An Understanding of Mechanism-Based Approaches for 1,3,4-Oxadiazole Scaffolds as Cytotoxic Agents and Enzyme Inhibitors

The world’s health system is plagued by cancer and a worldwide effort is underway to find new drugs to treat cancer. There has been a significant improvement in understanding the pathogenesis of cancer, but it remains one of the leading causes of death. The imperative 1,3,4-oxadiazole scaffold possesses a wide variety of biological activities, particularly for cancer treatment. In the development of novel 1,3,4-oxadiazole-based drugs, structural modifications are important to ensure high cytotoxicity towards malignant cells. These structural modification strategies have shown promising results when combined with outstanding oxadiazole scaffolds, which selectively interact with nucleic acids, enzymes, and globular proteins. A variety of mechanisms, such as the inhibition of growth factors, enzymes, and kinases, contribute to their antiproliferative effects. The activity of different 1,3,4-oxadiazole conjugates were tested on the different cell lines of different types of cancer. It is demonstrated that 1,3,4-oxadiazole hybridization with other anticancer pharmacophores have different mechanisms of action by targeting various enzymes (thymidylate synthase, HDAC, topoisomerase II, telomerase, thymidine phosphorylase) and many of the proteins that contribute to cancer cell proliferation. The focus of this review is to highlight the anticancer potential, molecular docking, and SAR studies of 1,3,4-oxadiazole derivatives by inhibiting specific cancer biological targets, such as inhibiting telomerase activity, HDAC, thymidylate synthase, and the thymidine phosphorylase enzyme. The purpose of this review is to summarize recent developments and discoveries in the field of anticancer drugs using 1,3,4-oxadiazoles.


