Efficient Synthesis and Anti-Tubercular Activity of a Series of Spirocycles: An Exercise in Open Science

Katrina A. Badiola; Diana H. Quan; James A. Triccas; Matthew H. Todd

doi:10.1371/journal.pone.0111782

Abstract

Tuberculosis afflicts an estimated 2 billion people worldwide and causes 1.3 million deaths annually. Chemotherapeutic solutions rely on drugs developed many years ago, with only one new therapeutic having been approved in the last 40 years. Given the rise of drug-resistant strains, there is an urgent need for the development of a more robust drug development pipeline. GlaxoSmithKline recently placed the structures and activities of 177 novel anti-tubercular leads in the public domain, as well as the results of ongoing optimisation of some of the series. Since many of the compounds arose from screening campaigns, their provenance was unclear and synthetic routes were in many cases not reported. Here we present the efficient synthesis of several novel analogues of one family of the GSK compounds—termed “Spiros”—using an oxa-Pictet–Spengler reaction. The new compounds are attractive from a medicinal chemistry standpoint and some were potent against the virulent strain, suggesting this class is worthy of further study. The research was carried out using open source methodology, providing the community with full access to all raw experimental data in real time.

Citation: Badiola KA, Quan DH, Triccas JA, Todd MH (2014) Efficient Synthesis and Anti-Tubercular Activity of a Series of Spirocycles: An Exercise in Open Science. PLoS ONE 9(12): e111782. https://doi.org/10.1371/journal.pone.0111782

Editor: Kamyar Afarinkia, Univ of Bradford, United Kingdom

Received: May 14, 2014; Accepted: September 30, 2014; Published: December 10, 2014

Copyright: © 2014 Badiola et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. General synthetic and analytical methods are detailed in Text S1. Raw NMR data for all compounds are available from The University of Sydney eScholarship Repository [http://hdl.handle.net/2123/10506]. The open electronic laboratory notebook for experiments carried out April–August 2013 is available from The University of Sydney eScholarship Repository [http://hdl.handle.net/2123/10461]; all other experiments are summarised in Spreadsheet S1.

Funding: This work was supported by the University of Sydney (KAB, MHT) and the National Health and Medical Research Council Centre of Research Excellence on Tuberculosis Control (APP1043225)(JAT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: Matthew Todd is a PLOS ONE Editorial Board Member (as an Associate Editor). This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.

Introduction

Infection by M. tuberculosis resulting in symptomatic tuberculosis (TB) can be fatal without treatment. In 2012, TB was responsible for the deaths of 1.3 million people and a further 8.6 million people were infected [1]. Globally, an estimated two billion people carry latent TB and are susceptible to developing active TB. Current first-line treatments include the “short-course-chemotherapy” regime, which involves combinations of rifampicin, isoniazid, pyrazinamide and ethambutol, taken over at least 6 months [2]. These drugs have been in use since the 1960s; the recent FDA approval of bedaquiline [3] makes this drug the first new treatment for TB to be approved in 40 years. The spread of partially- and totally drug resistant strains makes the development of new treatments (preferably targeting new cellular mechanisms) a priority [1].

GlaxoSmithKline (GSK) recently published the structures and anti-TB activities of 177 small molecules as part of a deposition of open data [4]. These leads were identified out of a pool of ∼20000 molecules, chosen from the GSK corporate compound collection based on favourable cell permeability and drug-like parameters. Of the 177 leads, seven compounds contained a thiophene spirocycle core; these were termed Spiros by GSK, represented by GSK2200150A (Figure 1).

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Figure 1.

The GSK HTS campaign identified GSK2200150A, which is representative of the GSK Spiros family of anti-TB leads (A). (B) The optimised Spiros analogue developed by GSK [5]. (C) Existing anti-tubercular candidates that have a mode of action that involves MmpL3.

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The members of the Spiros series are excellent starting points for the development of new anti-TB agents. The compounds were identified following a number of screens that evaluated their inhibition of the growth of mycobacteria, cytotoxicity and physical properties. The Spiros appear to affect an essential membrane transport protein (MmpL3) of M. tuberculosis [5]. There are no currently-approved drugs that target MmpL3, but four structurally dissimilar compounds (C, Figure 1) have been identified as acting on MmpL3 [6]–[9] as well as a more recent set of indoleamides [10]. In 2012 SQ109 completed a phase IIa clinical trial for pulmonary TB [11]. The Spiros analogues are not overtly similar in structure to these compounds; further investigation into the specific mode of action at MmpL3 is clearly required, but it is a desirable property of any new antitubercular compound that it should have a target different to existing therapeutics.

Screening campaigns frequently use commercial libraries that understandably lack synthetic provenance. The synthesis of the secondary amine spirocycle core has been incompletely reported in the patent literature and a synthesis of the GSK hit compound (GSK2200150A) was described in the academic literature but with incomplete data and limited information on analog synthesis. [4]–[5], [12]. We rationalised that Spiros analogues could be rapidly produced by first constructing the core using an oxa-Pictet–Spengler reaction followed by final stage diversification from the secondary amine 3 (Figure 2). This would allow the rapid synthesis of new Spiros analogues in three steps and the faster progression of this series in a hit-to-lead campaign.

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Figure 2. Potential for the rapid synthesis of Spiros analogues via a common 2° amine intermediate 3.

(A) An existing oxa-Pictet–Spengler reaction can be used to form the spirocycle core (blue) as the 2° amine 3 [12]. (B) Strategy to diversify from the 2° core 3 to produce Spiros analogues (C) with variation at the piperidine nitrogen (red).

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We herein describe this work which was conducted using an electronic laboratory notebook on the internet [13] and an open source research philosophy that had shown efficiency gains in the discovery of a synthetic route to a drug used in the treatment of schistosomiasis [14]. The licence governing such work is that the research may be used for any purpose, including for financial gain, provided the project is cited. The relevant laboratory notebook, containing a browseable snapshot of the experiments and all the data for the period April–August 2013, has been deposited online [15]. Data for remaining experiments (period late August–December 2013) are included in Spreadsheet S1 rather than the electronic laboratory notebook due to a local technical difficulty at the time these data were collected.

Results and Discussion

Synthesis of the Spiros Analogues

Our first synthetic approach was the use of compound 5 to make 3 with an oxa-Pictet Spengler reaction (Figure 3). We adapted the patent procedure (A, Figure 2) [12] by using 4-piperidone 5, instead of the corresponding ketal 2, since substitution of the ketal for the ketone in the oxa-Pictet–Spengler reaction should have a limited effect the reaction outcome [16]. The ketone is commercially available but was easily prepared from commercial 4-benzylpiperidinone 4 (A, Figure 3) [17] a compound that was itself later used. However, the harsh conditions of the cyclization (excess triflic acid) resulted in the formation of intractable mixtures.

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Figure 3. Synthesis of the core 70 was attempted using conditions adapted from the literature [12].

(A) N -Debenzylation of 4 was achieved under palladium-catalysed transfer hydrogenation conditions [17]. (B) Reaction of the ketone 5 with thiopheneethanol 1 in the presence of strong Brønsted acids did not produce the expected spirocycle 3.

