented for silencing the hormonal signaling in the breast (PDB Code-5NQR). The results were found to be consistent with the cytotoxic activity.

Inspired by the important role of antibiotics in the treatment and prevention of bacterial infections, the efficiency of the drugs is inadequate with the increase in the number of pathogens resistant to the antibiotics. The resistance to antibiotics is the main risk to public health and leads to an increase in the rate of morbidity and mortality in addition to the high cost of treatment 1 . The extensive use of antibiotics causes the accumulation of microbial resistance 2 . Thus, the current antivirulence approaches were established by genetic investigation to diagnose the virulence factors of numerous pathogens, where several methods were used to situate the pressure of the pathogens. Moreover, cancer is considered one of the primary causes of death in the world 3 . It is defined as the growth of the tumor cell through its ability to disperse through other cells in the body by a progression termed as metastasis that leads to death in most cases 4,5 . Cancer therapeutics include surgical treatment, radioactive treatment, immunotherapy, chemotherapy, etc. Chemotherapy is considered the most important step in the cancer treatment protocol. Nevertheless, the lack of selectivity of anticancer agents is the main limitation to the development of cancer medication. Thiadiazolopyrimidine derivatives are an important class of fused heterocyclic moieties with widespread biological effectiveness. The thiadiazolo-pyrimidine nucleus and its derivatives, belonging to the pseudo purine class, show interesting biological profiles, including antiviral 6 , anticancer 7,8 , antibiofilm 9 , antitumor 10 , antitubercular 11 , antiglycation 12 and antioxidant 13 activities. In the past few decades, these analogues were synthesized as PARP1 inhibitors 14 and STAT3 inhibitors 15 .
The thiadiazolo[3,2-a]pyrimidine derivative 3 was employed as a building unit for the construction of various functionalized tri-and tetra-cyclic compounds via reaction with nitrogen and carbon nucleophiles. The cyclization of thiadiazolopyrimidine 3 with hydrazine hydrate and/or phenylhydrazine was achieved by refluxing in EtOH/DMF mixture to produce the corresponding tricyclic compounds 3-aminopyrazolo[4,3-e]thiadiazolo[3,2a]pyrimidin-4-ones 4 and 5, respectively (Fig. 2). The chemical structures of 4 and 5 were characterized by IR, 1 H NMR, 13 C NMR, and MS analyses (experimental section). The IR spectra of compounds 4 and 5 did not show any absorption related to the nitrile function. The 1 H NMR spectrum of 5 showed singlet at δ 6.44 ppm for two protons corresponding to amino group (-NH 2 ). The aromatic protons were observed as multiplet at δ 7.42-7.57 ppm. The proton of thiadiazole ring resonates singlet at δ 8.51 ppm. The 13 C NMR spectrum displayed ten carbon signals corresponding to twelve carbon atoms. The characteristic carbon signal of conjugated cyclic carbonyl group was recorded at δ 164.38 ppm.
The tricyclic 6,8-diaminopyridothiadiazolo[3,2-a]pyrimidines 6 and 7 were obtained by the treatment of thiadiazolo[3,2-a]pyrimidine derivative 3 with active nitrile components (namely, malononitrile and ethyl cyanoacetate). The reaction was conducted by heating the reactants in acetic acid and ammonium acetate (Fig. 3). The structures of 6 and 7 were elucidated from the results of the spectral analyses. The proposed mechanism for the reaction of thiadiazolo[3,2-a]pyrimidine compound 3 with activated nitrile involves the nucleophilic addition of nitrile through its methylene group to the cyclic unsaturated nitrile of compound 3 to yield the intermediate Michael adduct (E). The heterocyclization of the intermediate (E) was assumed to occur by the addition of ammonia to the nitrile groups to produce the imino-perhydropyridine intermediate (F). The tautomerization leading to the aminodihydropyridine intermediate (G), followed by air oxidation (loss of H 2 ) results in the formation of the pyridothiadiazolo[3,2-a]pyrimidine compounds 6 and 7 (Fig. 3). The pyrano [3,4-e]thiadiazolo[3,2-a]pyrimidine tricyclic and tetracyclic compounds 8, 9 and 10 were obtained by the reaction of thiadiazolo[3,2-a]pyrimidine derivative 3 with acetylacetone and benzoyl acetone as examples from diketones, acetyl acetonitrile and benzoyl acetonitrile as examples from ketonitriles and 3-methylpyrazolin-5-ones, respectively (Fig. 4). The reaction was carried out by heating the reactants in tetrahydrofuran, which was initiated by using the 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) protocol. The suggested mechanism involves the Michael addition of the enolate carbonyl reagent to the β-carbon of the unsaturated nitrile system. The produced intermediate H undergoes intramolecular cyclization by the addition of enolic-OH functionality to the nitrile group in order to yield the pyranothiadiazolo[3,2-a]pyrimidine ring systems 8a and 8b. The structures of compounds 8, 9 and 10 were confirmed by the IR, 1 H NMR, 13 C NMR, and MS analyses. Accordingly, the 1 H NMR spectrum of 8a (as a typical example) exhibited two singlet signals at δ 2.18 and 2.34 ppm to identify the protons of methyl and acetyl groups, respectively. The proton of pyran ring is recorded at δ 5.26 ppm as singlet signal. The protons of amino function (-NH 2 ) resonate as singlet at δ 6.79 ppm. The singlet at δ 8.28 was attributed to the proton of thiadiazole ring. The 13 C NMR spectrum displayed eleven carbon signals. The characteristic carbon signal of acetyl-carbonyl carbon is observed at δ 195.91 ppm. The mass analysis recorded a molecular ion peak at m/z = 278.
