Antitubercular and antibacterial activities of isoxazolines derived from natural products: Isoxazolines as inhibitors of Mycobacterium tuberculosis InhA

Abstract

Isoxazoline derivatives of the natural products eugenol, 1’-S-acetoxychavicol acetate and sclareol are prepared through 1,3-dipolar cycloaddition reactions in an aqueous buffered system. The compounds are evaluated for their antitubercular and antibacterial activities. Compounds 2, 2a and 3f display strong antitubercular activity with minimum inhibitory concentration values of 26.68, 17.89 and 14.58 µM, respectively. Furthermore, derivative 3f exhibits antibacterial activity against Bacillus cereus (minimum inhibitory concentration value of 29.16 µM). Isoxazoline derivatives of 1’-S-acetoxychavicol acetate demonstrate improvements in cytotoxicity, and derivative 3f of sclareol demonstrates improved antitubercular and antibacterial activities. Isoxazolines derived from natural products exhibit Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) inhibitory activity, and molecular modelling predicts that they form hydrogen bonding and hydrophobic interactions with NADH and with the key residues of the InhA binding site.

Keywords

1,3-dipolar cycloaddition antibacterial activity antitubercular activity InhA inhibitor isoxazoline derivatives

Introduction

Tuberculosis (TB) is a contagious respiratory disease caused by Mycobacterium tuberculosis. According to the World Health Organization (WHO), TB was ranked among the top 10 most common causes of death due to an infectious agent, with approximately 10 million people being infected and 1.4 million people dying of TB in 2019; although both TB incidence and TB mortality rates slightly decreased globally in 2020, they did not reach the 2020 milestone.^1,2 Typically, TB is prevalent in tropical regions with dense populations, such as India, Indonesia, Nigeria, Bangladesh, the Philippines and Pakistan.^1–3 In addition, concern has grown with the emergence of multidrug-resistant strains that remain a public threat, with approximately >200,000 reported cases of resistance in 2019.^1,2,4–6 Therefore, identifying novel antitubercular agents is imperative.

Natural products, extracted and isolated from medicinal herbs, marine organisms, or microorganisms, display a variety of biological activities and are invaluable sources for drug discovery; examples of such products include artemisinin, penicillin, quinine, paclitaxel and amphotericin B.^7–9 Diverse primary or secondary metabolites can be derived from natural sources, leading to the discovery of new bioactive ingredients that are useful in medicinal chemistry and pharmaceutical chemistry research.^8,10 Chemical transformations involving the use of natural products as starting materials have been extensively applied in the fields of natural product research and medicinal chemistry.^11,12 Such transformations entail the use of natural products as building blocks, and the resulting natural product derivatives exhibit enhanced biological activities, thus leading to the discovery of new drug candidates. In nature, various natural products contain terminal alkene moieties, with some being reported to have antitubercular activities. These moieties can act as dipolarophiles, reacting with a nitrile oxide to form an isoxazoline ring, for example, phenylpropenes 1 and 2 and terpene 3 (Figure 1).^13–15 Studies have used isoxazolines as antitubercular scaffolds and report that they exhibit strong potencies against M. tuberculosis; for example, nitrofuranyl isoxazoline exhibited a minimum inhibitory concentration (MIC₉₀) value of 0.00005 µg/mL (1.16 × 10^–4), and an esterified isoxazoline derivative had an MIC₉₀ value of 1.56 µg/mL (3.96 µM) (Figure 1).^16–20 These results have inspired the incorporation of isoxazoline moieties into new molecular scaffolds. M. tuberculosis enoyl-acyl carrier protein reductase (InhA) is a promising targeted enzyme for investigating novel antitubercular drug candidates.^21,22 InhA catalyses trans-enoyl reduction in the type II fatty acid synthase (FAS II) system, leading to the synthesis of mycolic acids. Mycolic acids are found in the mycobacterial cell envelope of M. tuberculosis and are essential to its survival because they help protect it from antibiotics.

Figure 1.

Structures of antitubercular isoxazolines and examples of natural products containing an olefin moiety: eugenol (1), 1’-S-acetoxychavicol acetate (2) and sclareol (3).

According to our review of the literature, is oxazolines are heterocyclic compounds that are considered to be among the most potent antimicrobial agents.^23–27 However, studies on antitubercular agents containing an isoxazoline nucleus are scarce. In addition, the literature does not contain a molecular modelling study on isoxazoline derivatives as InhA inhibitors. Accordingly, in this study, natural compounds – namely, eugenol (1), 1’-S-acetoxychavicol acetate (2) and sclareol (3) – were structurally modified to synthesise isoxazoline derivatives bearing an olefin moiety through aqueous olefin–nitrile oxide cycloaddition reactions. The obtained isoxazoline derivatives were evaluated for their antitubercular and antibacterial activities, and molecular modelling was employed to further investigate the mechanisms underlying their activities.

Results and discussion

Chemistry

The isoxazoline derivatives of natural products 1–3 were prepared using a previously developed technique.²⁸ 1,3-Dipolar cycloaddition reactions of compounds 1–3 with nitrile oxides generated in situ from oxime chlorides a–f (Figure 2) in an aqueous buffered system resulted in the corresponding isoxazoline derivatives 1a–f, 2a–f and 3a–f in moderate to good yields (Table 1). All 18 isoxazoline derivatives 1a–f, 2a–f and 3a–f were novel and were characterised using ¹H and ¹³C NMR spectroscopy, infrared (IR) spectroscopy, and high-resolution mass spectrometry (HRMS). Notably, compounds (1a–f) are racemic, whereas mixtures of diastereomers were obtained for products 2a–f and 3a–f. All derivatives were used in biological evaluation studies without further purification.

Figure 2.

Structures of oxime chlorides a–f.

Table 1.

Synthesis of the isoxazoline derivatives.


R^1a	R^2b	Product	Yield (%)^c	d.r.^d
	CO₂Et	1a	75	–
	Ph	1b	63	–
	4-ClC₆H₄	1c	78	–
	4-MeOC₆H₄	1d	88	–
	4-O₂NC₆H₄	1e	85	–
	2-Py	1f	64	–
	CO₂Et	2a	51	1.04:1.00
	Ph	2b	40	1.02:1.00
	4-ClC₆H₄	2c	40	1.16:1.00
	4-MeOC₆H₄	2d	57	1.15:1.00
	4-O₂NC₆H₄	2e	55	1.04:1.00
	2-Py	2f	47	1.04:1.00
	CO₂Et	3a	65	1.05:1.00
	Ph	3b	59	1.11:1.00
	4-ClC₆H₄	3c	43	1.14:1.00
	4-MeOC₆H₄	3d	52	1.71:1.00
	4-O₂NC₆H₄	3e	73	1.13:1.00
	2-Py	3f	20	1.53:1.00

KP_i: K₂HPO₄/KH₂PO₄.

Starting material (1.0 equiv.).

Oxime chloride (1.2 equiv.).

Isolated yield.

d.r.: diastereoisomeric ratio (major/minor) based on ¹H NMR analysis.

All reactions were performed in aqueous phosphate buffer.