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Several reaction variables were explored to promote cyclisation, such as reducing the amount of acid, using the less acidic methanesulfonic acid [18]–[19], introducing a non-polar solvent (toluene or dioxane) and heating the reaction. These efforts resulted in either partial recovery of the thiophene starting material along with a complex product mixture or decomposition and the formation of polar compounds that were not identified.

The ketal 2 may therefore be crucial in this transformation where precise tuning of reactivity and conditions is necessary to promote conversion but not decomposition. However, formation of this ketal would unfavourably introduce another step in the synthesis (A, red, Figure 4). We therefore devised an alternate strategy for accessing the secondary amine 3: carrying out the oxa-Pictet–Spengler reaction using 4 prior to removal of the N-benzyl group (B).

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Figure 4. Re-evaluating the route to the 2° amine core 3 (blue).

(A) The attempted route based on the patent literature procedure [12]. The dotted lines represent the additional step (red) required to attempt cyclisation strictly under the patent conditions. (B) The revised strategy: cyclise to give 6 followed by debenzylation to give the 2° amine 3 (blue).

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The first step of the revised strategy proved effective; the N-benzylated core (6) was obtained in consistently good yield over a number of repeats (A, Figure 5). The reaction was promoted by 1.5 equivalents of methanesulfonic acid instead of triflic acid, the former being easier to handle. To counter the reduced acidity, the reaction temperature was increased. Lowering the acid loading or reaction time gave a mixture of the product 6 and starting material; the ketone 4 was inseparable from the product 6 in the post-reaction workup or by flash chromatography. The methanesulfonic acid-mediated reaction initially carried out was the most effective at cleanly obtaining the desired spirocycle 6.

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Figure 5. Executing the revised strategy towards the synthesis of spirocycle core as the 2° amine 3.

(A) The acid-mediated cyclisation. (B) Attempts to synthesise the 2° amine using catalytic hydrogenolysis. (C) 1-chloroethyl chloroformate was effective at producing the secondary amine 3. (D) 2-chloroethyl chloroformate resulted in incomplete deprotection of 6.

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The next step was the removal of the N-benzyl group from 6 to give the secondary amine 3 (B to D, Figure 5). The N-benzylated spirocycle 6 was stable to a variety of common palladium-catalysed hydrogenolysis conditions (B). The transfer hydrogenation conditions used in the preparation of 4-piperidinone 5 were ineffective; starting material 6 was recovered. Subsequent attempts were made using different combinations of pressure (hydrogen gas up to 8.3 bar), transfer hydrogenation and extended reaction times. In all attempts, starting material was recovered with minimal loss of material.

We turned instead to debenzylation conditions mediated by 1-chloroethyl chloroformate 7 (green), which gave the expected secondary amine 3 (blue) in excellent yield (C, Figure 5). The debenzylation proceeds presumably via a carbamate intermediate following the reaction of the starting material 6 with the chloroformate 7 and loss of benzyl chloride [20]–[21]. Subsequent decarboxylation, promoted by the excess of methanol and reflux conditions, produced the desired secondary amine 3 [20]–[21]. Isolation of the carbamate 9 when 2-chloroethyl chloroformate 8 was used is consistent with the proposed mechanism (D); the initial N-debenzylation step would be unaffected given the similar reactivity of the chloroformate functional groups, but methanol attack on the secondary carbon to lose the β-chloride would be less likely than attack on the tertiary carbon to lose the α-chloride. The secondary amine 3 was obtained, ready for final stage diversification in ∼84% yield over two steps.

Diversification from the secondary amine 3 using reductive amination and acylating conditions enabled the rapid synthesis of a variety of compounds in moderate to good yield (Figure 6). The sodium triacetoxyborohydride-mediated reductive amination procedure was adapted from the literature [22]–[23]. Acylation of the amine 3 was achieved using aroyl chlorides. All candidate compounds were designed to exhibit acceptable calculated logP values.

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Figure 6. Diversification in the final step.

The conditions for reductive amination (A) and acylation (B) of 3 to produce the final Spiros analogs 10–18 the yields reported are of pure material isolated following flash chromatography on silica.

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The acylation products 16 to 18 and the arylpyrrole 14 exhibited convoluted NMR spectra. The ¹³C{¹H}-NMR spectra of 16 to 18 contained the signals expected from the carbon atoms in the carbonyl groups (165 to 175 ppm in CDCl₃) indicating formation of the amide bond, but ¹H–¹³C{¹H} Heteronuclear Single Quantum Correlation (HSQC) spectroscopy was required to elucidate the piperidine ¹³C{¹H} region (A, Figure 7); the broad ¹H signals correlated to the aliphatic region of the ¹³C{¹H} spectrum (B) were consistent with the piperidine ring protons and all environments were accounted for. The broad signals observed for the methylene protons on the oxygen-containing ring in the compounds containing an exocyclic amide bond (i.e. compounds 16–18) most likely arise from the chirality exhibited by the individual rotamers (C and D), which, though in dynamic equilibrium via an achiral intermediate (B), make the protons attached to these carbons diastereotopic. Additional experiments were carried out on the acylated product 16, which showed temperature and magnetic field dependence (E) consistent with rapid rotation of the amide bond; at higher fields or lower temperatures peaks for the individual rotamers, and their more convoluted splitting patterns, became clear.

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Figure 7. The piperidine nitrogen signals of the acylated products can be visualised by HSQC experiments.

The ¹H-NMR and ¹³C{¹H}-NMR spectra respectively displayed on the x- and y-axes of the HSQC spectra are projections of the corresponding one-dimensional NMR experiments and are displayed for clarity. (A) The aliphatic region of the ¹H–¹³C{¹H} HSQC spectrum of 16; the red arrows indicate the ¹H signals corresponding to the piperidine protons (attached to the red ring of the structure). The corresponding piperidine carbon signals on the y-axis (circled red) are unclear in the ¹³C{¹H} spectrum. (B) Rotation around the amide bond generates chiral rotamers (C and D) dictated by the π-system of the benzamide, which makes the methylene protons in the oxygen-containing ring diastereotopic, (E) Variable magnetic field temperature and NMR experiments showing coalescence of methylene signals for compound 16. At lower temperature the diastereotopic oxygen-containing ring protons (E) exhibited higher order coupling, arising from the individual rotamers (Figure S39) and the ¹³C{¹H} NMR spectrum exhibited complete resolution of the piperidine ring carbon signals (Figure S40). Raw data may be found in Dataset S1.

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Activity of the Spiros Analogues

The activities of the compounds containing the spirocycle core (3, 6, 9–18) were determined against the virulent M.tuberculosis strain (H37Rv) (Table 1). Initially, M. tuberculosis H37Rv was exposed to a single compound dose of 100 µM for 7 days, and survival was determined in comparison to vehicle-treated bacterial cells using a Resazurin microtiter assay of growth inhibition [24]. The potency of compounds displaying activity at 100 µM was determined by calculating the concentration of drug inhibiting 50% of bacterial growth (IC₅₀). Excellent inhibitory activity against H37Rv for compounds containing the N-benzyl/pyrrole-type centres (6, 10, 11, 13 and 14) was observed other than for 12; the cyclohexyl-containing spirocycle 1, the secondary amine 3 and the N-acyl-type spirocycles (9, 16–18) were inactive. The presence of the N-CH₂-Ar group appeared to be a necessary but not sufficient condition for significant activity against H37Rv, but amide-type functionality was detrimental to the potency of the molecules. Novel compounds 13 and 14 were the most potent with activity comparable to the Spiros anti-TB compounds identified and developed by GSK [4]–[5].

