Targeting disease with benzoxazoles: a comprehensive review of recent developments

Abstract

Benzoxazole is an attractive scaffold in medicinal chemistry due to its diverse biological applications. From 2016 to 2023, a plethora of benzoxazole derivatives have been synthesized and evaluated for their pharmacological activities but a review on this topic was found lacking. This review thus aims to fill the gap and discusses the pharmacological activities of the synthesized derivatives, emphasizing their interactions with key biological targets implicated in diseases such as cancer, diabetes, pain, inflammation, and cardiovascular disorders. Relevant works were selected mainly from databases such as PubMed and Google Scholar to offer a comprehensive overview of derivatives with promising bioactivities. The limitations pertinent to some derivatives, such as poor in vitro or in vivo activities, were highlighted, while their prospects in drug discovery was discussed.

Introduction

Benzoxazole 1 (Fig. 1) is a heterocyclic aromatic compound whose structure comprises an aryl ring fused to an oxazole moiety. The parent benzoxazole scaffold is planar and is present in a wide range of natural products, pharmaceuticals, and functional materials [1, 2]. Many natural and synthetic benzoxazole-containing compounds exhibit a wide range of biological activities including antimicrobial [3, 4] antitumour [5], antioxidant [6], antiviral [7], antitubercular [8], and anthelmintic [9] properties, among others. The benzoxazole skeleton also forms the active component in many marketed drugs such as in the non-steroidal anti-inflammatory drug, funoxaprofen, the antibiotics calcimycin, the antibacterial boxazomycin B, and the muscle-relaxant chlorzoxazone [10, 11] (Fig. 1). Consequently, benzoxazoles continue to play an essential role in drug development.

Fig. 1

Structure of benzoxazole 1 and representatives of bioactive benzoxazoles

The structural make-up of the benzoxazole scaffold allows efficient interaction with biological targets. The planar benzene ring can form π-π stacking or π-cation interaction with the host molecule, whereas the 1-oxygen and 3-nitrogen of the oxazole moiety are hydrogen bond acceptors, hence, engage in non-covalent interactions. Furthermore, the lipophilic nature of benzoxazoles allows hydrophobic interaction with target proteins [12]. The benzoxazoles are also considered the bioisosteres of nucleic bases, adenine and guanine. This structural similarity enables favorable interaction between benzoxazoles and biopolymers in living systems. It may also explain the broad spectrum of biological activities observed in them [13].

In recent years, drug discovery efforts aimed at identifying novel compounds, including those containing the benzoxazole fragment, have increasingly relied on computational methodologies. Computational methods are more efficient and can significantly reduce the time and cost associated with traditional drug discovery process. In silico approaches such as virtual screening, molecular docking, and molecular dynamics simulation are some commonly applied modern drug discovery methods used in identifying novel drug molecules [14,15,16].

Benzoxazoles hold a prominent position in medicinal chemistry, with extensive research focused on their synthesis and elucidation of their biological activity. Nevertheless, a comprehensive review from 2016 onwards appears to be absent. Many novel benzoxazoles have reached clinical trials within the past decade with significant improvements in their potency, selectivity and/or physiochemical properties [13]. Since there are significant recent developments, this review aims to address this gap by documenting the benzoxazoles synthesized within this timeframe (2016–2023), discussing their pharmacological importance and shedding light on their modes of action. It also touches on the synthetic methods, current limitations of benzoxazoles, and their prospects in drug discovery.

Synthesis of benzoxazoles

Conventionally, benzoxazoles are synthesized through the condensation of 2-aminophenols with carbonyl compounds such as aldehydes [17], ketones [18], carboxylic acids [19], esters [20] and acyl chlorides [21]. Their synthesis has been achieved via different methodologies including (i) solution-phase, (ii) solid-phase, and (iii) green synthesis [22]. Several reaction conditions have also been employed leading to the benzoxazole nucleus. Although some methods/conditions are mild, others may involve the use of hazardous solvents and/or harsh reaction conditions. However, each methodology offers distinct advantages in terms of efficiency, yield, and environmental considerations as comprehensively outlined by Soni et al. [23]. Various synthetic strategies for benzoxazoles are briefly outlined in this section as the focus of this review is on the biological applications of benzoxazoles.

Solution-phase synthesis

Solution-phase synthesis involves conventional approaches, such as the condensation of 2-aminophenols with aldehydes, ketones, and carboxylic acids, among others. Wong and co-workers (Fig. 2) recently prepared some benzoxazoles through the oxidative cyclization of imines generated in situ from the condensation of 2-aminophenols and aldehydes. The reaction which was silver carbonate mediated, afforded the desired products in up to 95% yield even though it took 16 h of reflux to complete [24]. Similarly, Nguyen and co-workers (Fig. 2), reported the condensation reaction of o-nitrophenol and acetophenone in the presence of FeCl₂·4H₂O and sulfur to give benzoxazoles in moderate yield. Although the transformation was achieved at a mild temperature of 80 °C, the reaction time was 16 h [25]. A simple condensation reaction between carboxylic acids and amidino-substituted 2-aminophenols was also reported by Ptiček and co-workers (Fig. 2). Moderate yields of amidino-substituted 2-arylbenzoxazoles were obtained by carrying out the reaction in polyphosphoric acid (PPA) [26].

Fig. 2

Solution-phase synthesis of benzoxazoles

Solid-phase synthesis

Solid-phase synthesis covers the efficient methods leading to benzoxazole derivatives as a result of combinatorial assembly [27]. Radi and co-workers (Fig. 3), reported the synthesis of 2-substituted benzoxazoles using polymer-bound esters as solid-support. The desired benzoxazoles were obtained in a two-step one-pot procedure in good to excellent yields under 30 min of microwave irradiation [28]. Lim and co-workers (Fig. 3), used resin-bound esters to obtain substituted benzoxazoles at elevated temperatures but short reaction time [29]. Chanda and co-workers (Fig. 3), on the other hand used an ionic liquid-immobilized o-phenylenediamine support to synthesize a series of benzimidazole linked benzoxazole compounds under microwave irradiation. Notably, the products were obtained in a six-step procedure with yields of up to 95% [30].

Fig. 3

Solid-phase synthesis of benzoxazoles

Green synthesis

The synthesis of benzoxazoles via environmentally friendly and sustainable approaches is desirable, aiming to overcome the limitations associated with conventional methods, which often involve the use of toxic reagents and harsh reaction conditions. Nguyen and co-workers (Fig. 4), reported the synthesis of benzoxazoles using a magnetic nanomaterial Fe₃O₄-supported Lewis acidic ionic liquid (LAIL@MNP) as the green catalyst. The reaction was conducted under solvent-free conditions to afford the desired products in up to 82% yield [31]. Chang and co-workers (Fig. 4), reported a one-pot approach leading to benzoxazoles by using a molecular sieve as the catalyst. Notably, the reaction excluded the use of transition-metal catalyst, chemical oxidants, or strong acids which are associated with conventional condensation reactions of aminophenols and aldehydes. Interestingly, high yields were obtained by carrying out the reaction in xylene [32]. Recently, Patil and co-workers (Fig. 4), reported a green and mild route for the synthesis of benzoxazole derivatives using Indion 190 resin as the catalyst. Ultrasound irradiation was applied to drive the reaction, giving yields of up to 96% in 20 min [33].