For the eugenol series 1a–f, ¹H NMR spectroscopy confirmed the formation of an isoxazoline ring in the parent structure, indicating the presence of methylene proton signals at values of 2.83–3.03 ppm. Isoxazoline ring proton signals were also observed at 3.01 and 3.60 ppm for the 1’-S-acetoxychavicol acetate series 2a–f, which suggested that these products were obtained as mixtures of two diastereoisomers. The diastereoisomeric ratios of products 2a–f were determined based on the ratio of the CH proton signals at 5.89 and 6.02 ppm and are reported in Table 1. The cycloaddition reactions of 1’-S-acetoxychavicol acetate (2) with nitrile oxides a–f showed no stereoselectivity; the diastereoisomeric ratios of the products 2a–f were mostly determined as approximately 1:1, except for compounds 2c (1.16:1.00) and 2d (1.15:1.00). For the products 2a–f, the major diastereoisomers were inferred by a comparison of the vicinal coupling constants (³J_HH) for the major and minor products between the ring hydrogen next to the oxygen (H-5) and the adjacent exocyclic hydrogen next to the acetoxy group (H-1’), assuming that the syn configuration of the major diastereoisomers was formed by considering the magnitudes of the coupling constant (³J_HH) values between 5.89 and 5.95 Hz.²⁹ All sclareol derivatives 3a–f revealed isoxazoline ring proton signals at 3.05–3.56 ppm. ¹³C NMR spectroscopy revealed that the characteristic C=N carbon of the isoxazoline ring resonated at values ranging between 152.4 and 159.3 ppm in all the products 1a–f, 2a–f and 3a–f. Mixtures of two diastereoisomers of isoxazoline products 3a–f were also formed. Determination of the diastereoisomeric ratios of products 3a–f was carried out by analysis of the isoxazoline ring proton signals at 4.49 and 4.79 ppm (Table 1). The cycloaddition reactions between sclareol (3) and nitrile oxides a–f resulted in the formation of the corresponding products 3a–f with diastereoisomeric ratios of approximately 1.1:1, apart from compounds 3d (1.71:1.00) and 3f (1.53:1.00). The syn- or anti-configurations of the major diastereomers of products 3a–f were not assigned due to no direct evidence from proton signals and vicinal coupling constant (³J_HH) values. According to the literature, the regioselectivity of such 1,3-dipolar cycloaddition reactions between nitrile oxides and terminal alkenes can be controlled by steric effects, resulting in the formation of 2-isoxazoline rings as single regioisomers;^29,30 however, these reactions were not stereoselective as we did not detect the formation of predominant diastereoisomers.²⁸

The isoxazoline derivatives 1a–f, 2a–f and 3a–f were evaluated for their antitubercular and antibacterial activities (Table 2). The cytotoxic activity of the isoxazoline derivatives was also tested using a colorimetric assay (Table 3). Furthermore, the most active compounds against M. tuberculosis were evaluated for potential InhA inhibitory activity (Table 4). Molecular docking was performed to investigate the binding action of the isoxazoline derivatives.

Table 2.

Antitubercular and antibacterial activities of the isoxazoline derivatives.

Compound	Antitubercular and antibacterial activities (MIC, (µM))
Compound	Mtb ^a	Bacillus cereus ^b	Staphylococcus aureus ^c	Pseudomonas aeruginosa ^d	Escherichia coli ^e
1	–	–	–	–	–
1a	–	–	–	–	–
1b	–	–	–	–	–
1c	–	–	–	–	629.39
1d	–	–	–	–	–
1e	304.58	–	–	–	–
1f	351.73	–	–	–	–
2	26.68	426.90	1707.59	–	–
2a	17.89	–	–	–	–
2b	282.99	–	–	–	–
2c	257.85	–	–	–	–
2d	130.41	–	–	–	–
2e	125.51	251.02	1004.10	–	–
2f	141.10	–	–	–	–
3	81.04	162.07	162.07	–	324.15
3a	118.04	118.04	118.04	–	–
3b	–	–	–	–	–
3c	216.42	–	–	–	–
3d	109.25	–	–	–	–
3e	52.90	–	423.17	–	–
3f	14.58	29.16	116.66	–	–

Mtb: M. tuberculosis H37Ra strain: MIC (µM) of rifampin (0.01).

Bacillus cereus: MICs (µM) of chloramphenicol (12.38) and tetracycline∙HCl (4.16–8.32).

Staphylococcus aureus: MICs (µM) of chloramphenicol (1.55) and tetracycline∙HCl (0.26).

Pseudomonas aeruginosa: MICs (µM) of chloramphenicol (24.76) and tetracycline∙HCl (8.32).

Escherichia coli: MICs (µM) of chloramphenicol (6.19) and tetracycline∙HCl (2.08–4.16).

Inactive at >100 µg/mL for Mtb and >400 µg/mL for the four bacterial strains (maximum concentration).

Table 3.

Cytotoxic activity of the isoxazoline derivatives.

Compound	Cytotoxic activity (IC₅₀ (µM), mean ± SD, n = 3)
	HepG2^a	MOLT-3^a	HuCCA-1^a	A549^a
1	–	–	–	–
1a	–	–	–	–
1b	–	–	–	–
1c	111.87 ± 6.32	93.53 ± 4.40	–	–
1d	–	123.50 ± 3.99	–	–
1e	135.11 ± 10.05	90.61 ± 1.95	–	–
1f	–	–	–	–
2	26.38 ± 5.16	15.45 ± 3.76	25.14 ± 3.76	39.27 ± 2.43
2a	–	95.70 ± 2.17	–	–
2b	–	–	136.63 ± 2.89	–
2c	110.90 ± 5.29	–	–	–
2d	–	–	–	–
2e	–	46.74 ± 1.81	104.17 ± 1.78	–
2f	–	–	–	–
3	70.40 ± 2.01	49.89 ± 0.74	52.61 ± 2.07	49.95 ± 4.18
3a	59.63 ± 1.06	71.72 ± 4.63	72.07 ± 7.36	76.02 ± 3.09
3b	60.80 ± 4.09	34.02 ± 1.71	42.68 ± 6.92	39.75 ± 5.19
3c	59.25 ± 1.06	34.50 ± 1.95	40.45 ± 0.35	42.16 ± 2.66
3d	58.93 ± 3.45	34.55 ± 1.31	42.28 ± 3.65	40.93 ± 0.76
3e	13.20 ± 0.87	17.98 ± 0.25	21.16 ± 0.25	20.86 ± 1.21
3f	59.77 ± 5.32	38.59 ± 2.40	52.00 ± 1.94	82.41 ± 2.24
Doxorubicin	0.53 ± 0.03	0.012 ± 0.003	0.76 ± 0.02	0.40 ± 0.01
Etoposide	58.75 ± 7.61	0.054 ± 0.003	–	–

Inactive at 50 µg/mL (maximum concentration).

Table 4.

Relative inhibitory activity of the 1’-S-acetoxychavicol acetate isoxazoline series 2a–f against the InhA enzyme.

Compound	% Relative inhibition of the InhA enzyme at 1 µM
2	28.34 ± 3.28
2a	27.54 ± 5.18
2b	15.34 ± 0.25
2c	32.66 ± 5.66
2d	18.66 ± 1.42
2e	19.71 ± 8.42
2f	25.17 ± 6.55
Triclosan	50.49 ± 3.22

Biological evaluation

The derivatives were evaluated for their antitubercular and antibacterial activities (Table 2). Derivatives 1e and 1f exhibited antitubercular activities with MIC values of 304.58 and 351.73 µM, respectively. Moreover, 1’-S-acetoxychavicol acetate (2), a natural product from the rhizome of Alpinia galangal, displayed antitubercular activity with an MIC value of 26.68 µM (Table 2); furthermore, it demonstrated cytotoxic activity with IC₅₀ values of 15.45–39.27 µM (Table 3). Derivative 2a exhibited antitubercular activity with an MIC value of 17.89 µM, which was better than that of 1’-S-acetoxychavicol acetate (2), its parent compound; however, this derivative exhibited no cytotoxic activity against human hepatocellular carcinoma (HepG2), human cholangiocarcinoma (HuCCA-1), or human lung adenocarcinoma (A549) cell lines and exhibited very weak cytotoxic activity (IC₅₀ = 95.70 µM) against the human T-lymphoblastic leukaemia (MOLT-3) cell line (Table 3). Accordingly, derivative 2a demonstrated reduced toxicity compared with the parent natural product 2. Sclareol (3), a natural product, had antitubercular activity with an MIC value of 81.04 µM, but its isoxazoline derivatives 3a–e exhibited moderate activity with MIC values ranging from 52.90 to 216.42 µM. Among the isoxazoline derivatives of sclareol (3), derivative 3f, which had a 2-pyridyl moiety, exhibited the most potent antitubercular activity with an MIC value of 14.58 µM. Sclareol (3) had an MIC value of 81.04 µM; therefore, derivative 3f (MIC = 14.58 µM) was 5.5-fold more active than its parent natural product. However, the cytotoxic activity (IC₅₀ = 38.59–82.41 µM) of derivative 3f was comparable to that (IC₅₀ = 49.89–70.40 µM) of the parent natural product 3 (Table 3). Notably, the results obtained for derivatives 3a–f show their improved cytotoxicity against all four cancer cell lines compared with isoxazolines 1a–f and 2a–f. The structures of these derivatives may demonstrate greater hydrophobicity (due to the aliphatic skeleton) and H-bonding capacity (−OH), resulting in increased cellular uptake of compounds, leading to cytotoxicity.

