We demonstrated that the 3-substituted benzothiophene-1,1-dioxide class of compounds are effective inhibitors of Mycobacterium tuberculosis growth under aerobic conditions. We examined substitution at the C-3 position of the benzothiophene-1,1-dioxide series systematically to delineate structure–activity relationships influencing potency and cytotoxicity. Compounds were tested for inhibitory activity against virulent M. tuberculosis and eukaryotic cells. The tetrazole substituent was most potent, with a minimum inhibitory concentration (MIC) of 2.6 µM. However, cytotoxicity was noted with even more potency (Vero cell TC50 = 0.1 µM). Oxadiazoles had good anti-tubercular activity (MICs of 3–8 µM), but imidazoles, thiadiazoles and thiazoles had little activity. Cytotoxicity did not track with anti-tubercular activity, suggesting different targets or mode of action between bacterial and eukaryotic cells. However, we were unable to derive analogs without cytotoxicity; all compounds synthesized were cytotoxic (TC50 of 0.1–5 µM). We conclude that cytotoxicity is a liability in this series precluding it from further development. However, the series has potent anti-tubercular activity and future efforts towards identifying the mode of action could result in the identification of novel drug targets.
Tuberculosis (TB), which is caused by
The benzo[b]thiophene-1,1-dioxide (BTD) series was reported to have activity against
We used
CellTiter-Glo® Luminescent Cell Viability Assay (Promega) was used to measure ATP as a indicator of cell viability. The Vero cell line (ATCC CCL81) was grown in Dulbecco’s Modified Eagle Medium (DMEM), High Glucose, GlutaMAX™ (Invitrogen), 10% FBS (Fetal Bovine Serum), and 1x of Penicillin-Streptomycin Solution (100 units/mL of penicillin, 100 µ g/mL of streptomycin). Compounds were solubilized in DMSO (dimethyl sulfoxide) and assayed using a 10-point three-fold serial dilution starting at the highest concentration of 50 µ M. CellTiter-Glo® Reagent (Promega) was added to 96-well plates after 2 days of incubation at 37 °C, 5% CO2. Relative luminescent units (RLU) were measured using Perkin Elmer Wallac 1420 Victor2 plate reader. Inhibition curves were fitted using the Levenberg–Marquardt algorithm. Toxic concentration (TC50) was defined as the concentration of compound that gave 50% inhibition of growth. Selectivity index was calculated as MIC/TC50. For published data (
1H and NMR spectral data were recorded in CDCl3 or Acetone-d6 on a 300 MHz Bruker NMR spectrometer. Column chromatography was conducted on a Revelaris flash chromatography system. Reactions were monitored using thin-layer chromatography (TLC) on silica gel plates. HPLC analysis was conducted on an Agilent 1100 series LC system (Agilent ChemStation Rev.A.10.02; Phenomenex-Luna-C18, 4.8 mm × 150 mm, 5 µm, 1.0 mL/min, UV 254 nm, room temperature) with MeCN/H2O (0.05% TFA or HCOOH buffer) gradient elution. HPLC-MS was performed on a Gilson 321 HPLC with detection performed by a Gilson 170 DAD and a Finnigan AQA mass spectrometer operating in electrospray ionisation mode using a Phenomenex Gemini C18 150 × 4.6 mm column. Compounds
To a solution of
To a solution of 200 mg (0.82 mmol) of
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BTD analogs were synthesized as outlined in
We probed the consequences of having oxazoles and oxadiazoles as substituents at the C-3 position. Compounds were tested for efficacy against a virulent strain of
Compound | R-group | MIC (µM) |
TC50 (µM) |
SI |
---|---|---|---|---|
|
|
3.1 ± 0.07 | 0.1 ± 0 | 0.03 |
|
|
8.2 ± 0.6 | 0.2 ± 0 | 0.02 |
|
|
5.7 ± 2.9 | 0.2 ± 0.07 | 0.04 |
|
|
7.2 ± 0.3 | 0.3 ± 0.3 | 0.04 |
|
|
3.9 ± 1.7 | 0.3 ± 0.2 | 0.08 |
MIC is the minimum concentration required to inhibit growth of
TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two runs ± standard deviation.
SI is the selectivity index. Selectivity index is calculated as MIC/TC50.
For comparison, MIC of rifampicin is 0.003 µM and isoniazid is 0.2 µM (
Since we had seen good activity with the compounds, but significant cytotoxicity, we determined whether we could separate the two activities to generate potent, non-toxic compounds. We examined the influence of thiazoles and thiadiazoles on the biological activity and cytotoxicity of these BTD compounds. Anti-tubercular activity was diminished by the replacement of an oxazole with either a thiazole or a thiadiazole; these compounds showed MICs ≥20 µM (
Compound | R-group | MIC (µM) |
TC50 (µM) |
SI |
---|---|---|---|---|
|
|
20 | 1 | 0.05 |
|
|
20 | 3 ± 1.2 | 0.2 |
|
|
20 | 1 | 0.05 |
|
|
>20 | 1 ± 0.4 | NC |
|
|
9.0 ± 4.7 | 1 ± 0.1 | 0.1 |
|
|
>20 | 1 ± 0.4 | NC |
|
|
>20 | 3 ± 1 | NC |
|
|
20 | 3 ± 0.7 | 0.2 |
MIC is the minimum concentration required to inhibit growth of
TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two runs ±standard deviation.
SI is the selectivity index. Selectivity index is calculated as MIC/TC50. For comparison, MIC of rifampicin is 0.003 µM and isoniazid is 0.2 µM (
Not calculated
We then investigated the effect of C-3 imidazoles to see if we could improve the SI. Similar to the thiazoles and thiadiazoles, this resulted in diminished activity (MIC >20 µM) (
Compound | R-group | MIC (µM) |
TC50 (µM) |
SI |
---|---|---|---|---|
|
|
>20 | 1 | NC |
|
|
>20 | 5 | NC |
|
|
>20 | ND | NC |
|
|
>20 | 0.3 ± 0.07 | NC |
|
|
2.6 | 0.1 ± 0 | 0.004 |
MIC is the minimum concentration required to inhibit growth of
TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two runs ±standard deviation.
SI is the selectivity index. Selectivity index is calculated as MIC/TC50.
Not calculated Not determined
Finally, we explored the influence of having six membered heterocycles in the C-3 position. We synthesized compounds with pyrimidyl (
Compound | R-group | MIC (µM) |
TC50 (µM) |
SI |
---|---|---|---|---|
|
|
>20 | 10 ± 1 | NC |
|
|
>20 | ND | NC |
|
|
>20 | ND | NC |
|
|
>20 | ND | NC |
|
|
>20 | ND | NC |
|
|
>20 | ND | NC |
|
|
>20 | ND | NC |
MIC is the minimum concentration required to inhibit growth of
TC50 is the concentration required to inhibit growth of Vero cells by 50%. TC50 is the average of two runs ±standard deviation.
SI is the selectivity index. Selectivity index is calculated as MIC/TC50.
Not calculated Not determined
We conducted a systematic exploration of the aryl/heteroaryl thioether substituents at the C-3 position of the benzo[b]thiophene-1,1-dioxide compound series for its inhibitory activity against
General methods
We thank Alfredo Blakeley, David Roberts and Yulia Ovechkina for technical assistance.
Tanya Parish is an Academic Editor for PeerJ. The authors are employees at Infectious Disease Research Institute (IDRI).