Introduction

Tuberculosis (TB) is one of the persistent infectious diseases caused by the pathogen Mycobacterium tuberculosis (MTB), claiming about 1.8 million lives in 2015 as reported by the World Health Organization (WHO) [1]. The directly observed treatment short-course (DOTS) is one of the most efficient multidrug therapy programs developed by WHO against TB, yet the treatment success rate struggles to reach the WHO’s target of 85%. First-line TB drugs such as isoniazid, pyrazinamide, ethambutol, rifampicin and streptomycin require longer duration and have failed due to poor compliance which resulted in the appearance of multidrug-resistant (MDR) and extremely drug-resistant (XDR) MTB strains [2,3,4,5,6,7,8,9]. The treatment of MDR-TB requires second-line drugs that are less effective and are associated with serious side effects [10,11,12,13]. Hence, it is quite necessary to carry out further research into the development of new potent chemical agents with promising antimycobacterial properties [14, 15].

The strategy of combining two pharmacophores of known biological activity into a single molecule has emerged as a powerful tool in the hands of medicinal chemists. This strategy may result in either increased overall potency of the parent compounds or could result in an entirely new complementary biological activity [16,17,18]. Prompted by this idea, we adopted a similar strategy to design our target bis-spirochromanones as potential antimycobacterial agents.

Fig. 1
figure 1

Previously reported derivatives containing chroman-4-one pharmacophore with their antimycobacterial activity

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The chroman-4-one framework has been reported to be an integral part of several antimycobacterial agents (Fig. 1) [19,20,21]. Thus, we anticipated that the combination of two chroman-4-one moieties connected through an appropriate spacer in a single molecule may provide a new framework suitable for the design and identification of novel anti-tubercular agents (Fig. 2). Indeed, the strategy was found to be operative in our case. Herein, we report our preliminary findings on the synthesis and the pharmacological evaluation of bis-spirochromanones. To the best of our knowledge, pharmacological evaluation of this class of compounds, especially as anti-tubercular agents, has not been explored to date.

A literature search revealed that spirochromanone derivatives were synthesized from respective cyclic ketones, pyrrolidine and 2-hydroxyacetophenone using a Kabbe condensation approach [22, 23]. To achieve our target bis-spirochromanones, we initially used cyclohexane-1,4-dione as cyclic ketone and carried out a Kabbe condensation. However, this method appeared to be less attractive for the synthesis of target molecules as the isolation of bis-spirochromanones and the introduction of different 2-hydroxyacetophenones in a single molecule resulted to be difficult. To circumvent this problem, we envisioned that using 1,4-dioxaspiro[4.5]decan-8-one instead of cyclohexane-1,4-dione as cyclic ketone would lead to our desired molecules in three steps (Scheme 1).

Results and discussion

Chemistry

Accordingly, our synthetic sequence commenced with the preparation of dispiro[chromane-2,1\(^\prime \)-cyclohexane-4\(^\prime \),2\(^{\prime \prime }\)-[1,3]dioxolan]-4-one 3 by the Kabbe condensation of 2-hydroxyacetophenone 1 with 1,4-dioxaspiro[4.5]decan-8-one 2 in the presence of pyrrolidine [24]. Then, acetal deprotection with excess 5N HCl provided the corresponding ketone spiro[chromane-2,1\(^\prime \)-cyclohexane]-4,4\(^\prime \)-dione 4 (Scheme 2) in 86 % yield [25].

Thus, reacting spirodione 4 with different substituted acetophenones 5a–o in the presence of pyrrolidine at \(80\, {^{\circ }}\hbox {C}\) for 10 h in EtOH provided the desired bis-spirochromanones 6a–o in moderate to good yields (Scheme 3, Fig. 3).

The synthesized bis-spirochromanones were characterized by \(^{1}\hbox {H NMR}\), \(^{13}\hbox {C } \hbox {NMR}\), Mass and FTIR spectral analysis; X-ray diffractometry confirmed the structure of compound 6e (CCDC 1405780) as shown in Fig. 4.

Fig. 2
figure 2

Designed bis-spirochromanone framework for the identification of novel antimycobacterial agents

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Scheme 1
scheme 1

Retro-synthetic strategy

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Scheme 2
scheme 2

Synthesis of spiro[chromane-2,1\(^\prime \)-cyclohexane]-4,4\(^\prime \)-dione 4

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Scheme 3
scheme 3

Synthesis of bis-spirochromanones

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Fig. 3
figure 3

Bis-spirochromanones with isolated yields

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Fig. 4
figure 4

ORTEP drawing (30% probability level) of compound 6e (CCDC 1405780)

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Antimycobacterial activity

Our 15-compound library was screened for in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv using the agar dilution method for the determination of MIC (the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation). The MIC values (\(\upmu \hbox {M}\)) of the synthesized compounds and three standard anti-tubercular drugs are presented in Table 1. As observed from Table 1, our bis-spirochromanones were found to be more potent than mono-spirochromanones.