Introduction
Humankind faces one of the most challenging of public health challenges, in the form of cancer. The development of cancer involves the uncontrollable division of abnormal cells that can infiltrate and destroy normal tissues in the body. High morbidity and mortality rates characterize the disease; after cardiovascular disease, it is the second leading cause of death in many countries. In order to reduce cancer death rates worldwide, chemotherapy is currently the main treatment modality for cancer, whether used alone or combined with surgery and radiotherapy [1].
The American Cancer Society (ACS) provide data on cancer mortality in the United States. In 2016, there were an estimated 1,630,730 new cancer cases, and 589,390 cancerrelated deaths in the United States. In 2017, due to advances in early detection and treatment, and changes in population age distribution, the ACS expects that those estimates The American Cancer Society (ACS) provide data on cancer mortality in the United States. In 2016, there were an estimated 1,630,730 new cancer cases, and 589,390 cancerrelated deaths in the United States. In 2017, due to advances in early detection and treatment, and changes in population age distribution, the ACS expects that those estimates will increase by about 8%.. Figure 1 represents the significant share of individuals affected by cancer each year [2,3]. In 2018, about 9.6 million deaths and 19.1 million cancer diagnoses were reported globally, according to the International Agency for Research on Cancer (IARC). The North American Association of Central Cancer Registries (NAACCR), a professional cancer registry organization, has compiled data on the main cancers that led to thousands of deaths in the United States in 2021. Ten types of cancer are identified by NAACCR and IARC as the world's leading cause of death ( Figure 2). The treatment of cancer is a multidisciplinary effort involving physicians, oncologists, and surgeons and there are many different treatment strategies available worldwide. The treatment strategy for cancer depends on the type, stage, and the location of the cancer. This is determined by several factors including: age (children may need a different treatment plan to adults); how far the disease has progressed (stage); the stage of growth at the time surgery is performed (what has happened since diagnosis); how advanced the disease is at the time of treatment; and whether it can be cured or not (curable vs. incurable).The treatment of cancer has become a highly specialized technology, which involves diverse approaches, such as radiation therapy and biotechnology. In addition, novel methods of diagnosis and external therapies such as surgery, chemotherapy, radiotherapy, targeted therapies, and immune therapies such as interferon, are currently on going therapies in cancer hospitals and research laboratories across the world [4].
In recent years, research in the synthetic and semi-synthetic fields has become more prevalent, because of cancer's life-threatening nature. Therefore, the need for new cancer therapies is increasing as researchers and clinicians try to find solutions to the problem of drug shortages [5,6]. In 2018, about 9.6 million deaths and 19.1 million cancer diagnoses were reported globally, according to the International Agency for Research on Cancer (IARC). The North American Association of Central Cancer Registries (NAACCR), a professional cancer registry organization, has compiled data on the main cancers that led to thousands of deaths in the United States in 2021. Ten types of cancer are identified by NAACCR and IARC as the world's leading cause of death ( Figure 2). The treatment of cancer is a multidisciplinary effort involving physicians, oncologists, and surgeons and there are many different treatment strategies available worldwide. The treatment strategy for cancer depends on the type, stage, and the location of the cancer. This is determined by several factors including: age (children may need a different treatment plan to adults); how far the disease has progressed (stage); the stage of growth at the time surgery is performed (what has happened since diagnosis); how advanced the disease is at the time of treatment; and whether it can be cured or not (curable vs. incurable).The treatment of cancer has become a highly specialized technology, which involves diverse approaches, such as radiation therapy and biotechnology. In addition, novel methods of diagnosis and external therapies such as surgery, chemotherapy, radiotherapy, targeted therapies, and immune therapies such as interferon, are currently on going therapies in cancer hospitals and research laboratories across the world [4].
In recent years, research in the synthetic and semi-synthetic fields has become more prevalent, because of cancer's life-threatening nature. Therefore, the need for new cancer therapies is increasing as researchers and clinicians try to find solutions to the problem of drug shortages [5,6].
Heterocyclic compounds, i.e., 5-membered and 6-membered rings, or fused ring systems, play an imperative role in the finding and progress of new drug molecules with the highest potency and lower toxicity [7]. Despite this, rings containing an N, O, or S atom have become a magnet in the field of synthetic chemistry in order to develop new medicinal compounds because of their huge therapeutic potentials. Research investigating different types of heterocyclic moieties, i.e., H. pyrazoles, tetrazoles, oxadiazoles, thiadiazoles, triazoles, etc., has shown attraction in recent years [8]. The 1,3,4-oxadiazole has four different isomers ( Figure 3) and among them, the 1,2,3-isomer has shown instability due to the formation of diazoketone tautomers (ring opening) [9]. This heterocyclic ring was Pharmaceuticals 2023, 16, 254 3 of 42 synthesized for the first time by Ainsworth in 1965, by the thermal decomposition reaction of hydrazine. Oxadiazole moiety with the formula C 2 H 2 ON 2 , 70.05 g/mol molecular weight and water soluble [10]. Heterocyclic compounds, i.e., 5-membered and 6-membered rings, or fused ring systems, play an imperative role in the finding and progress of new drug molecules with the highest potency and lower toxicity [7]. Despite this, rings containing an N, O, or S atom have become a magnet in the field of synthetic chemistry in order to develop new medicinal compounds because of their huge therapeutic potentials. Research investigating different types of heterocyclic moieties, i.e., H. pyrazoles, tetrazoles, oxadiazoles, thiadiazoles, triazoles, etc., has shown attraction in recent years [8]. The 1,3,4-oxadiazole has four different isomers ( Figure 3) and among them, the 1,2,3-isomer has shown instability due to the formation of diazoketone tautomers (ring opening) [9]. This heterocyclic ring was synthesized for the first time by Ainsworth in 1965, by the thermal decomposition reaction of hydrazine. Oxadiazole moiety with the formula C2H2ON2, 70.05 g/mol molecular weight and water soluble [10]. The oxadiazole skeleton is also referred to as Azoximes, Diazoxol, Furadiazole, Biozol, Furoxanes, and Oxybiazole. The oxadiazole ring is thermostable due to its resonance energy near 167.4 kJ/mol, which increases its thermal stability with substitution at the 2nd position [11].
Due of their ability to form hydrogen bonds with the receptor site, oxadiazole or its isomers are of considerable interest to chemical, medical, and pharmaceutical research for the development of innovative drugs. Further, this molecular ring is commercially available in many important drugs, e.g., Furamizole, with potent antibacterial activity; Nesapidil, with antiarrhythmic activity; Raltegravir, as an antiviral drug; Tiodazosin, as an antihypertensive agent; and the preferred derivative of the FDA-approved anticancer drug, Zibotentan ( Figure 4) [12,13].  Heterocyclic compounds, i.e., 5-membered and 6-membered rings, or fused ring systems, play an imperative role in the finding and progress of new drug molecules with the highest potency and lower toxicity [7]. Despite this, rings containing an N, O, or S atom have become a magnet in the field of synthetic chemistry in order to develop new medicinal compounds because of their huge therapeutic potentials. Research investigating different types of heterocyclic moieties, i.e., H. pyrazoles, tetrazoles, oxadiazoles, thiadiazoles, triazoles, etc., has shown attraction in recent years [8]. The 1,3,4-oxadiazole has four different isomers ( Figure 3) and among them, the 1,2,3-isomer has shown instability due to the formation of diazoketone tautomers (ring opening) [9]. This heterocyclic ring was synthesized for the first time by Ainsworth in 1965, by the thermal decomposition reaction of hydrazine. Oxadiazole moiety with the formula C2H2ON2, 70.05 g/mol molecular weight and water soluble [10]. The oxadiazole skeleton is also referred to as Azoximes, Diazoxol, Furadiazole, Biozol, Furoxanes, and Oxybiazole. The oxadiazole ring is thermostable due to its resonance energy near 167.4 kJ/mol, which increases its thermal stability with substitution at the 2nd position [11].
Due of their ability to form hydrogen bonds with the receptor site, oxadiazole or its isomers are of considerable interest to chemical, medical, and pharmaceutical research for the development of innovative drugs. Further, this molecular ring is commercially available in many important drugs, e.g., Furamizole, with potent antibacterial activity; Nesapidil, with antiarrhythmic activity; Raltegravir, as an antiviral drug; Tiodazosin, as an antihypertensive agent; and the preferred derivative of the FDA-approved anticancer drug, Zibotentan ( Figure 4) [12,13]. The oxadiazole skeleton is also referred to as Azoximes, Diazoxol, Furadiazole, Biozol, Furoxanes, and Oxybiazole. The oxadiazole ring is thermostable due to its resonance energy near 167.4 kJ/mol, which increases its thermal stability with substitution at the 2nd position [11].
Due of their ability to form hydrogen bonds with the receptor site, oxadiazole or its isomers are of considerable interest to chemical, medical, and pharmaceutical research for the development of innovative drugs. Further, this molecular ring is commercially available in many important drugs, e.g., Furamizole, with potent antibacterial activity; Nesapidil, with antiarrhythmic activity; Raltegravir, as an antiviral drug; Tiodazosin, as an antihypertensive agent; and the preferred derivative of the FDA-approved anticancer drug, Zibotentan ( Figure 4) [12,13]. The 1,3,4-oxadiazole has been a well-known pharmacophore for about 85 years and is in high demand in numerous biological and chemical fields [14]. Due to the presence of an additional heteroatom in the ring, and its inductive effect, oxadiazole shows very weak basic properties. Because nitrogen atoms (=N-) are present in the oxadiazole ring, it behaves as a conjugated diene, thus reducing its aromaticity [15].
Due to the lower electron cloud density in the oxadiazole moiety, electrophilic reactions at carbon atoms are difficult, resulting in 1,3,4-oxadiazolium salts. However, nucleophilic substitution reactions proceed in halogenated oxadiazole by replacing the halogen atom, which is illustrated in Figure 5 [16][17][18].  The 1,3,4-oxadiazole has been a well-known pharmacophore for about 85 years an is in high demand in numerous biological and chemical fields [14]. Due to the presence o an additional heteroatom in the ring, and its inductive effect, oxadiazole shows very wea basic properties. Because nitrogen atoms (=N-) are present in the oxadiazole ring, it be haves as a conjugated diene, thus reducing its aromaticity [15].
Due to the lower electron cloud density in the oxadiazole moiety, electrophilic reac tions at carbon atoms are difficult, resulting in 1,3,4-oxadiazolium salts. However, nucle ophilic substitution reactions proceed in halogenated oxadiazole by replacing the haloge atom, which is illustrated in Figure 5 [16][17][18].   [19]. This method involves th reaction between different Boc and Cbz-Nα-protected amino acid hydrazides wit isoselenocyanato esters via cyclodeselenization, in the presence of THF and TEA at opt mized temperature ranges, which derived selenosemicarbazide (11) as intermediates fo  The 1,3,4-oxadiazole has been a well-known pharmacophore for about 85 years and is in high demand in numerous biological and chemical fields [14]. Due to the presence of an additional heteroatom in the ring, and its inductive effect, oxadiazole shows very weak basic properties. Because nitrogen atoms (=N-) are present in the oxadiazole ring, it behaves as a conjugated diene, thus reducing its aromaticity [15].
Due to the lower electron cloud density in the oxadiazole moiety, electrophilic reactions at carbon atoms are difficult, resulting in 1,3,4-oxadiazolium salts. However, nucleophilic substitution reactions proceed in halogenated oxadiazole by replacing the halogen atom, which is illustrated in Figure 5 [16][17][18].
Keshari K. Jha et al. reported a very simple and easy method of preparation for 1,3,4-oxadiazole (52-53) (Scheme 15) derivatives by the ring closure reactions of various acylhydrazides (51) with carbon disulphide in alkaline solution, and with aromatic acids in POCl3. This method produced a final product in high yield. Arylhydrazides (51) were synthesized from various aromatic acids in the presence of H 2 SO 4 as a catalyst, via esterification [35].

