Comparative Study of the Synthetic Approaches and Biological Activities of the Bioisosteres of 1,3,4-Oxadiazoles and 1,3,4-Thiadiazoles over the Past Decade

The bioisosteres of 1,3,4-oxadiazoles and 1,3,4-thiadiazoles are well-known pharmacophores for many medicinally important drugs. Throughout the past 10 years, 1,3,4-oxa-/thiadiazole nuclei have been very attractive to researchers for drug design, synthesis, and the study of their potential activity towards a variety of diseases, including microbial and viral infections, cancer, diabetes, pain, and inflammation. This work is an up-to-date comparative study that identifies the differences between 1,3,4-thiadiazoles and 1,3,4-oxadiazoles concerning their methods of synthesis from different classes of starting compounds under various reaction conditions, as well as their biological activities and structure–activity relationship.


Synthetic Approaches for the Preparation of Different Classes of 1,3,4-Oxadiazole and 1,3,4-Thiadiazole Derivatives
The synthetic approaches adopted for the preparation of 1,3,4-oxadiazole and 1,3,4thiadiazole derivatives can be classified according to the starting material as follows:
In 2019, Sekhar et al. [6] reported the cyclocondensation of E-aroylethene (14a-c) or E-arylsulfonylethene (15a-c) sulfonylacetic acid hydrazides with substituted cinnamic acid (16a-c) using the conventional method by reflux in POCl 3 to produce the 5-styryl-1,3,4oxadiazole derivatives (17a-c, 18a-c) in moderate yields. Interconversion of oxadiazole to thiadiazole was affected by the reaction of (17a-c, 18a-c) with thiourea in refluxing tetrahydrofuran to obtain 5-styryl-1,3,4-thiadiazole (19a-c, 20a-c) in moderate yields. However, all synthesized compounds were obtained in higher yields and in shorter reaction times by the ultrasound irradiation method when compared to the conventional method (Scheme 1). thiadiazole derivatives can be classified according to the starting material as follows:

Synthesis from Sulfonyl Acetic Acid Hydrazide
Sulphonylmethyl-1,3,4-oxadiazole derivatives are synthesized by cyclocondensati of sulfonyl acetic acid hydrazide with derivatives of carboxylic acids or sulfonyl ace acids. The most common reagent used for cyclocondensation is POCl3 (Figure 9). The 1,3 oxadiazole can then be interconverted to the 1,3,4-thiadiazole analog using thiourea a tetrahydrofuran (THF) [6]. . Proposed mechanism for the synthesis of 1,3,4-oxadiazole derivatives using sulfonyl ace acid hydrazide based on the reported mechanism of formation of oxadiazole from hydrazide a and carboxylic acid [49].
x FOR PEER REVIEW 12 of 59 Scheme 10. Synthetic pathway for compounds 62a-e and 63a-e.
The comparative analysis illustrated that, among oxadiazoles and thiadiazoles, the sulfur moiety in 1, 3, 4-thiadiazoles acts as a potent inhibitor, while compounds containing moderately electronegative elements such as sulfur and chlorine (170d,j) showed better in vitro activity than compounds with more electronegative elements, such as oxygen (169d,j) ( Figure 21) [84,89]. Long-chain 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles were synthesized and screened as antimicrobial agents against S. pyogenes, S. aureus, P. aeruginosa, K. pneumoniae, and E. coli bacterial strains and C. albicans, A. fumigatus, T. mentagrophytes, and P. marneffei fungal strains. The study results showed no difference in activity between the bioisosteres of oxadiazoles and thiadiazoles. Examination of SAR indicated that the presence of a hydroxyalkyl chain at the fifth position of the oxa-/thiadiazole ring increased the antibacterial activity in compounds 143c, 143d, 145c, and 145d, as they were equally as potent as the reference drug chloramphenicol (MIC = 6.25 μg/mL). Moreover, the compounds with an internal double bond in the long alkenyl substituent of synthesized oxa-/thiadiazoles 143b and 145b were found to be potent antifungal agents (Figure 22) [64]. A series of 5-[N-(p-flurobenzyl)indolyl]propyl-1,3,4-(oxa)thiadiazoles were tested in vitro for their antibacterial and antifungal activities. Only the thiadiazole derivatives showed promising results. The 2-phenylamino derivative 75d was more potent than the reference drug Gentamicin against Gram-negative E. coli, with MIC = 0.24 and 3.9 μg/mL, respectively. The 2-ethylamino analog 75b was equipotent to Gentamicin against E. coli (MIC = 3.9 μg/mL), and was four times more potent than Gentamicin against P. aeruginosa (MIC 3.9 μg/mL and 15.6 μg/mL, respectively). The 2-cyclohexyl amino counterpart 75c showed promising results against E. coli (MIC 0.49 μg/mL) compared to Gentamicin and displayed equivalent potency to Amphotericin B against the S. racemosum fungus (MIC = 3.9 μg/mL) (Figure 23) [61]. Long-chain 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles were synthesized and screened as antimicrobial agents against S. pyogenes, S. aureus, P. aeruginosa, K. pneumoniae, and E. coli bacterial strains and C. albicans, A. fumigatus, T. mentagrophytes, and P. marneffei fungal strains. The study results showed no difference in activity between the bioisosteres of oxadiazoles and thiadiazoles. Examination of SAR indicated that the presence of a hydroxyalkyl chain at the fifth position of the oxa-/thiadiazole ring increased the antibacterial activity in compounds 143c, 143d, 145c, and 145d, as they were equally as potent as the reference drug chloramphenicol (MIC = 6.25 µg/mL). Moreover, the compounds with an internal double bond in the long alkenyl substituent of synthesized oxa-/thiadiazoles 143b and 145b were found to be potent antifungal agents ( Figure 22) [64]. Long-chain 5-alkenyl/hydroxyalkenyl-2-phenylamine-1,3,4-oxadiazoles and thiadiazoles were synthesized and screened as antimicrobial agents