Biginelli Reaction Mediated Synthesis of Antimicrobial Pyrimidine Derivatives and Their Therapeutic Properties

Antimicrobial resistance was one of the top priorities for global public health before the start of the 2019 coronavirus pandemic (COVID-19). Moreover, in this changing medical landscape due to COVID-19, finding new organic structures with antimicrobial and antiviral properties is a priority in current research. The Biginelli synthesis that mediates the production of pyrimidine compounds has been intensively studied in recent decades, especially due to the therapeutic properties of the resulting compounds, such as calcium channel blockers, anticancer, antiviral, antimicrobial, anti-inflammatory or antioxidant compounds. In this review we aim to review the Biginelli syntheses reported recently in the literature that mediates the synthesis of antimicrobial compounds, the spectrum of their medicinal properties, and the structure–activity relationship in the studied compounds.


Introduction
Twelve years after 1881, the year in which the German Arthur Rudolf Hantzsch reported the multicomponent synthesis of dihydropyridine [1], in 1893, the Italian chemist Pietro Biginelli published the synthesis of 3,4-dihydropyrimidin-2(1H)-ones, by a simple one-pot condensation reaction of an aromatic aldehyde, urea, and ethyl acetoacetate in ethanol solution (Scheme 1) [2,3]. Both reactions proved to be "key methods" for the synthesis of pyridine and pyrimidine derivatives, respectively, which were greatly developed in the following period, especially due to the applications of the synthesized compounds. The Biginelli reaction has been intensively studied in the last two decades, especially due to the applications of synthesized dihydropyrimidinone compounds at the beginning, especially as calcium channel blockers of the nifedipine-type [4], and then as antitumor [5][6][7][8], antibacterial, antiviral [9,10], anti-inflammatory [11,12], analgesic [13], anti-Alzheimer [14], or antioxidant [15] compounds. The synthetic method initially reported by Biginelli has undergone changes, such as the "Atwal-modification" [4,5], and most often, the most efficient catalyst has been sought, which would lead to a higher product yield, milder reaction conditions, and efficient catalyst recovery [16,17]. In the last decade, several improved methods were reported for
Raj et al. reported the synthesis of dihydropyrimidinones 50a-50e, by a Biginelli reaction using Zn(ClO 4 ) 2 as catalyst (Scheme 17) [87]. In vitro antibacterial studies of dihydropyrimidones 50a-50e were carried out against Escherichia coli MTCC 119, Shigella flexneri MTCC 1457, Pseudomonas aeruginosa MTCC 741, and Staphylococcus aureus MTCC 740 strains, by disk-diffusion assay. Antifungal evaluations were also carried out against two fungal strains, Geotrichum candidum MTCC 3993 and Candida albicans MTCC 227 (Table 3). From the determined MIC values, it can be said that compound 50a had the best antimicrobial activity against all the strains tested.    Ahmad et al. reported the synthesis of some 2-amino-1,4-dihydropyrimidines by a Biginelli reaction, starting from guanidine HCl, benzaldehyde, and ethyl acetoacetate in DMF, and SnCl 2 ·2H 2 O or NaHCO 3 as catalyst, under ultrasonic irradiation [90].
The good antibacterial activities of compounds 64, 65, and 66 ( Figure 5), against S. aureus, B. subtilis, E. coli, and S. typhi, as well as the theoretical studies, have shown that these compounds may have acceptable pharmacokinetic/drug-like properties.  [91]. All the synthesized compounds exhibited significant activity against pathogenic bacteria Salmonella typhi and Staphylococcus aureus. These dihydropyrimidinone (DHPM) derivatives also focus on the bacterial ribosomal A site RNA as a drug target. Series of docking studies were also performed for human 40S rRNA as a target. It was found that amikacin drug exhibited higher binding affinity than compound 68e, which showed relatively low binding affinity towards human rRNA site ( Figure 6). Desai and Bhatt reported the synthesis of compounds 72a-72c, using, in the first step, a Biginelli reaction in the presence of SnCl 2 ·2H 2 O catalyst to obtain compound 69, which, in the presence of hydrazine hydrate, resulted in a hydrazide 70, from which Schiff bases 71a-71c were obtained by reaction with aromatic aldehydes. Cyclization of compounds 71 in the presence of triethylamine provided the desired β-lactams 72a-72c, as shown in Scheme 20 [92]. Compounds 72a-72c exhibited outstanding antimicrobial properties against almost all tested strains S. aureus, S. pyogenes, E. coli, P. aeruginosa, C. albicans, A. niger, and A. clavatus with MIC = 12.5-50 µg mL −1 for antibacterial activities and 25-100 µg mL −1 for antifungal activities, respectively. Thus, bis-derivatives 75a-75b were found to be more efficacious than their corresponding mono analogues 74a-74b. Compound 75b with two pyrimidithione rings showed high synergy with amphotericin-B and fluconazole, both followed by compounds 75a, 74b, and 74a. Rani et al. synthesized compounds 76a-76e by a Biginelli reaction from 3-oxo-Nphenylbutanamide, guanidine nitrate, an aldehyde, and HCl as catalyst [95] (Scheme 21), and compounds 77a-77e, from the reaction of derivatives 76 with 6-(hydroxymethyl)tetrahydro-2H-pyran-2,3,4,5-tetraol, ethyl acetoacetate, and monochloroacetic acid. It was found that compounds 77a and 77b had significant activity against S. aureus (MIC = 2.14 × 10 −2 µM mL −1 ), and compound 77c was most potent against B. subtilis (MIC = 0.58 × 10 −2 µM mL −1 ). Compound 77e displayed more potent activity against E. coli (MIC = 1.10 × 10 −2 µM mL −1 ), and compound 77d was found to be most potent against C. albicans and A. niger (MIC = 1.04 × 10 −2 µM mL −1 ).   (Table 4). All compounds exhibited excellent antibacterial activity against S. aureus -bold values (MIC = 0.2-6.25 µg mL −1 ), but in the case of B. subtilis, all compounds were less active. The efficacy of substituent at C-6 position decreased in the order -CH 3 > -7,8-Benzo > -Cl > -OCH 3 (b > e > c >a). Similarly, compounds 81a, 81c, and 81e were highly active against E. coli, whereas the other compounds showed significantly less activity.