Biochemical Mechanisms Leading to Cancer
The uncontrolled division of cancer cells is a hallmark of this deadly disease. In the current era, chemotherapy is the most common method of cancer treatment, either alone or in combination with surgery and radiotherapy. The demand for new cancer therapies is increasing as researchers and clinicians consider this solution to the drug shortage dilemma. Modern genomic and proteomic technologies have steadily increased the number of proposals for new cancer drug targets, based on the assessment of cancer-specific biological pathways. Many proteins and enzymes such as cyclin-dependent kinases (CDKs) participate in the regulation of cell proliferation and the phase of the cell cycle-G0/G1, S, G2, and M. Consequently, the regulation of cells and their phases is regarded as a promising target for the development of new anticancer molecules. Therefore, targeted therapies require an understanding of how cell-cycle and their regulations, genes regulations, and biochemical pathways affect cancer behavior. In this challenging disease, molecularly specific therapies have provided some of the most significant advances, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors. The findings of this study will demonstrate that cell proteins, growth factors, and numerous biological pathways could be selective targets to develop new anticancer drugs. The review also emphasizes

Biochemical Mechanisms Leading to Cancer
The uncontrolled division of cancer cells is a hallmark of this deadly disease. In the current era, chemotherapy is the most common method of cancer treatment, either alone or in combination with surgery and radiotherapy. The demand for new cancer therapies is increasing as researchers and clinicians consider this solution to the drug shortage dilemma. Modern genomic and proteomic technologies have steadily increased the number of proposals for new cancer drug targets, based on the assessment of cancer-specific biological pathways. Many proteins and enzymes such as cyclin-dependent kinases (CDKs) participate in the regulation of cell proliferation and the phase of the cell cycle-G0/G1, S, G2, and M. Consequently, the regulation of cells and their phases is regarded as a promising target for the development of new anticancer molecules. Therefore, targeted therapies require an understanding of how cell-cycle and their regulations, genes regulations, and biochemical pathways affect cancer behavior. In this challenging disease, molecularly specific therapies have provided some of the most significant advances, including monoclonal antibodies and small-molecule tyrosine kinase inhibitors. The findings of this study will demonstrate that cell proteins, growth factors, and numerous biological pathways could be selective targets to develop new anticancer drugs. The review also emphasizes the various anticancer properties of 1,3,4-oxadiazole scaffolds, which target primarily enzymes and kinases. Many targets exist, but few are here, namely:

Telomerase
Telomerase is specialized ribonucleoprotein that is found in mammalian cells, having a very specific function for the maintenance and stability of telomere, for the functionalization of chromosomal integration and cell proliferation [63][64][65]. In most somatic cells, during the DNA replication process, the telomere length decreases (known as the mitotic clock). When the telomere's length became too short, the metabolism becomes slower, causing genomic instability, thereby stopping the proliferation of cells. This phenomenon is called senescence. However, this ribonucleoprotein becomes activated in cancerous cells by adding extra nucleotide sequence, TTAGGG, and becomes stabilized ( Figure 8).

Telomerase
Telomerase is specialized ribonucleoprotein that is found in mammalian cells, having a very specific function for the maintenance and stability of telomere, for the functionalization of chromosomal integration and cell proliferation [63][64][65]. In most somatic cells, during the DNA replication process, the telomere length decreases (known as the mitotic clock). When the telomere's length became too short, the metabolism becomes slower, causing genomic instability, thereby stopping the proliferation of cells. This phenomenon is called senescence. However, this ribonucleoprotein becomes activated in cancerous cells by adding extra nucleotide sequence, TTAGGG, and becomes stabilized ( Figure 8). So, tumor progression may be prevented by inhibiting the addition of extra nucleotide sequence, TTAGGG, or by inhibiting the shortening of the telomere. Therefore, telomere may be considered a potential target for the development of telomerase inhibitors in carcinogenesis, due to its role in continuous cell division and preventing replicative senescence [66,67] (Figure 9). So, tumor progression may be prevented by inhibiting the addition of extra nucleotide sequence, TTAGGG, or by inhibiting the shortening of the telomere. Therefore, telomere may be considered a potential target for the development of telomerase inhibitors in carcinogenesis, due to its role in continuous cell division and preventing replicative senescence [66,67] (Figure 9).