against S. pyogenes, S. aureus, P. aeruginosa, K. pneumoniae, and E. coli bacterial strains and C. albicans, A. fumigatus, T. mentagrophytes, and P. marneffei fungal strains. The study results showed no difference in activity between the bioisosteres of oxadiazoles and thiadiazoles. Examination of SAR indicated that the presence of a hydroxyalkyl chain at the fifth position of the oxa-/thiadiazole ring increased the antibacterial activity in compounds 143c, 143d, 145c, and 145d, as they were equally as potent as the reference drug chloramphenicol (MIC = 6.25 μg/mL). Moreover, the compounds with an internal double bond in the long alkenyl substituent of synthesized oxa-/thiadiazoles 143b and 145b were found to be potent antifungal agents (Figure 22) [64]. A series of 5-[N-(p-flurobenzyl)indolyl]propyl-1,3,4-(oxa)thiadiazoles were tested in vitro for their antibacterial and antifungal activities. Only the thiadiazole derivatives showed promising results. The 2-phenylamino derivative 75d was more potent than the reference drug Gentamicin against Gram-negative E. coli, with MIC = 0.24 and 3.9 μg/mL, respectively. The 2-ethylamino analog 75b was equipotent to Gentamicin against E. coli (MIC = 3.9 μg/mL), and was four times more potent than Gentamicin against P. aeruginosa (MIC 3.9 μg/mL and 15.6 μg/mL, respectively). The 2-cyclohexyl amino counterpart 75c showed promising results against E. coli (MIC 0.49 μg/mL) compared to Gentamicin and displayed equivalent potency to Amphotericin B against the S. racemosum fungus (MIC = 3.9 μg/mL) (Figure 23) [61]. A series of 5-[N-(p-flurobenzyl)indolyl]propyl-1,3,4-(oxa)thiadiazoles were tested in vitro for their antibacterial and antifungal activities. Only the thiadiazole derivatives showed promising results. The 2-phenylamino derivative 75d was more potent than the reference drug Gentamicin against Gram-negative E. coli, with MIC = 0.24 and 3.9 µg/mL, respectively. The 2-ethylamino analog 75b was equipotent to Gentamicin against E. coli (MIC = 3.9 µg/mL), and was four times more potent than Gentamicin against P. aeruginosa (MIC 3.9 µg/mL and 15.6 µg/mL, respectively). The 2-cyclohexyl amino counterpart 75c showed promising results against E. coli (MIC 0.49 µg/mL) compared to Gentamicin and displayed equivalent potency to Amphotericin B against the S. racemosum fungus (MIC = 3.9 µg/mL) (Figure 23) [61].
various non-related bacteria [100]. The designed compounds were tested for antibacterial activity against a selection of 24 bacterial species, using KKL-35 as a reference. 6-Chloro-N- (5-(4-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)nicotinamide 153c, and its 3chlorobenzamide analog 153a exhibited a wide spectrum of action and strong antimicrobial activity. They were active against almost all of the tested Gram-positive strains except C. perfringens and S. pyogenes. However, the only Gram-negative strain affected was B. fragilis (by 153c). They also affected the growth of the mycobacterium, M. fortuitum, and 153c had an effect against M. abscessus. Compared to KKL-35, 153c displayed lower MICs for E. faecalis (MIC 2-4 mg/L), E. faecium (MIC 2 mg/L), and M. fortuitum (MIC 8 mg/L). In addition, 153a showed a superior effect against E. faecalis, E. faecium, and S. epidermidis (MICs = 0.0625-1 mg/L). Furthermore, these compounds show synergistic activity with conventional antibiotics and have low toxicities (Figure 26) [87]. Karabanovich et al., evaluated the activity of a series of substituted benzylsulfanyl-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as antitubercular agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis. The activity results revealed that there was no difference in activity between oxadiazoles and thiadiazole analogs. Most of the compounds bearing the 3,5-dinitrobenzylsulfanyl moiety (233 and 234 series) exhibited excellent activity against drug-susceptible and multidrugresistant M.tb. strains, with no cross-resistance with first-and second-line anti-TB drugs. An SAR study indicated that removal or substitution of one nitro group or changing the positions of both nitro groups resulted in a decrease or loss of activity ( Figure 27). The anti-TB effect of the compounds was selectivity with low toxicity, genotoxicity, and mutagenicity [101]. Karabanovich et al., evaluated the activity of a series of substituted benzylsulfanyl-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as antitubercular agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis. The activity results revealed that there was no difference in activity between oxadiazoles and thiadiazole analogs. Most of the compounds bearing the 3,5-dinitrobenzylsulfanyl moiety (233 and 234 series) exhibited excellent activity against drug-susceptible and multidrugresistant M.tb. strains, with no cross-resistance with first-and second-line anti-TB drugs. An SAR study indicated that removal or substitution of one nitro group or changing the positions of both nitro groups resulted in a decrease or loss of activity ( Figure 27). The anti-TB effect of the compounds was selectivity with low toxicity, genotoxicity, and mutagenicity [101]. According to the theory that the incorporation of different pharmacophores into a single molecule can change the activity of the newly obtained compound, a series of cyclic imides containing both 1,3,4-oxadiazole and 1,3,4-thiadiazole heterocycles was prepared and tested for their antimicrobial activity against four strains of bacteria (S. aureus, S. pyogenes, E. coli, and P. aeruginosa) and C. albicans fungi. The results indicated that compounds 235 (IZD = 14-20.5 mm), 236 (IZD = 12.6-21 mm), and 237 (IZD = 12.9-20.1 mm) were highly active against all types of tested bacteria compared to Ampicillin (IZD = 12-17 mm). Only compounds 