The MIC values of all the isoxazolines were determined using the broth microdilution method³¹ against both Gram-positive bacteria (Bacillus cereus and Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) (Table 2). The natural product eugenol (1) was inactive against the bacterial strains tested; however, its isoxazoline derivative 1c inhibited E. coli growth with an MIC value of 629.39 µM. In contrast to the isoxazoline derivatives of eugenol (1), which exhibited antibacterial activity against the Gram-negative bacterium E. coli, the natural product 1’-S-acetoxychavicol acetate (2) and its isoxazoline derivative 2e selectively exhibited activity against the Gram-positive bacteria B. cereus and S. aureus, with MIC values of 426.90/1707.59 µM and 251.02/1004.10 µM, respectively (Table 2). Sclareol (3) displayed antibacterial activity against both Gram-positive and Gram-negative bacteria, with MIC values against B. cereus, S. aureus, and E. coli of 162.07, 162.07 and 324.15 µM, respectively. However, the isoxazoline derivatives 3a, 3e and 3f selectively exhibited activity against the Gram-positive bacteria B. cereus and S. aureus (MIC = 29.16–423.17 µM). Among the derivatives of 3, compound 3f was the most active agent (MIC against B. cereus = 29.16 µM), which was 5.5-fold more active than the parent natural compound 3 (MIC against B. cereus = 162.07 µM). Therefore, derivative 3f exhibited stronger antibacterial and antitubercular activities than its parent natural product, sclareol (3); this phenomenon demonstrates that an isoxazoline moiety can improve antibacterial and antitubercular activities. These findings on the antibacterial activity demonstrate that most isoxazolines were inactive or exhibited poor activity against Gram-negative bacteria because these molecules might permeate very well but interact very poorly with the target protein. Compound 3f shows improved antitubercular and antibacterial activities compared with the parent natural compound 3; however, the two diastereoisomers of compound 3f should be separated to establish which diastereoisomer is the active compound.

InhA inhibitory activity

Many antitubercular agents inhibit InhA as the biomolecular target.^21,22 In particular, five-membered heterocyclic compounds exhibit potent antitubercular activity; however, studies investigating isoxazoline derivatives for their InhA inhibitory activities are scarce.³² Accordingly, in this study, the compounds were tested for their InhA inhibitory activities using the drug triclosan as a positive control (Table 4). The relative inhibitory potency of 1 µM of triclosan against the InhA enzyme was 50.49%. The 1’-S-acetoxychavicol acetate series 2a–f, which exhibited the most prominent antitubercular activities, was selected for our evaluation of the InhA inhibitory activity. At 1 µM, the derivatives exhibited a relative inhibitory activity of 15%–33%. Derivative 2c at 1 µM had the best inhibitory activity against InhA (33%); however, it demonstrated weak inhibitory activity (MIC = 257.85 µM) against the M. tuberculosis strain H37Ra. The inhibitory activity of derivative 2a against InhA was highly comparable to that of its parent compound 2 (28%); this derivative exhibited relatively strong activity against M. tuberculosis (MIC = 17.89 µM). The reason for the lack of a correlation between InhA inhibitory activity and in vitro antitubercular activity must be addressed in further analysis.

Molecular docking of isoxazoline derivatives to the InhA enzyme

Molecular modelling was employed to investigate the binding action of the isoxazoline derivatives. The docking protocol was first validated to ensure its reliability and reproducibility. The co-crystallised ligand was removed from the crystal structure of InhA, its energy was minimised, and it was redocked to the InhA binding site (see section “Experimental”). The ChemPLP scoring function was used for docking. The results revealed that ChemPLP demonstrated good overlays of heavy atoms between the co-crystallised ligand and the best ranked docked conformation with a root-mean-square deviation (RMSD) value of 1.9 Å calculated using DockRMSD³³ (Figure S24, Supporting Information). The RMSD value of <2.0 Å is considered acceptable and discriminates well-docked from poorly docked ligands with respect to crystallographic data.³⁴

Table S1 (Supporting Information) lists the calculated molecular docking scores, which were similar to those of triclosan. The molecular docking results provided insights into the possible binding interactions and binding modes of the isoxazoline derivatives. Compounds 2 and 2a exhibited the most prominent antitubercular activities, and showed 27%–28% InhA inhibition. Compound 2a can exhibit both S,R and R,R configurations at chiral carbons (C-5 and C-1’, respectively). However, hydrogen bonds, that are crucial for ligand–target interactions, were not observed with the 5R,1’R configuration. Thus, it is presumed that the 5S,1’R configuration is the active species. Hydrogen bonding interactions were observed between the phenylacetate moiety and nicotinamide adenine dinucleotide, reduced form, (NADH) for 2 and 2a (5S,1’R). In addition, the phenylacetate moiety of compound 2 was predicted to form hydrogen bonds with Tyr158, whereas the phenylacetate moiety of 2a (5S,1’R) was in close proximity to Tyr158, suggesting potential hydrogen bond formation under dynamical systems. The predicted binding modes of the most active compounds, 2 and 2a (5S,1’R), are illustrated in Figure 3. Derivative 2c was the strongest inhibitor, albeit exhibiting weak antitubercular activity. The phenylacetate moiety was in close proximity to the key residues Tyr158 and NADH, but hydrogen bonding interactions with the residues were not observed. Hydrogen bonding interactions were observed between the phenylacetate moiety and NADH and Tyr158. NADH and Tyr158 have been thoroughly documented for their involvement in enzyme–inhibitor complex stabilisation;^35,36 for example, Figure S25 (Supporting Information) illustrates a hydrogen bonding interaction of Tyr158 with co-crystallised triclosan. Furthermore, residues that formed hydrophobic interactions with triclosan included Gly96, Met103, Phe149, Tyr158, Met161, Ala198 and Met199. This is partially reflected in Figure 3, which depicts the Met103, Phe149, Tyr158, Ala198 and Met199 residues that form hydrophobic interactions with the derivatives. The three-dimensional (3D) representations of the predicted binding structures are illustrated in Figure 3. The same amino acids in the two-dimensional (2D) representations are seen in the 3D representations.

Figure 3.

(a) The predicted binding interactions of 1’-S-acetoxychavicol acetate (2), and (b) its derivative 2a (5S,1’R) with the InhA binding site based on the ChemPLP scoring function. Hydrogen bonding is depicted as green dotted lines, and the distances are shown in Å. Red lashes represent the hydrophobic interactions between the residues and the derivatives. (a) 3D representations: predicted binding interactions of 2 to the InhA binding site. Hydrogen bonding is depicted as green dotted lines. Amino acids Phe149, Pro156, Tyr158, Pro193, Met199 and NADH are shown as stick representations coloured black, brown, purple, olive, green and pink, respectively. (b) 3D representations: predicted binding interactions of 2a (5S,1’R) to the InhA binding site. Hydrogen bonding is depicted as green dotted lines. Amino acids Met103, Phe149, Met155, Pro156, Ala157, Tyr158, Ala198, Met199 and NADH are shown as stick representations coloured orange, black, blue, brown, navy blue, purple, yellow, green and pink, respectively.

Because the isoxazoline derivatives 2a–f were investigated for their potential as antitubercular agents, evaluating their physicochemical properties against benchmarks such as the Lipinski filters³⁷ and the Veber rule³⁸ was pertinent to assess the drug-like properties of the compounds. The molecular descriptors evaluated in this study were molecular weight (MW), hydrogen bond donor (HD), hydrogen bond acceptor (HA), rotatable bond (RB), topological polar surface area (TPSA) and Moriguchi log P (MlogP),³⁹ which is used to calculate lipophilicity. Table 5 presents the calculated molecular descriptors, revealing the derivatives and clinically approved triclosan exhibiting behaviour within the drug-like chemical space; that is, the numerical figures did not exceed the Lipinski or Veber limits. This finding demonstrates that the isoxazoline derivatives 2a–f are pharmacokinetically favourable scaffolds for drug development.

Table 5.

Physicochemical properties of the 1’-S-acetoxychavicol acetate (2) series and triclosan.