Telomeres Association Proteins
Telomeres protect exonucleolytic degradation and the fusion of chromosome by forming helical loop structures around chromosomes. The telomerase enzyme regulates telomere lengthening for chromosomal integrity via various chemical reactions in DNA, including reverse transcription and translocation [80]. Chromosomal integrity is regulated by telomerase via the other six protein subunits which are associated with it. The six proteins subunits of telomerase, i.e., hTERC, hsp90, hTERT, TEP1, p23, and dyskerin, together with  Figure 11 [80,81]. Telomerase activity is primarily regulated by human telomerase RNA components, and human telomerase reverse transcriptase subunits [82].

Telomeres Association Proteins
Telomeres protect exonucleolytic degradation and the fusion of chromosome by forming helical loop structures around chromosomes. The telomerase enzyme regulates telomere lengthening for chromosomal integrity via various chemical reactions in DNA, including reverse transcription and translocation [80]. Chromosomal integrity is regulated by telomerase via the other six protein subunits which are associated with it. The six proteins subunits of telomerase, i.e., hTERC, hsp90, hTERT, TEP1, p23, and dyskerin, together with their functions, are summarized in Figure 11 [80,81]. Telomerase activity is primarily regulated by human telomerase RNA components, and human telomerase reverse transcriptase subunits [82].  Figure 12) has shown a significant amount of cytotoxicity potential against the cell lines, i.e., the MID GI50 value of 2.09 (mean graph midpoint (arithmetical mean value of treated cancer cell lines)) was observed, which was  (Figure 12) has shown a significant amount of cytotoxicity potential against the cell lines, i.e., the MID GI50 value of 2.09 (mean graph midpoint (arithmetical mean value of treated cancer cell lines)) was observed, which was significant value as a comparison to marketed anticancer drugs, i.e., bendamustine with MID GI50 value 60, and chlorambucil with GI50 value 52, respectively [83]. significant value as a comparison to marketed anticancer drugs, i.e., bendamustine with MID GI50 value 60, and chlorambucil with GI50 value 52, respectively [83]. A new series of 1,4-benzodioxan moiety containing 1,3,4-oxadiazole derivatives (64) (Figure 13) was prepared by Zhang et al. and screened for telomerase inhibitory activity by TRAP-PCR-ELISA assay [84].   A new series of 1,4-benzodioxan moiety containing 1,3,4-oxadiazole derivatives (64) (Figure 13) was prepared by Zhang et al. and screened for telomerase inhibitory activity by TRAP-PCR-ELISA assay [84]. significant value as a comparison to marketed anticancer drugs, i.e., bendamustine with MID GI50 value 60, and chlorambucil with GI50 value 52, respectively [83]. A new series of 1,4-benzodioxan moiety containing 1,3,4-oxadiazole derivatives (64) (Figure 13) was prepared by Zhang et al. and screened for telomerase inhibitory activity by TRAP-PCR-ELISA assay [84].   The synthesized derivatives were tested against four different cancer cell lines, HEPG2, HELA, SW1116, and BGC823, compared with positive control 5-fluorouracil, a well-known anticancer agent. Among all derivatives, Compound 2- (65) was found to possess the most potent telomerase inhibitory action (IC 50 = 1.27 ± 0.05 µM).

Histone Deacetylase (HDAC) Functions and Its Inhibitors
Human histone deacetylases (HDACs) comprise 18 proteins that span four different protein classes. In carcinogenesis, these proteins play a role in transcription, gene regulation, mutation, and protein encoding. Therefore, HDAC inhibitors, are relatively new classes of anticancer agents that induce cell death, cell apoptosis, and seize cancer cell cycle. They play significant roles in gene regulation, cell death, and apoptosis in cancer cells [88,89]. Histone as a protein performs a very important role for acetylation and deacetylation by HDACs and HATs (histone acetyl-transferases), which eliminate acetyl groups from DNA-binding histone proteins, thereby lowering the uses of chromatin for transcription factors, and preventing transcription inside genetic material which regulate cell proliferation and cell death ( Figure 17) [90].  (73) was found to be the most potent inhibitor against four cancer cell lines (HEPG2, MCF7, SW1116, BGC823) with an IC 50 value of 1.18 ± 0.14 µM, which is lower than that of the positive control staurosporine (4.18 ± 0.05 µM) and ethidium bromide(2.71 ± 0.18 µM) [87].
Zheng et al. synthesized novel 2-chloropyridine derivatives possessing 1,3,4-oxadiazole moiety (69) ( Figure 15) and evaluated telomerase inhibitory activity against gastric cancer cell lines SGC-7901 by modified TRAP (telomere repeat amplification protocol). Among all derivatives, Compound 70 and 71 showed significant telomerase inhibitory activity (IC50 = 2.3 ± 0.07 µ M and 2.56 ± 0.11, respectively) as compared to the positive control ethidium bromide (IC50 = 2.5 ± 0.23). A docking binding model and structure activity relationship showed that electron-donating groups on the ortho position of the benzene ring had lower inhibitory activity than those on the para position [86].

Histone Deacetylase (HDAC) Functions and Its Inhibitors
Human histone deacetylases (HDACs) comprise 18 proteins that span four different protein classes. In carcinogenesis, these proteins play a role in transcription, gene regulation, mutation, and protein encoding. Therefore, HDAC inhibitors, are relatively new classes of anticancer agents that induce cell death, cell apoptosis, and seize cancer cell cycle. They play significant roles in gene regulation, cell death, and apoptosis in cancer cells [88,89]. Histone as a protein performs a very important role for acetylation and deacetylation by HDACs and HATs (histone acetyl-transferases), which eliminate acetyl groups from DNA-binding histone proteins, thereby lowering the uses of chromatin for transcription factors, and preventing transcription inside genetic material which regulate cell proliferation and cell death ( Figure 17) [90].

