Review Article

Benzopyran-Core as an Antimycobacterial Agent

Sandile B Simelane¹*, Paseka T Moshapo² and Raban W Masuka³

¹Department of Chemistry, Faculty of Science and Engineering, University of Eswatini, Eswatini

²Department of Chemical Sciences, University of Johannesburg, South Africa

³University of Zimbabwe, School of Pharmacy, Mt Pleasant Drive, Zimbabwe

Submission: October 18, 2020;Published: December 01, 2020

*Corresponding author: Sandile B Simelane, Department of Chemistry, Faculty of Science and Engineering, University of Swaziland, Private Bag 4, Kwaluseni, Swaziland

How to cite this article: Sandile B S, Paseka T M, Raban W M. Benzopyran-Core as an Antimycobacterial Agent. Organic & Medicinal Chem IJ. 2020; 10(2): 555784. DOI: 10.19080/OMCIJ.2020.10.555784.

Abstract

Tuberculosis (TB) is one of the world’s most deadly infectious diseases, causing 1.2 million deaths in 2018. TB is the leading cause of death from a single infectious agent, ahead of HIV/AIDS. The African continent bears the highest global TB/HIV burden and over 50% of TB cases in sub-Saharan Africa are co-infected with HIV. With an estimated 1.7 billion people (23% of the world’s population) with latent TB infection, there is an urgent need to develop drugs that will eradicate or control the disease. Moreover, the emergence of multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) have accelerated the need for new antitubercular agents with novel biological targets and different mechanism of action. Among the wide spectra of heterocyclic compounds, benzopyran derivatives have displayed diverse biological applications. Found in many natural products, this scaffold together with its synthetic analogs has intrigued medicinal chemists to explore its applicability as anti-TB drugs. To further intensify research in this area, there is need to gather the latest information on benzopyrans as antimycobacterial agents. This review presents an overview of recent developments (2000 -2018) in anti-TB applications of benzopyrans, both the synthetic analogs and isolated natural products. The objective of this review is to focus on active benzopyran analogs and structure activity relationship (SAR) analysis. We envisage that this review will be helpful in rational design of potent, less toxic benzopyran-based anti-TB drug candidates.

Keywords:Mycobacterium tuberculosis; Anti-TB agents; Medicinal chemistry; Benzopyran

Abbreviations: TB: Tuberculosis; HIV/AIDS: Human immunodeficiency virus infection/acquired immune deficiency syndrome; MDR-TB: multi-drug resistant tuberculosis; XDR-TB: extensively drug resistant tuberculosis; SAR: structure activity relationship; Mtb: Mycobacterium tuberculosis; INH: isoniazid; RIF: rifampicin; ETB: ethambutol; PZA: pyrazinamide; SADC: Southern African Development Community; CYP1B1: Cytochrome P450 Family 1 Subfamily B Member 1; MIC: minimum inhibitory concentration; FtsZ: filament temperature-sensitive mutant Z; MABA: micro-plate alamar blue assay; SI: selectivity index; sm-DNA: salmon milt-Deoxyribonucleic acid; A-T: adeninethiamide; MmpL3: Mycobacterial membrane protein Large; BCG: Bacillus Calmette-Guérin; MRA: microdilution resazurin assay; L-J: Lowenstein-Jensen; DprE1: decaprenylphosphoryl-β-D-ribose 2′-epimerase; TAACF: tuberculosis antimicrobial acquisition and coordinating facility; ATP: Adenosine triphosphate; HadAB: β-hydroxyacyl-ACP dehydratase

Introduction

Tuberculosis (TB) is a chronic and potentially fatal disease caused by Mycobacterium tuberculosis (Mtb) complex. The Mtb complex is made up of bacilli from M. tuberculosis, M. bovis, M. mungi, M. caprae, M. canettii, M. africanum, M. microti and M. pinnipedii [1-3]. Known as “white plague”, Mtb was first isolated and identified by Robert Koch in 1882 [4]. In 2017, TB caused an estimated 1.3 million deaths among HIV-negative people and there were an additional 300 000 deaths from TB among HIV-positive people [5]. One-third of the world’s population is affected with Mtb and an estimated 10 million people developed TB in 2017, with 488 000 of them affected by multi drug-resistant TB (MDR-TB). Multi drug-resistant TB is defined as tuberculosis whose bacteria are resistant to isoniazid (INH) and rifampicin (RIF). If the bacterium is resistant to isoniazid and rifampicin as well as any of the floroquinolones, it is called extensively drug resistant TB (XDR-TB) [6].