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Table 1. Anti-tubercular activity of the synthesised Spiros analogues.

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Toxicity of compounds displaying anti-H37Rv activity was assessed using the human monocytic cell line THP1 (Table 1). The IC₅₀ against THP was calculated and was compared to the IC₅₀ calculated against M. tuberculosis H37Rv. Compounds 6, 10, 11 and 13 displayed THP1 toxicity at relatively high concentrations (>50 µM), suggesting potential for the future development of these compounds. Compounds 6 and 11 were less toxic than the original GSK structure (10) yet were also less potent against H37Rv. Compound 14 was highly active against H37Rv however was toxic against THP1 in the low µM range.

Conclusion

A three-step synthesis of new TB drug leads is reported that will provide rapid access to potent compounds that may be used in a future assessment of this series. This should both aid in the synthesis of existing analogues for examination of their pharmacokinetic properties and the synthesis of diverse new analogues in this series to optimise potency and drug likeness as well as to further mechanism of action studies. Future participants in such efforts are encouraged to adopt the open platform that has already been developed for full data sharing and collaboration.

Materials and Methods

General Chemistry

General synthetic and analytical methods are detailed in Text S1. Raw NMR data for all compounds are available from The University of Sydney eScholarship Repository [25]. The open electronic laboratory notebook for experiments carried out April–August 2013 is available from The University of Sydney eScholarship Repository [15]; all other experiments are summarised in Spreadsheet S1.

6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3

This compound exists in the literature but no data are reported [22]. To a stirred solution of 6 (2.3 g, 7.7 mmol, 1 equiv.) in anhydrous THF (200 mL) under argon at −78°C was added 1-chloroethyl chloroformate (1.6 mL, 15 mmol, 2 equiv.). The reaction mixture was stirred for 30 min then allowed to warm to rt. THF was removed under reduced pressure leaving a residual volume of ∼10 mL which was diluted with methanol (200 mL) and heated at reflux for 20 min. The now clear brown solution was concentrated under reduced pressure to give the crude product as a brown foam (2.8 g). The crude material was purified by flash chromatography on silica (5∶100∶0.5, CH₃OH∶CH₂Cl₂∶NH₄OH, v/v to 10∶100∶1, CH₃OH∶CH₂Cl₂∶NH₄OH, v/v) to give the title compound as a pale brown solid (1.6 g, 99%); mp 86–88°C; ν_max (film)/cm⁻¹ 2953, 2923, 1072, 1050, 741, 654; δ_H (500 MHz; CDCl₃) 7.06 (d, J = 5.2 Hz, 1 H), 6.79 (d, J = 5.2 Hz, 1 H), 3.93 (t_app, J = 5.4 Hz, 2 H), 3.06–3.01 (m, 2 H), 2.90–2.87 (m, 2 H), 2.82 (t_app, J = 5.3 Hz, 2 H), 1.85–1.78 (m, 4 H), 1.66 (brs, 1 H) (Figure S1); δ_C (126 MHz; CDCl₃) 141.6, 132.5, 124.5, 122.4, 73.9, 59.1, 42.2, 37.0, 26.0 (Figure S2); ¹H –¹³C HSQC (Figure S3) and ¹H –¹³C{¹H} HMBC (Figure S4) spectra also supplied; Anal. Calcd. for C₁₁H₁₅NOS: C, 63.12; H, 7.22; N, 6.69. Found: C, 63.46; H, 7.56; N, 6.61.

1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6

To a vigorously stirred solution of thiopheneethanol (0.50 mL, 4.5 mmol, 1 equiv.) and 1-benzyl-4-piperidinone (0.80 mL, 4.5 mmol, 1 equiv.) in toluene (50 mL) was added methanesulfonic acid (0.29 mL, 4.5 mmol, 1 equiv.). The reaction mixture was heated at reflux (oil bath at 130°C) for 16 h and allowed to cool to rt. The mixture was diluted with ethyl acetate (50 mL) and washed with saturated NaHCO₃ (30 mL). The aqueous fraction was extracted with ethyl acetate (2×30 mL), further basified with solid sodium hydroxide (4.0 g) and extracted with ethyl acetate (2×30 mL). The combined organic fractions were dried (Na₂SO₄) and concentrated under reduced pressure to give the crude product as a dark brown oil (1.7 g). The crude material was purified by flash chromatography on silica (10–50% ethyl acetate/hexanes) to give the title compound as a pale yellow oil (1.2 g, 86%); ν_max (film)/cm⁻¹ 2936, 2813, 1075, 854, 672; δ_H (500 MHz; CDCl₃) 7.37–7.31 (m, 4 H), 7.27–7.24 (m, 1 H), 7.06 (d, J = 5.2 Hz, 1 H), 6.82 (d, J = 5.2 Hz, 1 H), 3.93 (t_app, J = 5.4 Hz, 2 H), 3.56 (s, 2 H), 2.82 (t_app, J = 5.4 Hz, 2 H), 2.74–2.71 (m, 2 H), 2.43–2.38 (m, 2 H), 2.01–1.95 (m, 2 H), 1.86–1.82 (m, 2 H) (Figure S5); δ_C (126 MHz; CDCl₃) 141.3, 138.6, 132.7, 129.4, 128.3, 127.1, 124.5, 122.3, 73.3, 63.5, 59.1, 49.2, 36.2, 26.0 (Figure S6); ¹H –¹³C HSQC (Figure S7) and ¹H –¹³C{¹H} HMBC (Figure S8) spectra also supplied; HRMS (ESI) 300.14161 ([M+H]⁺), calcd. for C₁₈H₂₂NOS⁺ 300.14166.

Reductive amination: general procedure 1

This procedure was adapted from the literature [20] [22]. Compounds 10–15 were prepared using this method. To a stirred solution of secondary amine 3 (1 equiv.) and the appropriate aldehyde or ketone (1.1 equiv.) in anhydrous dichloromethane (to 50 mM of 70) was added sodium triacetoxyborohydride (1.5 equiv.). The mixture was stirred at rt under argon for 18–24 h then quenched by pouring over saturated NaHCO₃ solution. The aqueous phase was extracted with dichloromethane (2 times). The combined organic fractions were dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica to give the corresponding tertiary amine product.