Histone Deacetylase (HDAC) Functions and Its Inhibitors
Human histone deacetylases (HDACs) comprise 18 proteins that span four different protein classes. In carcinogenesis, these proteins play a role in transcription, gene regulation, mutation, and protein encoding. Therefore, HDAC inhibitors, are relatively new classes of anticancer agents that induce cell death, cell apoptosis, and seize cancer cell cycle. They play significant roles in gene regulation, cell death, and apoptosis in cancer cells [88,89]. Histone as a protein performs a very important role for acetylation and deacetylation by HDACs and HATs (histone acetyl-transferases), which eliminate acetyl groups from DNAbinding histone proteins, thereby lowering the uses of chromatin for transcription factors, and preventing transcription inside genetic material which regulate cell proliferation and cell death (Figure 17) [90].

Thymidylate Synthase and Its Inhibitors
The DNA replication process is a very complex process and many enzymes are involved in its biosynthesis, transcription, and repair. Among all enzymes, thymidylate synthase is an essential enzyme that shows a significant contribution in its replication. Further, TS helps in the conversion of dUMP to dTMP ( Figure 20) for producing thymine via thymidylic acid, which is the building block of nucleic acid, an essential unit of DNA. This inactivation of TMP leads to a decrease in the dTTP, causing disturbance in the DNA biosynthesis, and slows down the growth and proliferation of the cell [94].
The DNA replication process is a very complex process and many enzymes are involved in its biosynthesis, transcription, and repair. Among all enzymes, thymidylate synthase is an essential enzyme that shows a significant contribution in its replication. Further, TS helps in the conversion of dUMP to dTMP ( Figure 20) for producing thymine via thymidylic acid, which is the building block of nucleic acid, an essential unit of DNA. This inactivation of TMP leads to a decrease in the dTTP, causing disturbance in the DNA biosynthesis, and slows down the growth and proliferation of the cell [94].  Platelet-derived endothelial cell growth factor is also known as thymidine phosphorylase (TP). This enzyme is responsible for the reversible conversion of thymidine to thymine (pyrimidine nucleoside degraded) inside the nucleic acid, and causes proliferation. Thymidine, thymine, and 2-deoxy-D-ribose 1-phosphate together form pyrimidine nucle- Platelet-derived endothelial cell growth factor is also known as thymidine phosphorylase (TP). This enzyme is responsible for the reversible conversion of thymidine to thymine (pyrimidine nucleoside degraded) inside the nucleic acid, and causes proliferation. Thymidine, thymine, and 2-deoxy-D-ribose 1-phosphate together form pyrimidine nucleoside, which gives 2-deoxy-D-ribose after dephosphorylation ( Figure 22). This is identified via in vitro as well as in vivo studies by the stimulation of chemotactic and VEGF (vascular endothelial growth factor) secretion for angiogenic activity [97,98]. Platelet-derived endothelial cell growth factor is also known as thymidine phosphorylase (TP). This enzyme is responsible for the reversible conversion of thymidine to thymine (pyrimidine nucleoside degraded) inside the nucleic acid, and causes proliferation. Thymidine, thymine, and 2-deoxy-D-ribose 1-phosphate together form pyrimidine nucleoside, which gives 2-deoxy-D-ribose after dephosphorylation ( Figure 22). This is identified via in vitro as well as in vivo studies by the stimulation of chemotactic and VEGF (vascular endothelial growth factor) secretion for angiogenic activity [97,98]. Therefore, a higher concentration of thymidine phosphorylase inside cell signaling may lead to uncontrolled cell division, or cancerous cells. Thymidine phosphorylase enzyme may also become activated by physical and chemical stress in the cancer tissue, that promotes the concentration of 2-deoxy-D-ribose, which is the causative factor for the progression of tumors. So, the platelet-derived endothelial cell growth factor becomes a very important target for the development of novel thymidine phosphorylase inhibitors as anticancer drugs [99,100].
Khan et al. prepared various 2,5-substituted 1.3.4-oxadiazoles and evaluated all molecules against thymidine phosphorylase activity. Among all of the tested derivatives, Compound 2,5-di(pyridin-3-yl)-1,3,4-oxadiazole (77) was the most potent as an enzyme inhibitor having 3-pyridyl substituent at positions 2 and 5, against 7-deazaxanthin taken as a standard drug [101]. Therefore, a higher concentration of thymidine phosphorylase inside cell signaling may lead to uncontrolled cell division, or cancerous cells. Thymidine phosphorylase enzyme may also become activated by physical and chemical stress in the cancer tissue, that promotes the concentration of 2-deoxy-D-ribose, which is the causative factor for the progression of tumors. So, the platelet-derived endothelial cell growth factor becomes a very important target for the development of novel thymidine phosphorylase inhibitors as anticancer drugs [99,100].
Ismail celiket al. designed and synthesized a number of novel 1,3,4-oxadiazole-benzimidazole derivatives. MTT assays were used to evaluate their cytotoxicity against five cancer cell lines, i.e., A549, MCF-7, C6, HepG2, and HeLa. Out of theses cancer cell lines, derivatives (130)(IC50 =6.554 ± 0.287 μM) and (131) (IC50 =5.132 ± 0.211 μM) were found to have satisfactory potencies and higher anticancer activities, as compare to doxorubicin (IC50 =10.525 ± 0.472 μM) against only MCF-7. An in vitro, aromatase (ARO) enzyme inhibition assay was also performed. Additionally, molecular docking studies were conducted to detect their binding sites and types of interactions with aromatases. Com-pounds130 and 131 were also investigated via molecular dynamic simulations for its possible binding mode to CYP19A1 and it was found that both derivatives had great potential as compared to others [124].
Ismail celiket al. designed and synthesized a number of novel 1,3,4-oxadiazole-benzimidazole derivatives. MTT assays were used to evaluate their cytotoxicity against five cancer cell lines, i.e., A549, MCF-7, C6, HepG2, and HeLa. Out of theses cancer cell lines, derivatives (130)(IC50 =6.554 ± 0.287 μM) and (131) (IC50 =5.132 ± 0.211 μM) were found to have satisfactory potencies and higher anticancer activities, as compare to doxorubicin (IC50 =10.525 ± 0.472 μM) against only MCF-7. An in vitro, aromatase (ARO) enzyme inhibition assay was also performed. Additionally, molecular docking studies were conducted to detect their binding sites and types of interactions with aromatases. Com-pounds130 and 131 were also investigated via molecular dynamic simulations for its possible binding mode to CYP19A1 and it was found that both derivatives had great potential as compared to others [124].