The standard first line therapy for the treatment of active TB is a cocktail of rifampicin (RIF), isoniazid (INH), ethambutol (ETB) and pyrazinamide (PZA). After two months the number of drugs is reduced to rifampicin and isoniazid for four months, (2HRZE/4HR) [7]. Treatment of MDR-TB is longer and requires more toxic drugs [8]. MDR-TB is treated by a combination of eight to ten drugs with therapies lasting up to 18-24 months. Extensively drug resistant TB (XDR-TB) is virtually untreatable [9]. Tuberculosis is a transmissible infection, and Mtb is mainly transmitted by airborne aerosols through coughing, sneezing, speaking or any other respiratory fluids in the air from people with active pulmonary TB [10]. Mtb typically affects the lungs, but it can also affect other parts of the body and it is known as extrapulmonary tuberculosis. Common extrapulmonary sites of infection include lymph nodes, pleura and osteoarticular areas [11]. Extrapulmonary TB is common in people with a weakened immune system like those with HIV co-infections [12]. In some cases, the infection causes no symptoms, and it is called latent TB.

The World Health Organization [13] once estimated that 1 billion new cases of Mtb infections will be reported in the next two decades. They also reported that in 2017, 87% of new TB cases occurred in the 30 high TB burden countries [5]. Amongst these 30 countries, 16 (53%) are African countries and 9 of those are in the Southern African Development Community (SADC) [14]. These statistics should stimulate medicinal chemists and pharmacologists to invent new drug candidates with novel mechanism of action to combat or control the spread of Mtb. Some of the review articles found in literature in this research area range from target identification studies, distinct class of molecules as well as clinical status of different types of anti-TB compounds, some of which are heterocyclic compounds [15-19].

Heterocycle-based organic derivatives offer potential for the design of new drug-like molecules. These molecules have had a profound impact on both chemical biology and drug discovery [20]. Among these heterocycles, one that has received considerable attention from medicinal chemists is the benzopyran structural motif [21]. Benzopyrans are polycyclic molecules in which a benzene ring and pyran ring are fused together with various levels of saturation [22]. The name benzopyran is widely used to refer to polycycles fused with a pyran ring (chromenes), but also applies to heterocycles bearing a dihydropyran (chromans) and some of the benzopyran skeletons are shown in Figure 1.

Nicolaou et al. [23] labelled the benzopyran scaffold as a privileged structure because it is found in a range of biologically active compounds, both synthetic and naturally occurring. The benzopyran structural motif plays an important role in numerous pharmaceutical molecules with a wide range of biological properties, which include anti-cancer [24], anti-inflammatory [25], antibacterial [26], anti-arthritis [27], anti-viral [28], anti-Alzheimer’s [29], anti-skin disease activities [30] and the treatment of type 2 diabetes [31]. In addition some benzopyran derivatives have demonstrated potential as alanine racemase inhibitors [32], cytochrome P450 1B1 (CYP1B1) inhibitors [33], monocarboxylate transporter 1 inhibitors [34], P-glycoprotein inhibitors [35] and α-glucosaminidase inhibitors [36]. Some benzopyran derived commercial drugs include warfarin (for treatment of retinal occlusion, pulmonary embolism, and cerebral embolism), flavoxate (for symptomatic relief of dysuria), dronabinol (for stimulating appetite), nebivolol (for treatment of essential hypertension) and vitamin E which is an antioxidant with demonstrated immune enhancing effects (Figure 2).

Xiu et al. [21] recently reviewed the therapeutic applications of benzopyrans. However, there is no review article that has detailed the significance of this scaffold as anti-TB agents. Benzopyrans with a carbonyl or phenyl group at position 2 are coumarins and flavonoids, respectively. Coumarins and flavonoids have been reviewed as antimycobacterial agents [37,38], hence are not part of this review. In the present article we focus on published reports (from 2000-2018) of benzopyran derivatives as potential anti-TB agents.

Synthetic anti-TB benzopyran derivatives

Raju et al. [39] synthesized a series of 4H-chromen-1,2,3,4- tetrahydropyrimidine-5-carboxylate derivatives and screened for their in vitro antimycobacterial activity against Mtb H37Rv, using the agar dilution method [40]. The results showed that six compounds 1(a-d), 2 and 3 exhibit excellent activity with minimum inhibitory concentration (MIC) values between 3.4- 7.4μM and were more potent than standard drugs ethambutol (MIC=7.6μM) and ciprofloxacin (MIC=9.5μM) (Figure 3). The electron withdrawing trifluoromethyl group at the 6th position of the 1,2,3,4-tetrahydropyrimidine-5-carboxylates enhances antimycobacterial activity better than methyl and phenyl substituents. Nitro substitution on the phenyl ring also play an important role in enhancing the activity compared to halides. Also, the furan ring fused to the phenyl moiety of the chromone 2 and 3 maintained potency of the 4H-chromen-1,2,3,4- tetrahydropyrimidine-5-carboxylate derivatives.

Amino alcohol fused spirochromone conjugates were prepared and evaluated for their potential anti-TB application against Mtb H37Rv. Compound (4a) was found to be the most active compound with MIC=3.13μg/mL (Figure 4). The structure activity relationship of the amino alcohol fused spirochromone conjugates revealed that compounds possessing cycloalkyl and more specifically cyclopentyl group at the 2nd position of the chromanone ring favor better activity than a piperidinyl moiety. Furthermore, the isopropyl group on the aromatic ring of the amino alcohol as well as halide substitution enhances activity [41]. Dongamanti et al. [42] reported synthesis of chemically modified bis-spirochromanones starting from 2-hydroxyacetophenone and 1,4-dioxaspiro [4.5] decan-8-one using the Kabbe condensation method [43].