Biginelli Reaction Mediated Synthesis of Antitubercular Pyrimidine Derivatives
Tuberculosis, resulting from infection by the bacterium Mycobacterium tuberculosis, is a major worldwide health problem [116]. Approximately 2 million people die every year. The emergence of multi-drug resistance has forced the development of new structural classes of antitubercular agents, with several of them showing promising activity against M. tuberculosis [117]. Virsodia et al. synthesized new N-phenyl-6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydro-pyrimidine-5-carboxamides using the Biginelli reaction by reacting acetoacetanilide derivatives, substituted aldehydes, and urea in methanol with a catalytic amount of HCl. Compound 114 showed 65% inhibition of M. tuberculosis, the best antitubercular activity ( Figure 15). Additionally, 3D-QSAR studies are reported. Trivedi et al. reported the synthesis of 30 dihydropyrimidines by a classical Biginelli reaction and their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv [118]. Two compounds, 115a and 115b, with MIC of 0.02 µg mL −1 against M. tuberculosis, were found to be the most active of all and more potent than isoniazid. Akhaja et al. found that compound 14d displayed promising antitubercular activity compared to standards Rifampicin and Izoniazid [75]. Yadlapalli implemeted a Biginelli reaction for the synthesis of 4-aryl-3,4-dihydro-2(1H)-pyrimidone esters possessing lipophilic carbamoyl groups [120]. Compounds 117a and 117b, with a MIC value of 0.125 and 0.25 µg mL −1 , were found to be the most potent in the series ( Figure 16). Ambre et al. reported the synthesis of 16 compounds, 4-(substituted) phenyl-2-thioxo-3,4-dihydro-1H-chromino [4,3-d]pyrimidin-5-one and 4-(substituted) phenyl-3,4-dihydro-1H-chromino [4,3-d]pyrimidine-2,5-dione analogs as antitubercular agents by a classical. Biginelli reaction between a substituted aldehyde, 6substituted-4-hydroxy coumarin, urea (or thiourea), and p-toluenesulfonic acid as catalyst. Compounds 118a and 118b, with MIC of 59% and 61%, respectively, were found to be the most potent in these series [121]. It was found that compounds 119a-119d exhibited an MIC between 0.08 and 0.09 µM, which is found to be better than the standard reference Isoniazid with MIC of 0.2 µM. The very good antitubercular activity was correlated with the presence of the fluorine atom in the molecule. Scheme 34. Synthesis of compounds 119a-119d.

Antioxidant Activity of Antimicrobial Pyrimidine Derivatives
Youseff et al. found that compounds 37a and 37b showed considerable inhibitory activity in the hemolysis assay (Table 11) [81]. Compounds 37c and 37d ( Figure 17) showed moderate antioxidant and inhibitory activity (Table 12). Additionally, the antioxidant assay by ABTS method showed that compounds 37a, 37b, 37c, and 37d showed potent antioxidant activity.    [86]. Attri et al. reported that that compounds 73a-73c possess good-to-moderate antioxidant activity in comparison to the standard gallic acid and quercetin [93]. Rani et al. reported that compounds 77f and 77g exhibited excellent in vitro antioxidant activity due to the presence of electron releasing groups on benzylidene portion ( Figure 18) [95].