The construction of pyrimidine nucleus fused with the building unit 3 has been explored through the reactions with various cyclic nitrogen 1,3-binucleophiles. Thus, the tetracyclic compounds 11, 12, and 13 were produced by the reaction of thiadiazolo[3,2-a]pyrimidine derivative 3 with different α-aminoazole reagents (namely, 5-aminotetrazole, 3-amino-1,2,4-triazole, and/or 5-amino-3-methylpyrazole) as 1,3-binucleophiles (Fig. 5)     Biological assessment. Antimicrobial and antiquorum-sensing assessment. The antimicrobial activity of the synthesized thiadiazolopyrimidine compounds was studied toward diverse pathogenic strains, such as gram-positive bacteria (Staphylococcus aureus ATCC 29213 and Bacillus cereus UW85), gram-negative bacteria (Escherichia coli ATCC 12435 and Pseudomonas aeruginosa ATCC 29260), and fungi (Candida albicans and Aspergillus fumigatus 293). The assessment was performed through the two-fold dilution technique using Ampicillin (antibacterial) and Fluconazole (antifungal) as the reference drugs 28,29 . The minimal inhibitory concentration (MICs, µg/mL) of the synthesized derivatives for prohibiting microbial growth was determined through the visual detection (no turbidity) technique. The obtained results (Table 1) for the four bacterial strains indicated that the compounds 8a-b and 9a-b demonstrated significant efficacy toward the two strains of gram-positive bacteria (S. aureus and B. cereus). Meanwhile, derivatives 16a-b and 17a-b revealed eminent effectiveness toward gram-negative bacteria, such as E. coli and P. aeruginosa. Furthermore, compounds 8a-b and 9a-b showed good effectiveness toward C. albicans but no marked activity against A. fumigatus ( Table 1). The synthesized thiadiazolopyrimidine-based compounds were screened for their antiquorum-sensing (anti-QS) inhibition activity by the Chromobacterium violaceum (ATCC 12472) technique using catechin as the standard compound [28][29][30] . The QS technique of C. violaceum was remarked by the detection of violacein (violet pigment) 31,32 . Meanwhile, the reactivity of the synthesized thiadiazolopyrimidine-containing derivatives as drugs depends on their efficiency to inhibit the liberation of violacein during the QS technique. The QS inhibition was determined by the following equation: QS inhibition (mm) = (r 2 − r 1 ), where r 1 is the inhibition radius of the bacterial growth and r 2 is the inhibition radius for growth as well as the release of the pigment. The compounds 8b, 9a, 16a-b, and 17a-b exhibited remarkable anti-QS activities ( Table 2).