Acylation: general procedure 2

Compounds 16–18 were prepared using this method. A stirred solution of secondary amine 3 (1 equiv.) and triethylamine (2 equiv.) in anhydrous dichloromethane (to 0.2 M of 70) under argon was cooled in a brine ice bath. The appropriate acid chloride (1 equiv.)—either benzoyl chloride, 2-bromobenzoyl chloride or 4-bromobenzoyl chloride—was slowly added. The mixture was stirred at temperature for 15 min, slowly allowed to warm to rt and stirred for a further for 12–15 h. The reaction was quenched by pouring over saturated NaHCO₃ solution. The aqueous phase was extracted with dichloromethane (3 times). The combined organic fractions were dried (Na₂SO₄) and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica to give the corresponding amide product.

2-Chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1- carboxylate 9

This procedure was adapted from the literature [22]. To a stirred solution of 3 (0.40 g, 1.4 mmol, 1 equiv.) in anhydrous THF (35 mL) under argon at −78°C was added 2-chloroethyl chloroformate (0.18 mL, 1.7 mmol, 1.3 equiv.). The reaction mixture was stirred at −78°C for 30 min then allowed to warm to rt. The solvent was removed under reduced pressure and the brownish residue was suspended in methanol (40 mL) and heated at reflux for 1 h. The now clear brown solution was concentrated under reduced pressure to give the crude product as a yellow oil (∼0.5 g). The crude material was purified by flash chromatography on silica (10% ethyl acetate/CH₂Cl₂) to give the title compound as a colourless oil that solidified when taken to 4°C (0.35 g, 82%); mp 57–60°C; ν_max (film)/cm⁻¹ 2953, 2923, 1072, 1050, 741, 654; δ_H (500 MHz; CDCl₃) 7.08 (d, J = 5.2 Hz, 1 H), 6.72 (d, J = 5.2 Hz, 1 H), 4.36 (t_app, J = 5.7 Hz, 2 H), 4.04 (brs, 2 H), 3.94 (t_app, J = 5.4 Hz, 2 H), 3.71 (t, J = 5.6 Hz, 1 H), 3.21 (s, 1 H), 2.83 (t, J = 5.4 Hz, 1 H), 1.84–1.81 (m, 1 H) (Figure S9); δ_C (126 MHz; CDCl₃) 154.9, 140.3, 132.9, 124.1, 122.7, 73.2, 65.0, 59.4, 42.5, 39.9, 25.9 (Figure S10); HRMS (ESI) 280.10010 ([M−Cl]⁺) calcd. for C₁₄H₁₈NO₃S⁺ 280.10019; ¹H –¹³C HSQC (Figure S11) and ¹H –¹³C{¹H} HMBC (Figure S12) spectra also supplied; HRMS (ESI) 338.05867 ([M+Na]⁺), calcd. for C₁₄H₁₈ClNO₃SNa⁺ 338.05881; Anal. Calcd. for C₁₄H₁₈ClNO₃S: C, 53.24; H, 5.74; N, 4.44. Found: C, 53.21; H, 5.77; N, 4.40. ¹³C{¹H}-NMR signals are missing or obscured due to rotamers around the carbamate.

1-((2,3-Dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10

Prepared according to general procedure 1. Following purification by flash chromatography (1∶100∶0.1, CH₃OH∶CHCl₃∶NH₄OH), the title compound was obtained as a colourless oil that solidified to a colourless solid when taken to -20°C (52 mg, 58%); mp 106–109°C; ν_max (film)/cm⁻¹ 2926, 2813, 1505, 1068, 886, 648; δ_H (500 MHz; CDCl₃) 7.06 (d, J = 5.1 Hz, 1 H), 6.88 (s_app, 1 H), 6.84–6.79 (m, 3 H), 4.25 (s, 4 H), 3.92 (t_app, J = 5.3 Hz, 2 H), 3.45 (s, 2 H), 2.82 (t_app, J = 5.3 Hz, 2 H), 2.71 (brd_app, J = 11.2 Hz, 2 H), 2.37 (t_app, J = 11.9 Hz, 2 H), 1.97 (td_app, J = 13.2, 4.0 Hz, 2 H), 1.84 (d, J = 13.0 Hz, 2 H) (Figure S13); δ_C (126 MHz; CDCl₃) 143.4, 142.7, 141.4, 132.7, 132.0, 124.6, 122.40, 122.30, 118.1, 117.0, 73.4, 64.51, 64.49, 62.9, 59.1, 49.1, 36.3, 26.0 (Figure S14); ¹H –¹³C HSQC (Figure S15) and ¹H –¹³C{¹H} HMBC (Figure S16) spectra also supplied; HRMS (ESI) 358.14712 ([M+H]⁺), calcd. for C₂₀H₂₄NO₃S⁺ 358.14714; Anal. Calcd. for C₂₀H₂₃NO₃S: C, 67.20; H, 6.49; N, 3.92. Found: C, 66.55; H, 6.54; N, 3.88. Discrepancy in CHN analysis noted, but data consistent with 4M+ H₂O, i.e. calcd. for C₈₀H₉₄N₄O₁₃S₄ 66.36; H, 6.54; N, 3.87.

1-(Benzo[d] [1], [3]dioxin-5-ylmethyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno [3,2-c]pyran] 11

Prepared according to general procedure 1. Following purification by flash chromatography (2∶100∶0.2, CH₃OH∶CH₂Cl₂∶NH₄OH), the title compound was obtained as a colourless oil that solidified to a colourless solid when taken to 4°C (37 mg, 44%); mp 81–84°C; ν_max (film)/cm⁻¹ 2923, 2813, 1501, 1487, 1440, 1242, 1074, 1036, 934, 853, 648; δ_H (500 MHz; CDCl₃) 7.06 (d, J = 5.2 Hz, 1 H), 6.89 (d_app, J = 1.0 Hz, 1 H), 6.81 (d, J = 5.2 Hz, 1 H), 6.78 (dd, J = 8.0, 1.0 Hz, 1 H), 6.75 (d_app, J = 8.0 Hz, 1 H), 3.92 (t_app, J = 5.4 Hz, 2 H), 3.47 (s, 2 H), 2.82 (t_app, J = 5.4 Hz, 2 H), 2.72–2.70 (m, 2 H), 2.40–2.35 (m, 2 H), 2.00–1.94 (m, 2 H), 1.86–1.83 (m, 2 H) (Figure S17); δ_C (126 MHz; CDCl₃) 147.7, 146.7, 141.3, 132.73, 132.56, 124.5, 122.48, 122.33, 109.8, 108.0, 101.0, 73.4, 63.3, 59.1, 49.1, 36.3, 26.0 (Figure S18); HRMS (ESI) 344.13146 ([M+H]⁺), calcd. for C₁₉H₂₂NO₃S⁺ 344.13149; Anal. Calcd. for C₁₉H₂₁NO₃S: C, 66.45; H, 6.16; N, 4.08. Found: C, 66.59; H, 6.26; N, 3.98.