The bis-spirochromanones were evaluated for their potential in vitro antimycobacterial activity against H37Rv strain using the agar dilution method to determine minimum inhibitory concentration (MIC) values. Compounds 5 and 6 with MIC values of 5.30 and 6.40μM respectively, were found to be more potent than the first line antitubercular drug ethambutol (MIC=7.64 μM) (Figure 4). Furthermore, the bis-spirochromanones e.g. compound 5 were found to be more potent than mono-spirochromanones and compound 6 with three chroman-4-one pharmacophores was amongst the most potent bulky molecules. This was further supported by docking studies into the filament temperaturesensitive mutant Z (FtsZ) domain of Mtb H37Rv. Compounds with a higher surface area in the active site also exhibited higher docking scores. For compound 6, the docking score and surface area were -6.52Kcal/mol and 408 Å2 respectively.

Singh et al. [44] described the synthesis of 3-furanochromones and assessed their antitubercular activity against the H37Rv strain of Mtb. The MIC values of the compounds were determined using the micro-plate alamar blue assay (MABA) [45]. The compounds 7(a-d) showed moderate antitubercular activity with MIC values ranging between 10 and 12 μg/mL (Figure 5). Das et al. [46] reported the synthesis of 3-benzylidine- 4-chromanones and evaluated them for growth inhibition of Mtb H37Rv in BACTEC 12B medium and the growth inhibition was determined by the broth dilution assay [47]. Compound (8a) with a meta-bromo substituent on the benzylidine moiety showed highest inhibition (99%) at a concentration of 10μg/mL (Figure 5). The IC50 and IC90 values of compounds with greater than 90% inhibition were generated using the microplate alamar blue assay. All these compounds 8(a-c) showed IC50 and IC90 values less than 1μg/mL with a selectivity index (SI) greater than 20. Meta-substituted 3-benzylidine-4-chromanone derivatives were more potent than their ortho- and para-substituted counterparts. Furthermore, the 3-benzylidine-4-chromanones were more active than 2-benzylidine cyclohexanones, confirming the importance of the oxygen for activity.

Feng et al. [48] synthesized a series of chemically modified 4-chromanones and evaluated them against Mtb H37Rv. The 2-propyl-4-chromanol 9 showed better activity in the entire series with an MIC value of 12.5μg/mL (Figure 5). Increasing the aliphatic chain led to loss of activity and oxidation of the hydroxyl group at the 4th position was deleterious for activity. 2,10-dihydro- 4aH-chromeno [3,2-c] pyridin-3-yl analogs were synthesized and evaluated for their activity against Mtb H37Rv and multi-drug resistant M. tuberculosis (MDR-TB) [49]. Three compounds 10 (ac) inhibited Mtb with MIC values of less than 1μM and compound 10a (MIC=0.22μM) was more potent than isoniazid (MIC=0.36μM) (Figure 6). All the 2,10-dihydro-4aH-chromeno[3,2-c]pyridin- 3-yl analogs inhibited the MDR-TB clinical isolate with MIC values ranging from 0.07 to 7.09μM and were found to be more active than standard drugs isoniazid (MIC=45.57μM), ofloxacin (MIC=34.39μM) and ethambutol (MIC=122.36μM).

In this series, antimycobacterial activity was enhanced by the introduction of an electron donating methyl group on the pyridyl moiety, whereas introduction of weakly deactivating and electron withdrawing dibromo groups decreased the activity. Replacement of phenyl ring with naphthyl moiety enhanced the activity 4–8 times, and further introduction of a methyl group on the naphthyl moiety enhanced the activity 2-fold. Replacement of phenyl ring with phenoxymethyl group was detrimental to activity. Compound 10a was found to be non-toxic up to 62.5μg/mL with selectivity index (IC50/MIC) of more than 629 for Mtb and 1977 for MDRTB. Subsequently, compound 10a was tested for in vivo efficacy against Mtb at a dose of 25mg/kg in CD-1 mice. The compound decreased bacterial load in lung and spleen tissues with 1.11 and 2.94-log10 protections respectively but was found to be less active than isoniazid at the same dose level.

Nalla et al. [50] described the synthesis of chromone embedded [1,2,3]-triazole derivatives and screened for their in vitro antitubercular activity against Mtb H37Rv using the MABA method. Compound 11 was the most potent compound in vitro with an MIC value of 1.56μg/mL, 4.8 times more active than the standard drug ethambutol (MIC, 7.64 μg/mL) (Figure 6). Modification on the triazole core by changing the substituents from aromatic to aliphatic groups (cyclic or acyclic) enhances the activity against Mtb. Furthermore, substitution on the phenyl ring with alkyl groups such as 4-methyl, 4-ethyl, 4-propyl and 4-pentyl do not favor better activity, except for 4-t-butyl group. The same group also performed molecular docking studies which identified enoyl acyl carrier protein reductase as the potential target. This enzyme plays a role in mycolic acid biosynthesis. Khan et al. [51] demonstrated the synthesis of (2-Amino-3-cyano-4H-chromen-4- yl)-phosphonates and screened for their antitubercular activity against Mtb H37Rv using disc diffusion susceptibility method [52].