Fig. 4

Green synthesis of benzoxaozles

Biological activities of benzoxazoles

Benzoxazoles as anticancer agents

Benzoxazole is a commonly found scaffold in anticancer compounds due to its ability to target a wide range of metabolic pathways and cellular processes in cancer pathology [34]. Recent studies have shown benzoxazole derivatives to possess potent anticancer activity [35,36,37]. These derivatives elicit their function by targeting various enzymes or proteins such as DNA topoisomerases [38], protein kinases [39], histone deacetylases [40], cyclooxygenases [41], cholinesterases [42], etc. that are involved in the pathway of cancer formation and proliferation. The next sections shall discuss these molecular targets and their relevance to cancer treatment.

DNA topoisomerases

DNA topoisomerases are a group of enzymes which play important roles in DNA topology including DNA relaxation and supercoiling. DNA compaction is such that the entire genome of a cell needs to be squeezed into the nucleus. It is therefore supercoiled with the help of the enzyme topoisomerase in a way that avoids knots and tensions. Type I and II are the two main types of topoisomerases. Topoisomerase-I breaks one strand of the double helix at its end and makes it rotate around the other intact strand while topoisomerase-II breaks and uncoils both strands simultaneously to allow free rotation. These actions are necessary for DNA replication, transcription, recombination and chromatin remodeling. Therefore, targeting and inhibiting DNA topoisomerase is an effective strategy in the treatment of cancer [43, 44].

Karatas et al. [45] in 2021 synthesized some novel antitumour agents targeting human DNA topoisomerase enzymes (hTopo I and hTopo IIα). Among the compounds synthesized, 5-nitro-2-(4- butylphenyl)benzoxazole 2 and 2-(4-butylphenyl)oxazolo[4,5-b]pyridine 3, (Fig. 5), showed the highest inhibitory effect against DNA topoisomerases I and IIα. Although the two compounds were found to be more effective (IC₅₀ = 2 µM) than the standard drug, etoposide (IC₅₀ = 10 µM) in inhibiting topoisomerases, they were not effective when tested on the human epithelial cervix adenocarcinoma (HeLa), human epithelial colorectal adenocarcinoma (WiDR), human lung Carcinoma (A549), and human breast adenocarcinoma (MCF-7). Since the topoisomerase inhibition assay was carried out in a cell-free system, the researchers opined that in order to observe a similar potent effect against the cancer cells, sufficient amount of the compounds must enter the cells and reach the nucleus stably. They suggested therefore that the compounds could not enter the cells at sufficient concentrations hence their poor activity.

Fig. 5

Structures of compounds 2 and 3 and their anticancer activities

In continuation of the work [45], Foto et al. [38] synthesized a series of (5 or 6)-amino-2-(substituted phenyl and benzyl)benzoxazoles in order to study if any changes would occur in cytotoxicity and topoisomerase I and II inhibitory effects. The compounds 4a–g and 5a–e (Fig. 6), were synthesized and tested against the cancer cell lines HeLa, MCF-7, and A549.

Fig. 6

Structures of compounds 3a–g and 4a–g and their anticancer activities

The majority of the compounds were effective on the HeLa cells but showed poor activity in inhibiting the human lung cancer cells (A594) or the human breast cancer cells (MCF-7). The results, summarized in Table 1 shows that the best compounds also have good selectivity against cancerous cells. Surprisingly, it was observed that the compounds showed poor inhibitory activity against topoisomerase I, indicating that the anticancer activity of the compounds stems from targeting other pathways.

Table 1 Cytotoxicity of compounds 4a–g and 5a–e on HeLa and L929 cells

In a similar work, Zilifdar et al. [46] studied the DNA damaging effect as well as the cytotoxic activity of some benzoxazole derivatives and their primary metabolites. Both compounds 6 and 8 are metabolites of benzoxazole 7 (Fig. 7), formed after the hydrolysis of the oxazole ring [47]. Therefore, evaluating their in vitro cytotoxicity will shed light on the in vivo activity of the parent benzoxazole compound 7. Both compounds 6 and 8 seem to preserve the activity demonstrated by compound 7, indicating the high probability of compound 7 being active in vivo.

Fig. 7

Anticancer activities of compounds 6, 7 and 8

Kinases

Kinases catalyze the transfer of phosphate group from adenosine triphosphate (ATP) to substrates such as proteins, lipids, carbohydrates, and nucleic acids [48]. Kinases are involved in a wide range of cellular processes, including cell growth, cell division, apoptosis and signal transduction. They have become important targets for drug development, especially in cancer therapy because aberrant kinase activity is frequently associated with the development and progression of cancer [49]. Examples of well-known kinases include the Epidermal Growth Factor Receptor (EGFR) kinase, Cyclin-Dependent Kinases, Janus kinases, Phosphoinositide 3-Kinase, and Vascular Endothelial Growth Factor Receptor (VEGFR). The majority of anticancer research in recent years are focused on the VEGFR and EGFR receptor kinases. Both VEGFR and EGFR are cell surface receptors which are often dysregulated, mutated or overexpressed in certain types of cancer, such as lung [50, 51] and breast [52, 53] cancers.

The VEGFR-2 plays an important role in tumor angiogenesis and its inhibition has proven an effective strategy in cancer therapy. A series of 2-thioacetamide linked benzoxazole-benzamide conjugates were synthesized recently [54] as potential inhibitors of VEGFR-2. Most of the compounds exhibited sub-micromolar IC₅₀ values against VEGFR-2. Compound 9 (Fig. 8) with IC₅₀ = 0.268 µM, was more potent against VEGFR-2 than the clinically used kinase inhibitor, sorafenib IC₅₀ = 0.352 µM. Interestingly, the compound also displayed excellent cytotoxicity against both colorectal carcinoma (HCT-116) and MCF-7 cancer cell lines. Its interaction with crucial amino acid residues such as Leu840, Lys868, Cys919, Asp1046 and Phe1047 at the active site of VEGFR were important for its observed biological activity (Fig. 9).

Fig. 8

Cytotoxicity of Compound 9 and Sorafenib on VEGFR-2, MCF-7 and HCT-116 cancer cell lines

Fig. 9

Interaction of compound 9 with amino-acids Leu840, Lys868, Cys919, Asp1046 and Phe1047 (adapted from Eissa et. al., 2022) [54]

El-helby et al. [55] synthesized some benzoxazole derivatives as potential VEGFR-2 inhibitors and tested their anticancer activity against hepatocellular carcinoma (HepG2), breast cancer (MCF‐7), and colorectal carcinoma (HCT‐116) cells. Among the synthesized molecules, compound 10 (Fig. 10) was found to be the most potent with IC₅₀ = 4.13 ± 0.2, 6.93 ± 0.3, and 8.67 ± 0.5 µM, against HepG2, HCT‐116, and MCF‐7 cells, respectively. Compound 10 also inhibited VEGFR-2 at a lower IC₅₀ value (0.07 ± 0.01 µM) compared with sorafenib (IC₅₀ = 0.1 ± 0.02 µM). Similarly, El-kady et al. [56] evaluated the cytotoxicity as well as the VEGFR-2 inhibition potential of some novel benzoxazole derivatives. ELISA results revealed compound 11 (Fig. 10) as the best VEGFR-2 inhibitor (VEGFR-2 protein concentration = 586.3 ± 16.1 pg/ml) while compound 12 (Fig. 10), was the most cytotoxic on MCF-7 and HepG2 cancer cells. Compound 13 (Fig. 10) [36], also targeting VEGFR-2 gave IC₅₀ values of 10.50 µM and 15.21 µM against HepG2 and MCF-7, respectively. It also had the most promising VEGFR-2 inhibitory activity (IC₅₀ = 97.38 nM).