235 (IZD = 18.6 mm) and 237 (IZD = 22.2 mm) were also According to the theory that the incorporation of different pharmacophores into a single molecule can change the activity of the newly obtained compound, a series of cyclic imides containing both 1,3,4-oxadiazole and 1,3,4-thiadiazole heterocycles was prepared and tested for their antimicrobial activity against four strains of bacteria (S. aureus, S. pyogenes, E. coli, and P. aeruginosa) and C. albicans fungi. The results indicated that compounds 235 (IZD = 14-20.5 mm), 236 (IZD = 12.6-21 mm), and 237 (IZD = 12.9-20.1 mm) were highly active against all types of tested bacteria compared to Ampicillin (IZD = 12-17 mm). Only compounds 235 (IZD = 18.6 mm) and 237 (IZD = 22.2 mm) were also highly active against C. albicans fungi (Fluconazole IZD = 18 mm) ( Figure 28) [102]. According to the theory that the incorporation of different pharmacophores into a single molecule can change the activity of the newly obtained compound, a series of cyclic imides containing both 1,3,4-oxadiazole and 1,3,4-thiadiazole heterocycles was prepared and tested for their antimicrobial activity against four strains of bacteria (S. aureus, S. pyogenes, E. coli, and P. aeruginosa) and C. albicans fungi. The results indicated that compounds 235 (IZD = 14-20.5 mm), 236 (IZD = 12.6-21 mm), and 237 (IZD = 12.9-20.1 mm) were highly active against all types of tested bacteria compared to Ampicillin (IZD = 12-17 mm). Only compounds 235 (IZD = 18.6 mm) and 237 (IZD = 22.2 mm) were also highly active against C. albicans fungi (Fluconazole IZD = 18 mm) ( Figure 28) [102]. This study supports the same theory, whereby the potential antibacterial activity of a series of 1,3,4-thiadiazole derivatives tagged to oxadiazole and thiadiazole units were evaluated against E. coli, S. typhimurium, L. monocytogenes, K. pneumonia, S. typhi, S. aureus, and B. subtilis species. The assay results revealed that compounds 238 and 239, with a strong electron-withdrawing fluorine atom at the para position, were more potent than Ampicillin on all tested strains (MIC = 62.5-100 μg/mL and 100-250 μg/mL, respectively). This study supports the same theory, whereby the potential antibacterial activity of a series of 1,3,4-thiadiazole derivatives tagged to oxadiazole and thiadiazole units were evaluated against E. coli, S. typhimurium, L. monocytogenes, K. pneumonia, S. typhi, S. aureus, and B. subtilis species. The assay results revealed that compounds 238 and 239, with a strong electron-withdrawing fluorine atom at the para position, were more potent than Ampicillin on all tested strains (MIC = 62.5-100 µg/mL and 100-250 µg/mL, respectively). It was also noticed that the 1,3,4-thiadiazoles linked with 1,3,4-thiadiazole units 239 were more active than 1,3,4-thiadiazoles linked with 1,3,4-oxadiazole 238 (Figure 29) [103].   A series of 2,5-disubstituted-1,3,4-thiadiazole tethered 1,3,4-(oxa)thiadiazole derivatives were synthesized as antimicrobial agents. Their activities were evaluated against a panel of standard strains of pathogenic microorganisms, including Gram-positive bacteria (S. pneumonia, B. subtilis, and S. aureus), Gram-negative bacteria (P. aeruginosa, E. coli, and K. pneumonia), and fungi (A. fumigatus, C. albicans, and G. candidum), and against reference drugs Ciprofloxacin and Fluconazole. The antimicrobial activity of the thiadiazoles (102a-c) revealed that the tested compounds showed promising activity against all bacterial and fungal strains at 8-31.25 µg/mL. However, the oxadiazole isosteres (101a-c) showed greater antibacterial activity against Gram-positive bacteria, at MIC = 8-16 µg/mL, but they exhibited lower antifungal activity (MIC = 31.25-62.5 µg/mL). Oxadiazole 240 functionalized with a thiol group at position 2 exhibited excellent antibacterial activities against all bacterial strains at MIC = 4-8 µg/mL and good activity towards fungal strains at MIC =16-31.28 µg/mL (Figure 30) [76].

Antiviral Activity
A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives containing 1,3,4oxa(thia)diazole rings as a part of the metal chelation motif were screened for their in vitro anti-HIV-1 activity by determining their ability to inhibit the replication of HIV-1 in Hela cell cultures. All of the tested compounds were safe with no cytotoxicity at a concentration

Antiviral Activity
A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives containing 1,3,4-oxa(thia) diazole rings as a part of the metal chelation motif were screened for their in vitro anti-HIV-1 activity by determining their ability to inhibit the replication of HIV-1 in Hela cell cultures. All of the tested compounds were safe with no cytotoxicity at a concentration of 100 µM. Compounds containing thiadiazole rings were more potent than the corresponding oxadiazole analogs. The highest degrees of inhibition against HIV-1 (NL4-3) were reported for the thiadiazoles 63b and 63c: 51% and 48%, respectively. The docking study revealed that the anti-HIV activity of these compounds might involve a metal chelating mechanism ( Figure 37) [59]. The antiviral activity of a series of thioether/sulfone compounds containing 1,2,3thiadiazole and 1,3,4-oxa(thia)diazole rings was tested against tobacco mosaic virus TMV using Ningnanmycin as a reference drug. The data results revealed that the compounds containing 1,2,3-thiadiazole incorporating 1,3,4-oxadiazole ring 248a, 248b, 248c, and 250a showed equipotent activities to Ningnanmycin with curative effects ranging from 46.8% to 54.1%. However, a slight lowering of activity was noticed with the 1,3,4-thiadiazole isosteres (249,251). Oxidation of thioethers (248,249) to their corresponding sulfones (250,251) slightly reduced the values of antiviral activities ( Figure 38) [105].