Similarly, another antiproliferative study via MTT assay was performed by M. M alam and his colleagues by synthesizing a novel series of 1,2,3-triazole-incorporated thymol-1,3,4-oxadiazole conjugates (136). They tested all 14 derivatives against three differ-Twenty-seven derivatives of 5-substituted 2-amino-1,3,4-oxadiazole and 2-amino-1,3,4thiadiazoles were synthesized. A diffusion method was used to determine the antibacterial and antifungal activities, while MTT assay was used to determine the anticancer activities. The researchers found that conjugate (133) displayed remarkable cytotoxic activity against HepG2 cell line (IC 50 = 8.6 µM), which is comparable to the activity of paclitaxel, and is non-toxic on LLC-PK1 normal cell line. According to the structure-activity relationship and the molecular docking study of the synthesized compound, ethoxy, halogen, and nitro derivatives possess significant antimicrobial and cytotoxic properties [126]. Twenty-seven derivatives of 5-substituted 2-amino-1,3,4-oxadiazole and 2-amino-1,3,4-thiadiazoles were synthesized. A diffusion method was used to determine the antibacterial and antifungal activities, while MTT assay was used to determine the anticancer activities. The researchers found that conjugate (133) displayed remarkable cytotoxic activity against HepG2 cell line (IC50= 8.6 μM), which is comparable to the activity of paclitaxel, and is non-toxic on LLC-PK1 normal cell line. According to the structure-activity relationship and the molecular docking study of the synthesized compound, ethoxy, halogen, and nitro derivatives possess significant antimicrobial and cytotoxic properties [126].
Similarly, another antiproliferative study via MTT assay was performed by M. M alam and his colleagues by synthesizing a novel series of 1,2,3-triazole-incorporated thymol-1,3,4-oxadiazole conjugates (136). They tested all 14 derivatives against three different cancer cell lines, i.e., MCF-7, HCT-116, and HepG2, and found derivative (137)  Twenty-seven derivatives of 5-substituted 2-amino-1,3,4-oxadiazole and 2-amino-1,3,4-thiadiazoles were synthesized. A diffusion method was used to determine the antibacterial and antifungal activities, while MTT assay was used to determine the anticancer activities. The researchers found that conjugate (133) displayed remarkable cytotoxic activity against HepG2 cell line (IC50= 8.6 μM), which is comparable to the activity of paclitaxel, and is non-toxic on LLC-PK1 normal cell line. According to the structure-activity relationship and the molecular docking study of the synthesized compound, ethoxy, halogen, and nitro derivatives possess significant antimicrobial and cytotoxic properties [126].
Ibrahim H. Eissa et al. synthesized a series of 14 compounds of 1,3,4-oxadiazolenaphthalene hybrids (141) and tested their cytotoxicity against MCF-7 (human breast cancer cell line) and HepG2 (human hepatocellular carcinoma cell line) via MTTS assay. 06 derivatives (142, 143, 144, 145, 146, and 147) showed the highest potential among the 14 synthesized derivatives and their efficacy against VEGFR-2 also evaluated for further studies. A good antiproliferative effect was observed for Compound (142) against both cell lines and inhibitory activity was demonstrated against VEGFR-2. It also induced 22.86% apoptosis compared to 0.51% apoptosis in control (HepG2) cells [129].
A new series of furo[2,3-d]pyrimidine-1,3,4-oxadiazole hybrid derivatives were synthesized and evaluated for their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A549). Data showed that Compound 8f exhibits moderate cytotoxicity, with IC50 values ranging from 13.89 to 19.43 μM. Besides, Compound (148) induced apoptosis through caspase 3/7 activation, cell death independently of the mitochondrial pathway, and cell cycle arrest in the S phase for HT1080 cells and the G1/M phase for A549 cells [130].
A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed, synthesized and evaluated for cytotoxicity by the MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, the results of TP assay identified that 1,3,4-oxadiazole molecules displayed anticancer activity partially by the inhibition of phosphorylation of thymidine. The TP assay identified (149) and (150) as potential inhibitors with anticancer activity against both the cell lines [131].
A.E Mansouri and his collaborators designed and synthesized a series of homonucleosides containing theophylline,1,3,4-oxadiazole derivatives (151). Each compound was A good antiproliferative effect was observed for Compound (142) against both cell lines and inhibitory activity was demonstrated against VEGFR-2. It also induced 22.86% apoptosis compared to 0.51% apoptosis in control (HepG2) cells [129].
A new series of furo[2,3-d]pyrimidine-1,3,4-oxadiazole hybrid derivatives were synthesized and evaluated for their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A549). Data showed that Compound 8f exhibits moderate cytotoxicity, with IC 50 values ranging from 13.89 to 19.43 µM. Besides, Compound (148) induced apoptosis through caspase 3/7 activation, cell death independently of the mitochondrial pathway, and cell cycle arrest in the S phase for HT1080 cells and the G1/M phase for A549 cells [130]. A good antiproliferative effect was observed for Compound (142) against both cell lines and inhibitory activity was demonstrated against VEGFR-2. It also induced 22.86% apoptosis compared to 0.51% apoptosis in control (HepG2) cells [129].
A new series of furo[2,3-d]pyrimidine-1,3,4-oxadiazole hybrid derivatives were synthesized and evaluated for their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A549). Data showed that Compound 8f exhibits moderate cytotoxicity, with IC50 values ranging from 13.89 to 19.43 μM. Besides, Compound (148) induced apoptosis through caspase 3/7 activation, cell death independently of the mitochondrial pathway, and cell cycle arrest in the S phase for HT1080 cells and the G1/M phase for A549 cells [130].
A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed, synthesized and evaluated for cytotoxicity by the MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, the results of TP assay identified that 1,3,4-oxadiazole molecules displayed anticancer activity partially by the inhibition of phosphorylation of thymidine. The TP assay identified (149) and (150) as potential inhibitors with anticancer activity against both the cell lines [131].
A.E Mansouri and his collaborators designed and synthesized a series of homonucleosides containing theophylline,1,3,4-oxadiazole derivatives (151). Each compound was A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed, synthesized and evaluated for cytotoxicity by the MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, the results of TP assay identified that 1,3,4-oxadiazole molecules displayed anticancer activity partially by the inhibition of phosphorylation of thymidine. The TP assay identified (149) and (150) as potential inhibitors with anticancer activity against both the cell lines [131]. A good antiproliferative effect was observed for Compound (142) against both cell lines and inhibitory activity was demonstrated against VEGFR-2. It also induced 22.86% apoptosis compared to 0.51% apoptosis in control (HepG2) cells [129].