Anticancer Activity of Antimicrobial Pyrimidine Derivatives
Yadlapalli reported that compound 117a, with excellent antitubercular activity against MTB H37Rv, showed moderate anticancer activity against MCF-7 breast cancer cell lines [120]. Compound 77h was found to be the most potent anticancer agent (IC 50

Anti-Inflammatory Activity of Antimicrobial Pyrimidine Derivatives
Gelatinases are present in the physiologic system and play a key role in inflammation and autoimmunity states. Activated inflammatory cells and dermal fibroblasts can express several proteinases designated as matrix metalloproteinases (MMPs) able to degrade all connective tissue macromolecules [98]. Among these are gelatinases, e.g., MMP-2 and MMP-9, which, together with interstitial collagenase, have been assumed to be of importance in connective tissue remodeling after inflammation. The obtained results revealed that all compounds 80a-80e and 81a-81e were highly active against MMP-2 (72 kDa gelatinase A). Similarly, the compounds 80e, 81a, 81b, 81d, and 81e were highly active against MMP-9 (92 kDa gelatinase B), whereas the compounds 80b and 80d showed slight inhibitory activity, and rest of the compounds were not active against MMP-9 (Table 13).  [79]. From the anti-inflammatory activity result analysis, Viveka et al. observed that compounds 92a, 92b, 92d, 92e, 92f, 92g, and 92h showed good activity, with 67.61 to 85.33% inhibition of the edema (Figure 19). The compounds 92a (85.33), 92d (81.32), and 92h (80.75) showed potent anti-inflammatory activity compared with the other test compounds and are comparable with the standard, indomethacin (86.76). This emphasizes the presence of the 3F-4CH 3 -substituted phenyl ring on the 5th position of the 3-oxothiazolopyrimidine nucleus in this pyrazole series [101]. Additionally, El-Emary et al. found that compounds 110b and 112b (Scheme 29) had the most anti-inflammatory activity, comparable to that of Indomethacin [113].

Analgesic Activity of Antimicrobial Pyrimidine Derivatives
Alam et al. reported that compounds 31c, 31d, and 31e with analgesic activity (expressed as % protection) of 44.35%, 47.01%, and 50.36%, respectively, have analgesic properties, considering Indomethacin as the standard (60.30%) [79]. Khalifa Figure 21A). Compound 4a exhibited at least three different long-distance hydrogen-bond interactions. An O1 atom showed with the NH 2 atom of Arg259 with a distance of 3.06 Å, an O3 atom showed with the NE2 atom of His93 with a distance of 2.87 Å, and a Cl atom showed with the O atom of Pro69 with a distance of 2.87 Å ( Figure 21B). Additionally, other atoms of the molecule showed several important hydrophobic interactions against Asp256, Asn94, Met414, Asp71, and Tyr411 with different distances. These results showed that the relatively higher antiviral activity of compound 48a than 47a may due to lower docking and binding energies, as well as higher hydrogen and hydrophobic interactions.

Antiparasitic Activity of Antimicrobial Pyrimidine Derivatives
Rajanarendar reported that compounds 6b and 6d are proved to be lethal for mosquito larvae, with LC 50 concentration, representing the concentration in ppm that killed 50%, of 0.85 and 0.88, respectively (Scheme 4) [72]. Thus, pyrimidine compounds 6b and 6d can be useful as more toxic substances to kill mosquito larvae. Fatima et al. reported the synthesis of three Biginelli compounds 120, 121, and 122 more potent than the standard drug Chloroquine against K1 strains of P. falciparum, with an IC 50 (µg mL −1 ) of 0.56, 0.5 and 0.5, respectively ( Figure 23) [123]. These compounds with three different pharmacophores have potential to be exploited in medicinal chemistry.

Conclusions
This review summarizes the recent Biginelli syntheses of pyrimidine compounds with antimicrobial properties, as well as their biological activities mentioned in the literature. Regarding the Biginelli synthesis of pyrimidine compounds, the presentation clearly shows that the catalyst has an important role in the development of the reaction and in obtaining a high yield. Derivatization of Biginelli compounds leads in most cases to compounds with stronger antimicrobial properties than the initial Biginelli dihydropyrimidines. Additionally, the presence of another heterocycle in the final molecules, such as pyrazole, thiazole, isoxazole, imidazole, benzothiazole, phenothiazine, 1,3,4-thiadiazole, coumarin, chromene, indole, and quinoline, potentiate the antimyrobial activity of pyrimidine compounds. In general, thiopyrimidine compounds have a stronger antimicrobial activity than pyrimidinone compounds. Selenopyrimidine compounds generally have better antimicrobial activity than pyrimidinone. Additionally, the presence of certain groups grafted on the benzimidazole and pyrazole nuclei, such as -NO 2 , -CN, -F, -CF 3 , -CN, -COOCH 3 , -NHCO, -CHO, Cl, -OH, OCH 3 , OC 2 H 5 , and -N(CH 3 ) 2 , increases the antimicrobial activity of the compounds [124][125][126][127]. However, there are quite a few studies performed on the structureproperties relationship for these compounds, as well as few studies of molecular mechanics, DFT, through which to achieve the directed synthesis of some biologically active molecules. Therefore, we hope that this article will be a starting point for conducting new theoretical studies and syntheses of new compounds for the synthesis of improved antimicrobial compounds that possess other biological activities.