Compound 12a exhibited strong antitubercular activity with an MIC value of 10μg/mL, equaling that of standard antitubercular drug isoniazid (Figure 7). It is evident from the SAR that electron withdrawing groups on the phenyl ring; particularly, the nitro group enhances antitubercular activity of the chromenylphosphonates, while electron donating groups result in loss of potency. Molecular docking calculations predicted strong affinity of 12a towards salmon milt (sm-DNA) with a binding affinity (ΔG) -7.4Kcal/ mol [51]. The compound exhibited selective affinity towards adeninethiamine (A-T) base pairs. Non-covalent interactions, H-bonding and van der Waals forces were predicted as the driving forces of interaction.

Laxmi et al. [53] reported the synthesis of 6-methyl-4,8- dioxo-4,8-dihydropyrano[3,2-g] chromenes and evaluated their antitubercular properties against Mtb H37RV in BACTEC 12B medium. Analog 13 was identified as the most potent inhibitor against Mtb, with an MIC value (IC90) of 5.90μg/mL in the MABA assay. Its antitubercular activity was superior to that of the antitubercular drug, pyrazinamide (PZA; IC90>20 μg/mL) (Figure 7). However, the compound was toxic to Vero cell lines with selectivity index (SI) of 2.41. Interestingly, the inactivity of the structurally related analogs indicates that the presence of the C8- methyl group in 13 may be critical for antitubercular activity. Thus, compound 13 could be a lead for subsequent optimization in the search for novel antitubercular agents. Tantry et al. [54] identified a novel class of spirochromenes from a whole cell-based screen of spiropiperidine series comprising of three closely related classes of compounds namely, spiroindenes, spiroindolones and spirochromenes to identify leads against Mtb.

This was followed by re-synthesis of the active compounds and profiling for antitubercular activity. Among these compounds spirochromene 14 was potent with an MIC value of 0.8μM (Figure 7). Since spiroindenes were the largest subseries with good potency spread widely in the spiropiperidine series, it was further investigated ahead of the spirochromenes. However, whole genome sequencing efforts of the spiroindene resistant mutants resulted in the identification of I292L mutation in Mycobacterial membrane protein Large (MmpL3) required for the assembly of mycolic acid into the cell wall core of Mtb.

Khan et al. [55] reported a facile multicomponent one pot synthesis of spiro [chromene-2, 2-pyrollidin]-4’-one derivatives. The antitubercular activity of synthesized compounds was investigated against Mtb (MTCC, 300) using the agar dilution method. The electron withdrawing groups in the phenyl ring, particularly the nitro group reinforced the antitubercular activity of the chromeno derivatives. The higher activity could be due to the nitro group contributing hydrogen bonding atoms resulting in favorable substrate-inhibitor interactions. The hydroxyl groups could also be improving the potency in a similar fashion. Among the nitro derivatives 15 (a-c) the para-nitro substituted derivatives are more active compared to ortho substituted ones. Enzyme docking studies of 15 also indicated a high affinity for the enoyl acyl carrier protein reductase. Warekar et al. [56] illustrated the synthesis of 1,2-bis(4-nitrophenyl)-1H-benzo[f]chromen-3-amine derivatives and were evaluated for their in vitro antitubercular activity against Mtb H37RV strain.

Compound 16 was the most potent with MIC=1.6μg/mL, better than standard drugs pyrazinamide and streptomycin (Figure 8). Angelova et al. [57] reported the synthesis of 2H-chromene derivatives with various substituted hydrazide-hydrazone pharmacophore attached to the 3rd position of the chromene ring. The synthesized compounds were evaluated in vitro against Mtb H37Rv, using the Canetti method [58]. Compound 17 was amongst the most potent compounds in the series with an MIC value of 0.17μM, better than isoniazid (MIC=1.45μM). Furthermore, the potent compounds were investigated for cytotoxicity against human embryonal kidney cell line HEK-293T and compound 17 displayed the second-best selectivity index of 448.

Tripathi et al. [59] reported synthesis of a series of hybrid molecules bearing a benzopyran skeleton and evaluated them against both avirulent (H37Ra) and virulent (H37Rv) strains of Mtb using the agar microdilution method. Most of the compounds were active against the virulent strain, Mtb H37Rv with MIC values ranging from 1.56-12.5μg/mL, while the same compounds were inactive against the avirulent strain, indicating the selectivity of these compounds towards the virulent strain. From the 4-(1-(2,2-dimethylchroman-6-yl)-3-phenylprop-2-ynyl) cyclic amines series, compounds 18 (a-c) had the most improved antitubercular activity with MIC values of 1.56, 3.12 and 6.12μg/ mL respectively (Figure 8). From the SAR, the morpholine moiety was bad for activity compared to piperidine. This enhanced activity with the piperidine could be due to increased lipophilicity since it is devoid of the polar oxygen. Also, the aromatic ring resulted in loss of activity compared to aliphatic chains.