oxadiazoles bioisosteres, where only one oxadiazole compound 80b showed sel moderate activity against NUGC and DLD1 cell lines. Among 1,3,4-thiadiazoles, on compounds bearing 4-chlorophenyl (81b) and 4-bromophenyl 81c pharmacophores found to be active, which proves that the electronegativity of substituents pla essential role in the activity. Compound (81c) showed nearly equipotent activity a the NUGC cell line compared to the standard CHS 828, with IC50 = 0.028 μM, whi also showed 4-fold higher activity against HA22T and HEPG2 cell lines compared analog 81b (IC50 = 0  Rajak et al., prepared two series of hydroxamic acid-based 1,3,4-oxa-/thiadiazoles as histone deacetylase inhibitors and examined their antiproliferative activities in vitro using histone deacetylase inhibitory assay and MTT assay. They were also tested for antitumor activity against Ehrlich ascites carcinoma cells in Swiss albino mice. The results showed that among the oxadiazole series, compound 252 was the most potent, displaying the maximum HDAC inhibitory activity with an IC 50 = 0.017 µM against HDAC-1 and an IC 50 = 0.28 µM in HCT-116 cell proliferation assay, as well as %TWI = 85.7 and %TCI = 77.7 against Ehrlich ascites carcinoma cells in Swiss albino mice. In the thiadiazole series, compound 253 was the most potent, displaying the maximum HDAC inhibitory activity, with an IC 50 = 0.018 µM against HDAC-1 and an IC 50 = 0.31 µM in HCT-116 cell proliferation assay, and %TWI = 75.0; %TCI = 76.8 against Ehrlich ascites carcinoma cells. An overview of the results shows that thiadiazoles were found to be more active than the corresponding oxadiazole ( Figure 40) [106].
Molecules 2022, 27, x FOR PEER REVIEW 46 Rajak et al., prepared two series of hydroxamic acid-based 1,3,4-oxa-/thiadiazo histone deacetylase inhibitors and examined their antiproliferative activities in vitro histone deacetylase inhibitory assay and MTT assay. They were also tested for antit activity against Ehrlich ascites carcinoma cells in Swiss albino mice. The results sh that among the oxadiazole series, compound 252 was the most potent, displayin maximum HDAC inhibitory activity with an IC50 = 0.017 μM against HDAC-1 and a = 0.28 μM in HCT-116 cell proliferation assay, as well as %TWI = 85.7 and %TCI = against Ehrlich ascites carcinoma cells in Swiss albino mice. In the thiadiazole s compound 253 was the most potent, displaying the maximum HDAC inhibitory act with an IC50 = 0.018 μM against HDAC-1 and an IC50 = 0.31 μM in HCT-116 proliferation assay, and %TWI = 75.0; %TCI = 76.8 against Ehrlich ascites carcinoma An overview of the results shows that thiadiazoles were found to be more active tha corresponding oxadiazole ( Figure 40) [106]. The in vitro antiproliferative activity of a series of benzosuberones embedded 1,3,4-oxa(thia)diazole moieties was examined against four human cancer cell (cervical He La, breast MDA-MB-231, pancreatic PANC1, and alveolar A549), w Paclitaxel, Nocodazole, Colchicine, Combretostatin, and Doxorubicin were use standard drugs. The reported data showed that compounds 204a, 254, and 255 sh potent activity, with GI50 values ranging from 0.079 µM to 0.957 µM against the human cancer cell lines. However, compound 254 was nearly equipotent to Colch against the HeLa cell line with GI50 = 0.079 µM. Based on the overall results, it concluded that benzosuberones attached to oxadiazoles were more active than corresponding thiadiazoles (Figure 41) [98]. The in vitro antiproliferative activity of a series of benzosuberones embedded with 1,3,4-oxa(thia)diazole moieties was examined against four human cancer cell lines (cervical He La, breast MDa-mB-231, pancreatic PANC1, and alveolar A549), where Paclitaxel, Nocodazole, Colchicine, Combretostatin, and Doxorubicin were used as standard drugs. The reported data showed that compounds 204a, 254, and 255 showed potent activity, with GI 50 values ranging from 0.079 µM to 0.957 µM against the four human cancer cell lines. However, compound 254 was nearly equipotent to Colchicine against the HeLa cell line with GI 50 = 0.079 µM. Based on the overall results, it was concluded that benzosuberones attached to oxadiazoles were more active than the corresponding thiadiazoles ( Figure 41) [98].