The synthesized products have MIC values in the micromolar range, varying from \(5.3\,\hbox { to }\,68.3\, \upmu \hbox {M}\). Among them, nine compounds 6a, 6b, 6c, 6h, 6j, 6l, 6m,   6n and 6o with MIC values varying from \(5.3\,\hbox { to }\,35.9 \upmu \hbox {M}\) possess more inhibitory efficiency compared to that of standard pyrazinamide (\(\hbox {MIC} = 50.77\, \upmu \hbox {M}\)). The inhibitory efficiency of compound 6h with a MIC value 8.6 \(\upmu \hbox {M}\) was close to that of standard ethambutol (\(\hbox {MIC} = 7.64\, \upmu \hbox {M}\)). Compounds 6o and 6n with MIC values of 5.3 and \(6.4 \,\upmu \hbox {M}\), respectively, were found to be more potent than the first-line anti-tubercular drug ethambutol (\(\hbox {MIC} = 7.64 \,\upmu \hbox {M}\)). However, all our compounds exhibited poorer inhibitory efficiency compared to isoniazid (\(\hbox {MIC} = 0.72 \,\upmu \hbox {M}\)) and rifampicin (\(\hbox {MIC} = 0.24 \,\upmu \hbox {M}\)).

The antimycobacterial activity study results revealed that compounds 6h (with methyl substitution), 6o (with benzofuran scaffold) and 6n (with three chroman-4-one pharmacophores) exhibit promising antimycobacterial activity compared to the remaining substituted bis-spirochromanones synthesized in this study.

Table 1 MIC values of bis-spirochromanones against Mycobacterium tuberculosis
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Fig. 5
figure 5

Antimycobacterial activity comparison between compounds possessing different number of chroman-4-one pharmacophores

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Fig. 6
figure 6

Docking poses of II (pink color), 6a (green color) and 6n (orange color) deeply entrenched into the active site of FtsZ. (Color figure online)

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Fig. 7
figure 7

Docking pose and ligand interaction diagram of compound II, showing H-bond interaction with ASP-184

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Fig. 8
figure 8

Docking pose and ligand interaction diagram of compound 6a, showing H-bond interaction with GLY-105

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Fig. 9
figure 9

Docking pose and ligand interaction diagram of compound 6n, showing H-bond interactions with GLY-104, THR-106, ARG-140 and ARG-181

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Overall, the antimycobacterial activity of compound II, having one chroman-4-one pharmacophore, has been enhanced by tethering it with another chroman-4-one (Fig. 5). This strategy was further supported by docking studies.

Molecular docking studies

Melak et al. [26] reported 14 prospective drug targets of M. tuberculosis H37Rv prioritized based on their flow to resistance genes, among them glcB, katG, dnaN, sirA and FtsZ proteins have solved 3D structures. In order to get a suitable target to predict binding interactions, compounds II, 6a and 6n were docked against the above five protein targets, resulting in higher binding affinities against FtsZ.

FtsZ (filamenting temperature-sensitive mutant Z) is a prokaryotic homologue of the eukaryotic cytoskeleton tubulin [27], plays a crucial role in bacterial cell division and is highly preserved in a wide range of bacteria [28,29,30]. In the course of bacterial cytokinesis, FtsZ accumulates into a highly dynamic cytoskeleton scaffold (the Z-ring) by undergoing GTP-dependent polymerization, producing protofilaments at the site of septum formation [31, 32]. In addition, FtsZ activates other downstream proteins which are responsible for the invagination of cell membrane and septum formation, completing the bacterial cell division [33, 34]. Thus, FtsZ has been recognized as an attractive target to develop novel antibacterial agents [35, 36].

In order to explain the effect of the number of chroman-4-one units on binding activity, compounds II (one chroman-4-one), 6a (two chroman-4-ones) and 6n (three chroman-4-ones) were docked into the FtsZ domain of M. tuberculosis H37Rv, and the overlay of docked poses of II, 6a and 6n is shown in Fig. 6. The docked poses with ligand interactions between target protein and these compounds can be identified by docking analysis and are depicted in Figs. 7, 8 and 9. It should be noted that compound 6n (\(\hbox {docking score} =-6.52 \hbox { Kcal/mol}\)) showed more hydrogen bond interactions compared to 6a (\(\hbox {docking score} = -5.76 \hbox { Kcal/mol}\)) and compound II (\(\hbox {docking score} = -4.99 \hbox { Kcal/mol}\)). In addition, the surface area of compounds 6n, 6a and II in the active site cavity was found to be 408, 301 and \(207\,{\AA }^{2}\), respectively (Fig. 10). It is clearly showed that due to the small size of compound II, it is located at one corner of cavity that leads to less binding interactions with FtsZ. On the other hand, due to the large size of compound 6n, it is well accommodated in the active site cavity and engages in more binding interactions with target. Thus, the addition of chroman-4-one rings is expected to contribute with more binding interactions with the receptor. On the whole, an increase in the number of chroman-4-one rings on the core nucleus contributed to more potent activity and is also supported by the docking results.