Structural activity relationship. The relationship between the structures of the synthesized thiadiazolopyrimidine-containing compounds and achieved antimicrobial results was discussed as follow: (1) The incorporation of a pyrazole ring to the thiadiazolopyrimidine skeleton to produce 3-aminopyrazolo-thiadiazolopyrimidinone and 3-amino-phenylpyrazolothiadiazolo-pyrimidinone (compounds 4 and 5) did not boost the activity toward all the screened bacterial strains. (2) The introduction of a pyran ring to the thiadiazolopyrimidine skeleton in compounds 8a-b, 9a-b and 10a-b promoted promising antibacterial effectiveness against S. aureus and B. cereus. In addition, they demonstrated remarkable antifungal efficacy toward C. albicans and no significant activity against A. fumigatus. (3) The tetracyclic pyrazolopyrimido-thiadiazolopyrimidinone derivatives 16a-b and 17a-b displayed good results against E. coli and P. aeruginosa, which may be attributed to the existence of  In vitro cytotoxicity evaluation. The cytotoxicity of the prepared tri-and tetra-cyclic thiadiazolopyrimidine compounds were examined toward various cancer cell lines, such as liver and breast cancer (MCF-7), prostate cancer (PC3), laryngeal carcinoma (Hep-2), carcinoma (HepG2), and standard normal fibroblast cells (WI38) using the MTT assay 33 at the National Research Centre (Egypt). The cytotoxicity ( The suggested structural activity relationship (SAR) of the thiadiazolopyrimidine derivatives suggested the structural countenance associated with the anticancer efficacy. (1) The pyrazole ring fused with the thiadiazolopyrimidinone skeleton in compounds 4 and 5 caused strong activity against the HepG2 cells and low effectiveness against the other three tested cells. (2) The incorporation of pyridine to the thiadiazolopyrimidine skeleton in compounds 6 and 7 did not offer the desired activity against the tested cell lines. In contrast, the fusion of the pyran ring to the thiadiazolopyrimidine skeleton in compounds 8b (substituted with benzoyl group) and 9b (substituted with phenyl group) presented strong anticancer efficacy against the MCF-7 and Hep-2 cells and reasonable activity against the HepG2 and PC3 cell lines. (3) The construction of the pyrazolopyrimidine moiety fused with the thiadiazolopyrimidine skeleton to produce tetracyclic compounds led to the enhancement of the anticancer activity against the MCF-7 cell lines. In addition, compounds 16a and 16b possessing an aminopyrazole nucleus exhibited higher cytotoxic efficacy against the MCF-7 cell lines than their corresponding compounds 17a and 17b containing a hydroxypyrazole nucleus. Also, the derivatives 16b and 17b (substituted with the 4-anisyl group) displayed higher reactivity than their conjugates 16a and 17a containing the 4-tolyl group. This is supported by order of biological anticancer activity toward MCF-7 cell lines on tuning the substituents 34 . (4) The results of the cytotoxicity examination on normal cells (WI38) indicated that compounds 16 and 17 displayed the lowest cytotoxicity with the IC 50 values ranging from 57.86 to 62.26 µM. (5) The tetracyclic compounds 16a Table 2. Quorum-sensing inhibitor efficacy of the synthesized thiadiazolopyrimidine derivatives. a No effectiveness (-, < 2 mm inhibition zone); weak effectiveness (2-9 mm); moderate effectiveness (10-15 mm); strong effectiveness (> 15 mm). Bleomycin-dependent DNA damage. The prepared polycyclic thiadiazolopyrimidine-based compounds were examined through the bleomycin-dependant DNA damage, and the results were compared to that of ascorbic acid as a positive control. The obtained data reflected the ability of these derivatives to protect the DNA from damage. The capability of compounds 16a-b and 17a-b to manifest the best protective effect against DNA damage was indicated by the corresponding absorbance values ranging from 0.031 to 0.053 (Table 4) 35,36 .  www.nature.com/scientificreports/ Molecular docking. The in silico molecular docking studies were conducted to evaluate the types of requisite interaction between the thiadiazolopyrimidine-based compounds and the crystal structure of the potent inhibitors of NUDT5 silence hormone signaling in breast cancer (PDB Code-5NQR) 37 . The thiadiazolopyrimidine derivative 3 displayed two types of intermolecular interactions with low binding effects. The first type of interaction bonds the S atom of the thiadiazole ring with Asp 194, and the second interaction bonds the N-atom in the nitrile group with Gly 61 over a binding score S of -4.3922 kcal/mol (Fig. S1). The tricyclic compound 4 (pyrazolothiadiazolopyrimidine substituted with the amine functional group at position-3) showed two H-bonds resulting from the bonding of the N atom of pyrimidine with Arg 84 (2.92 Å) (Fig. S2) and that of the O atom of the carbonyl group with Arg 84. The resultant binding score S was found to be -4.4293 kcal/mol. The pyrazolothiadiazolopyrimidine compound 5 exhibited a better binding score (S = -4.2102 kcal/mol) through the formation of four H-bonds (Fig. S3). One of the H-bond resulted from the bonding of the S atom of the thiadiazole ring with Glu 169, the second bond formed between the N atom of the aminopyrazole moiety and Ser 172, while the third and fourth bonds were π-π interactions of the thiazole and pyrimidine rings with Ile 171. Nonetheless, the pyridothiadiazolopyrimidine derivative 6 presented two H-bonds corresponding to the bonding of the N atom of aminopyridine with Arg 51 and that of the S atom of thiadiazole with Glu 65 (Fig. S4). The derivative 6 displayed weak interactions with the 5NQR amino acids (S = -4.5643 kcal/mol). Meanwhile, the pyridothiadiazolopyrimidine compound 7 revealed two H-bonds between the N atom of the aminopyridine moiety with Asp 194, and the O atom of the ester group with Arg 51 of 5NQR (S = -6.6614 kcal/mol) (Fig. S5). An H-bonding was formed by the N atom of the amidic moiety with Asp 347, and a π-π bond was observed between the pyridine ring and Arg 84 (3.40 Å).