Prado et al. [60] reported the synthesis of benzofuro[3,2-f] [1] benzopyrans and screened them for antimycobacterial activity against various strains of Mtb. Both 3,3-dimethyl-3H benzofuro[3,2-f][1]benzopyran and 1,2-dihydro-3,3-dimethyl-3H benzofuro[3,2-f][1]benzopyran displayed significant activities when tested against Mtb H37Rv with MIC99 values of 5 μg/mL. Both compounds 19 and its reduced analog 20 were found to be more potent than isoniazid (INH) when tested for inhibitory activity on M. Smegmatis mc2 155 strain. In addition, similar MIC99 values were obtained for compounds 19 and 20 on other mycobacterial strains such as Mtb H37Ra as well as BCG (Bacillus Calmette- Guérin) (Figure 9).

Functionalization with the aim of improving solubility in biocompatible solvents, such as dihydroxylation of the pyranodibenzofuran ring and subsequent conversion into esters resulted in complete loss of activity. The same research group carried out structure activity relationship studies on compounds 19 and 20 in a bid to improve the physicochemical properties of the scaffold [61]. The compounds were screened for their antimycobacterial activity on the fast-growing saprophyte M. smegmatis mc2 155 and on the virulent strain Mtb H37Rv, using the microdilution resazurin assay (MRA) [62]. However, their efforts were rather unsuccessful as the linear tetracyclic analogs were less active and cytotoxic compared to their isomers 19 and 20. Only compound 21 among the tetracyclic analogs showed moderate activity (MIC99=35μg/mL) (Figure 9). The same research team further expanded their SAR studies towards the synthesis of furo[3,2-f] chromenes derivatives.

The synthesized compounds were also screened for antimycobacterial activity on the same strains following the MRA method. From the screened furo[3,2-f] chromenes derivatives, two compounds (22 (a-b)) showed improved activity from 19 and 20 (Figure 9). From the SAR, replacement of the pyridyl ring with a phenyl or methyl group resulted in loss of activity. The introduction of aliphatic chains on C3 of the furan ring led to disappointing antitubercular activity [63]. Termentzi et al. [64] further reported the synthesis of 8-, 9-, 10-, and 11-halo, hydroxy, and methoxy substituted derivatives of the antimycobacterial 3,3-dimethyl- 3H-benzofuro[3,2-f][1]benzopyran (19). The antimycobacterial activities of the substituted benzofurobenzopyrans were screened on Mycobacterium bovis BCG and Mtb H37Rv using the MRA method.

All the compounds substituted on the phenyl ring were more potent than the reference compound 19 against Mtb H37RV strain, indicating that substitution on the benzofurobenzopyran core with hydroxy, methoxy and halogen groups enhanced the antimycobacterial activity. The good activity of hydroxylated compounds 23 (a-b), was unfortunately, accompanied by strong in vitro cytotoxicity against vero cells (Figure 9). The order of activity with this substituted compound is Hydroxy > Methoxy > Halides. These biological results suggest that the substitution on the phenyl ring by an electron-withdrawing group leads to less active compounds. On the contrary, substitution with electron donating hydroxy or methoxy groups significantly enhances the antimycobacterial activity. Enzyme inhibition studies of these compounds suggest a mode of action that involves the inhibition of mycobacterial cell wall lipid biosynthesis.

Ganihigama et al. [65] reported the synthesis of densely functionalized chromenes with various substitutions such as dimedone, indole and benzotriazole. The chromenes were screened for antimycobacterial activity against the non-virulent H37Ra strain of Mtb following the MABA method. Compound 24 was the most active chromene with an MIC value of 25.0μg/ mL (Figure 10). Substituting the naphthalene ring for a phenyl resulted in loss of activity. Furthermore, substitution on the phenyl ring with hydroxyl or methoxy groups resulted in 3-fold loss in activity. Kamdar et al. [66] reported the design, synthesis and antitubercular activity of chromeno[2,3-d] pyrimidine-2-thiones. The compounds were evaluated for their in vitro antitubercular activity against Mtb H37Rv. Quinoline substituted compound 25 had the highest activity, exhibiting inhibition at 62.5μg/mL which was comparable to that of standard drug rifampicin (40μg/mL). Replacement of the quinolone moiety with a naphthalene resulted in loss of activity. Also, phenyl and phenol replacements did not improve the antitubercular activity. The same research group also described the synthesis of 4H-chromeno[2,3-d] pyrimidines and evaluated them for their in vitro antitubercular activity against Mtb H37Rv. From the biological data, pyrimidin-4-one 26 showed the best activity (MIC=62.5μg/mL) against mycobacteria (Figure 10) [67].