Fig. 10

Structures of compound 10–15 and their inhibitory activity against VEGFR and cancer cells

Other researches aimed at modulating the vascular endothelial growth factor (VEGFR) receptor pathway to induce apoptosis include compound 14 [57] and 15 [58] (Fig. 10). Both compounds exerted an exceptional VEGFR-2 inhibitory activity with IC₅₀ values of 0.0554 µM and 97.3 nM, respectively. The structures of benzoxazoles 10–15 are presented in Fig. 6 as well as their anticancer and VEGFR inhibitory activities.

The epidermal growth factor receptor (EGFR, also known as erbB1 or HER1) belongs to the ErbB family of receptor tyrosine kinases responsible for cell growth and proliferation. It is often overexpressed, especially in breast, lung, and colon cancers [59, 60]. Therefore, EGFR inhibitors may serve an effective strategy to treat the mentioned cancers [61]. A series of benzoxazole-appended piperidine derivatives[62], were investigated as potential inhibitors of the aromatase enzyme (ARO) and EGFR. The benzoxazoles 16 and 17 (Fig. 11) were the promising candidates which demonstrated potent EGFR inhibition and apoptosis-promoting properties.

Fig. 11

Inhibitory activities of compound 16–23 on various cancer cells

Philoppes and Lamie [63] also reported a series of benzoxazole and benzothiazole derivatives incorporated with phthalide as potential EGFR inhibitors. The compounds 18a and 18b (Fig. 11), were demonstrated to possess the best cytotoxic activity against HepG2 and MCF-7 cell lines with IC₅₀ = 0.011 and 0.006 μM, respectively. Meanwhile, compounds 19a and 19b (Fig. 11), by Kumar and co-workers, displayed potent cytotoxic activities when screened against breast (MCF-7), lung (A549), and melanoma (A375) cancer cell lines. In addition, their molecular docking results revealed strong binding interaction with EGFR receptor [64]. Compound 20 and 21 (Fig. 11), are anti-breast cancer agents which exert their activities through the inhibition of the EGFR and ARO enzymes respectively [65]; while compound 22 and 23 (Fig. 11), were cytotoxic on MDA-MB-231 and MCF-7 breast cancer cells, respectively [66].

Protein Kinase B also known as Akt is a family of serine/threonine kinases that have emerged as a key regulator in various cellular processes such as differentiation, proliferation, metabolism and apoptosis. Activated protein kinase B has been identified in various forms of cancer [67]. Compound 24 (Fig. 12) synthesized from thymoquinone inhibited the phosphorylation and Insulin-like Growth Factor-1 Receptor (IGF1R β) in HeLa and HepG2 cells with IC₅₀ = 4.13 and 9.36 μM respectively. Another compound, 25 (Fig. 12), exhibited tyrosine kinase inhibitory properties (IC₅₀ = 0.56 ± 0.24 μM), and was found to be potent against various cancer cell lines [68].

Fig. 12

Structure of compound 24 and the inhibitory activity of compound 25 on A594, MCF-7 and MDA MB-231 cell lines

Auroras are serine/threonine kinases that are crucial regulators in the control of the cell cycle and mitosis. Growing research suggests that Aurora kinases are significantly overexpressed in diverse human cancer cell lines. They are also key players in tumourigenesis and hence, are promising targets for cancer therapy [69]. An et al. [70] synthesized a series of benzoxazole derivatives as type II kinase inhibitors and evaluated their inhibitory activities against Aurora Kinases and tumor growth. Compound 26 (Fig. 13) displayed the most potent Aurora B Kinase inhibitory activity and suppressed cell proliferation in breast (MDA-MB-231), Prostate (PC-3), and colon (HCT-15) cancer cells. Furthermore, the molecular docking of compound 26 against Aurora B kinase gave a pose similar to VX-680, the reference compound, which explained the selectivity and potency of compound 26 in inhibiting Aurora B kinase.

Fig. 13

Growth inhibitory activity of compound 26 on MDA-MB-231, PC-3, and HCT-15

A series of benzoxazole multi-kinase inhibitors were synthesized and tested against EGFR, HER2, VEGFR-2, and the CDK2 protein kinase enzymes. Compound 27 (Fig. 14) demonstrated the best inhibitory activity on the enzymes with IC₅₀ values of 0.279, 0.224, 0.565, and 0.886 µM, respectively. Compound 27 also revealed a broad range of antitumour activity pattern against HepG2 (IC₅₀ = 6.83 µM), MCF-7 (IC₅₀ = 3.64 µM), MDA-MB-231 (IC₅₀ = 2.14 µM), and HeLa (IC₅₀ = 5.18 µM). An important advantage of targeting more than one kinase, is an increase in potency, due to the synergistic effect. Moreover, this approach can reduce the possibility of developing drug resistance [71].

Fig. 14

Structure of compound 27

Cholinsterases

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes within the serine hydrolase group and are crucial in the development of Alzheimer’s disease due to their peptidase activity [42]. However, the involvement of AChE has also been observed in non-neural actions such as cell proliferation, differentiation and apoptosis. Abnormal expression of AChE has been reported in different types and stages of cancers, implying the involvement of AChE in the regulation of tumor development. This suggest that it might serve as a potential therapeutic target for cancer therapy [72]. Compound 28 (Fig. 15), synthesized by Skrzypek et al. [42] exhibited both AChE and BChE inhibition properties at low µM levels. Additionally, 28 was evaluated against various cancer cells, revealing substantial potency.

Fig. 15

Inhibitory activity of compound 28 on various cancer cells

Cyclooxygenases

Cyclooxygenases (COXs), also known as prostaglandin–endoperoxide synthase are enzymes that play a crucial role in the body’s inflammatory response and in the synthesis of prostaglandins [73]. COXs enzymes serve as molecular targets for all NSAIDs but have also been considered as targets in cancer treatment [74]. Furthermore, COX-2 selective inhibitors have been proven as anticancer agents [75]. The COXs inhibitors, compound 29a and 29b (Fig. 16), were synthesized recently [41] and tested against various cancer cell lines. Cytotoxicity of the compounds on the cancer cells ranged from 4.3 to 9.2 µm while the COXs enzyme inhibition was between 1.92 µM and 8.21 µM. The cytotoxicity of the compounds against five cancer cells is presented in Table 2.

Fig. 16

Inhibitory activity of compounds 29a, 29b, and 30 on cancer cells and COXs enzyme

Table 2 Cytotoxicity of compounds 29a and 29b on various cancer cells

A series of benzothiazole/pyrazole and benzoxazole/pyrazole hybrid compounds were synthesized [76] with the aim of evaluating their COXs inhibitory potential as well as their anticancer activity. Compound 30 (Fig. 16), was the most potent in inhibiting COX-2 activity and also demonstrated the most significant anti-proliferative effects on A594 lung cancer cells.

Histone deacetylases

Sirtuins (SIRTs), are NAD⁺ dependent class III histone deacetylases that have emerged as potential therapeutic targets due to their roles in diverse physiological and pathological processes [77]. Of the seven mammalian sirtuins identified (SIRT1–7), SIRT1–3 have received the most attention. These three sirtuins are often associated with cancer, as alterations in their functions are frequently observed in cancer cells [78]. A study carried out in 2020 [77] identified compound 31a and 31b (Fig. 17) as small‐molecule SIRT1 modulators. These compounds induced apoptosis in A594 human lung adenocarcinoma with IC₅₀ values of 46.66 ± 11.54 (31a) and 55.00 ± 5.00 µM (31b). In order to elucidate their mechanism of action, the SIRT1 activity of the compounds were evaluated on C6 rat glioma cells. Compound 31a decreased SIRT1 levels in a dose‐dependent manner whereas compound 31b increased the SIRT1 levels. This suggested that both compounds induced apoptosis following different mechanisms (inhibition or activation of SIRT1).