Paclitaxel, Nocodazole, Colchicine, Combretostatin, and Doxorubicin were used as standard drugs. The reported data showed that compounds 204a, 254, and 255 showed potent activity, with GI50 values ranging from 0.079 µM to 0.957 µM against the four human cancer cell lines. However, compound 254 was nearly equipotent to Colchicine against the HeLa cell line with GI50 = 0.079 µM. Based on the overall results, it was concluded that benzosuberones attached to oxadiazoles were more active than the corresponding thiadiazoles ( Figure 41) [98].  In the same year, two series of novel diosgenin derivatives bearing 1,3,4-ox or 1,3,4-thiadiazole moieties were evaluated for their cytotoxicity in four huma cell lines (HepG2, A549, MCF-7, and HCT-116) and normal human gastric epithe (GES-1) using the MTT assay in vitro. The data revealed that the 1,3,4-thiadiazo were more active than the 1,3,4-oxadiazole series against HepG2 and A549 cells. the 1,3,4-thiadiazoles, compound 60d ( Figure 43) with a 3-pyridyl group at the C5 was the most potent. It was 6.7-fold more potent than the parent drug diosgen against the A549 cell line (IC50 = 3.93 µM and 26.41 µM, respectively, as well as b fold more potent than DG against the HepG2 cell line (MIC = 11.73 μM and 33 respectively). Moreover, compound 60d displayed low toxicity against non-can GES-1 cells (IC50 = 420.4 µM), showing high selectivity [58]. On the basis that combining different heterocyclic nuclei may show syn effects, Kamal and Sobhy decided to merge the two bioisosteres, 1,3,4-oxadiaz 1,3,4-thiadiazole, in one system to enhance the biological activity of the syn In the same year, two series of novel diosgenin derivatives bearing 1,3,4-oxadiazole or 1,3,4-thiadiazole moieties were evaluated for their cytotoxicity in four human cancer cell lines (HepG2, A549, MCF-7, and HCT-116) and normal human gastric epithelial cells (GES-1) using the MTT assay in vitro. The data revealed that the 1,3,4-thiadiazole series were more active than the 1,3,4-oxadiazole series against HepG2 and A549 cells. Among the 1,3,4-thiadiazoles, compound 60d ( Figure 43) with a 3-pyridyl group at the C5 position was the most potent. It was 6.7-fold more potent than the parent drug diosgenin (DG) against the A549 cell line (IC 50 = 3.93 µM and 26.41 µM, respectively, as well as being 2.9-fold more potent than DG against the HepG2 cell line (MIC = 11.73 µM and 33.87 µM, respectively). Moreover, compound 60d displayed low toxicity against non-cancer cells GES-1 cells (IC 50 = 420.4 µM), showing high selectivity [58]. In the same year, two series of novel diosgenin derivatives bearing 1,3,4-oxa or 1,3,4-thiadiazole moieties were evaluated for their cytotoxicity in four human cell lines (HepG2, A549, MCF-7, and HCT-116) and normal human gastric epithe (GES-1) using the MTT assay in vitro. The data revealed that the 1,3,4-thiadiazo were more active than the 1,3,4-oxadiazole series against HepG2 and A549 cells. the 1,3,4-thiadiazoles, compound 60d ( Figure 43) with a 3-pyridyl group at the C5 was the most potent. It was 6.7-fold more potent than the parent drug diosgen against the A549 cell line (IC50 = 3.93 µM and 26.41 µM, respectively, as well as be fold more potent than DG against the HepG2 cell line (MIC = 11.73 μM and 33 respectively). Moreover, compound 60d displayed low toxicity against non-can GES-1 cells (IC50 = 420.4 µM), showing high selectivity [58]. On the basis that combining different heterocyclic nuclei may show syn effects, Kamal and Sobhy decided to merge the two bioisosteres, 1,3,4-oxadiaz 1,3,4-thiadiazole, in one system to enhance the biological activity of the synt compounds and evaluated their cytotoxicity against a human colon carcinoma (HCT-116) using Doxorubicin as a reference drug. Among all the tested compou On the basis that combining different heterocyclic nuclei may show synergistic effects, Kamal and Sobhy decided to merge the two bioisosteres, 1,3,4-oxadiazole and 1,3,4thiadiazole, in one system to enhance the biological activity of the synthesized compounds and evaluated their cytotoxicity against a human colon carcinoma cell line (HCT-116) using Doxorubicin as a reference drug. Among all the tested compounds, the most active derivatives were 257d and 258c (IC 50 = 0.73 and 0.86 µg/mL, respectively), while the other compounds showed moderate to poor activity compared to Doxorubicin IC 50 = 0.42 µg/mL. The assay results and an examination of the structure-activity relationship revealed that, as a substituent at position 2 of 1,3,4-thiadiazole, the acetyl group (COCH 3 ) (257a-f) resulted in higher activity than the anilide group (CONHPh) (258a-c), which was better than the phenyl moiety (Ph) 256. Additionally, for substituents at position 4 of 1,3,4thiadiazole, 4-ClC 6 H 4 > 4-NO 2 C6H 4 > 4-OCH 3 C 6 H 4 > 4-CH 3 C 6 H 4 > 4-BrC 6 H 4 > C 6 H 5 ( Figure 44) [107]. Based on the same idea, a series of novel hybrid molecules containing 1,3,4oxadiazole and 1,3,4-thiadiazole bearing the Schiff base moiety were designed and evaluated with respect to their in vitro antitumor activities against SMMC7721, MCF-7, and A549 human tumor cell lines by CCK-8 assay. The data revealed that some compounds were more potent than the positive control, 5-Fluorouracil (5-FU), against various cell lines. Among these compounds, compound 259b (4-chloro) was the most potent against SMMC-7721 cells, with IC50 =2.84 μM. Compounds 259c (4-methoxy) and 259d (4-nitro) displayed highly effective antitumor activities against MCF-7 cells, with IC50 = 4.56 and 4.25 μM, respectively. The unsubstituted phenyl derivative 259a and the 4nitro-substituted derivative 259d showed significant activity against A549 cells, with IC50 = 4.11 and 4.13 μM, respectively. The pharmacological results suggest that the substituents of the phenyl ring on the 1,3,4-oxadiazole are important for modulating antiproliferative activities against various tumor cell lines ( Figure 45) [108]. In 2015, a series of 2,5-Bis[(2-substituted-1,3,4-oxa(thia)diazol-5-yl)propylthio]-1,3,4thiadiazoles were tested