Haveliwa et al. [68] further reported the synthesis of chromone fused cytosine analogs derivatives and were investigated against Mtb H37Rv using the Lowenstein-Jensen (L-J) method. Compound 27 was the most effective against Mtb H37Rv strain with 99% inhibition at 40μg/mL concentration. The introduction of electron withdrawing groups such as the 7-chloro substitution enhanced activity while electron donation groups were deleterious for activity. Haveliwa et al. [69] synthesized a series of chromone-fused thiopyrimidines and investigated their antitubercular activity against Mtb H37Rv at a concentration of 62.5μg/mL. Compound 28 with the dibromo group on the phenyl ring of the chromone depicted the highest inhibition (Figure 11). Similarly, electron withdrawing halogen substituents improved the inhibitory activity compared to methyl substituents. The same research team further reported the synthesis of functionalized H- [1] benzopyrano [2,3- b] pyridine derivatives by the Friedländer reaction of 2-amino-4- oxo-4H-chromene-3-carbonitriles with ethyl cyanoacetate. The compounds were investigated for their antitubercular activity against Mtb H37Rv at a concentration of 62.5μg/mL, using the L-J method. In this experiment, compound 29 was the most effective against Mtb H37Rv strain with 99% inhibition [70].

Balasubramania et al. [71] reported the synthesis of triazolechromene hybrids and screened them for their in vitro activity against Mtb H37Rv following the MABA method. Naphthoquinonetriazole- chromene hybrid 30 with IC90=9.86μM was more potent than standard anti-TB drugs cycloserine and pyrimethamine (Figure 12). The results demonstrate that triazole-chromene hybrids with a fluoro substituted aniline are more active than the other compounds and 6-chloro substitution on the chromene ring improves activity while 6-bromo substitution was unfavorable for antitubercular activity. Khare et al. [72] synthesized 1,2,3-triazolechromene conjugates and evaluated them for their in vitro antitubercular activity against Mtb H37Rv strain. Compounds 31 (a-b) were the most effective in terms of inhibiting mycobacterial growth with MIC=12.5μg/mL.

The antitubercular activity depends upon substituents present on phenyl ring, as well as the position of the substituents on the phenyl ring. Electron withdrawing substituents such as R=4-Cl and R=NO2 demonstrated improved antitubercular activity, compared to electron donating groups such as R=4-OMe and R=4-Me. Furthermore, the position of the substituent was found to be important for activity as observed with the nitro substituents, favoring the para position ahead of the ortho and meta substitutions. Docking studies showed significant binding affinity in the active site of Mycobacterium tuberculosis DprE1 enzyme. Triazole-fused spirochromenes were synthesized and evaluated against Mtb H37Rv strain following the MABA assay [73].

Compounds 32 (a-c) possess higher inhibitory activity than standard drug ethambutol (7.64μM) (Figure 12). No evident SAR trend could be deduced from the different substituents in the phenyl ring. However, substituting the phenyl ring for a benzyl moiety by the triazole side of the prototype led to 8 to 15-fold loss in activity. Muthukhrishnan et al. [74] synthesized a series of 1,2,3-triazole fused spirochromone conjugates and screened them for their in vitro antimycobacterial activity against M. tuberculosis H37Rv. The compounds displayed significant in vitro activity against Mtb, with compound 33 the most potent (MIC=0.78μg/ mL) and found to be better than first line antimycobacterial drug ethambutol (1.56μg/mL). The SAR reveals that compounds with a cyclohexyl group at the 2nd position of the chromone ring show better activity than piperidinyl moiety. Furthermore, aromatic substitution at the 4th position of the triazole was favorable than alkyl substitution. Moreover, alkyl substituents on the aromatic ring improve activity.

Alvey et al. [75] reported the synthesis of furo[3,2-f] chromanes and investigated their effect on the growth of Mycobacterium bovis BCG as well as on the virulent strain Mtb H37Rv, using the MRA method. Among the synthesized furo[3,2-f] chromanes, chromane 34, was the most potent with MIC95=0.6μg/mL (Figure 12). However, many analogs which inhibited the growth of M. tuberculosis in the 0.6-5μg/mL range (including chromane 34) turned out to be cytotoxic on vero cells at the same concentration range. Li et al. [76] described the synthesis of 2-azacyclo-5- trifluoromethyl-8-nitrobenzopyran-4-one derivatives (35-37) and assessed their minimum inhibitory concentration (MIC) to Mtb H37Rv strain using the MABA method. The compounds exhibited good antitubercular activity, which is comparable with standard drugs, but were inferior to their benzothiopyran-4-one counterparts (Figure 13). However, both the benzothiopyranones and benzopyranones together with benzoxazinones were designed through scaffold morphing from benzothiazinones to target DprE1.