Fig. 17

Compound 31a and 31b

Beyond the targets discussed so far, benzoxazoles have also demonstrated therapeutic potential through their modulation of other molecular targets implicated in cancer pathogenesis. Noteworthy among these biotargets include Poly (ADP-ribose) polymerase [79], Mitogen-Activated Protein Kinase Kinase (MEK) [80], and Cytochrome P450 enzyme [35]. The structures of the most active benzoxazoles corresponding to these targets mentioned is provided in Fig. 18, along with their activities against relevant cancer cells.

Fig. 18

Cytotoxicity of compounds 32–34 on various cancer cells

Benzoxazoles as anti-microbial agents

The increasing global public health problem of infectious diseases, which encompass a wide range of bacterial and fungal infections, have been worsened by the growing threat of antimicrobial drug resistance [81]. A decline in the development of new antimicrobials bearing a novel scaffold compounded the complexity of this growing health threat [82]. Thus, drugs capable of overcoming such resistance are still actively sought.

Benzoxazoles are believed to exhibit enhanced interaction with biological targets due to their structural similarity with adenine and guanine bases. In studies conducted by Oksuzoglu and co-workers [83], it was demonstrated that benzoxazoles effectively inhibit DNA topoisomerases, and this inhibition is closely associated with their antibacterial activity. In addition, the presence of DNA gyrase in all bacteria and its absence in higher eukaryotes makes it an effective target in antibacterial studies [3]. Several research has been conducted on the potential of benzoxazoles as antimicrobial agents. This section discusses the relevant findings, particularly of benzoxazoles targeting certain microbes including bacteria, fungi and mycobacteria.

Antibacterial benzoxazoles

Mishra et al. [84] synthesized compound 35 (Fig. 19), an azo-linked benzoxazole and subsequently evaluated its antibacterial activity against Staphylococcus aureus and Escherichia Coli strains. Unfortunately, the compound displayed poor activity against both strains when compared to the standard ciprofloxacin. Compound 36 (Fig. 19), screened against both gram positive and gram negative bacterial strains showed MIC values in the micromolar range [85]. Meanwhile, the Ag(I) complex–compound 37 (Fig. 19), was particularly outstanding in inhibiting Pseudomonas aeruginosa with an MIC value of 0.7 µM [86].

Fig. 19

Activities of compounds 35–41 against various bacterial strains

Kincses et al. [87] synthesized some benzoxazole-metal complexes with the aim to target multi-drug bacteria. Compound 38 and its metal complex 39 (Fig. 19), were found to be synergistic with ciprofloxacin against methicillin-resistant S. aureus (MRSA). On their own, the compounds exhibited strong inhibition against S. aureus and MRSA. 38 and 39 each recorded an MIC of 12.5 µM against S. aureus while against MRSA was 3.125 µM for each of the compounds, respectively.

Compound 40 (Fig. 19), is a representative molecule among the many benzoxazole scaffolds synthesized by Gorla et al., [88] against Francisella tularensis—the causative agent of tularemia. Compound 40 was bactericidal against all tested strains including Francisella tularensis (F.tularensis), Mycobacterium tuberculosis (M.tuberculosis) and Bacillus anthracis (B.anthracis) with IC₅₀ values < 10 µM. The antimycobacterial activity of 40 was linked to the inhibition of IMP dehydrogenase (IMPDH).

The utility of benzoxazoles also extends to agents against phytopathogenic bacteria. The bis-benzoxazole compound 41 (Fig. 19) protected Nicotiana benthamiana against wildfire disease caused by Pseudomonas syringae pv. Tabaci. This was confirmed by a reduction in diameter of bacterial lesions by about 52% in plants pre-treated with 50 μg/ml of compound 41 [89]. In 2023, Zou and his colleagues provided a summary of various agricultural biological activities demonstrated by benzoxazoles, including their herbicidal, insecticidal and antiviral properties [90].

Antifungal benzoxazoles

Fungal infections remain a serious medical problem especially in immune-impaired patients. This coupled with the resistance of fungi to available drugs (e.g., C. albicans resistance to azoles) necessitates the search for new and effective compounds [91]. Cavalho et al. [92] synthesized and evaluated the biological activity for a benzoxazole type derivatives of eugenol as antifungal agents. The compounds, 42a–d (Fig. 20), displayed moderate activity in inhibiting five species of candida spp. (Table 3).

Fig. 20

Structure of Compound 42a-d

Table 3 Inhibitory activity of compounds 42a-d against various fungal strains

Similar to phytopathogenic bacteria, disease in plants caused by phytopathogenic fungi leads to huge agricultural losses and poses a threat to global food security. A series of benzoxazole derivatives compound 43a–d (Fig. 21), were among the compounds screened against eight phytopathogenic fungi species (Table 4), including Fusarium oxysporum f. sp. niveum (F.n.), Fusarium. graminearum (F.g.), Fusarium solani (F.s.), Alternaria solani (A.s.), Colletotrichum gloeosporioides (C.g.), Valsa mali, Magnaporthe oryzae (M.o.), and Botrytis cinereal (B.c.). The compounds were found to be more active against two fungi, F. solani (F.s.) and B. cinereal (B.c.) than to other species [93]. Compound 43a displayed the best activity against B. cinerea with IC₅₀ of 19.92 µg/mL, while 43c was the most potent inhibitor of F. Solani (IC₅₀ = 4.34 µg/mL).

Fig. 21

Structure of compounds 43a–d

Table 4 Inhibitory activity of compounds 43a–d against various fungal strains

In an effort to discover new antifungal agents, Fan et al. [94] synthesized a series of 2-amino benzoxazole derivatives. Compounds 44a–d (Fig. 22) displayed the most potent antifungal activity, with EC₅₀ values ranging from 1.48 to 16.6 µg/mL against eight phytopathogenic fungal strains [Fusarium sulphureum (F.s.), Thanatephorus cucumeris (T.c.), Fusarium oxysporum (F.o.), Fusarium graminearum (F.g.), Botrytis cinerea (B.c.), Valsa mali (V.m.), Sclerotiua sclerotiorum (S.s.) and Alternaria solani (A.s.)] (Table 5). The compounds also displayed good preventive effect against Botrytis cinerea in vivo at the concentration of 100 µg/mL.

Fig. 22

Structure of compounds 44a-d

Table 5 Inhibitory activity of compounds 44a–d against various fungal strains

Inspired by the prevalence of pyridine, amide and benzoxazole in agrochemicals, Wang et al. synthesized some benzoxazoles bearing a pyridine ring and an amide moiety. The biological activity of the compounds was evaluated against eight phytopathogenic fungi including Fusarium solani, Colletotrichum gloeosporioides, Mycosphaerella melonis, Alternaria brassicae, Pyricularia grisea, Curvularia lunata, Alternaria solani, and Fusarium graminearum. The compounds mostly exhibited moderate antifungal activity. Compound 45a–c (Fig. 23), showed good antifungal activity with 45a and 45b providing over 50% inhibition rate against five fungi species. 45c especially inhibited Mycosphaerella melonis at rates of up to 76.4% [95].