4-((6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12

Prepared according to general procedure 1. Following purification by flash chromatography (2∶100∶0.1, CH₃OH∶CH₂Cl₂∶NH₄OH), the title compound was obtained as a colourless solid (41 mg, 44%); mp 170–172°C; ν_max (film)/cm⁻¹ 2927, 1517, 1277, 1265, 1071, 800, 732; δ_H (500 MHz; CDCl₃) 7.04 (d, J = 5.2 Hz, 1 H), 6.90 (s_app, 1 H), 6.83–6.77 (m, 2+1 H), 3.92 (t_app, J = 5.4 Hz, 2 H), 3.83 (s, 3 H), 3.50 (s, 2 H), 2.82 (t_app, J = 5.4 Hz, 2 H), 2.76–2.74 (m, 2 H), 2.41–2.36 (m, 2 H), 2.83–1.83 (m, 2 H), 2.02–1.96 (m, 2 H) (Figure S19); δ_C (126 MHz; CDCl₃) 146.7, 144.9, 141.1, 132.6, 129.9, 124.4, 122.38, 122.19, 114.2, 112.0, 73.3, 63.3, 59.0, 55.8, 49.0, 42.0, 35.9, 27.0, 25.9, 25.0 (Figure S20); ¹H –¹³C HSQC (Figure S21) spectrum also supplied; HRMS (ESI) 346.14711 ([M+H]⁺), calcd. for C₁₉H₂₄NO₃S⁺ 346.14714; Anal. Calcd. for C₁₉H₂₃NO₃S: C, 66.06; H, 6.71; N, 4.05. Found: C, 65.88; H, 6.91; N, 3.87.

1-(4-Chlorobenzyl)-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 13

Prepared according to general procedure 1. Following purification by flash chromatography (3∶100∶0.3, CH₃OH∶CHCl₃∶NH₄OH), the title compound was obtained as a colourless oil that solidified when taken to 4°C (79 mg, 64%); mp 106–109°C; ν_max (film)/cm⁻¹ 2923, 2816, 1489, 1076, 1016, 854, 646; δ_H (500 MHz; CDCl₃) 7.29 (s_app, 4 H), 7.07 (d, J = 5.2 Hz, 1 H), 6.81 (d, J = 5.2 Hz, 1 H), 3.92 (t_app, J = 5.4 Hz, 2 H), 3.52 (s, 2 H), 2.82 (t_app, J = 5.4 Hz, 2 H), 2.70–2.67 (m, 2 H), 2.42–2.36 (m, 2 H), 1.99–1.93 (m, 2 H), 1.86–1.83 (m, 2 H) (Figure S22); δ_C (126 MHz; CDCl₃) 141.2, 137.3, 132.8, 130.6, 128.5, 124.5, 122.4, 59.1, 49.2, 36.3, 26.0 (Figure S23); HRMS (ESI) 334.10268 ([M+H]⁺), calcd. for C₁₈H₂₁ClNOS⁺ 334.10269; Anal. Calcd. for C₁₈H₂₀ClNOS: C, 64.75; H, 6.04; N, 4.20. Found: C, 64.56; H, 6.08; N, 4.16.

1-((1-(4-Fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-6′,7′- dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] hydrochloride 14

Prepared according to general procedure 1. Following purification by flash chromatography (2∶100∶0.2, CH₃OH∶CHCl₃∶NH₄OH), the title compound was obtained as a brown solid (23 mg, 22%); ν_max (film)/cm⁻¹ 2922, 1511, 1224, 1075, 852; δ_H (500 MHz; CDCl₃) 7.18–7.14 (m, 3 H), 6.88 (s, 1 H), 6.02 (s, 1 H), 3.93 (t_app, J = 5.4 Hz, 2 H), 3.57 (brs, 2 H), 2.97 (brs, 1 H), 2.83 (t_app, J = 5.3 Hz, 2 H), 2.54 (s, 2 H), 2.23 (s, 1 H), 1.99 (s, 4 H), 1.91–1.88 (m, 3 H), 1.68 (s, 3 H) (Figure S24); δ_C (126 MHz; CDCl₃) 163.0, 161.0, 140.6, 135.0, 135.0, 132.7, 130.1, 130.1, 124.7, 122.6, 116.3, 116.1, 108.6, 72.9, 59.4, 54.4, 48.5, 35.3, 29.8, 26.0, 12.9, 11.1 (Figure S25); δ_F (471 MHz; CDCl₃) -113.8; HRMS (ESI) 411.19012 ([M−Cl]⁺), calcd. for C₂₄H₂₈FN₂OS 411.19009; Anal. Calcd. for C₂₄H₂₈ClFN₂OS: C, 64.49; H, 6.31; N, 6.27. Found: C, 64.05; H, 6.14; N, 5.95.

1-Cyclohexyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 15

Prepared according to general procedure 1. Following purification by flash chromatography (0.8∶100∶0.08, CH₃OH∶CHCl₃∶NH₄OH) the title compound was obtained as a colourless solid (67 mg, 84%); mp 170–172°C; ν_max (film)/cm⁻¹ 2925, 2854, 1076, 647; δ_H (500 MHz; CDCl₃) 7.05 (d, J = 5.2 Hz, 1 H), 6.84 (d, J = 5.2 Hz, 1 H), 3.92 (t, J = 5.4 Hz, 2 H), 2.82 (t, J = 5.4 Hz, 2 H), 2.78–2.76 (m, 2 H), 2.65 (t, J = 11.6 Hz, 2 H), 2.38–2.34 (m, 1 H), 2.04–1.81 (m, 8 H), 1.66–1.62 (m, 1 H), 1.32–1.21 (m, 4 H), 1.15–1.08 (m, 1 H) (Figure S26); δ_C (126 MHz; CDCl₃) 141.2, 132.7, 124.7, 122.3, 73.6, 64.3, 59.1, 44.8, 36.5, 29.0, 26.5, 26.2, 26.0 (Figure S27); HRMS (ESI) 292.17301 ([M+H]⁺), calcd. for C₁₇H₂₆NOS⁺ 292.17296; Anal. Calcd. for C₁₇H₂₅NOS: C, 70.06; H, 8.65; N, 4.81. Found: C, 69.51; H, 8.67; N, 4.70. Discrepancy in CHN analysis noted, but data consistent with 5M + H₂O i.e. calcd. for C₈₅H₁₂₇N₅O₆S₅: C, 69.20; H, 8.68; N, 4.75.

6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16

Prepared according to general procedure 2. Following purification by flash chromatography (1∶100∶0.1, CH₃OH∶CHCl₃∶NH₄OH), the title compound was obtained as a colourless solid (60 mg, 66%); mp 170–172°C; ν_max (film)/cm⁻¹ 2951, 1432, 1050, 709; δ_H (500 MHz; CDCl₃) 7.45–7.40 (m, 5 H), 7.10 (d, J = 5.2 Hz, 1 H), 6.76 (d, J = 5.2 Hz, 1 H), 4.67–4.64 (m, 1 H), 3.97–3.94 (m, 2 H), 3.65–3.60 (m, 1 H), 3.50–3.45 (m, 1 H), 3.22–3.16 (m, 1 H), 2.85 (t, J = 5.4 Hz, 2 H), 2.01–1.75 (m, 4 H) (Figure S28); δ_C (126 MHz; CDCl₃; 300 K) 170.5, 140.1, 136.4, 133.0, 129.7, 128.6, 127.1, 124.1, 122.9, 73.5, 59.5, 25.9 (Figure S29); δ_C (126 MHz; CDCl₃; 270 K) 170.5, 139.9, 136.0, 133.0, 129.7, 128.6, 127.0, 124.1, 122.9, 73.4, 59.5, 43.7, 38.1, 36.7, 35.5, 25.8; HRMS (ESI) 336.10287 ([M+Na]⁺), calcd. for C₁₈H₁₉NO₂SNa⁺ 336.10287; Anal. Calcd. for C₁₈H₁₉NO₂S: C, 68.98; H, 6.11; N, 4.47. Found: C, 68.03; H, 6.05; N, 4.44. Discrepancy in CHN analysis noted, but data consistent with 4M + H₂O i.e. calcd. for C₇₂H₇₈N₄O₉S₄: C, 68.00; H, 6.18; N, 4.41. ¹³C{¹H} signals are missing or obscured due to rotamers around the amide bond. The signals were visualised using ¹H –¹³C HSQC spectroscopy (Figure S30) and completely resolved at 270 K (Figure S40). Variable temperature NMR data for this compound may be found in Dataset S1.