Similarly, the aminopyranothiadiazolopyrimidine derivative 8a displayed two H-bonds resulting from the bonding of the S atom of thiadiazole ring with Asp 194 and N atom of the aminoxazine moiety with Cys 139 (S = -5.7796 kcal/mol) (Fig. S6). While, compound 8b showed three intermolecular forces resulting from the thiadiazole ring, one H-bond formed by the S atom of thiadiazole with Cys 139, and two π-π interactions with Arg 51 and Met 132 over a binding score S of -5.9214 kcal/mol (Fig. S7). Also, derivative 9a demonstrated two H-bonds resulting from the bonding of the S atom of thiadiazole with Asp 194 and N atom of the nitrile group with Gly along with a binding energy score, S of -5.3557 kcal/mol (Fig. S8). Compound 9b exhibited two H-bonds between the N atom of the amino group bonded to Ala 96 and Arg 84 through a good score S of -6.1407 kcal/mol (Fig. S9). Moreover, the aminopyrazolopyrano-thiadiazolopyrimidines 10a and 10b exhibited H-bonds and π-π interactions. The derivative 10a demonstrated three H-bonds between the N atom of the amino group and Val 62, N1 of pyrazole and Ala 96, and N2 of pyrazole and Arg 84 in addition to the π-π interaction between the thiadiazole ring and Arg 51. The bonds resulted in an overall energy score (S) of -4.9886 kcal/mol (Fig. S10). Besides the three π-π interactions displayed by thiadiazole with Val 170, pyrazole with Ile 171, and phenyl with Ile 171, the derivative 10b demonstrated two H-bonds formed by the S atom in thiadiazole with Thr 117, and N1 of the pyrazole ring with Ser 172. The binding score of 10b was found to be -5.2193 kcal/mol, as shown in Fig. S11. Moreover, the tetracyclic structures 11-13 revealed reasonable binding scores from − 5.2800 to -5.4772 kcal/mol resulting from different hydrogen bonds and π-π interactions ( Table 5, Figs. S12, S13, and S14). Furthermore, the aminopyrazolopyrimidothiadiazolopyrimidines 16 and 17 displayed remarkable binding scores. For instance, derivative 16a exhibited a binding score of -6.2989 kcal/mol (Fig. 6) attributed to the H-bonding of the N atom of pyrimidine with Arg 51, and the two π-π interactions of Ter 28 with pyrimidothiadiazole and pyrimidopyrimidine, respectively.
Compound 16b exhibited two intermolecular hydrogen bonds resulting from the N atom of aminopyrazole with Ala 96 and Gln 82 through a binding score (S) of -7.7053 kcal/mol (Fig. 7).
Alternatively, derivative 17a showed a binding score of -7.4560 kcal/mol (Fig. S15) resulting from the H-bond of the O atom in the hydroxyl group with Asp 194. Finally, derivative 17b showed π-π binding between the phenyl ring and Arg 51, and an H-bond between the N atom of the aminopyrimidine moiety and Asp 194 over a binding score of -7.5846 kcal/mol (Fig. S16). The standard reference drug 5-fluorouracil was subjected to 5NQR for a comparative study of the synthesized derivatives. The drug presented an intermolecular hydrogen bond with a binding score of -7.4560 kcal/mol (Fig. S17).
Finally, the docking technique showed that the derivatives 16a, 16b, 17a, and 17b gave respectable binding scores of -6.2989, -7.7053, -7.4560, and -7.5846 kcal/mol, respectively, in contrast to 5-Fluorouracil exhibiting a binding score of -3.8546 kcal/mol with 5NQR. The two-and three-dimensional images of most of the derivatives presented two intramolecular hydrogen bonds resulting from the thiadiazole and pyrimidine moieties. The challenge in the docking method is the development of the level of conformation for the ligand interactions in distinct compounds depending on the binding scores. All the synthesized derivatives possess thiadiazole and pyrimidine moieties that form hydrogen bonds with the receptors and chemically disparate amino acids of 5NQR. The large pocket size of 5NQR was constrained by typically few polar residues with specific binding sites (Asp 194, Ter 28, Met 132, Arg 84, Gly 97 and Cys139), as observed from the three-dimensional images, and offered a proper cavity for the synthesized thiadiazolopyrimidine-based compounds.