A new series of furo[2,3-d]pyrimidine-1,3,4-oxadiazole hybrid derivatives were synthesized and evaluated for their cytotoxic activity in four human cancer cell lines: fibrosarcoma (HT-1080), breast (MCF-7 and MDA-MB-231), and lung carcinoma (A549). Data showed that Compound 8f exhibits moderate cytotoxicity, with IC50 values ranging from 13.89 to 19.43 μM. Besides, Compound (148) induced apoptosis through caspase 3/7 activation, cell death independently of the mitochondrial pathway, and cell cycle arrest in the S phase for HT1080 cells and the G1/M phase for A549 cells [130].
A series of novel 1,3,4-oxadiazole derivatives with substituted phenyl ring were designed, synthesized and evaluated for cytotoxicity by the MTT method against two breast cancer cell lines (MCF-7 and MDA-MB-231). Further, the results of TP assay identified that 1,3,4-oxadiazole molecules displayed anticancer activity partially by the inhibition of phosphorylation of thymidine. The TP assay identified (149) and (150) as potential inhibitors with anticancer activity against both the cell lines [131].
Merccaptoacetamide-linked pyrimidine-1,3,4-oxadiazole hybrids (153) were designed and synthesized by Arbaz Sujat Shaikh and colleagues. The novel pyrimidine-1,3,4-oxadiazole hybrids were evaluated for their in vitro cytotoxic potential against 04 cancer cell lines, i.e., lung cancer cells (A549), prostate cancer cells (PC-3, DU-145), and human embryonic kidney cells (HEK) [133].  Further, a mixture of molecular modeling and target-based assay studies proved that Compound (154) was well accommodated at the active site of the DNA topoisomerase II complex, and exhibited favorable physiochemical and ADME/T properties in silico. Similarly, a series of imidazo[1,2-a]pyridine-oxadiazole hybrids (155) was synthesized and tested for their in vitro anticancer potential against lung cancer cells (A549) and prostate cancer cells (PC-3, DU-145) by Dilep Kumar Sigalapalli and his team. Further, a mixture of molecular modeling and target-based assay studies proved that Compound (154) was well accommodated at the active site of the DNA topoisomerase II complex, and exhibited favorable physiochemical and ADME/T properties in silico. Similarly, a series of imidazo[1,2-a]pyridine-oxadiazole hybrids (155) was synthesized and tested for their in vitro anticancer potential against lung cancer cells (A549) and prostate cancer cells (PC-3, DU-145) by Dilep Kumar Sigalapalli and his team.
As a result of screening the all-synthesized compounds, derivative (156) was found to be the most potent on A549 cells with an IC50 value of 2.8 ± 0.02 μM, as a comparison to podophyllotoxin (standard drug), with an IC50 value of 0.09 ± 0.01. The effect of derivative (156) on different phases of the cell cycle was also determined by annexin-v/PI dual staining (economical dye for apoptotic cell investigation) and it as found that Compound (156) induces apoptosis in A549 cells. The molecular modeling studies also revealed that derivative (156) had a significant affinity towards the tubulin receptor, binding with significant physico-chemical properties. Structure-activity relationships also revealed that halogen substituents and EDGs have an important impact on the anticancer potential of synthesized derivatives. Halogen atoms at para position on phenyl rings linked to 1,3,4-oxadiazole decreases anticancer potential, and EDGs increases the anticancer potential of designed derivatives [134].
As a result of screening the all-synthesized compounds, derivative (156) was found to be the most potent on A549 cells with an IC 50 value of 2.8 ± 0.02 µM, as a comparison to podophyllotoxin (standard drug), with an IC 50 value of 0.09 ± 0.01. The effect of derivative (156) on different phases of the cell cycle was also determined by annexinv/PI dual staining (economical dye for apoptotic cell investigation) and it as found that Compound (156) induces apoptosis in A549 cells. The molecular modeling studies also revealed that derivative (156) had a significant affinity towards the tubulin receptor, binding with significant physico-chemical properties. Structure-activity relationships also revealed that halogen substituents and EDGs have an important impact on the anticancer potential of synthesized derivatives. Halogen atoms at para position on phenyl rings linked to 1,3,4-oxadiazole decreases anticancer potential, and EDGs increases the anticancer potential of designed derivatives [134].
Pharmaceuticals 2023, 16, x FOR PEER REVIEW Figure 23. The structure-activity relationship of pyrimidine-1,3,4-oxadiazole analogues Further, a mixture of molecular modeling and target-based assay studies p Compound (154) was well accommodated at the active site of the DNA topois complex, and exhibited favorable physiochemical and ADME/T properties in s ilarly, a series of imidazo[1,2-a]pyridine-oxadiazole hybrids (155) was synthe tested for their in vitro anticancer potential against lung cancer cells (A549) an cancer cells (PC-3, DU-145) by Dilep Kumar Sigalapalli and his team.
As a result of screening the all-synthesized compounds, derivative (156) to be the most potent on A549 cells with an IC50 value of 2.8 ± 0.02 μM, as a com podophyllotoxin (standard drug), with an IC50 value of 0.09 ± 0.01. The effect of (156) on different phases of the cell cycle was also determined by annexin-v/PI ing (economical dye for apoptotic cell investigation) and it as found that Comp induces apoptosis in A549 cells. The molecular modeling studies also revealed ative (156) had a significant affinity towards the tubulin receptor, binding with physico-chemical properties. Structure-activity relationships also revealed th substituents and EDGs have an important impact on the anticancer potential sized derivatives. Halogen atoms at para position on phenyl rings linked to 1,3 zole decreases anticancer potential, and EDGs increases the anticancer poten signed derivatives [134].
A new series of 1,2,3-triazole/thioacetamide linked benzimidazole-based 1 azole derivatives (157) has been prepared by Syed Nazreen and colleagues. MDA-MB-231, SKOV3, and A549, were studied for the cytotoxicity of the sy conjugates cytotoxicity via MTTS assay, and EGFR inhibition was investigated Compounds (158) and (159) showed the most promising cell cycle distribution, which provided insight into their intracellular mechanism of action. The A549 cells treated with Compound (157) exhibited a decrease in S phase distribution and an increase in G1 and G2 phases, while Compound (158) exhibited an increase in G1 phase distribution and a decrease in S phase distribution. Compound (158) increased the number of cells in the G1 phase in MDA-MB-231 breast cells, while decreasing the number of cells in the G2 phase. SKOV3 cells showed an increase in S phase cell distribution, but a decrease in G1 phase cell In non-small cell lung cancer, erlotinib impairs the G0/G1 phase; in hepatocellular carcinoma, it impairs the G1/S checkpoint; and in esophageal cancer, it impairs the G1/G0 phase. The S, G1, and G2 phases of the cell cycle are arrested by Compounds (158) and (159) [135].