Fig. 10
figure 10

Surface area and length of compounds 6n (orange), 6a (green) and II (pink) in the active site of FtsZ. (Color figure online)

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Cytotoxicity

Compounds 6b, 6c, 6h, 6l, 6m, 6n and 6o exhibited good in vitro antimycobacterial potency and were further evaluated for their toxicity in a HEK cell line at a concentration of \(50\, \upmu \hbox {g/mL}\) using the Promega Cell Titer 96 non-radioactive cell proliferation assay [37]. The most promising anti-TB compounds 6h, 6n and 6o showed 17, 19 and 17% cytotoxicity, respectively.

Conclusion

In summary, a 15-compound library of spirochromanones containing more than one chroman-4-one group was synthesized using a Kabbe condensation approach. The identity of compound 6e was established by single crystal X-ray diffraction. The in vitro antimycobacterial activity results revealed that compounds 6h, 6n and 6o showed promising potency. Molecular docking studies against FtsZ revealed that the addition of chromone groups in compounds 6a and 6n contributed to more interactions with the receptor than compound II.

Experimental section

General information

All the reactions were performed using oven-dried apparatus. All chemicals and reagents used in this work were purchased from Sigma-Aldrich and were used as received without further purification. Reactions were monitored by thin layer chromatography (TLC) on silica gel plates \((60 \hbox { F}_{254})\), visualizing using ultraviolet light. Column chromatography was performed on silica gel (60–120 mesh) using distilled hexane, ethyl acetate and acetone. \(^{1}\hbox {H } \hbox {NMR}\) and \(^{13}\hbox {C} \hbox { NMR}\) spectra were recorded on a Bruker Avance II 400 MHz in \(\hbox {CDCl}_{3}\). Proton chemical shifts (\(\updelta \)) are relative to tetramethylsilane (TMS, \(\updelta = 0.00\)) as internal standard and expressed in ppm. Spin multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and bs (broad). Coupling constants (J) are given in Hertz. Mass spectra were recorded on a GCMS-QP 1000 EX mass spectrometer. Melting points were determined using a Stuart SMP3 melting point apparatus and are uncorrected.

General experimental procedure

To a solution of spiro[chromane-2,1\(^\prime \)-cyclohexane]-4,4\(^\prime \)-dione (4) (1 mmol) in dry ethanol, a catalytic amount of pyrrolidine was added followed by a substituted 2\(^\prime \)-hydroxyacetophenone analog (5a–o) (1 mmol). The reaction mixture was refluxed for 10 h with constant stirring. The solvent was then removed under reduced pressure, and the residue was extracted with ethyl acetate. The organic extract was washed with 1M HCl, 1M NaOH and finally brine. After this, the organic extract was dried over \(\hbox {Na}_{2}\hbox {SO}_{4}\), filtered and concentrated under reduced pressure. The resulting residue was purified by column chromatography (hexane/acetone 7:3) to afford desired dispiro[chromane-2,1\(^\prime \)-cyclohexane-4\(^\prime \),2\(^{\prime \prime }\)-chromane]-4,4\(^{\prime \prime }\)-diones (6a–o).

Dispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6a):

Colorless solid. Yield: 70%. Melting point: \(88\text {--}90 \,{^{\circ }}\hbox {C}\);IR (\(\hbox {KBr}, \hbox {cm}^{-1})\): 3352, 2932, 1720, 1682, 1456, 1228; \(^{1}\hbox {H} \hbox { NMR}\) (\(400 \hbox { MHz}, \hbox {CDCl}_{3})\) \(\delta \) ppm 1.84–1.90 (m, 4H), 1.96–2.01 (m, 4H), 2.75 (s, 4H), 6.96–7.02 (m, 4H, Ar–H), 7.47–7.51 (m, 2H, Ar–H), 7.85–7.87 (\(\hbox {d}\), \(J = 7.52 \hbox { Hz}\), 2H, Ar–H); \(^{13}\hbox {C} \hbox { NMR}\) (\(100 \hbox { MHz}, \hbox {CDCl}_{3})\) \(\delta \) ppm 29.3, 48.5, 78.3, 118.2, 120.7, 121.1, 126.6, 136.2, 159.1, 191.9; \(\hbox {MS } (\hbox {ESI mass}): {m/z}\,349\, [\hbox {M}+\hbox {H}]^{+}\).