Fig. 23

Structure of compounds 45a–c

Multifunctional antimicrobial benzoxazoles

The broad spectrum of biological activities exhibited by benzoxazoles means that in most cases, they interact with multiple molecular targets involved in disease pathway. In fact, most antibiotics with an established history of clinical success rarely target only one specific molecule [96]. This section discusses antimicrobial benzoxazoles effective against more than one microbe.

Meryem Erol et al. [97] synthesized four 2,5-substituted benzoxazole derivatives (Compound 46a–d (Fig. 24)) and evaluated their antimicrobial activity against a variety of bacterial and fungal strains. Compound 46a against Staphylococcus aureus isolate, and 46a and 44b against Escherichia coli isolate showed more potent antimicrobial activity with MIC values of 16 μg/mL respectively, than some of the reference drugs. The compounds were however less effective on the fungal strains compared to the reference drugs (Table 6). In another related work, Erol reported compound 47 (Fig. 25), as having the maximum activity (MIC = 8 μg/mL) against S. aureus, E. faecalis and their drug-resistant isolates. 47 was also reported to have substantial effect against C. albicans but was still less effective than amphotericin B, the reference drug [98]. However, compound 48 (Fig. 25), synthesized by the same group recorded the closest activity to fluconazole against Candida albicans with MIC = 16 μg/mL [99]. The remaining compounds in the series showed rather weak activity against the gram positive and negative bacterial strains. Compound 49 (Fig. 25) against Candida albicans was also 16 μg/ml but showed weak activity against the bacterial strains tested [100].

Fig. 24

Structure of compound 46a–d

Table 6 Antimicrobial activity of compounds 46a–d on bacterial and fungal strains compared to reference drugs

Fig. 25

Antimicrobial activity of compounds 47–49

The benzoxazoles 50a–c (Fig. 26), recorded excellent antimicrobial properties when they were assessed against a panel of four bacterial and two fungal strains (Table 7). Compound 50b displayed the strongest antimicrobial activity, followed by 50a and then 50c [101]. Rodrigues et al. [102] screened the bioactivity of ten benzoxazoles synthesized from methoxy cyano acrylate. Among these, compound 51a and 51b (Fig. 27) demonstrated the best antimicrobial activity against fungi, gram positive, and gram negative bacteria (Table 8). 51b showed good inhibitory capacity at 25 μg/mL against all the organisms except S. typhi, while 51a was effective against E. coli, S. aureus, and P. syringae.

Fig. 26

Structure of compound 50a–c

Table 7 Antimicrobial activity of compounds 50a–c on bacterial and fungal strains

Fig. 27

Structures of compounds 51a and 51b

Table 8 Antimicrobial activity of compound 51a and 51b on bacterial and fungal strains screened at 25 μg/mL

In an effort to explore the synergistic effect of combining different pharmacophores, Jasiewicz and co-workers synthesized a series of indoles bearing imidazole, benzothiazole-2-thione, or benzoxazole-2- thione moiety at the C3 position. The compounds were then tested for their antibacterial and fungicidal activities. The benzoxazole derivative (compound 52, Fig. 28), was less effective against the bacterial and fungal strains compared to the other compounds bearing the imidazole or thiazole moiety, adjudged from the microbial zone of inhibition assay. However, its fungicidal activity against P. placenta at a concentration of 0.01% was quite promising (64%) [103]. Compound 53 (Fig. 28), exhibited a strong gram positive and gram negative bacterial effect but was rather weak on C. albicans [104]. The antifungal activity of compound 54 (Fig. 28), was also weak, this was in addition to its poor activity against various bacterial strains [105]. Compound 55 (Fig. 28), showed moderate activity against both the gram negative and gram positive organisms, but was also rather weak against C. albicans [106].

Fig. 28

Antimicrobial activity of compounds 52–55

Padmini et al. worked on the synthesis of some benzoxazole derivatives and subsequently evaluated their antimicrobial and cytotoxic activities. Compounds 56 and 57a–e (Fig. 29) emerged as potent antimicrobial agents. They showed a remarkable zone of bacterial growth inhibition comparable to the standard tetracycline. The antifungal properties were also similar to that of fluconazole [107]. The bioactivity of the compounds against some selected bacterial and fungal strains is given in Table 9. The benzoxazole derivative (compound 58, Fig. 30), exhibits a broad antibacterial activity with MIC values in the range of 64–256 μg/ml [108]. It was effective against P. aeruginosa which is known to be resistant to ampicillin. Furthermore, it displayed a much better activity against E. coli isolate and P. aeruginosa isolate in comparison with gentamycin (Table 10).

Fig. 29

Structure of compound 56 and 57a–e

Table 9 Antimicrobial activity of compound 56 and 57a–e against bacterial and fungal strains

Fig. 30

Structure of compound 58

Table 10 Antimicrobial activity of compound 58 on bacterial and fungal strains

Compounds 59a–c (Fig. 31) demonstrated potent antibacterial, antifungal and antitubercular properties when they were screened against some microbial strains. Notably, the compounds showed the comparable activity with standard drug sulfamethoxazole, against M. tuberculosis and drug-resistant M.tuberculosis with MIC value of 8 μg/mL (Table 11) [109]. Meanwhile, in another study, compound 60a (Fig. 32), was found to be the most potent against A. niger (MIC = 2.40 nM), while 60b (Fig. 32) recorded the best activity against C.albicans (MIC = 0.34 nM). The two compounds also exhibited excellent antibacterial properties ranging from 2.40–4.80 nM [11]. Lastly, the antifungal, as well as the cytotoxic properties of some 2-mercaptobenzoxazole derivatives were also examined by Staniszewska et al. [110]. The cell growth inhibition of the compounds revealed compound 61a as the most active against C.glabrata, while 61b, was the most active against C.albicans (Fig. 32). The compounds did not totally inhibit the growth of the pathogens up till 16 μg/mL

Fig. 31

Structure of compounds 59a–c

Table 11 Antimicrobial activity of compound 59a–c against bacterial and fungal strains

Fig. 32

Antimicrobial activity of compound 60a–b and 61a–b

Benzoxazoles as antiviral agents

In recent years, the surge in global outbreaks of viral diseases have become a challenging phenomenon. Viruses are obligate intracellular pathogens consisting of a DNA or RNA genome enclosed within a proteinaceous capsid [111] and are able to infect all types of organisms. Prior research have highlighted benzoxazoles as having antiviral potential [7, 112, 113]. However, in the years spanning 2016–2023, literature search revealed that only very few papers have focused on the use of benzoxazoles as therapeutic agents against viral infections, mostly utilizing computational approaches in rationalizing activities.

Cheerala et al. reported the combination of two scaffolds, benzoxazole and thiazolidinone (B-T hybrids) as inhibitors of SARS-CoV-2. Molecular docking was performed on the hybrid compounds to determine their interaction with 3CLp (the 3-chymotrypsin-like protease) and PLp (the papain-like protease) of SARS-CoV-2. Compounds 62a and 62b (Fig. 33), interacted best with the catalytic dyad residue of 3CLp (Cys145 and His41) through hydrogen bonds. The compounds were appropriately positioned within the binding pocket of 3CLp, similar to Tipranavir as shown in Fig. 34. In the case of PLp, several types of interactions were observed, including hydrogen bonding, π–π stacking, π–cation stacking, and hydrophobic interactions in its active site. Docking result revealed that compounds 63a and 63b (Fig. 33), interacted and formed hydrogen bonds with the residue His272 of the catalytic triad, similar to Tipranavir. Molecular dynamics simulations showed that compound 62b exhibited the best binding free energy (ΔG = −6.83 kcal mol⁻¹) with 3CLp while 63a exhibited the best ΔG (−7.76 kcal mol⁻¹) with PLp. These results indicated that the compounds could serve as potential antiviral agents against SARS-CoV-2 [114].