(4-Bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17

Prepared according to general procedure 2. Following purification by flash chromatography (1∶100∶0.1, CH₃OH∶CHCl₃∶NH₄OH), the title compound was obtained as a colourless solid (60 mg, 66%); mp 170–172°C; ν_max (film)/cm⁻¹ 2923, 1628, 1433, 1072; δ_H (500 MHz; CDCl₃) 7.56–7.52 (m, 2 H), 7.39–7.30 (m, 2 H), 7.10 (d, J = 5.2 Hz, 1 H), 6.74 (d, J = 5.2 Hz, 1 H), 4.65–4.58 (m, 1 H), 3.95–3.92 (m, 2 H), 3.62–3.44 (m, 2 H), 3.23–3.15 (m, 1 H), 2.85 (t_app, J = 5.4 Hz, 2 H), 2.02–1.83 (m, 2 H), 1.83–1.72 (m, 2 H) (Figure S31); δ_C (126 MHz; CDCl₃) 169.5, 139.9, 135.1, 133.1, 131.8, 128.8, 124.00, 123.97, 122.9, 73.4, 59.5, 25.9 (Figure S32); ¹H –¹³C HSQC (Figure S33) and ¹H –¹³C{¹H} HMBC (Figure S34) spectra also supplied; HRMS (ESI) 416.01134 ([M+Na]⁺), calcd. for C₁₈H₁₈⁸¹BrNOSNa⁺ 416.01134; Anal. Calcd. for C₁₈H₁₈BrNO₂S: C, 55.11; H, 4.62; N, 3.57. Found: C, 55.88; H, 4.89; N, 3.45. ¹³C{¹H} signals are missing or obscured due to rotamers around the amide bond. Discrepancy in CHN analysis noted; data match sample containing <10% des-brominated compound, but this was not observed in the ¹H NMR spectrum for this sample.

(2-Bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18

Prepared according to general procedure 2. Following purification by flash chromatography (1∶100∶0.1, CH₃OH∶CH₂Cl₂∶NH₄OH), the title compound was obtained as a colourless solid (81 mg, 73%); mp 48–52°C; ν_max (film)/cm⁻¹ 2932, 1634, 1436, 1073, 768; δ_H (500 MHz; CDCl₃) 7.60–7.54 (m, 1 H), 7.38–7.31 (m, 2 H), 7.24 (dd, J = 13.3, 6.7 Hz, 2 H), 7.08 (dd, J = 4.8, 2.5 Hz, 1 H), 6.72 (d, J = 5.2 Hz, 1 H), 4.73–4.66 (m, 1 H), 3.93 (tq, J = 10.0, 4.9 Hz, 2 H), 3.55 (td, J = 12.9, 3.2 Hz,), 3.42–3.34 (m, 1 H), 3.28–3.12 (m, 2 H), 2.83 (t, J = 4.9 Hz, 2 H), 2.10 (td, J = 13.5, 5.0 Hz, 1 H), 1.99 (dd, J = 9.8, 3.6 Hz, 1 H), 1.94–1.90 (m, 1 H), 1.81–1.69 (m, 2 H), 1.26 (t, J = 7.2 Hz, 1 H), 1.05 (t, J = 7.1 Hz, <1 H) (Figure S35); δ_C (126 MHz; CDCl₃) 167.8, 167.6, 140.0, 139.9, 138.9, 138.5, 138.4, 133.4, 132.9, 132.8, 132.8, 130.3, 130.2, 130.0, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 124.1, 123.9, 122.8, 122.8, 119.4, 119.2, 73.4, 73.3, 59.5, 43.5, 42.8, 42.5, 39.0, 37.7, 37.5, 36.6, 36.3, 35.6, 35.5, 31.0, 25.8, 14.0, 12.6 (Figure S36); ¹H –¹³C HSQC (Figure S37) and ¹H –¹³C{¹H} HMBC (Figure S38) spectra also supplied; HRMS (ESI) 414.01338 ([M+Na]⁺), calcd. for C₁₈H₁₈BrNO₂SNa⁺ 414.01338; Anal. Calcd. for C₁₈H₁₈BrNO₂S: C, 55.11; H, 4.62; N, 3.57. Found: C, 55.36; H, 4.36; N, 3.59. The additional ¹³C{¹H} and ¹H signals are due to rotamers around the amide bond.

Resazurin assay of growth inhibition

Compounds were tested for activity at either single concentration (100 µM) or serially diluted in 10 µL of purified H₂O in triplicate in 96 well microtiter plates. M. tuberculosis H37Rv was grown in complete Middlebrook 7H9 media (Bacto, Australia) containing albumin, dextrose and catalase (ADC), 20% Tween 80 and 50% glycerol (Sigma-Aldrich, Australia). A bacterial suspension (90 µL) at OD_600nm of 0.001 was added to the wells and incubated for 7 days. Resazurin (10 µL; 0.05%(w/v); Sigma-Aldrich, Australia) was then added, incubated for 24 h at 37°C, and fluorescence measured at 590 nm using a FLUOstar Omega microplate reader (BMG Labtech, Germany). After subtraction of background fluorescence from all wells, the percentage mycobacterial survival was determined by comparing the fluorescence of wells containing compounds compared to control wells not treated with compound.

Compound intracellular efficacy and toxicity

THP1 cells (TIB-202R), a human monocyte cell line (American Type Culture Collection, USA), were grown in complete Dulbecco's Modified Eagle Media (DMEM; LifeTechnologies, Australia) including 10% fetal bovine serum (FBS), 200 µM L-glutamine (LifeTechnologies, Australia), and 1 mM HEPES buffer solution (LifeTechnologies, Australia). Cells (1×10⁵) in media containing 50 ng/mL phorbol 12-myristate 13-acetate (PMA) were added to 96-well plates and were then incubated for 48 h at 37°C to allow adherence and differentiation. Compounds (1.56–200 µM) were added to the wells and incubated for 4 days at 37°C. Then 0.05%(w/v) resazurin (4 h) was added and the fluorescence measured. Cell viability was calculated as percentage fluorescence in comparison to untreated cells.