6-Bromodispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^\prime \) ,2 \(^{\prime \prime }\) -dione (6b):

White solid. Yield: 90%. \(\hbox {Melting point}:\,176\text {--}178 \,{^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3336, 2915, 1724, 1683, 1448, 1215; \(^{1}\hbox {H} \hbox { NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3})\) \(\delta \) ppm 1.82–1.87 (m, 4H), 1.94–2.02 (m, 4H), 2.74 (s, 4H), 6.88–6.90 (\(\hbox {d}, J = 8.78 \hbox { Hz}, 1\hbox {H}, \hbox {Ar}{-}\hbox {H}\)), 6.96–7.03 (m, 2H, Ar–H), 7.48–7.51 (m, 1H, Ar–H), 7.55–7.58 (dd, \(J = 8.78\hbox {Hz}\), 1.75Hz, 1H, Ar–H), 7.85–7.87 (d, \(J = 7.52 \hbox { Hz}\), 1H, Ar–H), 7.97 (d, \(J = 2.0\hbox {Hz}\), 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3})\,\delta \) ppm 29.2, 48.2, 48.5, 78.2, 78.9, 113.8, 118.1, 120.2, 120.7, 121.2, 121.9, 126.6, 129.1, 136.3, 138.8, 157.9, 159.0, 190.6, 191.8; \(\hbox {MS } (\hbox {ESI mass}): {m/z}\, 427 \,[\hbox {M}+\hbox {H}]^{+}\).

6-Chlorodispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6c):

Light yellow solid. Yield: 92%. \(\hbox {Melting point}: 188\text {--}190\, {^{\circ }}\hbox {C};\hbox {IR}\, (\hbox {KBr}, \hbox {cm}^{-1})\): 3324, 2916, 1732, 1684, 1463, 1217; \(^{1}\hbox {H} \hbox { NMR}\, (400 \hbox { MHz}, \hbox {CDCl}_{3})\) \(\delta \) ppm 1.81–1.87 (m, 4H), 1.97–2.02 (m, 4H), 2.74 (s, 4H), 6.93–7.02 (m, 3H, Ar–H), 7.42–7.45 (dd, \(J = 8.78\hbox { Hz}\), 2.76 Hz, 1H, Ar–H), 7.47–7.52 (m, 1H, Ar–H), 7.82 (d, \(J = 2.51 \hbox { Hz}\), 1H, Ar–H), 7.85–7.87 (dd, \(J = 7.78 \hbox { Hz}\), 1.75 Hz, 1H, Ar–H); \(^{13}\hbox {C} \hbox { NMR } (100 \hbox { MHz}, \hbox {CDCl}_{3})\,\delta \) ppm 29.27, 29.29, 48.2, 48.5, 78.2, 78.9, 118.1, 119.9, 120.7, 121.2, 121.4, 126.0, 126.6, 126.7, 136.1, 136.3, 157.5, 159.0, 190.8, 191.8; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 383\, [\hbox {M}+\hbox {H}]^{+}\).

6-Chloro-7-methyldispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6d):

White solid. Yield: 88%. \(\hbox {Melting point}: 217\text {--}218 {^{\circ }}\hbox {C}; \hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3361, 2920, 1723, 1688, 1462, 1225; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}\), \(\hbox {CDCl}_{3})\, \delta \) ppm 1.78–1.89 (m, 4H), 1.93–2.04 (m, 4H), 2.38 (s, 3H), 2.71 (s, 2H), 2.74 (s, 2H), 6.88 (s, 1H, Ar–H), 6.96–7.03 (m, 2H, Ar–H), 7.49–7.50 (m, 1H, Ar–H), 7.81 (s, 1H, Ar–H), 7.85–7.88 (dd, \(J = 7.78 \hbox { Hz}\), 1.50 Hz, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR} (100 \hbox { MHz}, \hbox {CDCl}_{3})\, \delta \) ppm 20.8, 29.3, 48.2, 48.5, 78.2, 78.7, 118.2, 119.7, 120.3, 120.7, 121.1, 126.4, 126.6, 127.3, 136.3, 145.3, 157.3, 159.0, 190.6, 191.9; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 397\, [\hbox {M}+\hbox {H}]^{+}\).