for their in vitro antiproliferative activities in four different human cancer cell lines (human breast adenocarcinoma, MCF7, human ductal breast epithelial tumor, T47D, human epithelial colorectal adenocarcinoma, Caco-2, and human epithelial carcinoma, He La). All compounds demonstrated relatively high activities against the examined cell lines. There was no difference in activity related to the replacement of oxadiazole with a thiadiazole ring, while changing methyl and phenyl groups in (101a,c) and (102a,c) into ethyl groups 101b and 102b significantly enhanced the cytotoxic activity, with LD50 values ranging from 376 ng/µL to 438 ng/µL, which suggests a steric factor mediating either transport or molecular interaction of these compounds with cellular targets. Furthermore, the addition of one more Cl atom into the structure of compound 260a (LD50 = 648-690 ng/µL) gave compound 260b, with double the activity Based on the same idea, a series of novel hybrid molecules containing 1,3,4-oxadiazole and 1,3,4-thiadiazole bearing the Schiff base moiety were designed and evaluated with respect to their in vitro antitumor activities against SMMC7721, MCF-7, and A549 human tumor cell lines by CCK-8 assay. The data revealed that some compounds were more potent than the positive control, 5-Fluorouracil (5-FU), against various cell lines. Among these compounds, compound 259b (4-chloro) was the most potent against SMMC-7721 cells, with IC 50 = 2.84 µM. Compounds 259c (4-methoxy) and 259d (4-nitro) displayed highly effective antitumor activities against MCF-7 cells, with IC 50 = 4.56 and 4.25 µM, respectively. The unsubstituted phenyl derivative 259a and the 4-nitro-substituted derivative 259d showed significant activity against A549 cells, with IC 50 = 4.11 and 4.13 µM, respectively. The pharmacological results suggest that the substituents of the phenyl ring on the 1,3,4oxadiazole are important for modulating antiproliferative activities against various tumor cell lines ( Figure 45) [108]. Based on the same idea, a series of novel hybrid molecules containing 1,3,4oxadiazole and 1,3,4-thiadiazole bearing the Schiff base moiety were designed and evaluated with respect to their in vitro antitumor activities against SMMC7721, MCF-7, and A549 human tumor cell lines by CCK-8 assay. The data revealed that some compounds were more potent than the positive control, 5-Fluorouracil (5-FU), against various cell lines. Among these compounds, compound 259b (4-chloro) was the most potent against SMMC-7721 cells, with IC50 =2.84 μM. Compounds 259c (4-methoxy) and 259d (4-nitro) displayed highly effective antitumor activities against MCF-7 cells, with IC50 = 4.56 and 4.25 μM, respectively. The unsubstituted phenyl derivative 259a and the 4nitro-substituted derivative 259d showed significant activity against A549 cells, with IC50 = 4.11 and 4.13 μM, respectively. The pharmacological results suggest that the substituents of the phenyl ring on the 1,3,4-oxadiazole are important for modulating antiproliferative activities against various tumor cell lines ( Figure 45) [108]. In 2015, a series of 2,5-Bis[(2-substituted-1,3,4-oxa(thia)diazol-5-yl)propylthio]-1,3,4thiadiazoles were tested for their in vitro antiproliferative activities in four different human cancer cell lines (human breast adenocarcinoma, MCF7, human ductal breast epithelial tumor, T47D, human epithelial colorectal adenocarcinoma, Caco-2, and human epithelial carcinoma, He La). All compounds demonstrated relatively high activities against the examined cell lines. There was no difference in activity related to the replacement of oxadiazole with a thiadiazole ring, while changing methyl and phenyl groups in (101a,c) and (102a,c) into ethyl groups 101b and 102b significantly enhanced the cytotoxic activity, with LD50 values ranging from 376 ng/µL to 438 ng/µL, which suggests a steric factor mediating either transport or molecular interaction of these compounds with cellular targets. Furthermore, the addition of one more Cl atom into the structure of compound 260a (LD50 = 648-690 ng/µL) gave compound 260b, with double the activity In 2015, a series of 2,5-Bis[(2-substituted-1,3,4-oxa(thia)diazol-5-yl)propylthio]-1,3,4thiadiazoles were tested for their in vitro antiproliferative activities in four different human cancer cell lines (human breast adenocarcinoma, MCF7, human ductal breast epithelial tumor, T47D, human epithelial colorectal adenocarcinoma, Caco-2, and human epithelial carcinoma, He La). All compounds demonstrated relatively high activities against the examined cell lines. There was no difference in activity related to the replacement of oxadiazole with a thiadiazole ring, while changing methyl and phenyl groups in (101a,c) and (102a,c) into ethyl groups 101b and 102b significantly enhanced the cytotoxic activity, with LD 50 values ranging from 376 ng/µL to 438 ng/µL, which suggests a steric factor mediating either transport or molecular interaction of these compounds with cellular targets. Furthermore, the addition of one more Cl atom into the structure of compound 260a (LD 50 = 648-690 ng/µL) gave compound 260b, with double the activity (LD 50 = 356-398 ng/µL) ( Figure 46) [76]. Ozdemir et al. designed and evaluated several oxadiazole and thiadiazole derivatives with respect to their anticancer effects on A549 human lung adenocarcinoma and C6 rat glioma cell lines and evaluated their inhibitory effects on matrix metalloproteinases (MMPs). Compounds 261, 263, and 264 showed high cytotoxic activity against the C6 cell line, with IC50 ranging from 0.0128 mM to 0.157 mM, compared to Cisplatin, with IC50 = 0.103 mM. Only the benzodioxole-substituted oxadiazoles 263 and 264 had a cytotoxic effect on the A549 cell line, with IC50 values of 0.125 and 0.349 mM, without causing any toxicity towards the NIH/3T3 mouse embryonic fibroblast cell line, which suggests that the (1,3-benzodioxol-5-ylmethyl) amino group enhances the antitumor activity against A549 cells. At the same time, the benzodioxole-substituted thiadiazole analog 262 was inactive