Compound	MW (g/mol)	HD	HA	RB	TPSA (Å²)	MlogP
2	234.27	0	4	6	52.60	2.50
2a	349.37	0	8	9	100.49	1.60
2b	353.40	0	6	7	74.19	3.10
2c	387.84	0	6	7	74.19	3.60
2d	383.43	0	7	8	83.42	2.84
2e	398.40	0	8	8	120.01	3.22
2f	354.39	0	7	7	87.08	2.12
Triclosan	289.54	1	2	2	29.46	3.84

MW: molecular weight; HD: hydrogen bond donor; HA: hydrogen bond acceptor; RB: rotatable bond; TPSA: topological polar surface area; MlogP: Moriguchi log P.

Conclusion

In this study, 18 isoxazoline derivatives of natural products 1–3 were successfully prepared, and their biological activities were then evaluated. The eugenol (1) series displayed minimal biological activities. The 1’-S-acetoxychavicol acetate (2) and sclareol (3) series exhibited relatively good antitubercular activities but showed weak antibacterial and cytotoxic activities. An exception was compound 2, which exhibited the strongest cytotoxic activity and had results comparable to those of derivative 3e; that is, it had IC₅₀ values of 15–39 µM across all four cancer cell lines. The newly synthesised derivative 3f was the most promising because it demonstrated an approximately 5.5-fold higher activity against B. cereus than did its parent compound 3 and an approximately 14-fold higher activity than that of compound 2. In addition, derivative 3f exhibited the best inhibitory activities against S. aureus growth, with the MIC value being 29.16 µM. The 1’-S-acetoxychavicol acetate (2) series clearly demonstrated inhibitory activity against InhA in the in vitro assay. Furthermore, molecular docking demonstrated hydrogen bonding and hydrophobic interactions of the derivatives with NADH and the amino acid residues of the InhA binding site. Calculations of the molecular descriptors indicated that the compounds are pharmacokinetically suitable for further development as drug candidates.

Experimental

Chemicals and instrumentation

All commercially available chemicals and solvents were purchased from Sigma Aldrich and Santa Cruz and were used without purification. 1’S-acetoxychavicol acetate (2) and oxime chlorides a–f were obtained from the compound library of the Natural Products Laboratory at Chulabhorn Research Institute.

The NMR spectra of the studied compounds were recorded on a Bruker Avance 300 spectrometer (¹H NMR at 300 MHz and ¹³C NMR at 75 MHz). The values of the chemical shifts (δ) are reported in ppm, with coupling constants (J) being reported in Hertz. The abbreviations used in the NMR assignments are as follows: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad. HRMS data were obtained using a Bruker micro time-of-flight mass spectrometer. IR spectra were recorded on a Perkin Elmer Spectrum ONE spectrophotometer using the universal attenuated total reflectance technique. Melting points (m.p.) were determined in open capillary tubes using a Büchi 535 melting point apparatus without correction. Flash column chromatography was executed using columns packed with silica gel 60 (Merck, 0.063–0.200 mm), eluting with the stated solvent system. Thin-layer chromatography was performed on Merck DC-Alufolien Kieselgel 60F₂₅₄ aluminium-backed silica plates. Each plate, after being developed with an appropriate solvent system, was visualised under ultraviolet light (λ = 254 or 365 nm).

Synthesis of isoxazoline derivatives 1a–f, 2a–f and 3a–f

Oxime chloride a, b, c, d, e or f (0.3–0.6 mmol, 1.2 equiv.) was added to a stirred solution of compound 1, 2 or 3 (0.25–0.50 mmol, 1.0 equiv.) in acetone (0.15–0.25 mL, ~5% co-solvent). A solution of 0.1 M K₂HPO₄/KH₂PO₄ (pH = 4.0, 3.0–5.0 mL) was added to the reaction mixture and allowed to stir at room temperature for 20 h. The reaction mixture was extracted with EtOAc (3 × 20 mL). The organic phases were combined, washed with brine (50 mL), dried over Na₂SO₄, filtered and then concentrated in vacuo. The crude residue was purified using flash column chromatography (20% EtOAc/hexane) to give the desired product. The structures of the products were confirmed through spectroscopic analysis (¹H and ¹³C NMR, IR and HRMS).

Ethyl 5-(4-hydroxy-3-methoxybenzyl)-4,5-dihydroisoxazole-3-carboxylate (1a)

Colourless oil (52.5 mg, 75%). IR (UATR): 3449, 2940, 1717, 1589, 1515, 1465, 1433, 1378, 1269, 1254, 1125 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.44 (s, 1H, OH), 6.92 (d, J = 1.8 Hz, 1H, ArH), 6.84–6.60 (m, 2H, 2×ArH), 5.04 (ddt, J = 6.5, 8.3, 10.8 Hz, 1H, CH), 4.25 (q, J = 7.1 Hz, 2H, OCH₂), 3.83 (s, 3H, OCH₃), 3.21 (dd, J = 10.8, 17.5 Hz, 1H, CH₂), 2.96 (dd, overlapping, 1H, CH₂), 2.95 (dd, overlapping, 1H, CH₂), 2.84 (dd, J = 6.7, 13.9 Hz, 1H, CH₂), 1.28 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 161.3, 152.4, 148.3, 146.3, 128.9, 122.8, 115.7, 113.8, 85.2, 62.0, 56.2, 40.8, 38.5, 14.3. HRMS (APCI): m/z [M + H]⁺ calcd for C₁₄H₁₈NO₅: 280.1180; found: 280.1190.

2-Methoxy-4-((3-phenyl-4,5-dihydroisoxazol-5-yl)methyl)phenol (1b)

Off-white solid (44.8 mg, 63%); m.p. 90–91 °C (lit.⁴⁰ m.p. 100–101 °C). IR (UATR): 3421, 2937, 1765, 1602, 1514, 1448, 1432, 1356, 1268, 1237, 1153 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.71–7.63 (m, 2H, 2×ArH), 7.47–7.35 (m, 3H, 3×ArH), 7.40 (s, overlapping, 1H, OH), 6.96 (d, J = 1.1 Hz, 1H, ArH), 6.77 (d, J = 1.1 Hz, 2H, 2×ArH), 4.95 (dddd, J = 6.2, 6.8, 8.0, 10.3 Hz, 1H, CH), 3.84 (s, 3H, OCH₃), 3.39 (dd, J = 10.3, 16.8 Hz, 1H, CH₂), 3.14 (dd, J = 8.0, 16.9 Hz, 1H, CH₂), 3.00 (dd, J = 6.2, 13.8 Hz, 1H, CH₂), 2.83 (dd, overlapping, 1H, CH₂). ¹³C NMR (75 MHz, acetone-d₆): δ 157.0, 148.2, 146.1, 131.2, 130.5, 129.6, 129.4 (2), 127.3 (2), 122.7, 115.7, 113.8, 83.0, 56.2, 41.2, 39.7. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₁₇H₁₇NO₃Na: 306.1101; found: 306.1100.

4-((3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)-2-methoxyphenol (1c)

Yellow oil (62.0 mg, 78%). IR (UATR): 3415, 2936, 2884, 1597, 1514, 1495, 1432, 1403, 1351, 1268, 1236, 1152 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.73–7.63 (m, 2H, 2×ArH), 7.52–7.41 (m, 2H, 2×ArH), 7.39 (s, 1H, OH), 6.95 (s, 1H, ArH), 6.76 (d, J = 1.1 Hz, 2H, 2×ArH), 4.97 (dddd, J = 6.2, 6.8, 8.0, 10.3 Hz, 1H, CH), 3.84 (s, 3H, OCH₃), 3.40 (dd, J = 10.3, 16.9 Hz, 1H, CH₂), 3.15 (dd, J = 8.0, 16.9 Hz, 1H, CH₂), 3.00 (dd, J = 6.2, 13.9 Hz, 1H, CH₂), 2.84 (dd, overlapping, 1H, CH₂). ¹³C NMR (75 MHz, acetone-d₆): δ 156.2, 148.2, 146.1, 135.7, 129.9, 129.6 (2), 129.5, 128.9 (2), 122.7, 115.7, 113.8, 83.3, 56.2, 41.1, 39.5. HRMS (APCI): m/z [M + H]⁺ calcd for C₁₇H₁₇NO₃Cl: 318.0891; found: 318.0897.