6,8-Dichlorodispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6e):

White solid. Yield: 86%. \(\hbox {Melting point}: 236\text {--}238 \,{^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3340, 2936, 1745, 1690, 1452, 1210; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3})\) \(\delta \) ppm 1.89–2.02 (m, 8H), 2.76–2.79 (m, 4H), 6.96–7.03 (m, 2H, Ar–H), 7.47–7.51 (m, 1H, Ar–H), 7.56 (s, 1H, Ar–H), 7.75 (s, 1H, Ar–H), 7.86–7.88 (d, \(J=\) 7.52 Hz, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \, \delta \) ppm 29.3, 47.9, 48.5, 78.1, 80.4, 118.1, 120.7, 121.2, 122.3, 124.5, 124.8, 126.4, 126.7, 135.7, 136.2, 153.3, 159.0, 190.0, 191.8; \(\hbox {MS} (\hbox {ESI mass}): {m/z }\, 417 \,[\hbox {M}+\hbox {H}]^{+}\).

7-Ethoxydispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6f):

Cream solid. Yield: 85%. \(\hbox {Melting point}: 144\text {--}146 \,{^{\circ }}\hbox {C}; \hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3328, 2942, 1718, 1678, 1468, 1232; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3})\, \delta \) ppm 1.42–1.45 (t, \(J = 7.02 \hbox {Hz}, \hbox {3H}\)), 1.80–1.89 (m, 4H), 1.96–2.03 (m, 4H), 2.68 (s, 2H), 2.74 (s, 2H), 4.05–4.10 (q, \(J = 7.02 \hbox { Hz}\), 2H), 6.39–6.40 (d, \(J = 2.51 \hbox { Hz}\), 1H, Ar–H), 6.53–6.56 (dd, \(J = 8.78 \hbox { Hz}\), 2.25 Hz, 1H, Ar–H), 6.96–7.02 (m, 2H, Ar–H), 7.47–7.51 (m, 1H, Ar–H), 7.78–7.80 (d, \(J=8.78\) Hz, 1H, Ar–H), 7.85–7.87 (dd, \(J = 7.78 \hbox { Hz}\), 1.50 Hz, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR } (100 \hbox { MHz}, \hbox {CDCl}_{3})\, \delta \) ppm 14.6, 29.41, 29.48, 48.2, 48.5, 64.0, 78.3, 78.7, 101.7, 109.8, 114.4, 118.2, 120.7, 121.1, 126.6, 128.3, 136.3, 159.1, 161.1, 165.7, 190.4, 192; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 393 \,[\hbox {M}+\hbox {H}]^{+}\).

6-Fluorodispiro[chromane-2,1 \(^\prime \) -cyclohexane-4 \(^\prime \) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6g):

White solid. Yield: 88%. \(\hbox {Melting point}: 116\text {--}118\, {^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3348, 2917, 1741, 1687, 1460, 1224; \(^{1}\hbox {H} \hbox { NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3})\, \delta \) ppm 1.82–1.86 (m, 4H), 1.97–2.01 (m, 4H), 2.746–2.749 (m, 4H), 6.94–7.02 (m, 3H, Ar–H), 7.19–7.24 (m, 1H, Ar–H), 7.47–7.53 (m, 2H, Ar–H), 7.85–7.87 (dd, \(J = 7.78 \hbox { Hz}\), 1.50 Hz, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3})\,\delta \) ppm 29.2, 29.3, 48.2, 48.5, 78.2, 78.6, 111.6, 111.9, 118.2, 119.7, 119.8, 120.7, 121.1, 123.6, 123.8, 126.6, 136.3, 155.2, 155.8, 158.2, 159.0, 191.1, 191.8; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 367 \,[\hbox {M}+\hbox {H}]^{+}\).

6-Methyldispiro[chromane-2,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) -dione (6h):

White solid. Yield: 84%. \(\hbox {Melting point}: 160\text {--}162 \,{^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3354, 2918, 1728, 1685, 1466, 1230; \(^{1}\hbox {H} \hbox { NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.83–1.85 (m, 4H), 1.97–1.99 (m, 4H), 2.30 (s,3H), 2.72–2.74 (m, 4H), 6.87–6.88 (m, 1H, Ar–H), 6.96–7.00 (m, 2H, Ar–H), 7.30–7.31 (s, 1H, Ar–H), 7.48–7.50 (m, 1H, Ar–H), 7.65 (s, 1H, Ar–H), 7.85–7.87 (m, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \, \delta \) ppm 20.3, 29.30, 29.34, 48.5, 48.6, 78.1, 78.4, 118.0, 118.2, 120.3, 120.7, 121.1, 126.2, 126.6, 130.5, 136.2, 137.3, 157.1, 159.1, 192.0, 192.2; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 363 \,[\hbox {M}+\hbox {H}]^{+}\).