Supporting Information

Figure S1.

¹H NMR (500 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3.

https://doi.org/10.1371/journal.pone.0111782.s001

(PDF)

Figure S2.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3.

https://doi.org/10.1371/journal.pone.0111782.s002

(EPS)

Figure S3.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3.

https://doi.org/10.1371/journal.pone.0111782.s003

(EPS)

Figure S4.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3.

https://doi.org/10.1371/journal.pone.0111782.s004

(EPS)

Figure S5.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6.

https://doi.org/10.1371/journal.pone.0111782.s005

(PDF)

Figure S6.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6.

https://doi.org/10.1371/journal.pone.0111782.s006

(EPS)

Figure S7.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 1-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6.

https://doi.org/10.1371/journal.pone.0111782.s007

(EPS)

Figure S8.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of 1-benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6.

https://doi.org/10.1371/journal.pone.0111782.s008

(EPS)

Figure S9.

¹H NMR (500 MHz, CDCl₃) spectrum of 2-chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-carboxylate 9.

https://doi.org/10.1371/journal.pone.0111782.s009

(EPS)

Figure S10.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 2-chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-carboxylate 9.

https://doi.org/10.1371/journal.pone.0111782.s010

(EPS)

Figure S11.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 2-chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-carboxylate 9.

https://doi.org/10.1371/journal.pone.0111782.s011

(EPS)

Figure S12.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of 2-chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-carboxylate 9.

https://doi.org/10.1371/journal.pone.0111782.s012

(EPS)

Figure S13.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-((2,3-dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10.

https://doi.org/10.1371/journal.pone.0111782.s013

(EPS)

Figure S14.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-((2,3-dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10.

https://doi.org/10.1371/journal.pone.0111782.s014

(EPS)

Figure S15.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 1-((2,3-dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10.

https://doi.org/10.1371/journal.pone.0111782.s015

(EPS)

Figure S16.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of 1-((2,3-dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10.

https://doi.org/10.1371/journal.pone.0111782.s016

(EPS)

Figure S17.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-(benzo[d][1], [3]dioxin-5-ylmethyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno [3,2-c]pyran] 11.

https://doi.org/10.1371/journal.pone.0111782.s017

(EPS)

Figure S18.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-(benzo[d][1], [3]dioxin-5-ylmethyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno [3,2-c]pyran] 11.

https://doi.org/10.1371/journal.pone.0111782.s018

(EPS)

Figure S19.

¹H NMR (500 MHz, CDCl₃) spectrum of 4-((6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.

https://doi.org/10.1371/journal.pone.0111782.s019

(EPS)

Figure S20.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 4-((6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.

https://doi.org/10.1371/journal.pone.0111782.s020

(EPS)

Figure S21.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 4-((6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12.

https://doi.org/10.1371/journal.pone.0111782.s021

(EPS)

Figure S22.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-(4-chlorobenzyl)-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 13.

https://doi.org/10.1371/journal.pone.0111782.s022

(EPS)

Figure S23.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-(4-chlorobenzyl)-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 13.

https://doi.org/10.1371/journal.pone.0111782.s023

(EPS)

Figure S24.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-((1-(4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-6′,7′- dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] hydrochloride 14.

https://doi.org/10.1371/journal.pone.0111782.s024

(EPS)

Figure S25.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-((1-(4-fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-6′,7′- dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] hydrochloride 14.

https://doi.org/10.1371/journal.pone.0111782.s025

(PDF)

Figure S26.

¹H NMR (500 MHz, CDCl₃) spectrum of 1-cyclohexyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 15.

https://doi.org/10.1371/journal.pone.0111782.s026

(EPS)

Figure S27.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 1-cyclohexyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 15.

https://doi.org/10.1371/journal.pone.0111782.s027

(EPS)

Figure S28.

¹H NMR (500 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.

https://doi.org/10.1371/journal.pone.0111782.s028

(EPS)

Figure S29.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.

https://doi.org/10.1371/journal.pone.0111782.s029

(EPS)

Figure S30.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16.

https://doi.org/10.1371/journal.pone.0111782.s030

(EPS)

Figure S31.

¹H NMR (500 MHz, CDCl₃) spectrum of (4-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17.

https://doi.org/10.1371/journal.pone.0111782.s031

(EPS)

Figure S32.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of (4-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17.

https://doi.org/10.1371/journal.pone.0111782.s032

(EPS)

Figure S33.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of (4-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17.

https://doi.org/10.1371/journal.pone.0111782.s033

(EPS)

Figure S34.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of (4-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17.

https://doi.org/10.1371/journal.pone.0111782.s034

(EPS)

Figure S35.

¹H NMR (500 MHz, CDCl₃) spectrum of (2-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18.

https://doi.org/10.1371/journal.pone.0111782.s035

(EPS)

Figure S36.

¹³C{¹H} NMR (126 MHz, CDCl₃) spectrum of (2-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18.

https://doi.org/10.1371/journal.pone.0111782.s036

(EPS)

Figure S37.

¹H –¹³C HSQC (500 MHz, CDCl₃) spectrum of (2-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18.

https://doi.org/10.1371/journal.pone.0111782.s037

(EPS)

Figure S38.

¹H –¹³C{¹H} HMBC (500 MHz, CDCl₃) spectrum of (2-bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18.

https://doi.org/10.1371/journal.pone.0111782.s038

(EPS)

Figure S39.

¹H NMR (CDCl₃) spectra (4.5–2.5 ppm) of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16 at variable temperature and magnetic field strength.

https://doi.org/10.1371/journal.pone.0111782.s039

(EPS)

Figure S40.

Overlay of the ¹³C{¹H} NMR (126 MHz, CDCl₃) spectra of 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16 at 300 K and 270 K.

https://doi.org/10.1371/journal.pone.0111782.s040

(EPS)

Text S1.

General synthetic and chemical analysis methods.

https://doi.org/10.1371/journal.pone.0111782.s041

(RTF)

Spreadsheet S1.

Summary of the experimental the experimental data.

https://doi.org/10.1371/journal.pone.0111782.s042

(XLSX)

Dataset S1.

Variable Temperature NMR Data for Compound 16.

https://doi.org/10.1371/journal.pone.0111782.s043

(ZIP)

Acknowledgments

Synthesis of the novel arylpyrrole 14 was possible through the kind provision of the relevant aldehyde by Dr. Alice Williamson (The University of Sydney). We are grateful to technical assistance from Dr. Ian Luck (NMR spectroscopy), Mr. John Twyman (electronic lab notebook) and Dr. Nick Proschogo (mass spectrometry); elemental analyses were performed at The Campbell Microanalytical Laboratory, University of Otago. The electronic lab notebook used was the open source Labtrove software developed at the University of Southampton (www.labtrove.org); we thank the Labtrove team for help in creating the browseable snapshot.

Author Contributions

Conceived and designed the experiments: KAB JAT MHT. Performed the experiments: KAB DHQ. Analyzed the data: KAB DHQ JAT MHT. Contributed reagents/materials/analysis tools: KAB DHQ. Wrote the paper: KAB JAT MHT.