Fig. 33

Structure of compounds 62a–b, 63a–b

Fig. 34

The molecular interaction of B–T hybrids within the active site of the 3CLp (A) Surface binding view of Tipranavir and compounds 62a and 62b on 3CLp (salmon-Tipranavir, orange-62a, blue-62b). The molecular interactions of (B) Tipranavir, (C) 62a, and (D) 62b are shown in 3D representations. The yellow dotted are the hydrogen bonding interactions of the ligands with active site residues. (Adapted from Cheerala et al. 2021) [114]

The molecular docking of 2-(p-chloro-benzyl)-5-[3-(4-ethly-1-piperazynl)propionamido]-benzoxazole, 64 (Fig. 35), with COVID-19 main protease (M-pro) was performed by using the optimized geometry and the experimentally determined three-dimensional structure of the main protease. The main protease plays an important role in mediating viral replication and transcription and could serve as a potential target for the inhibition of COVID-19 replication. The binding energy of compound 64 in the active site of the main protease was determined to be −6.1 kcal/mol. As there was strong affinity between compound 64 and the protease, compound 64 was hypothesized to exhibit antiviral activity [115].

Fig. 35

Structure of compound 64

Benzoxazoles as antiprotozoal agents

Protozoan diseases are widespread in tropical countries and have an important impact on public health [116]. Among these diseases is malaria which is caused by plasmodium species, leishmaniasis, attributed to leishmania species and human African trypanosomiasis, caused by various species of Trypanosoma [117]. There has always been a constant need to discover newer and more effective drugs especially due to the drug resistance posed by various pathogens. Interestingly, compounds containing the benzoxazole scaffold have been reported as active agents against protozoan diseases, and may serve to contribute to the on-going efforts to finding effective treatment.

Abdelgewad et al. [118] synthesized a series of benzoxazole derivatives and evaluated their potential as antimalarial, antileishmanial, antitrypanosomal, and antimicrobial agents. Three compounds, 65a–c (Fig. 36), showed promising antimicrobial and antiprotozoal activities. The 2-chloroacetamide 65b, exhibited substantial antimicrobial activity, better than 65a or 65c. However, all the three compounds displayed antiprotozoal activity. 65b exhibited the most prominent activity against both resistant (W2) and chloroquine-sensitive (D6) strains of plasmodium falciparum with IC₅₀ values of 5.1 and 2.2 μM, respectively. Compound 65c showed moderate antileishmanial activity against both L. donovani amastigotes and promastigotes along with potent antitrypanosomal activity. A summary of activities against the microbial strains is provided in Table 12.

Fig. 36

Structures of compound 65a–c

Table 12 Antiprotozoal activity of compounds 65a–c

Folquitto et al. [119] evaluated the in vitro antileishmanial activity of ten benzoxazole compounds against the amastigote and promastigote form of Leishmania amazonensis. Compounds 66, (Fig. 37) exhibited satisfactory activities and low cytotoxicity against both forms of L. amazonensis. Selectivity (5.22) towards the promastigote form was 9-fold greater compared with pentamidine (0.58). Similarly, Escudero-Martínez et al. [120], described the bioactivity of several series of semisynthetic podophyllotoxin derivatives on Leishmania infantum, the etiological agent responsible for visceral leishmaniasis. Compound 67 (Fig. 37) was the most potent against L. infantum amastigote (EC₅₀ = 4.5 ± 0.1 µM). The EC₅₀ value against the promastigote form was 19.4 ± 2.1 µM. The cytotoxicity of 67 was also assayed on murine splenocytes which results revealed high selectivity (22.2) against amastigote-infected splenocytes.

Fig. 37

Antiprotozoal activity of compounds 66 – 70

Abdeen et al. [121] functionalized the benzoxazole derivative 68 (Fig. 37), obtained from the PubChem database thereby creating various analogs with variable sulfonamide groups. The parent compound 68 and the analogs were screened as potential inhibitors of HSP60/10 chaperonin system of Trypanosoma brucei, the causative agent of African sleeping sickness. The E,coli HSP60/10 also known as GroEL/Es were used to study the inhibition potential of the synthesized compounds. Thus, two biochemical assays based on the inhibition of GroEL/Es were employed. Compound 69 and 70 (Fig. 37) were the most potent against the GroEL/ES-dMDH refolding and ATPase activity with IC₅₀ values more potent than that of the parent compound.

Benzoxazoles as anthelmintic agents

Helminthiasis is a significant public health problem which leads to a high morbidity and mortality rate especially in the developing world [122]. In addition, infection by helminths to livestock accounts for huge economic losses particularly in areas where overgrazing is practiced. This is further exacerbated by the growing helminthic resistance to existing drugs, lack of vaccines and difficulty in treatment [9]. The N-methylbenzo[d]oxazol-2-amine 71, (Fig. 38), was synthesized recently through the methylation of benzoxazole-2-thiol followed by a nucleophilic addition–elimination reaction with methylamine. Compound 71 was further evaluated for its anthelmintic properties and was found to be very active against Caenorhabditis elegans (C.elegans), Trichinella spiralis (T.Spiralis), and Gnathostoma spinigerum (G.spinigerum). Results also indicated that 71 was comparatively potent as albendazole, an orally administered anthelmintic drug [123].

Fig. 38

Anthelmintic activity of 71 against C.elegans, T.spiralis and G.spinigerum

Benzoxazoles as antidiabetic agents

Type 2 diabetes mellitus is the most common form of the disease accounting for around 90% of all cases [124]. Several molecular targets including Dipeptidylpepdidase 4, Alpha-glucosidase, Sodium glucose transporter, Peroxisome proliferator-activated receptor-gamma, etc., have been explored as strategies in treating the disease[125]. However, despite the successes recorded, treatment of diabetes remains a challenge.

Elevated levels of α-glucosidase and α-amylase in the body have been linked to diabetes [126]. Inhibitors of α-glucosidase and α-amylase thus represent a strategy for treating the disease. Rodrigues et al. obtained the compounds 72a and 72b (Fig. 39) as the most potent inhibitors of the enzymes. Although the compounds exhibited poor α-glucosidase inhibitory activities at lower concentrations, they were highly effective at 1000 μg/ml. At this concentration, the α-glucosidase inhibitory activity for 72a was about 85% while 72b was over 70%. The compounds also recorded remarkable results for α-amylase inhibition, above 90% for 72a and close to 90% for 72b at 100 μg/ml [127]. Khan et al. also demonstrated that compound 73 (Fig. 39) exhibits strong α-amylase, α-glucosidase inhibitory activity with IC₅₀ values at 1.10 ± 0.20 μM, 1.20 ± 0.30 μM, respectively. A greater potency than the standard acarbose (11.12 ± 0.15 μM and 11.29 ± 0.07 μM respectively) against both enzymes [128]. Similarly, the α-amyloglucosidase inhibitory activity of Compound 74 (Fig. 39) was found to be the most potent (IC₅₀ = 0.24 ± 0.01 μM) among the series of benzoxazolyl linked benzylidene based rhodamine compounds synthesized by Singh and co-workers [129].