against A549 cell lines, which proves that the oxadiazole scaffold is essential for activity against A549 cells. Compounds 263 and 264 were also the most effective MMP-9 inhibitors in this series. Moreover, docking studies pointed out that compounds 263 and 264 had a good affinity for the active site of the MMP-9 enzyme ( Figure 47) [109]. The structure-activity relationship of the anticancer activity of substituted 1,3,4-oxa-/thiadiazoles is summarized in Figure 48. Ozdemir et al. designed and evaluated several oxadiazole and thiadiazole derivatives with respect to their anticancer effects on A549 human lung adenocarcinoma and C6 rat glioma cell lines and evaluated their inhibitory effects on matrix metalloproteinases (MMPs). Compounds 261, 263, and 264 showed high cytotoxic activity against the C6 cell line, with IC 50 ranging from 0.0128 mM to 0.157 mM, compared to Cisplatin, with IC 50 = 0.103 mM. Only the benzodioxole-substituted oxadiazoles 263 and 264 had a cytotoxic effect on the A549 cell line, with IC 50 values of 0.125 and 0.349 mM, without causing any toxicity towards the NIH/3T3 mouse embryonic fibroblast cell line, which suggests that the (1,3benzodioxol-5-ylmethyl) amino group enhances the antitumor activity against A549 cells. At the same time, the benzodioxole-substituted thiadiazole analog 262 was inactive against A549 cell lines, which proves that the oxadiazole scaffold is essential for activity against A549 cells. Compounds 263 and 264 were also the most effective MMP-9 inhibitors in this series. Moreover, docking studies pointed out that compounds 263 and 264 had a good affinity for the active site of the MMP-9 enzyme ( Figure 47) [109]. Ozdemir et al. designed and evaluated several oxadiazole and thiadiazole derivatives with respect to their anticancer effects on A549 human lung adenocarcinoma and C6 rat glioma cell lines and evaluated their inhibitory effects on matrix metalloproteinases (MMPs). Compounds 261, 263, and 264 showed high cytotoxic activity against the C6 cell line, with IC50 ranging from 0.0128 mM to 0.157 mM, compared to Cisplatin, with IC50 = 0.103 mM. Only the benzodioxole-substituted oxadiazoles 263 and 264 had a cytotoxic effect on the A549 cell line, with IC50 values of 0.125 and 0.349 mM, without causing any toxicity towards the NIH/3T3 mouse embryonic fibroblast cell line, which suggests that the (1,3-benzodioxol-5-ylmethyl) amino group enhances the antitumor activity against A549 cells. At the same time, the benzodioxole-substituted thiadiazole analog 262 was inactive against A549 cell lines, which proves that the oxadiazole scaffold is essential for activity against A549 cells. Compounds 263 and 264 were also the most effective MMP-9 inhibitors in this series. Moreover, docking studies pointed out that compounds 263 and 264 had a good affinity for the active site of the MMP-9 enzyme ( Figure 47) [109]. The structure-activity relationship of the anticancer activity of substituted 1,3,4-oxa-/thiadiazoles is summarized in Figure 48. The structure-activity relationship of the anticancer activity of substituted 1,3,4-oxa-/thiadiazoles is summarized in Figure 48.

Antidiabetic Activity
In 2019, Taha et al., reported a series of 5-(4-hydroxy-2-methoxyphenyl)-1,3,4oxadiazoles coupled to 5-aryl-1,3,4-thiadiazole motifs through a phenyl ring as potent βglucuronidase inhibitors. Several analogs were more potent enzyme inhibitors (IC50 = 0.96-28.10 μM) than the standard D-saccharic acid 1,4-lactone (IC50 = 48.40 μM). Study of the structure-activity relationships indicated that the nature and relative position of substituents on the phenyl ring attached to thiadiazole greatly affected the inhibitory potency. The 2,4,6-trichlorophenyl derivative 265 displayed superior activity (IC50 = 0.96 μM) compared to monochloro-substituted analogs, regardless of the position (o,m or p-). Moreover, compound 266a with the 3,4-dihydroxyphenyl moiety (IC50 = 1.40 μM) was four times more potent than the 2,5-dihydroxyphenyl analog 267a (IC50 = 6.20 μM). A docking study revealed that the hydroxyl groups in 6 had higher potential to be engaged in proper hydrogen bonding interaction with the enzyme active site than in 7. In contrast, compound 267b having 2-hydroxy-5-methoxy groups on the phenyl ring was much more potent than the 3-hydroxy-4-methoxy phenyl derivative 266b, with IC50 = 12.30 and 28.10 μM, respectively. The steric effect of the methoxy group next to the hydroxy function in 266b decreased the ability of the hydroxyl group to be involved in H-bonding with the enzyme and reduced the activity (Figure 49) [109].

Antidiabetic Activity
In 2019, Taha et al., reported a series of 5-(4-hydroxy-2-methoxyphenyl)-1,3,4-oxadiazoles coupled to 5-aryl-1,3,4-thiadiazole motifs through a phenyl ring as potent β-glucuronidase inhibitors. Several analogs were more potent enzyme inhibitors (IC 50 = 0.96-28.10 µM) than the standard D-saccharic acid 1,4-lactone (IC 50 = 48.40 µM). Study of the structureactivity relationships indicated that the nature and relative position of substituents on the phenyl ring attached to thiadiazole greatly affected the inhibitory potency. The 2,4,6trichlorophenyl derivative 265 displayed superior activity (IC 50 = 0.96 µM) compared to monochloro-substituted analogs, regardless of the position (o,m or p-). Moreover, compound 266a with the 3,4-dihydroxyphenyl moiety (IC 50 = 1.40 µM) was four times more potent than the 2,5-dihydroxyphenyl analog 267a (IC 50 = 6.20 µM). A docking study revealed that the hydroxyl groups in 6 had higher potential to be engaged in proper hydrogen bonding interaction with the enzyme active site than in 7. In contrast, compound 267b having 2-hydroxy-5-methoxy groups on the phenyl ring was much more potent than the 3-hydroxy-4-methoxy phenyl derivative 266b, with IC 50 = 12.30 and 28.10 µM, respectively. The steric effect of the methoxy group next to the hydroxy function in 266b decreased the ability of the hydroxyl group to be involved in H-bonding with the enzyme and reduced the activity (Figure 49) [109].