References

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[ref1] 1. World Health Organisation (2013) World Health Organisation Global Tuberculosis Report 2013. Available: http://www.who.int/tb/publications/global_report/gtbr13_executive_summary.pdf?u=1. Accessed 30 Jan 2014.

[ref2] 2. Zumia A, Nahid P, Cole ST (2013) Advances in the development of new tuberculosis drug and treatment regimens. Nat. Rev. Drug Discovery 12:388–404.
View Article
Google Scholar

[3] View Article

[4] Google Scholar

[ref3] 3. Interim guidance on the use of bedaquiline to treat MDRTB (2012) World Health Organisation. Available: http://www.who.int/tb/challenges/mdr/bedaquiline/en/index.html. Accessed 8 May 2014.

[ref4] 4. Ballell L, Bates RH, Young RJ, Alvarez-Gomez D, Alvarez-Ruiz E, et al. (2013) Fueling open-source drug discovery: 177 small-molecule leads against tuberculosis. ChemMedChem 8:313–321.
View Article
Google Scholar

[7] View Article

[8] Google Scholar

[ref5] 5. Remuiñán MJ, Pérez-Herrán E, Rullás J, Alemparte C, Martínez-Hoyos M, et al. (2013) Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-69,79-Dihydrospiro[Piperidine-4,49- Thieno[3,2-c]Pyran] Analogues with Bactericidal Efficacy against Mycobacterium tuberculosis Targeting MmpL3. PLoS ONE 8:e60933.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref6] 6. Tahlan K, Wilson R, Kastrinsky DB, Arora K, Nair V, et al. (2012) SQ109 Targets MmpL3, a Membrane Transporter of Trehalose Monomycolate Involved in Mycolic Acid Donation to the Cell Wall Core of Mycobacterium tuberculosis. Antimicrob. Agents Chemother 56:1797–1809.
View Article
Google Scholar

[13] View Article

[14] Google Scholar

[ref7] 7. Grzegorzewicz AE, Pham H, Gundi VAKB, Scherman MS, North EJ, et al. (2012) Inhibition of mycolic acid transport across the Mycobacterium tuberculosis plasma membrane. Nat. Chem. Biol 8:334–341.
View Article
Google Scholar

[16] View Article

[17] Google Scholar

[ref8] 8. Stanley SA, Grant SS, Kawate T, Iwase N, Shimizu M, et al. (2012) Identification of Novel Inhibitors of M. tuberculosis Growth Using Whole Cell Based High-Throughput Screening. ACS Chem. Biol 7:1377–1384.
View Article
Google Scholar

[19] View Article

[20] Google Scholar

[ref9] 9. La Rosa V, Poce G, Canseco JO, Buroni S, Pasca MR, Biava M, et al. (2012) MmpL3 Is the Cellular Target of the Antitubercular Pyrrole Derivative BM212. Antimicrob. Agents Chemother 56:324–331.
View Article
Google Scholar

[22] View Article

[23] Google Scholar

[ref10] 10. Lun S, Guo H, Onajole OK, Pieroni M, Gunosewoyo H, et al. (2013) Indoleamides Are Active Against Drug-Resistant Mycobacterium tuberculosis. Nat. Commun 4:2907.
View Article
Google Scholar

[25] View Article

[26] Google Scholar

[ref11] 11. Clinical Trial number NCT01218217. Available: http://clinicaltrials.gov/ct2/show/NCT01218217. Accessed 6 May 2014.

[ref12] 12. Shao L, Campbell JE, Hewitt MC, Campbell U, Hanania TG (2011) Multicyclic Compounds and Methods of use Thereof. WO2011069063A2.

[ref13] 13. Robertson MN, Ylioja PM, Williamson AE, Woelfle M, Robins M, et al. (2014) Open Source Drug Discovery – A Limited Tutorial. Parasitology 141:148–157.
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Google Scholar

[33] View Article

[34] Google Scholar

[ref15] 15. Badiola K, Todd M (2013) Electronic Lab Notebook for Synthesis of Tuberculosis Drug Leads. The University of Sydney eScholarship Repository. Available: http://hdl.handle.net/2123/10461. Accessed 7 May 2014.

[ref16] 16. Bouguerne B, Hoffmann P, Lherbet C (2010) Bismuth Triflate as a Safe and Readily Handled Source of Triflic Acid: Application to the Oxa–Pictet–Spengler Reaction. Synth. Commun 40:915–926.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref17] 17. Ram S, Spicer LD (1987) Debenzylation of N-Benzylamino Derivatives by Catalytic Transfer Hydrogenation with Ammonium Formate. Synth. Commun 17:415–418.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref18] 18. McCallum C, Pethybridge AD (1975) Conductance of Acids in Dimethyl-Sulphoxide-II. Conductance of Some Strong Acids in DMSO at 25°C. Electrochim. Acta 20:815–818.
View Article
Google Scholar

[43] View Article

[44] Google Scholar

[ref19] 19. Bordwell FG (1988) Equilibrium Acidities in Dimethyl Sulfoxide Solution. Acc. Chem. Res 21:456–463.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref20] 20. Olofson RA, Martz JT, Senet J–P, Piteau M, Malfroot T (1984) A New Reagent for the Selective, High-Yield N-Dealkylation of Tertiary Amines: Improved Syntheses of Naltrexone and Nalbuphine. J. Org. Chem 49:2081–2082.
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Google Scholar

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[ref23] 23. Abdel-Magid AF, Carson KG, Harris BD, Maryanoff CA, Shah RD (1996) Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures. J. Org. Chem 61:3849–3862.
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Google Scholar

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[59] View Article

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[ref25] 25. Badiola K, Todd M (2013) NMR Data for Synthesis of Spiro Tuberculosis Drug Leads. The University of Sydney eScholarship Repository. Available: http://hdl.handle.net/2123/10506. Accessed 14 May 2014.

Figures

Abstract

Introduction

Results and Discussion

Synthesis of the Spiros Analogues

Activity of the Spiros Analogues

Conclusion

Materials and Methods

General Chemistry

6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 3

1-Benzyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 6

Reductive amination: general procedure 1

Acylation: general procedure 2

2-Chloroethyl 6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1- carboxylate 9

1-((2,3-Dihydrobenzo[b][1], [4]dioxin-6-yl)methyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno[3,2-c]pyran] 10

1-(Benzo[d] [1], [3]dioxin-5-ylmethyl)-6′,7′-dihydrospiro[piperidine- 4,4′-thieno [3,2-c]pyran] 11

4-((6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) (2-methoxyphenol) 12

1-(4-Chlorobenzyl)-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 13

1-((1-(4-Fluorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)-6′,7′- dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] hydrochloride 14

1-Cyclohexyl-6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran] 15

6′,7′-Dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl(phenyl) methanone 16

(4-Bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 17

(2-Bromophenyl)(6′,7′-dihydrospiro[piperidine-4,4′-thieno[3,2-c]pyran]-1-yl) methanone 18

Resazurin assay of growth inhibition

Compound intracellular efficacy and toxicity

Supporting Information

Acknowledgments

Author Contributions

References