8 \(^{\prime \prime }\),8 \(^{\prime \prime }\) -Dimethyl- 7 \(^{\prime \prime }\),8 \(^{\prime \prime }\) -dihydro-6 \(^{\prime \prime }\) H -dispiro[chromane-2,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\) ,2 \(^{\prime \prime }\) -pyrano[3,2-g]chromene]-4,4 \(^{\prime \prime }\)(3 \(^{\prime \prime }\) H)- dione (6i:)

Light yellow solid. Yield: 89%. \(\hbox {Melting point}: 166\text {--}168\, {^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3364, 2921, 1727, 1675, 1445, 1236; \(^{1}\hbox {H} \hbox { NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.34 (s, 6H), 1.78–7.85 (m, 6H), 1.96–1.99 (m, 4H), 2.65 (s, 2H), 2.72–2.75 (m, 4H), 6.31 (s, 1H, Ar–H), 6.94–7.01 (m, 2H, Ar–H), 7.46–7.50 (m, 1H, Ar–H), 7.61 (s, 1H, Ar–H), 7.85–7.87 (d, \(J = 7.52 \hbox { Hz}\), 1H, Ar–H); \(^{13}\hbox {C} \hbox { NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 21.5, 27.0, 29.42, 29.49, 32.6, 48.4, 48.6, 75.9, 78.2, 78.5, 104.8, 114.2, 115.3, 118.2, 120.7, 121.0, 126.6, 127.9, 136.2, 159.0, 159.1, 161.4, 190.8, 192.0; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 433 \,[\hbox {M}+\hbox {H}]^{+}\).

9 \(^{\prime \prime }\),9 \(^{\prime \prime }\) -Dimethyl-8 \(^{\prime \prime }\),9 \(^{\prime \prime }\) -dihydro-3 \(^{\prime \prime }\) H -dispiro[chromane-2,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\),2 \(^{\prime \prime }\) -pyrano[3,2-h]chromene]-4,4 \(^{\prime \prime }\)(7 \(^{\prime \prime }\) H)-dione (6j):

Light yellow solid. Yield: 86%. \(\hbox {Melting point}: 122\text {--}124\, {^{\circ }}\hbox {C}; \hbox {IR}\, (\hbox {KBr}, \hbox {cm}^{-1})\): 3362, 2923, 1729, 1677, 1448, 1234; \(^{1}\hbox {H } \hbox {NMR}\, (400 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.36 (s, 6H), 1.74–1.83 (m, 6H), 2.01–2.02 (m, 4H), 2.63–2.75 (m, 6H), 6.43–6.45 (m, 1H, Ar–H), 6.995–6.998 (m, 2H, Ar–H), 7.49–7.50 (m, 1H, Ar–H), 7.64–7.66 (m, 1H, Ar–H), 7.86–7.87 (m, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 21.5, 26.7, 29.5, 29.7, 31.7, 48.0, 48.8, 75.5, 78.3, 78.6, 109.1, 111.4, 114.2, 115.3, 118.2, 120.7, 121.1, 125.5, 136.4, 159.0, 159.1, 160.8, 190.6, 192.1; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 433 \,[\hbox {M}+\hbox {H}]^{+}\).

Dispiro[benzo[h]chromene-2,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\) ,2 \(^{\prime \prime }\) -chromane]-4,4 \(^{\prime \prime }\) (3 H )-dione (6k):

White solid. Yield: 88%. \(\hbox {Melting point}: 204\text {--}206 \,{^{\circ }}\hbox {C}; \hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3342, 2912, 1712, 1680, 1454, 1212; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3}) \, \delta \) ppm 1.84–1.96 (m, 4H), 2.01–2.12 (m, 4H), 2.76 (s, 2H), 2.86 (s, 2H), 6.98–7.02 (m, 2H, Ar–H), 7.10–7.12 (d, \(J = 9.03 \hbox { Hz}\), 1H, Ar–H), 7.39–7.43 (m, 1H, Ar–H), 7.48–7.52 (m, 1H, Ar–H), 7.60–7.64 (m, 1H, Ar–H), 7.73–7.75 (d, \(J = 8.03 \hbox { Hz}\), 1H, Ar–H), 7.85–7.88 (dd, \(J = 7.78 \hbox { Hz}\), 1.75 Hz, 1H, Ar–H), 7.93–7.95 (d, \(J = 9.03 \hbox { Hz}\), 1H, Ar–H), 9.41–9.43 (d, \(J = 8.53 \hbox { Hz}\), 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \, \delta \) ppm 29.0, 29.4, 48.5, 49.7, 78.3, 78.5, 112.0, 118.2, 119.0, 120.7, 121.1, 124.7, 125.6, 126.6, 128.3, 129.0, 129.6, 131.3, 136.3, 137.6, 159.1, 161.1, 192.0, 193.0; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 399 \,[\hbox {M}+\hbox {H}]^{+}\).