2-Methoxy-4-((3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)phenol (1d)

White solid (69.1 mg, 88%); m.p. 75–76 °C. IR (UATR): 3422, 2936, 2839, 1607, 1514, 1462, 1432, 1357, 1253, 1177, 1153 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.65–7.57 (m, 2H, 2×ArH), 7.37 (s, 1H, OH), 6.99–6.94 (m, 3H, 3×ArH), 6.76 (d, J = 1.1 Hz, 2H, 2×ArH), 5.01–4.78 (m, 1H, CH), 3.84 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.35 (dd, J = 10.2, 16.7 Hz, 1H, CH₂), 3.10 (dd, J = 8.0, 16.7 Hz, 1H, CH₂), 2.98 (dd, J = 6.2, 13.8 Hz, 1H, CH₂), 2.84 (dd, overlapping, 1H, CH₂). ¹³C NMR (75 MHz, acetone-d₆): δ 161.8, 156.5, 148.2, 146.1, 129.7, 128.8 (2), 123.6, 122.7, 115.7, 114.8 (2), 113.8, 82.6, 56.2, 55.6, 41.2, 40.0. HRMS (APCI): m/z [M + H]⁺ calcd for C₁₈H₂₀NO₄: 314.1386; found: 314.1398.

2-Methoxy-4-((3-(4-nitrophenyl)-4,5-dihydroisoxazol-5-yl)methyl)phenol (1e)

Yellow solid (70.1 mg, 85%); m.p. 128–129 °C. IR (UATR): 3495, 2937, 2846, 1600, 1574, 1513, 1432, 1343, 1268, 1236, 1152 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.31–8.16 (m, 2H, 2×ArH), 7.98–7.85 (m, 2H, 2×ArH), 7.42 (s, 1H, OH), 6.96 (d, J = 1.2 Hz, 1H, ArH), 6.77 (d, J = 1.1 Hz, 2H, 2×ArH), 5.06 (ddt, J = 6.5, 7.9, 10.5 Hz, 1H, CH), 3.83 (s, 3H, OCH₃), 3.48 (dd, J = 10.5, 16.9 Hz, 1H, CH₂), 3.22 (dd, J = 8.1, 16.9 Hz, 1H, CH₂), 3.03 (dd, J = 6.2, 13.9 Hz, 1H, CH₂), 2.88 (dd, overlapping, 1H, CH₂). ¹³C NMR (75 MHz, acetone-d₆): δ 156.1, 149.1, 148.2, 146.2, 137.2, 129.3, 128.2 (2), 124.6 (2), 122.8, 115.7, 113.8, 84.1, 56.2, 41.0, 39.1. HRMS (APCI): m/z [M + H]⁺ calcd for C₁₇H₁₇N₂O₅: 329.1132; found: 329.1141.

2-Methoxy-4-((3-(pyridin-2-yl)-4,5-dihydroisoxazol-5-yl)methyl)phenol (1f)

Yellow oil (45.5 mg, 64%). IR (UATR): 3397, 2937, 1581, 1514, 1470, 1431, 1363, 1269, 1236, 1208, 1153 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.58 (ddd, J = 1.0, 1.8, 4.9 Hz, 1H, ArH), 7.94 (dt, J = 1.1, 8.0 Hz, 1H, ArH), 7.81 (td, J = 1.8, 7.7 Hz, 1H, ArH), 7.45 (s, 1H, OH), 7.37 (ddd, J = 1.2, 4.9, 7.5 Hz, 1H, ArH), 6.95 (s, 1H, ArH), 6.77 (s, 2H, 2×ArH), 4.99 (ddt, J = 6.5, 8.1, 10.4 Hz, 1H, CH), 3.83 (s, 3H, OCH₃), 3.45 (dd, J = 10.4, 17.6 Hz, 1H, CH₂), 3.22 (dd, J = 8.1, 17.6 Hz, 1H, CH₂), 3.00 (dd, J = 6.3, 13.9 Hz, 1H, CH₂), 2.86 (dd, J = 6.7, 13.9 Hz, 1H, CH₂). ¹³C NMR (75 MHz, acetone-d₆): δ 159.1, 150.7, 150.2, 148.2, 146.1, 137.2, 129.5, 124.9, 122.8, 121.8, 115.7, 113.8, 83.6, 56.2, 41.1, 39.5. HRMS (ESI+): m/z [M + H]⁺ calcd for C₁₆H₁₇N₂O₃: 285.1250; found: 285.1247.

Ethyl (R/S)-5-((R)-acetoxy(4-acetoxyphenyl)methyl)-4,5-dihydroisoxazole-3-carboxylate (2a)

Colourless oil (89.8 mg, 51% (major/minor, 1.04:1.00)). IR (UATR): 2985, 2938, 1742, 1719, 1594, 1508, 1371, 1194, 1124 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.59–7.41 (m, 4H, 4×ArH), 7.24–7.07 (m, 4H, 4×ArH), 5.99 (d, J = 4.4 Hz, 1H, CH, minor), 5.90 (d, J = 6.5 Hz, 1H, CH, major), 5.32–5.05 (m, 2H, 2×CH), 4.26 (qd, J = 1.3, 7.1 Hz, 4H, 2×CH₂), 3.33–3.16 (m, overlapping, 3H, 2×CH₂), 3.01 (dd, J = 7.8, 17.8 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, minor), 2.25 (s, 3H, OAc, major), 2.09 (s, 3H, OAc, minor), 2.07 (s, 3H, OAc, major), 1.28 (t, J = 7.1 Hz, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 169.9, 169.5, 161.0, 160.9, 152.5 (2), 152.1, 151.9, 135.0, 134.6, 129.6, 128.9, 122.8 (2), 85.4, 85.0, 76.0, 74.9, 62.2 (2), 36.7, 35.5, 21.0, 20.9, 20.8, 14.3. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₁₇H₁₉NO₇Na: 372.1054; found: 372.1051.

4-((R)-Acetoxy((R/S)-3-phenyl-4,5-dihydroisoxazol-5-yl)methyl)phenyl acetate (2b)

Colourless oil (35.8 mg, 40% (major/minor, 1.02:1.00)). IR (UATR): 3059, 2934, 1746, 1607, 1508, 1447, 1370, 1195, 1167 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.72–7.62 (m, 4H, 4×ArH), 7.60–7.49 (m, 4H, 4×ArH), 7.46–7.39 (m, 6H, 6×ArH), 7.22–7.11 (m, 4H, 4×ArH), 5.98 (d, J = 4.8 Hz, 1H, CH, major), 5.91 (d, J = 6.8 Hz, 1H, CH, minor), 5.22–5.04 (m, 2H, 2×CH), 3.57–3.31 (m, overlapping, 3H, 2×CH₂), 3.19 (dd, J = 7.6, 17.2 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, minor), 2.25 (s, 3H, OAc, major), 2.06 (s, 3H, OAc, minor), 2.05 (s, 3H, OAc, major). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 170.0, 169.5, 157.1, 157.0, 152.0, 151.8, 135.5, 135.3, 130.7 (2), 129.6, 129.5 (2), 129.0, 127.4, 122.7, 122.6, 83.4, 83.0, 76.4, 75.4, 37.7, 36.7, 20.9 (3). HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₀H₁₉NO₅Na: 376.1155; found: 376.1150.

4-((R)-Acetoxy((R/S)-3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)methyl)phenyl acetate (2c)

Light yellow oil (39.4 mg, 40% (major/minor, 1.16:1.00)). IR (UATR): 2936, 1743, 1598, 1508, 1495, 1432, 1403, 1369, 1194, 1167 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.71–7.64 (m, 4H, 4×ArH), 7.58–7.50 (m, 4H, 4×ArH), 7.45 (dq, J = 2.5, 9.1 Hz, 4H, 4×ArH), 7.19–7.10 (m, 4H, 4×ArH), 5.98 (d, J = 4.7 Hz, 1H, CH, minor), 5.91 (d, J = 6.8 Hz, 1H, CH, major), 5.22–5.04 (m, 2H, 2×CH), 3.50–3.33 (m, overlapping, 3H, 2×CH₂), 3.19 (dd, J = 7.6, 17.2 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, minor), 2.25 (s, 3H, OAc, major), 2.06 (s, 3H, OAc, minor), 2.05 (s, 3H, OAc, major). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 170.0, 169.5, 156.3 (2), 152.0, 151.8, 136.0, 135.3, 135.1, 129.6 (2), 129.4, 129.0, 128.9, 122.7, 122.6, 83.7, 83.3, 76.3, 75.3, 37.5, 36.5, 20.9 (3). HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₀H₁₈NO₅ClNa: 410.0766; found: 410.0759.