Patents
The research and development of oxadiazole-based drugs has gained popularity in the last decades due to their properties such as low toxicity, good efficacy and safety, fine biochemical diversity. 1,3,4 oxadiazole and its derivatives have long been known for their abilities to inhibit cancer-causing cells, growth factors, kinases, and a number of other biological enzymes. As shown in Figure 7, 1,3,4 oxadiazole rings have proven their potential in numerous therapeutic drugs. In this way, the 1,3,4 oxadiazole ring played an important role in the development of a variety of anticancer lead structures. As a continuation of the 1,3,4 oxadiazole ring potential, a large number of anticancer patents have been filed/published.

Conclusions
In order to treat cancer more effectively, and with a low-level of toxicity, scientists are developing new anticancer agents. The treatment of drug-resistant cancer is limited due to the lack of anticancer drugs. Therefore, new antitumor drugs are urgently needed. Thus, the 1,3,4-oxadiazole scaffold remains an important target in modern medicinal chemistry for the discovery of new therapeutic leads. Further, 1,3,4-oxadiazole consists of two atoms of nitrogen interconnected with two atoms of carbon that are electron-deficient, and twoelectron pairs with an active oxygen atom attached to them. This review has compiled numerous synthetic chemical schemes over the past decades, and various anticancer activities of 1,3,4-oxadiazole derivatives were described. Aside from the molecular targets and pathways involved in carcinogenesis, the review explored mechanisms of action (MOAs), docking, and patents granted.
The docking of 1,3,4-oxadiazoles as antiproliferative agents is a very significant discovery in medical research. Molecular docking is important for designing specific drug delivery systems. Molecular docking study plays a significant role in the anticancer study of 1,3,4-oxadiazoles derivatives. In this review article, we summarized how molecular docking study helps to provide information in the binding of 1,3,4-oxadiazoles derivatives with different protein sites for their anticancer potentials.
This structure-activity relationship study contributes to the development of new 1,3,4oxadiazole-heterocycle hybrids with low toxicity to healthy cells, due to the presence of more than one pharmacophore in a hybrid. During the last 10 years, hybrids of 1,3,4oxadiazoles and heterocycles have shown huge anticancer potential due to extensive study on their structure modifications. A clear idea of how nontoxic, effective, anticancer drugs can be developed has also been provided through a review of the SAR and mechanisms of action of the hybrids. Therefore, the study of the 1,3,4-oxadiazole scaffold as anticancer agent has been well demonstrated and identify a possible pharmacophore that can be combined with existing compounds to boost anticancer activity. Several researchers have been interested in exploring the many potential applications of oxadiazole derivatives due to their broad pharmacological profiles. Future therapeutic molecules are likely to contain an oxadiazole motif, which helps in the development of novel medicines for the treatment of patients suffering from various tumors. Funding: The APC for this article was supported by the University of Oradea, Romania.

Institutional Review Board Statement:
The study did not require ethical approval because it focused on the review-based study of a particular disorder, there were no risks to participants, and it was absolutely harmless.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data sharing is not applicable to this article. It is the author's opinion that the study has no DOI, since no new data were created or analyzed in this review article study.