Fig. 39

Structure of compounds 72–76

Elevated levels of trimethylamine N-oxide produced through the conversion of choline to trimethylamine by gut microbiota have been linked to various types of diseases such as type 2 diabetes, fatty liver disease and cardiovascular diseases. Inhibition of choline TMA-lyase (CutC), the enzyme responsible for the conversion, serves as a strategy for managing diabetes. In vitro CutC inhibitory assay revealed compound 75 (Fig. 39) as a CutC inhibitor with an IC₅₀ value of 2.4 ± 0.3 μM [130].

Abeed et al. [131], tested some benzoxazole compounds on hyperglycemic rats to assess their anti-diabetic effects. The hyperglycemia was related with an increase in the serum cholesterol and triglyceride (TG) levels. The administration of gliclazide (Glic) restored the normal cholesterol and TG levels (Fig. 33). Similarly, normal serum cholesterol and TG levels were observed in groups treated with 76a and 76b (structure Fig. 39, and activity Fig. 40).

Fig. 40

Bar chart showing the serum cholesterol and triglyceride level in hyperglycemic rats treated with Glic, compound 76a and 76b Normal: rats treated with the vehicle; STZ: rats treated with streptozotocin; STZ-Glic: rats treated with streptozotocin and Glic; STZ-T(x): rats treated with STZ and the tested compound of the corresponding number (x). a: significantly different from normal group at p < 0.05; b: significantly different from STZ group at p < 0.05

Benzoxazoles as anti-inflammatory agents

Inflammation is a protective mechanism by the body’s immune system in response to invading pathogens, foreign bodies and/or injury. However, prolonged inflammatory response often leads to organ pathology and the amplification of certain diseases such as arthritis, neurodegenerative diseases, cancer, asthma, allergy, diabetes etc. [132]. Today, the treatment of inflammation is predominantly centered on interrupting the synthesis or action of mediators that drive the host’s response to injury. Nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and biologic therapies like tumor necrosis factor α (TNFα) inhibitors, were developed based on this approach [133].

Macrophages are a critical component of the inflammatory response, and can be activated by lipopolysaccharide (LPS) to produce various inflammatory cytokines and mediators, including nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and inducible nitric oxide synthase (iNOS) which intensify the immune response. The inactivation of LPS-induced microphages is therefore a strategy to stop inflammation. In a study by Zhao et al. they synthesized the benzoxazole compound 77 (Fig. 41) and evaluated its anti-inflammatory properties. In a dose dependent manner, 77 could inhibit LPS-stimulated nitric oxide (NO), interleukin (IL)-6, (TNF)-α, and 3-nitrotyrosine (3-NT) production. The suppression of these cytokines was achieved through the phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) (Ser9) which then reduced GSK-3β activation in LPS-induced RAW264.7 macrophages [134].

Fig. 41

Structure of compounds 77–79

The inhibition of p38α MAP kinase blocks the production of COX-2 thereby preventing tissue destruction and inflammation [135]. A series of benzoxazole and benzothiazole derivatives were evaluated for their anti-inflammatory and p38α MAP inhibitory activity [136]. The benzothiazole compound 78 (Fig. 41) showed the highest potency (IC₅₀ = 0.031 ± 0.14 µM) against p38α MAP kinase. Isosteric replacement of the ring sulfur in the benzothiazole with oxygen gave 79 (Fig. 41), which had a slightly reduced potency (IC₅₀ = 0.038 ± 0.12 µM). However, both compounds were more active than the standard SB203580 (IC₅₀ = 0.043 ± 0.14 µM).

As previously mentioned [134], nitric oxide is a critical factor in the onset of inflammation. The in vitro evaluation of the inhibitory effects of benzoxazoles 80 and 81 (Fig. 42), on nitric oxide (NO) in LPS-induced RAW 264.7 cells showed promising results. The LPS-induced cells showed significant NO reduction when treated with both compounds in a concentration-dependent manner[137].

Fig. 42

Structure of compounds 80–89

Most NSAIDs elicit their anti-inflammatory activity through the nonselective inhibition of COX enzyme [138]. Compounds 82 and 83 (Fig. 42), were predicted to have strong binding affinity to the COX-2 enzyme when assessed with computational modeling. When tested in vitro, both compounds exhibited superior proteinase inhibition as well as membrane stabilizing inhibitory activities when compared to the standard drugs aceclofenac and etodolac. [139]. Another compound 84 (Fig. 42), also demonstrated a strong COX inhibitory activity with IC₅₀ values of 0.04 μM and 1.02 μM against COX-2 and COX-1 respectively [140]. Moreover, 84 also displayed anti-inflammatory activity (84.09% inhibition at 20 mg/kg b.wt.) from the rat paw edema assay.

The TNFα, interleukin 1β (IL1β), and interleukin 6 (IL6) are cytokines produced by microphages and are associated with inflammation. Ayaz et al. synthesized a series of bis-benzoxazole compounds and tested their potential towards reducing these pro-inflammatory cytokines in LPS-stimulated macrophages. Compound 85 (Fig. 42), was the most potent as it caused a dramatic decrease in TNFα and IL1β production whereas the production of IL-6 was completely knocked down even at low concentrations (Fig. 43) [141]. Similarly, some benzoxazoles targeting IL-6 were synthesized, out of which compound 86 and 87 (Fig. 42), demonstrated the highest activity [142]. At a concentration of 20 μg/ml, both 86 and 87 effectively suppressed IL-6 with an inhibitions rate of 97.5% and 93.1%, respectively. Moreover, both 86 and 87 exhibited remarkable IC₅₀ values of 18.9 and 5.8 μM, respectively. In comparison, madindolin A, a known selective inhibitor of IL-6, was 20.5 μM.

Fig. 43

Bar Chart showing the anti-inflammatory activity of compound 85 against necrosis factor α (TNFα), interleukin 1β (IL1β), and interleukin 6 (IL6). IL6 is completely knocked down in C; Control + : 1 μg/ml of LPS and DMSO; Group A, B, C: Effect of 50 μg/cm³ of compound 85 on cytokines present in RAW 264.7 cells

Interferon-γ (IFN-γ), is a cytokine produced by T cells and plays an important role in the pathogenesis of inflammatory diseases [143]. Recently, two novel benzoxazoles, compound 88 and 89, (Fig. 42) were synthesized and examined for their cytokine inhibitory activity. It was observed that both compounds could suppress the production of IFN-γ in CD4 + T cells in a dose-dependent manner (Fig. 44).

Fig. 44

Bar chart showing the suppression of interferon-γ by compound 88 and 89

Benzoxazoles against neurodegenerative diseases

In an increasingly aged society, the prevalence of neurodegenerative disorders like Alzheimer’s disease (AD) and Parkinson’s disease (PD) can affect as much as 1% of the population aged 60 and over [144]. While Alzheimer’s and Parkinson’s disease exhibit distinct pathological characteristics, several similarities between both diseases such as gene mutation, α-synuclein and tau protein aggregation and neuroinflammation are common [145]. AD is especially characterized by plaques of β-amyloid (Aβ), intracellular neurofibrillary tangles of hyperphosphorylated tau protein and loss of cholinergic neurons in the basal forebrain [146]. Neurodegeneration is partly caused by inadequate cholinergic transmission; thus, many treatment regimens are based on increasing the levels of acetylcholine through the inhibition of the acetylcholinesterase enzyme [147].