4-((R)-Acetoxy((R/S)-3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)methyl)phenyl acetate (2d)

Colourless oil (54.5 mg, 57% (major/minor, 1.15:1.00)). IR (UATR): 2938, 1747, 1608, 1515, 1369, 1306, 1252, 1230, 1196 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.68–7.55 (m, 4H, 4×ArH), 7.57–7.49 (m, 4H, 4×ArH), 7.21–7.09 (m, 4H, 4×ArH), 7.02–6.89 (m, 4H, 4×ArH), 5.96 (d, J = 4.8 Hz, 1H, CH, minor), 5.89 (d, J = 6.8 Hz, 1H, CH, major), 5.16–4.96 (m, 2H, 2×CH), 3.83 (s, 3H, OCH₃), 3.83 (s, 3H, OCH₃), 3.55–3.29 (m, overlapping, 3H, 2×CH₂), 3.14 (dd, J = 7.6, 17.1 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, major), 2.25 (s, 3H, OAc, minor), 2.06 (s, 3H, OAc, minor), 2.05 (s, 3H, OAc, major). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 170.0, 169.5, 162.0, 156.5 (2), 151.9, 151.7, 135.5, 135.4, 132.6, 130.2, 129.6, 129.0, 123.0, 122.7, 122.6, 114.9 (2), 83.1, 82.6, 76.5, 75.4, 55.7, 37.9, 37.0, 20.9 (3). HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₁H₂₁NO₆Na: 406.1261; found: 406.1253.

4-((R)-Acetoxy((R/S)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-5-yl)methyl)phenyl acetate (2e)

Yellow oil (55.0 mg, 55% (major/minor, 1.04:1.00)). IR (UATR): 3079, 2938, 1744, 1608, 1579, 1518, 1435, 1369, 1345, 1194, 1167 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.27 (dq, J = 2.3, 9.3 Hz, 4H, 4×ArH), 7.99–7.87 (m, 4H, 4×ArH), 7.61–7.49 (m, 4H, 4×ArH), 7.23–7.10 (m, 4H, 4×ArH), 6.02 (d, J = 4.5 Hz, 1H, CH, minor), 5.95 (d, J = 6.7 Hz, 1H, CH, major), 5.32–5.14 (m, 2H, 2×CH), 3.60–3.47 (m, overlapping, 3H, 2×CH₂), 3.31 (dd, J = 7.6, 17.3 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, major), 2.25 (s, 3H, OAc, minor), 2.07 (s, 3H, OAc, minor), 2.06 (s, 3H, OAc, major). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 170.0, 169.5, 156.3, 156.2, 152.0, 151.8, 149.3 (2), 136.6 (2), 135.2, 134.9, 129.6, 128.9, 128.5, 124.6 (2), 122.7 (2), 84.5, 84.0, 76.2, 75.2, 37.2, 36.1, 20.9 (2), 20.8. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₀H₁₈N₂O₇Na: 421.1006; found: 421.0997.

4-((R)-Acetoxy((R/S)-3-(pyridin-2-yl)-4,5-dihydroisoxazol-5-yl)methyl)phenyl acetate (2f)

Colourless oil (41.7 mg, 47% (major/minor, 1.04:1.00)). IR (UATR): 3056, 2931, 1745, 1584, 1508, 1472, 1440, 1367, 1193, 1167 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.59 (dddd, J = 1.0, 1.8, 4.9, 8.4 Hz, 2H, 2×ArH), 7.92 (ddt, J = 1.2, 6.6, 7.9 Hz, 2H, 2×ArH), 7.86–7.78 (m, 2H, 2×ArH), 7.61–7.48 (m, 4H, 4×ArH), 7.39 (dddd, J = 1.3, 2.3, 4.9, 7.4 Hz, 2H, 2×CH), 7.25–7.10 (m, 4H, 4×ArH), 6.01 (d, J = 4.7 Hz, 1H, CH, minor), 5.93 (d, J = 6.8 Hz, 1H, CH, major), 5.29–5.06 (m, 2H, 2×CH), 3.58–3.37 (m, overlapping, 3H, 2×CH₂), 3.25 (dd, J = 7.7, 17.9 Hz, 1H, CH₂), 2.26 (s, 3H, OAc, minor), 2.25 (s, 3H, OAc, major), 2.07 (s, 3H, OAc, minor), 2.06 (s, 3H, OAc, major). ¹³C NMR (75 MHz, acetone-d₆): δ 170.1, 170.0, 169.5, 159.0, 151.9, 151.8, 150.3, 150.1, 137.3 (2), 135.4, 135.1, 129.6, 129.0, 125.2 (2), 122.7, 122.6, 121.9 (2), 83.9, 83.5, 76.4, 75.3, 37.5, 36.5, 20.9 (2), 20.8. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₁₉H₁₈N₂O₅Na: 377.1108; found: 377.1102.

Ethyl (R/S)-5-((R)-2-hydroxy-4-((1R,2R,4aS,8aS)-2-hydroxy-2,5,5,8a-tetramethyldecahydronaphthalen-1-yl)butan-2-yl)-4,5-dihydroisoxazole-3-carboxylate (3a)

White solid (68.4 mg, 65% (major/minor, 1.05:1.00)); m.p. 58–59 °C. IR (UATR): 3375, 2927, 2869, 1720, 1592, 1464, 1387, 1256, 1128 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 4.74–4.67 (m, 1H, CH, major), 4.66–4.60 (m, 1H, CH, minor), 4.27 (qd, J = 1.0, 7.1 Hz, 4H, 2×OCH₂), 4.05 (br s, 1H, OH, minor), 3.90 (br s, 1H, OH, major), 3.52 (br s, 1H, OH, minor), 3.43 (br s, 1H, OH, major), 3.33–3.05 (m, 4H, 2×CH₂), 1.90–1.16 (m, overlapping, 28H, 14×CH₂), 1.29 (t, J = 7.1 Hz, 6H, 2×CH₃), 1.22 (s, 3H, CH₃, minor), 1.15 (s, 3H, CH₃, major), 1.14 (s, 3H, CH₃, major), 1.11 (s, 3H, CH₃, minor), 1.07–0.90 (m, 4H, 4×CH), 0.87 (s, 6H, 2×CH₃), 0.83 (s, 6H, 2×CH₃), 0.81 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 161.4, 152.5, 152.4, 90.6, 90.2, 89.7, 74.2, 74.1, 73.6, 73.5, 62.7 (2), 61.9 (2), 57.0, 45.0, 44.9, 42.8, 42.7, 42.4, 41.9, 40.5, 40.4, 39.9 (2), 34.6, 33.8, 33.7, 24.5, 24.4, 22.6, 22.0, 21.8, 21.2 (2), 21.1, 19.6, 19.1, 15.8 (2), 14.3. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₄H₄₁NO₅Na: 446.2877; found: 446.2866.