Analgesic and Anti-inflammatory Activities
In 2013, some N-(tetrazol-1H-5-yl)-6,14-endoethenotetrahydrothebaine 7α-substituted 1,3,4-(oxa)thiadiazoles were prepared and screened for analgesic activity in rats, applying hot-plate and tail-flick tests and employing Morphine as a reference drug. At 0.5 and 1 h after administration, the phenylamino-oxadiazole derivative 268 (reaction time in hot plate test = 9.78 s, tail-flick test = 21.45 s) was found to be more potent than Morphine (reaction time in hot plate test = 6.28 s, tail-flick test = 15.90 s). Conversely, the thiadiazole analog 269 was moderately active. The results demonstrated that the oxadiazole group on the 7α position of thebaine increased the analgesic activity ( Figure 54) [60].
Molecules 2022, 27, x FOR PEER REVIEW 54 of 59 rats, applying hot-plate and tail-flick tests and employing Morphine as a reference drug. At 0.5 and 1 h after administration, the phenylamino-oxadiazole derivative 268 (reaction time in hot plate test = 9.78 s, tail-flick test = 21.45 s) was found to be more potent than Morphine (reaction time in hot plate test = 6.28 s, tail-flick test = 15.90 s). Conversely, the thiadiazole analog 269 was moderately active. The results demonstrated that the oxadiazole group on the 7α position of thebaine increased the analgesic activity ( Figure  56) [60]. A series of polyheterocyclic thioethers containing 1,3,4-(oxa)thiadiazoles were examined for anti-inflammatory activity using carrageenan-induced rat paw edema assay and Diclofenac as a reference drug. Compounds 270, 271a, and 271b were more effective than Diclofenac in alleviating carrageenan-induced edema after 2, 3, and 4 h of treatment, with inhibition percentages of 37.5-55%, 30-35%, and 38-48%, respectively. In this study, oxadiazole and thiadiazole isosteres were presented as potent anti-inflammatory agents with no difference in their activities ( Figure 57) [110].

Conclusions
This study is a review article summarizing most of the published work about the medicinally important pharmacophores 1,3,4-oxadiazoles and 1,3,4-thiadiazoles throughout the past 10 years. The review presents different synthetic approaches to the two bioisosteres using different starting compounds, comparing the differences in the reagents used, the conditions, and the yields of the two classes. This comparative study also identified their pharmacological activities, as well as introducing deduced collective structure-activity relationship charts for antimicrobial, anticancer, and antioxidant activities. In most cases, it was concluded that 1,3,4-thiadiazoles are more active antimicrobial agents than 1,3,4oxadiazoles, in contrast to antioxidant activity, where 1,3,4-oxadiazoles are more active agents than 1,3,4-thiadiazoles. Concerning anticancer activity, it was noticed that combining the 1,3,4oxadiazole ring with the 1,3,4-thiadiazole ring enhanced the activity.  A series of polyheterocyclic thioethers containing 1,3,4-(oxa)thiadiazoles were examined for anti-inflammatory activity using carrageenan-induced rat paw edema assay and Diclofenac as a reference drug. Compounds 270, 271a, and 271b were more effective than Diclofenac in alleviating carrageenan-induced edema after 2, 3, and 4 h of treatment, with inhibition percentages of 37.5-55%, 30-35%, and 38-48%, respectively. In this study, oxadiazole and thiadiazole isosteres were presented as potent anti-inflammatory agents with no difference in their activities ( Figure 55) [110].  ). Conversely, the thiadiazole analog 269 was moderately active. The results demonstrated that the oxadiazole group on the 7α position of thebaine increased the analgesic activity ( Figure  56) [60]. A series of polyheterocyclic thioethers containing 1,3,4-(oxa)thiadiazoles were examined for anti-inflammatory activity using carrageenan-induced rat paw edema assay and Diclofenac as a reference drug. Compounds 270, 271a, and 271b were more effective than Diclofenac in alleviating carrageenan-induced edema after 2, 3, and 4 h of treatment, with inhibition percentages of 37.5-55%, 30-35%, and 38-48%, respectively. In this study, oxadiazole and thiadiazole isosteres were presented as potent anti-inflammatory agents with no difference in their activities ( Figure 57) [110].

Conclusions
This study is a review article summarizing most of the published work about the medicinally important pharmacophores 1,3,4-oxadiazoles and 1,3,4-thiadiazoles throughout the past 10 years. The review presents different synthetic approaches to the two bioisosteres using different starting compounds, comparing the differences in the reagents used, the conditions, and the yields of the two classes. This comparative study also identified their pharmacological activities, as well as introducing deduced collective structure-activity relationship charts for antimicrobial, anticancer, and antioxidant activities. In most cases, it was concluded that 1,3,4-thiadiazoles are more active antimicrobial agents than 1,3,4oxadiazoles, in contrast to antioxidant activity, where 1,3,4-oxadiazoles are more active agents than 1,3,4-thiadiazoles. Concerning anticancer activity, it was noticed that combining the 1,3,4oxadiazole ring with the 1,3,4-thiadiazole ring enhanced the activity.

Conclusions
This study is a review article summarizing most of the published work about the medicinally important pharmacophores 1,3,4-oxadiazoles and 1,3,4-thiadiazoles throughout the past 10 years. The review presents different synthetic approaches to the two bioisosteres using different starting compounds, comparing the differences in the reagents used, the conditions, and the yields of the two classes. This comparative study also identified their pharmacological activities, as well as introducing deduced collective structure-activity relationship charts for antimicrobial, anticancer, and antioxidant activities. In most cases, it was concluded that 1,3,4-thiadiazoles are more active antimicrobial agents than 1,3,4oxadiazoles, in contrast to antioxidant activity, where 1,3,4-oxadiazoles are more active agents than 1,3,4-thiadiazoles. Concerning anticancer activity, it was noticed that combining the 1,3,4-oxadiazole ring with the 1,3,4-thiadiazole ring enhanced the activity.