Dispiro[benzo[f]chromene-3,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\) ,2 \(^{\prime \prime }\) -chromane]-1,4 \(^{\prime \prime }\) (2H )-dione (6l):

White solid. Yield: 86%. \(\hbox {Melting point}: 218\text {--}220 \,{^{\circ }}\hbox {C};\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3339, 2913, 1716, 1686, 1450, 1208; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox {MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.95–2.18 (m, 8H), 2.80 (s, 2H), 2.86 (s, 2H), 6.99–7.03 (m, 2H, Ar–H), 7.39–7.41 (m,1H, Ar–H), 7.49–7.53 (m,1H, Ar–H), 7.54–7.58 (m, 1H, Ar–H), 7.62–7.66 (m, 1H, Ar–H), 7.80–7.82 (d, \(J = 8.03 \hbox { Hz}\), 1H, Ar–H), 7.84–7.85 (m, 2H, Ar–H), 8.23–8.25 (m, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR } (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 29.5, 29.8, 48.0, 48.7, 78.3, 79.6, 115.0, 118.2, 120.6, 120.7, 121.2, 121.5, 122.8, 125.3, 126.5, 126.6, 128.1, 129.6, 136.3, 137.7, 157.0, 159.1, 191.5, 192.0; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 399 \,[\hbox {M}+\hbox {H}]^{+}\).

4 \(^{\prime \prime }\) -Methyl-2 \(^{\prime \prime }\) H -dispiro[chromane-2,1 \(^{\prime }\)-cyclohexane-4 \(^{\prime }\),8 \(^{\prime \prime }\) -pyrano[2,3-f]chromene]- 2 \(^{\prime \prime }\),4,10 \(^{\prime \prime }\) (9 \(^{\prime \prime }\) H )-trione (6m):

Colorless solid. Yield: 68%. \(\hbox {Melting point}: 202\text {--}204 \,{^{\circ }}\hbox {C};\hbox {IR}\, (\hbox {KBr}, \hbox {cm}^{-1})\): 3355, 2940, 1745, 1680, 1673, 1458, 1206; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox {MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.70–2.08 (m, 8H), 2.40 (s, 3H), 2.75 (s, 1H), 2.78 (s, 1H), 2.81 (s, 1H), 2.88 (s, 1H), 6.20 (s, 1H, Ar–H), 6.92–7.03 (m, 3H, Ar–H), 7.48–7.53 (m, 1H, Ar–H), 7.68–7.70 (d, \(J = 8.78 \hbox { Hz}\), 1H, Ar–H), 7.85–7.87 (m, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 19.0, 29.26, 29.29, 30.3, 30.6, 48.4, 49.5, 78.1, 79.6, 109.3, 112.6, 113.9, 114.4, 114.6, 118.2, 121.2, 126.7, 131.0, 136.4, 151.8, 153.2, 159.0, 159.9, 162.1, 188.3, 191.8; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 431 \,[\hbox {M}+\hbox {H}]^{+}\).

Trispiro[chromane-2,1 \(^{\prime }\) -cyclohexane-4 \(^{\prime }\),2 \(^{\prime \prime }\) -pyrano[3,2-g]chromene-8 \(^{\prime \prime }\) ,1 \(^{\prime \prime \prime }\) -cyclohexane]-4,4 \(^{\prime \prime }\) ,6 \(^{\prime \prime }\) (3 \(^{\prime \prime }\) H 7 \(^{\prime \prime }\) H )-trione (6n):

White solid. Yield: 80%. \(\hbox {Melting point}: 178\text {--}180 \,{^{\circ }}\hbox {C}; \hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3362, 2938, 1720, 1701, 1684, 1460, 1232; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 1.48–1.56 (m, 4H), 1.63–1.75 (m, 4H), 1.83–2.03 (m, 10H), 2.68 (s, 2H), 2.72 (s, 2H), 2.75 (s, 2H), 6.52 (s, 1H, Ar–H), 6.96–7.03 (m, 2H, Ar–H), 7.48–7.52 (m, 1H, Ar–H), 7.85–7.88 (dd, \(J = 7.78 \hbox { Hz}\), 1.50 Hz, 1H, Ar–H), 8.47 (s, 1H, Ar–H); \(^{13}\hbox {C} \hbox { NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 21.4, 25.0, 29.3, 29.6, 35.0, 47.9, 48.3, 48.5, 78.1, 79.5, 81.4, 105.4, 115.5, 116.0, 118.2, 120.7, 121.2, 126.6, 127.8, 136.3, 159.0, 164.5, 165.2, 189.8, 190.4, 191.8; \(\hbox {MS } (\hbox {ESI mass}): {m/z }\, 487 \,[\hbox {M}+\hbox {H}]^{+}\).