(1R,2R,4aS,8aS)-1-((R)-3-Hydroxy-3-((R/S)-3-phenyl-4,5-dihydroisoxazol-5-yl)butyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (3b)

White solid (63.0 mg, 59% (major/minor, 1.11:1.00)); m.p. 79–80 °C. IR (UATR): 3365, 2924, 2868, 1600, 1463, 1446, 1388, 1359 1264, 1134 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.73–7.68 (m, 4H, 4×ArH), 7.45–7.40 (m, 6H, 6×ArH), 4.66–4.59 (m, 1H, CH, major), 4.59–4.53 (m, 1H, CH, minor), 3.92 (br s, 1H, OH, minor), 3.70 (br s, 1H, OH, major), 3.59 (br s, 1H, OH, minor), 3.54–3.20 (m, 4H, 2×CH₂), 3.39 (br s, overlapping, 1H, OH, major), 1.85–1.25 (m, 28H, 14×CH₂), 1.22 (s, 3H, CH₃, minor), 1.17 (s, 3H, CH₃, major), 1.15 (s, 3H, CH₃, minor), 1.14 (s, 3H, CH₃, major), 1.03–0.88 (m, 4H, 4×CH), 0.86 (s, 6H, 2×CH₃), 0.83 (s, 6H, 2×CH₃), 0.80 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 157.1 (2), 131.3, 131.2, 130.4 (2), 129.4 (2), 127.3, 88.3, 88.1, 74.2, 74.1, 73.7 (2), 62.8 (2), 57.0, 45.0, 44.9, 42.8, 42.7, 42.0, 40.5, 40.4, 39.9 (2), 35.8, 33.8, 33.7, 24.5 (2), 22.9, 22.0, 21.8, 21.2, 21.1, 19.7, 19.2, 19.1 (2), 15.9. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₇H₄₁NO₃Na: 450.2979; found: 450.2969.

(1R,2R,4aS,8aS)-1-((R)-3-((R/S)-3-(4-Chlorophenyl)-4,5-dihydroisoxazol-5-yl)-3-hydroxybutyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (3c)

Light yellow oil (50.0 mg, 43% (major/minor, 1.14:1.00)). IR (UATR): 3367, 2924, 2868, 1598, 1495, 1462, 1403, 1388, 1263, 1133 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 7.72 (d, J = 8.5 Hz, 4H, 4×ArH), 7.48–7.44 (m, 4H, 4×ArH), 4.69–4.62 (m, 1H, CH, major), 4.61–4.55 (m, 1H, CH, minor), 3.97 (br s, 1H, OH, minor), 3.75 (br s, 1H, OH, major), 3.63 (br s, 1H, OH, minor), 3.52–3.27 (m, 4H, 2×CH₂), 3.41 (br s, overlapping, 1H, OH, major), 1.99–1.20 (m, 28H, 14×CH₂), 1.23 (s, 3H, CH₃, minor), 1.16 (s, 3H, CH₃, major), 1.15 (s, 3H, CH₃, minor), 1.14 (s, 3H, CH₃, major), 1.01–0.88 (m, 4H, 4×CH), 0.86 (s, 6H, 2×CH₃), 0.83 (s, 6H, 2×CH₃), 0.80 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 156.3 (2), 135.7(2), 130.1, 130.0, 129.6 (2), 128.9, 128.8, 88.7, 88.4, 74.2, 74.1, 73.7 (2), 62.8, 62.7, 57.0, 45.0, 44.8, 42.8, 42.7 (2), 42.1, 40.4, 39.9 (2), 35.6, 33.8, 33.7, 24.6, 24.5, 22.9, 22.0, 21.8, 21.2, 21.1, 19.7, 19.2, 19.1, 15.9. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₇H₄₀NO₃ClNa: 484.2589; found: 484.2584.

(1R,2R,4aS,8aS)-1-((R)-3-Hydroxy-3-((R/S)-3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)butyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (3d)

Yellow oil (60.0 mg, 52% (major/minor, 1.71:1.00)). IR (UATR): 3367, 2924, 2868, 1609, 1517, 1462, 1359, 1307, 1253, 1176 cm⁻¹. 1H NMR (300 MHz, acetone-d₆): δ 7.69–7.59 (m, 4H, 4×ArH), 6.97 (dd, J = 1.9, 8.9 Hz, 4H, 4×ArH), 4.57 (dd, J = 9.3, 11.0 Hz, 1H, CH, major), 4.49 (dd, J = 8.7, 11.1 Hz, 1H, CH, minor), 3.95 (br s, 1H, OH, minor), 3.83 (s, 6H, 2×OCH₃), 3.72 (br s, 1H, OH, major), 3.48–3.19 (m, 4H, 2×CH₂), 1.88–1.18 (m, 28H, 14×CH₂), 1.16 (s, 6H, 2×CH₃), 1.14 (s, 6H, 2×CH₃), 1.00–0.87 (m, 4H, 4×CH), 0.85 (s, 6H, 2×CH₃), 0.83 (s, 6H, 2×CH₃), 0.80 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 161.7, 156.6, 156.5, 128.8 (2), 123.7, 123.6, 114.8 (2), 88.1, 87.8, 74.3, 74.1, 73.8, 73.6, 62.8, 62.7, 56.9, 55.6, 44.9, 44.7, 42.7, 42.6, 41.9, 40.4, 39.9 (2), 36.1, 33.8, 33.7 (2), 24.6, 24.5, 22.9, 22.0, 21.8, 21.2, 21.1, 19.6, 19.1, 15.9. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₈H₄₃NO₄Na: 480.3084; found: 480.3072.

(1R,2R,4aS,8aS)-1-((R)-3-Hydroxy-3-((R/S)-3-(4-nitrophenyl)-4,5-dihydroisoxazol-5-yl)butyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (3e)

Off-white powder (86.2 mg, 73% (major/minor, 1.13:1.00)); m.p. 88–89 °C. IR (UATR): 3369, 2925, 2868, 1600, 1578, 1520, 1462, 1388, 1341, 1133, 1109 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.30 (dq, J = 2.4, 9.3 Hz, 4H, 4×ArH), 8.03–7.92 (m, 4H, 4×ArH), 4.79–4.73 (m, 1H, CH, major), 4.72–4.66 (m, 1H, CH, minor), 4.02 (br s, 1H, OH, minor), 3.82 (br s, 1H, OH, major), 3.57 (br s, 1H, OH, minor), 3.56–3.38 (m, 4H, 2×CH₂), 3.37 (br s, 1H, OH, major), 1.96–1.19 (m, overlapping, 28H, 14×CH₂), 1.25 (s, 3H, CH₃, minor), 1.18 (s, 3H, CH₃, major), 1.15 (s, 3H, CH₃, minor), 1.14 (s, 3H, CH₃, major), 0.98–0.87 (m, 4H, 4×CH), 0.86 (s, 3H, CH₃, major), 0.85 (s, 3H, CH₃, minor), 0.83 (s, 6H, 2×CH₃), 0.80 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 156.3, 156.2, 149.1, 137.3 (2), 128.3, 128.2, 124.7, 124.6, 89.5 (2), 89.2, 88.7, 74.2, 74.1, 73.8, 73.7, 62.8 (2), 57.0, 45.3, 45.0, 44.9, 42.8 (2), 42.6, 42.3, 42.1, 40.6, 40.5, 39.9 (2), 35.3, 33.8, 33.7, 24.6, 24.5, 22.9, 22.8, 22.1, 21.8, 21.2, 21.1, 19.8, 19.7, 19.2, 19.1, 15.9. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₇H₄₀N₂O₅Na: 495.2829; found: 495.2813.

(1R,2R,4aS,8aS)-1-((R)-3-Hydroxy-3-((R/S)-3-(pyridin-2-yl)-4,5-dihydroisoxazol-5-yl)butyl)-2,5,5,8a-tetramethyldecahydronaphthalen-2-ol (3f)

Light yellow oil (21.0 mg, 20% (major/minor, 1.53:1.00)). IR (UATR): 3375, 2925, 2868, 1708, 1583, 1566, 1471, 1441, 1388, 1365, 1254, 1151, 1133 cm⁻¹. ¹H NMR (300 MHz, acetone-d₆): δ 8.66–8.56 (m, 2H, 2×ArH), 7.99–7.89 (m, 2H, 2×ArH), 7.92–7.76 (m, 2H, 2×ArH), 7.45–7.35 (m, 2H, 2×ArH), 4.73–4.66 (m, 1H, CH, major), 4.65–4.57 (m, 1H, CH, minor), 3.55–3.32 (m, 4H, 2×CH₂), 1.94–1.15 (m, 28H, 14×CH₂), 1.17 (s, 6H, 2×CH₃), 1.14 (s, 6H, 2×CH₃), 1.03–0.92 (m, 4H, 4×CH), 0.87 (s, 6H, 2×CH₃), 0.84 (s, 6H, 2×CH₃), 0.81 (s, 6H, 2×CH₃). ¹³C NMR (75 MHz, acetone-d₆): δ 159.3, 150.2 (2), 137.2, 124.9, 124.8, 121.7, 89.0, 88.9, 88.6, 82.8, 74.0, 73.8, 73.7 (2), 62.9, 62.8, 62.7 (2), 57.1, 57.0, 45.3, 45.1, 42.8 (2), 42.7, 42.2 (2), 40.6, 40.5 (2), 40.0, 39.9, 36.9, 35.8, 35.6, 33.8 (2), 33.7, 24.5, 22.9, 22.6, 22.2, 21.8, 21.2, 19.7, 19.2, 19.1, 15.9. HRMS (ESI+): m/z [M + Na]⁺ calcd for C₂₆H₄₀N₂O₃Na: 451.2931; found: 451.2927.