Chatterjee et al. [77] reported the synthesis of substituted thiophene heterocycles and investigated them as potential drugs for treatment of drug resistant and persistent tuberculosis. These compounds were evaluated against Mtb H37Rv strain following the MABA method. Compounds 38-40 showed great potency with MIC values less than 3.0μg/mL (Figure 13). Interestingly, these compounds were not cytotoxic with selectivity index greater than 50 when evaluated on Vero cells. From the SAR, reduction of the pyran ring resulted in partial loss of activity. Whole genome sequencing of the genomic DNA of the resistant mutants revealed that they all have a single point mutation resulting in the amino acid replacement Tyr321Cys in MSMEG 6382 and Tyr314Cys in rv3790. rv3790, encodes DprEl, a component of the essential decaprenylphosphoryl-B-D-ribofuranose 2’-epimerase (DprEl / DprE2) required for cell wall arabinan biosynthesis.

Ananthan et al. [78] reported anti-TB activities of a library of compounds obtained from the tuberculosis antimicrobial acquisition and coordinating facility (TAACF). Amongst the active compounds, identified via whole cell high throughput screening, was a series of multi-substituted benzopyran-4-ones. In total, 68 compounds were screened against Mtb H37Rv strain and 51% of these showed above 90% bacterial inhibition in the primary single dose assay. Compounds 41 (a-e) displayed potent antitubercular activity with MICs ranging from 0.43-1.8μg/mL (Figure 14). In general, compounds with substituents on C3 and C7 were the most potent and any substituent could be tolerated. Aromatic substituents directly attached to C3 through carbon were more favourable than those attached through oxygen.

A variety of esters composed of methyl, ethyl, cyclohexyl, and benzyl groups were found to be good for activity. Change in potency was not observed when C2 was substituted with a methyl group. Also, replacing the hydrogen on C8 with a methyl did not improve the potency of the compounds. Swaroop et al. [79] reported the synthesis of diaportheone B, one of the two benzopyranones isolated from endophytic fungus Diaporthe sp. P133 [80]. Several close analogs of diaportheone B were synthesized and their anti- TB potential was determined against Mtb H37Rv using the MABA method. Diaportheone B (42), its enantiomer (43) and the racemic mixture showed very similar activity (10-12.5μg/mL). With an MIC value of 6.25 μg/mL, compound 44 is the only analog that exhibit superior activity compared to the natural product, diaportheone B (Figure 14).

Benzopyran-based natural products as anti-TB agents More than half of the current drugs in use comprise of naturally occurring compounds and their derivatives. Natural products have also inspired scientists to synthesize about half of the synthetically produced medicinal compounds. Statistically, 90000 known naturally occurring compounds contribute about 40% of bioactive new chemical entities [81]. Even though plant species have continually demonstrated that they are a rich source of novel bioactive compounds, only a few species have been extensively investigated for their medicinal properties [82]. Hence, the search for bioactive metabolites has been rejuvenated in recent years. Benzopyrans and their benzo-fused derivatives are widely distributed in nature and are isolated from various medicinal plants [83]. Their occurrence has been partly attributed to numerous prenylation and cyclization reactions in many polyketide biosynthesis pathways [23]. The 2,2-dimethylbenzopyran system is popular in many naturally occurring benzopyrans and exhibits a variety of biological properties [84]. In this section, we discuss natural product extracts bearing the benzopyran scaffold as anti- TB agents.

Gupta et al. [85] observed the antimycobacterial activity of Helichrysum melanacme root ethanolic extract to be 500μg/mL against Mtb H37Ra and H37Rv strains through BACTEC assay. Bioactivity guided phytochemical analysis of the roots resulted in the identification of glabridin (45) as the active constituent against Mtb. The antitubercular activity of glabridin was found to be MIC=29.16μg/mL against both strains of Mtb. Kuete et al. [86] isolated 5 polyphenols from twigs of Dorstenia barteri and screened them for antimycobacterial activity against M. Smegmatis and Mtb H37Rv strains. Among the polyphenols, 4-hydroxylonchocarpin (46) exhibited pronounced antimycobacterial activity (MIC=9.76μg/ mL) against both mycobacterial strains.

Lall et al. [87] isolated 3 compounds from Helichrysum melanacme and investigated their antitubercular potential against the drug-sensitive strain of M. tuberculosis H37Rv. Chalcone (47) was the most active compound with an MIC value of 50μg/mL. Chiang et al. [88] isolated 19 compounds from the whole plant of Fatoua pilosa. The antimycobacterial activity of 13 compounds were tested in vitro against Mtb H37Rv using the Middlebrook-7H10 agar method to determine the MIC values [89]. Benzopyran (48) exhibited the strongest antimycobacterial activity (MIC=30μg/ mL) against Mtb H37Rv in vitro. Wu et al. [90] isolated pisonin B (49) and a plethora of other compounds from the methanol extract of the combined stem and root of Pisonia aculeate shrub. The in vitro antitubercular activity of each compound was evaluated using the Mtb strain H37Rv. Pisonin B was among the most active isolates with an MIC value of 25μg/mL.