2 \(^{\prime \prime }\) -(4-Bromobenzoyl)- 3 \(^{\prime \prime }\) -methyldispiro[chromane-2,1 \(^{\prime }\) - cyclohexane-4 \(^{\prime }\) ,7 \(^{\prime \prime }\) -furo[3,2-g]chromene]-4,5 \(^{\prime \prime }\) (6 \(^{\prime \prime }\) H )-dione (6o):

White solid. Yield: 75%. \(\hbox {Melting point}: 218\text {--}250 \,{^{\circ }}\hbox {C}\);\(\hbox {IR} (\hbox {KBr}, \hbox {cm}^{-1})\): 3368, 2948, 1725, 1686, 1670, 1444, 1222; \(^{1}\hbox {H } \hbox {NMR} (400 \hbox { MHz}\), \(\hbox {CDCl}_{3}) \,\delta \) ppm 1.87–2.01 (m, 8H), 2.63 (s, 3H), 2.76 (s, 2H), 2.83 (s, 2H), 6.97–7.04 (m, 2H, Ar–H), 7.09 (s, 1H, Ar–H), 7.48–7.53 (m, 1H, Ar–H), 7.66–7.68 (m, 2H, Ar–H), 7.86–7.88 (dd, \(J = 7.78 \hbox { Hz}\), 1.50 Hz, 1H, Ar–H), 7.93–7.96 (m, 2H, Ar–H), 8.31 (s, 1H, Ar–H); \(^{13}\hbox {C } \hbox {NMR}\, (100 \hbox { MHz}, \hbox {CDCl}_{3}) \,\delta \) ppm 10.0, 29.3, 29.5, 48.5, 48.6, 78.2, 79.2, 100.5, 118.1, 120.7, 121.2, 121.3, 124.3, 126.7, 127.9, 128.3, 131.1, 131.2, 131.7, 136.3, 158.8, 159.0, 159.6, 161.3, 184.1, 191.4, 191.8; \(\hbox {MS} (\hbox {ESI mass}): {m/z}\, 585 \,[\hbox {M}+\hbox {H}]^{+}\).

Antimycobacterial activity

In vitro MTB screening

Twofold serial dilutions of each test compound/drug were prepared and incorporated into Middlebrook 7H11 agar medium with oleic acid, albumin, dextrose and catalase (OADC) growth supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and \(0.78\,\upmu \hbox {g/mL}\). Inoculum of M. tuberculosis H37Rv ATCC 27294 was prepared from fresh Middlebrook 7H11 agar slants with OADC (Difco) growth supplement adjusted to 1 mg/mL (wet weight) in Tween 80 (0.05%) saline diluted to \(10^{-2}\) to give a concentration of \({\sim }10^{7} \hbox { cfu/mL}\). Five microliters of this bacterial suspension was spotted onto 7H11 agar tubes containing different concentrations of the drug as discussed above. The tubes were incubated at \(37\, {^{\circ }}\hbox {C}\), and final readings (MIC in \(\upmu \hbox {g/mL}\)) were determined after 28 days. Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. This method is similar to that recommended by the National Committee for Clinical Laboratory Standards for the determination of MIC in duplicates.

In vitro cytotoxicity screening

The compounds displaying good in vitro potency in the MTB MIC were further examined for toxicity in a HEK cell line at a concentration of \(50 \,\upmu \hbox {g/mL}\). After 72 h of exposure, viability was assessed on the basis of cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 non-radioactive cell proliferation assay [37].

Molecular docking-experimental protocol

For docking of ligands into a target’s active site, and to estimate the binding interactions of docked conformations, GLIDE version 5.6 [38] was used in this study. For our studies, the X-ray crystal structure of FtsZ domain of M. tuberculosis was retrieved from the Protein Data Bank (PDB ID: 4KWE) [39, 40]. The protein was prepared using the protein preparation wizard in Maestro 9.0 applying default parameters; a grid was centered around the active site by selecting the co-crystallized ligand and the receptor van der Waals scaling for nonpolar atoms was kept at 0.9. Molecules were built using Maestro’s build panel and prepared using LigPrep 2.0. The lowest energy conformation of the ligands was selected and docked into the generated grid using an Extra Precision (XP) docking mode.

Supplementary material

\(^{1}\hbox {H}\), \(^{13}\hbox {C}-\hbox {NMR}\) spectra and supplementary crystallographic data of CCDC 1405780 are available in the supplementary material.

Note: CCDC 1405780 contains supplementary crystallographic data for the compound 6e. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.