Anwar et al. recently investigated some novel benzoxazole-oxadiazole hybrid derivatives as potential cholinesterase inhibitors. Compound 90a-c (Fig. 45) exhibited moderate AChE and BChE inhibitory activities. Compound 90b, bearing di-Cl substitution at the 3,4-position of both aryl rings was the most potent inhibitor of targeted AChE and BChE enzymes. Against AChE, 90b established significant interaction with the enzyme’s active site including residues HIS-A-305, LEU-A-162, LEU-A-162, and HIS A-101, while against BChE, key interactions were observed with residues such as ARG-A-552, PHE-A-476, ASP-A-568, and ASP-A-568 (Fig. 46). The electron withdrawing di-Cl groups facilitated the interaction with the enzyme’s active site by removing electron density from the phenyl rings [148].

Fig. 45

Structure of compounds 90–92 and their bioactivities

Fig. 46

Protein–ligand interaction of compound 90b against (A) acetylcholinesterase (pdb: 1ACL), and (B) butyrylcholinesterase (pdb:1P0P) enzymes (adapted from Anwar et al.2023) [148]

Another strategy for treating AD and PD involves targeting and inhibiting the A_2A Adenosine receptor [149]. The A_2A receptor is a subtype of the four adenosine receptors (A1, A2B and A3) which is coupled to the G-protein. It has been established that A_2AR antagonists not only enhance spatial memory but also reduce β-amyloid accumulation, Tau hyperphosphorylation, and neurotoxicity [150]. Duroux et al. identified the compound 91 (Fig. 45), as an effective A_2A antagonist with IC₅₀ values in the nanomolar range. 91 also showed low cytotoxicity and high solubility [151]. The same group had previously synthesized some 2-arylbenzoxazoles containing an amino functional group as A_2A antagonists with the aim to increase compound solubility. Compound 92 (Fig. 45) was the most active in the series with K_i value of 1 μM. Furthermore, it had an excellent aqueous solubility (184 μM) without being cytotoxic at 100 μM. Both compound 91 and 92 are potent antagonists of the A_2A enzyme, display good ADME properties, and therefore, can be relevant starting points for further hit-to-lead optimization [151].

Benzoxazoles against cardiovascular diseases

Cardiovascular diseases (CVDs) are the major cause of morbidity and mortality in the developed countries. Moreover, as the population ages, the number of patients with diseases like hypertension, coronary heart disease and heart failure are on the rise [152]. The identified benzoxazoles for the treatment of CVDs in the timeframe under this review included those targeting the renin angiotensin system, and agents against hyperlipidemia.

Wu et al. synthesized a series of angiotensin II receptor 1 antagonists and tested their antihypertensive characteristics. The benzoxazoles 93 and 94 (Fig. 47) displayed nanomolar affinity to AT₁ receptor and could decrease blood pressure efficiently in hypertensive rats. In their study, the antihypertensive activity of 93 and 94 was greater than that of losartan [153].

Fig. 47

Structure of compounds 93 – 96 and their bioactivities

Hyperlipidemia is a leading risk factor of cardiovascular diseases and is characterized by the abnormal levels of one or more plasma lipids, such as triglycerides (TG), total cholesterol (TC), cholesterol esters, phospholipids, and plasma lipoproteins [154]. Zaib et al. evaluated the antihyperlipidemic activity of some imine benzoxazole derivatives as well as their binding affinities to relevant targets involved in lipid regulation. Compound 95 and 96 (Fig. 47) were predicted to have strong binding affinities to HMGCR, APOB and VCAM-1. (E-values = −7.4, −9.0, −7.9 and −7.5, −9.3, −8.3 kcal/mol respectively). In vitro, the compounds also significantly reduced the serum levels of TG, TC and other biomarkers that lead to the progression of cardiovascular diseases [155].

Benzoxazoles as analgesic agents

Pain is an unpleasant and emotional experience that is linked with actual or potential tissue damage or expressed in the context of such harm [156]. Current methods to ameliorate pain include the administration of NSAIDs (as pain is often associated with inflammation), selective suppressors of the cyclooxygenase-2 enzyme (COX-2), corticosteroids, and immune-suppressor candidates [157]. Non-steroidal anti-inflammatory drugs (NSAIDs) are still the most widely used therapeutic agents for the treatment of pain. However, they exhibit unfavorable gastrointestinal side effects [158], and as such, newer drugs are being explored.

The COX and LOX enzymes are key regulators of pain and inflammatory response and their inhibition has been recognized to be a beneficial treatment for inflammatory conditions [159]. Such a dual-type inhibitor was reported by Khadri., et al. in 2022. The compound 97 (Fig. 48), emerged as the best COX inhibitor with IC₅₀ values of 876 μM and 39.43 μM for COX-1 and COX-2 enzymes, respectively. Moreover, it also exhibited the highest selectivity index value at 21.92. The in vivo analgesic activity in mice revealed 97 as the best analgesic agent; offering protection of up to 86.5% as against 32.0% and 85.1% offered by celecoxib and indomethacin respectively. The 5-LOX inhibition by 97 was also comparable to those of the standard drugs [10].

Fig. 48

Structure of compounds 97 and 98

The benzoxazole 98 (Fig. 48) targets the H₃ receptor, which is a subtype of the histamine receptor that offers a wide range of neurological modulations [160, 161]. 98 was synthesized for the treatment of neuropathic pain for which it demonstrated excellent affinity for H₃R (Ki = 19.7 nM) and negligible effects on human ether-a-go-go-related gene and other off-target receptors. The compound also dose-dependently reversed formalin-evoked pain (Phase I, ED₅₀ = 6.0 mg/kg; Phase II, ED₅₀ = 7.8 mg/kg) and CCI-induced neuropathic pain (chronic constriction injury, ED₅₀ = 15.6 mg/kg). 98 thus, serves as a promising candidate for the treatment of neuropathic pain.

Conclusion and future perspective

A total of ten distinct pharmacological activities demonstrated by the benzoxazoles synthesized between 2016 and 2023 have been documented. In numerous instances, these compounds exhibited effects equal to or even more potent activities than standard drugs. This indicates the suitability of the benzoxazole scaffold for the drug discovery and development process in a myriad of diseases.

Within the context of the approximately 100 different compounds examined in this review, effort was made to highlight the interaction of the compounds with various molecular targets. Most of the compounds were designed by taking into consideration their ability to interact favorably with biological targets. Steps such as incorporating alkyl groups at certain positions, the addition of heteroatoms, or the inclusion of halides, were observed in many cases. These are common practices in rational drug design aimed at improving lipophilicity, solubility, binding affinity, and the overall pharmacological profile of drug molecules. Nonetheless, in most cases reported, only the in vitro assays were performed with only few instances reporting in vivo evaluations. Further modifications and evaluations of these compounds are required to make them applicable for future clinical studies. Assessing both in vivo efficacy and pharmacokinetic profiles is crucial for expediting the advancement of promising molecules to the lead candidate stage. It is imperative that future research incorporate these elements to ensure that medicinal chemists benefit from a more refined selection of potential drug candidates.

These limitations notwithstanding, the information presented in this review is aimed at providing an insight concerning target-oriented drug discovery efforts related to benzoxazoles. By documenting the recent progress, advancements could be made in the development of new benzoxazole-based therapeutics. Moreover, considering the volume of research being carried out, it is anticipated that benzoxazole-based drug discovery will continue to experience growth and success.