Tuntiwachwuttikul et al. [91] isolated four new chromones, perforamone A, B, C, and D together with six known compounds, peucenin-7-methyl ether, O-methylalloptaeroxylin, perforatic acid, eugenin, saikochromone A and greveichromenol, from the branches of Harrisonia perforata. The antimycobacterial activity of the isolates was assessed against Mtb H37Ra using the MABA method. Perforamone D (50) was the most active isolate (MIC=25μg/mL). Namdaung et al. [92] isolated six, structurally diverse phenolic compounds and screened them for their antimycobacterial activity against Mtb H37Ra. All compounds showed antitubercular activity, with compound 51 being the most active compound (MIC=6.25μg/mL). Other prenylated bioactive natural products were isolated from the fruit hulls of Garcinia mangostana [93]. Among these, Garcinone B (52) was the most active isolate against Mtb H37Ra (Figure 15). Joycharat et al. [94] isolated 11 compounds from the leaves of Aglaia forbesii. The benzopyran flavagline (53) exhibited the highest activity against Mtb H37Ra with an MIC value of 25μg/mL.

In conclusion, benzopyrans isolated from natural products are a significant class of antitubercular agents. Moreover, the target identification and mode of action studies on these phytochemicals may lead to identification of novel targets for further drug discovery work and synthetic modifications of these compounds could result into more potent compounds and lead drug candidates.

Benzopyran: Structural requirements for anti-TB activity

From the published data, it is evident that the benzopyran nucleus substituted at all positions (except position 1) with varied substituents produced potent anti-TB candidates. All the positions tolerated no substitution or substitution with differing substituents. At the 2nd position of the benzopyran, substituents may vary from amines, methyl and spirocycloalkyls. Among them the 2,2-dimethyl and spirocycloalkyl substituted derivatives demonstrated excellent anti-TB activity. The 3rd position may be substituted with phenyl and heterocyclic rings. Amides and heterocyclic rings at the 3rd position enhance anti-TB activity. The 4th position can be substituted with carbonyl and hydroxyl groups as well as phosphonates and heterocycles. Benzopyrans with carbonyl group at the 4th position were potent against Mtb. The phenyl ring of the benzopyran could either be substituted or fused with aromatic and heterocyclic rings such as furan and pyridine. Generally, the fused derivatives demonstrated improved antitubercular activity. Benzopyran with a methyl group at the 5th position evinced better activity, consistent with other methyl substitutions at position 6, 7 and 8. Other functional group substitutions in the phenyl ring that lead to improved anti-TB activity include halogens, alkyl ethers, triflouromethyl, hydroxyl, nitro group and phenyl ring. Reducing the double bond of the pyran ring generally leads to loss of activity (Figure 16).

Efforts towards target identification

An increase in efforts to discover antitubercular therapeutics has brought insights into the biology of Mycobacterium tuberculosis. Promising new drugs such as bedaquiline, which inhibits ATP synthase [95], and the nitroimidazoles delamanid and pretomanid, which inhibit both mycolic acid synthesis and energy production [96] have brought hope in the discovery of new drugs with novel mechanisms of action, capable of inhibiting multi- and extensively drug -resistant Mtb and, potentially nonreplicating Mtb with the hope of shortening the therapy duration. Although, not enough progress on investigating the mechanism action of benzopyrans, similar compounds such as coumarins have been reported to inhibit the fatty acyl-AMP ligase essential for mycobacterial growth, FadD32 [97]. Benzothiazinethiones, (delivered a preclinical candidate, SKLB-TB1001) from which some benzopyrans were derived from through scaffold morphing have been reported to be decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) inhibitors [98]. Similarly, flavonoids which are structurally like Pisonin B (49) are known to inhibit the β-hydroxyacyl-ACP dehydratase (HadAB) complex [99]. These new targets provide an avenue for exploration away from the known mechanisms of action of the standard drugs in the market.

Conclusion and future aspects

TB is still the leading cause of death from a single infectious agent, ahead of HIV/AIDS. With an estimated 1 million children developing TB in 2017, there is no doubt this statistic provokes the global scientific community to treat this deadly disease. However, current control strategies have little impact on TB control and some M. tuberculosis strains are resistant to all existing medication used for the treatment of TB, highlighting the need for new drugs with novel modes of action. The new drug should have increased capacity to inhibit bacterial growth and shorten treatment time, new mechanism of action, be less toxic and cheap to make so that it can be easily accessible to the developing world. Benzopyrans and its derivatives are ubiquitous natural products and have displayed a broad spectrum of pharmaceutical activities. Current literature clearly depicts substituted benzopyrans to play a vital role in the development of new anti-TB chemotypes. Multiple authors have reported benzopyran-anchored derivatives as potent antitubercular agents. This review has provided collective information on various benzopyran-based compounds with antitubercular activity that will be useful to initiate further drug discovery efforts using these benzopyran derivatives as a starting point to develop potent anti-TB agents. The identification of biological targets and mode of action of most reported active compounds has not been fully explored. This area together with SAR-based study will play an important role in guiding researchers to design safe and potent benzopyran drugs for TB treatment.

Acknowledgement

The authors would like to thank Sandeep Ghorpade for proofreading the article.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. However, the authors would like to thank their respective employers which are; University of Eswatini, University of Johannesburg and University of Zimbabwe.

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