Antitubercular activity assay

The MIC values used to determine the activities of the compounds against M. tuberculosis were determined using a microplate Alamar Blue assay.⁴¹ All the tested compounds were individually dissolved in dimethyl sulfoxide (DMSO) as stock solutions (10,000 µg/mL). Each solution (100 µL) of the tested compound in Middlebrook 7H9GC (concentration 400 µg/mL) was diluted twofold in 100 µL of Middlebrook 7H9GC in 96-well microplates and subsequently diluted serially. The prepared M. tuberculosis suspension (100 µL; at approximately 10⁵–10⁶ CFU/mL) was added to each microplate well (the highest concentration 100 µg/mL and the lowest concentration 0.1 µg/mL), and the microplates were incubated at 37 °C for 7 days. Both 20% Tween 80 (12.5 µL) and Alamar Blue (20 µL) were added to each microplate well, and the microplates were reincubated at 37 °C for 16–24 h. Mycobacterial growth was determined by a change in colour from blue to pink. If no colour change occurred, the MIC value was recorded as the lowest concentration. The M. tuberculosis strain H37Ra used in this study was obtained from a stock culture at Ramathibodi Hospital, Bangkok, Thailand. Rifampin was used as a standard drug for comparison.

Antibacterial activity assay

The MIC values were determined using the broth microdilution method in accordance with the guidelines of the European Society of Clinical Microbiology and Infectious Diseases.³¹ All synthetic compounds were evaluated on both Gram-positive bacteria (B. cereus and S. aureus) and Gram-negative bacteria (E. coli and P. aeruginosa). The standard drugs chloramphenicol and tetracycline were used as reference compounds.

Cytotoxic activity assay

The cytotoxic activities of all synthetic compounds against four cancer cell lines were evaluated. The cytotoxic activities against the adhesive cell lines HuCCA-1, A549 and HepG2 were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay.⁴² Cytotoxicity against the non-adhesive MOLT-3 cell line was evaluated using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay.^43,44 Doxorubicin and etoposide were used as standard drugs for comparison.

Expression and purification of InhA

Both the expression and purification of InhA were conducted using the previously reported method.⁴⁵ A culture of E. coli BL21(DE3) single competent cells carrying an InhA gene-inserted pET15b plasmid that contained N-terminal 6xHis-tag was grown in a large-scale Lysogeny Broth (LB) medium (5 L) containing ampicillin (100 µg/mL). The culture was incubated at 37 °C until an optical density (OD₆₀₀) value of 0.6 was observed. Proteins were subsequently induced by adding a solution of 50 µM isopropyl-β-d-1-thiogalactopyranoside and the obtained mixture was further shaken at 29 °C for 4 h. The culture of bacterial cells was harvested by centrifugation at 4000 r/min and 4 °C for 10 min. Bacterial cell pellets were resuspended in 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5% v/v glycerol and 0.5% v/v Triton X-100, followed by cell lysis using a French press at 2000 psi and centrifugation at 14,000 r/min and 4 °C for 30 min. The supernatant was loaded on a Ni-NTA column and subsequently washed with a gradient elution of 250 mM imidazole to isolate the purified InhA. The fractions containing InhA were combined and exchanged in 30 mM piperazine-N,N’-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6.8) containing 150 mM NaCl, 2 mM 1,4-dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM PMSF and 10% v/v glycerol; PD-10 desalting columns equilibrated with 30 mM PIPES buffer (pH 6.8) containing 150 mM NaCl were used to remove imidazole. The elution fractions were combined and concentrated using a Vivaspin centrifugal concentrator. The purified InhA was analysed through sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Nanodrop to determine the protein purity and protein concentration, respectively.

InhA inhibitory activity assay

The kinetic assay for InhA inhibitory activity was conducted using the previously reported method.⁴⁵ In brief, a stock solution of each inhibitor was prepared in DMSO. The final concentration of DMSO relative to the final volume (100 μL) for all kinetic reactions was 1%. The relative inhibitory activities of all the tested compounds against InhA were evaluated in 30 mM PIPES buffer (pH 6.8) containing 150 mM NaCl, 2 mM DTT, 1 mM EDTA, 1 mg/mL bovine serum albumin, 10% v/v glycerol, 50 μM NADH, 75 μM trans-2-dodecenoyl-CoA (DD-CoA), and 1 μM inhibitor. The prepared 96-well microplate for all kinetic reactions was shaken for 1 min before 20 nM (final concentration) InhA was added. NADH was added to the reaction mixture as a substrate and incubated at 25 °C for 5 min, followed by the addition of the test compound and further incubation at 25 °C for 20 min. A solution of 75 μM DD-CoA was added to the reaction, which was monitored over 10 min at 25 °C and measured by following the oxidation of NADH to NAD⁺ at λ_ex = 340 nm and λ_em = 420 nm. The fluorescence intensity of each well was read and recorded every 30 s. The relative inhibitory activity of each compound was determined as the percent of relative inhibition of InhA with respect to the control reaction, as indicated in equation (1)

\begin{array}{l} % relative inhibition = \\ 100 \times (\frac{\begin{array}{l} (F_{positive control} - F_{negative control}) \\ - (F_{compound} - F_{negative control}) \end{array}}{F_{positive control} - F_{negative control}}) \end{array}

(1)

where F_compound is the fluorescence intensity of the InhA inhibitory activity for each compound concentration, F_{positive control} is the fluorescence intensity of the InhA inhibitory activity in the absence of any compound, and F_{negative control} is the fluorescence intensity of the NADH oxidation reaction in the absence of the InhA enzyme.

Molecular modelling

Ligands were constructed using Scigress (version 2.8.1); the chemical structures were drawn and the energy minimised using the MM2⁴⁶ force field, and all were saved in 3D format. The crystal structure of M. tuberculosis enoyl-ACP reductase (InhA) was obtained from the Protein Data Bank⁴⁷ ID:2B35⁴⁸ with a resolution of 2.30 Å. Discovery studio (version 3.5) was used to prepare the crystal structure for docking. Specifically, in this process, hydrogen atoms were added, and co-crystallised ligands were removed. The NADH substrate was retained at the binding site because a study documented that InhA inhibitors were bound in an NADH-dependent manner.⁴⁹ The centre of the InhA binding site was defined at coordinates (x = 23.6.17, y = 20.702, z = 57.042) with 10 Å radii. The basic amino acids lysine and arginine were deemed to be protonated. Furthermore, aspartic and glutamic acids were assumed to be deprotonated. The ChemPLP⁵⁰ scoring function was implemented to dock the ligands to InhA using the Genetic Optimisation for Ligand Docking software package (GOLD, version 5.8.1).⁵¹ GOLD has been reported to be one of the best docking software packages available, demonstrating the best accuracy in identifying ligand pose in protein–ligand interactions.⁵² Molecular docking was conducted at 100% efficiency at 50 docking runs per ligand. In addition, the physicochemical properties of the ligands were calculated using Dragon (version 7.0).⁵³

Footnotes

Acknowledgements

The authors thank P. Intachote, S. Sengsai and B. Saimanee for their help in cytotoxic activity testing. The authors also appreciate P. Poolchanuan’s advice regarding antibacterial activities. In addition, HRMS spectra and IR data were kindly provided by the staff at Chulabhorn Research Institute.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by the Center of Excellence on Environmental Health and Toxicology (EHT), Science & Technology Postgraduate Education and Research Development Office, Ministry of Education. C.K. expresses thanks for the postdoctoral grant provided by the EHT.

ORCID iDs

Anuchit Phanumartwiwath

Poonpilas Hongmanee

Supplemental material

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