Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.


Raw inhibition data of Mtb
[Polyprenylphosphate] (µg/mL) Initial Rate (µg/mL/min) reported when the ratio of the rotamers is smaller than 1.5:1. For cases in which the ratio is greater or equal to 1.5, only the major rotamer was reported.

Inhibition of MurX activity by dihydrosansanmycin analogues Supplementary
Low resolution mass spectra were recorded on a Finnigan LCQ Deca ion trap mass spectrometer (ESI). High resolution mass spectra were recorded on a Bruker 7T

Materials
Analytical thin layer chromatography (TLC) was performed on commercially prepared silica plates (Merck Kieselgel 60 0.25 mm F254). Flash column chromatography was performed using 230-400 mesh Kieselgel 60 silica eluting with distilled solvents as described. Ratios of solvents used for TLC and column chromatography are expressed in v/v as specified. Compounds were visualised by UV light at 254 nm or using vanillin or cerium molybdate stain. Commercial materials were used as received unless otherwise noted. DCM and MeOH were distilled from calcium hydride, and THF and diethyl ether were distilled over sodium/benzophenone. tert-butanol was dried over activated 3 Å molecular sieves at least 24 h before use. Anhydrous DMF was purchased from Sigma Aldrich.

General procedure 3: Fmoc-strategy solid phase peptide synthesis
Fmoc deprotection: A solution of 10 vol.% piperidine/DMF (5 mL) was added to the resin and shaken for 4 min (´ 2). The resin was subsequently washed with DMF (5´ 3 mL), CH 2 Cl 2 (5´ 3 mL) and DMF (5´ 3 mL). The efficiency of the previous amino acid coupling was determined by spectroscopic measurement of the resulting fulvene piperidine adduct at λ = 301 nm.
The treatment was then repeated once.
Capping: A solution of 10 vol.% acetic anhydride in pyridine (5 mL) was added to the resin and shaken for 3 min. The resin was then washed with DMF (5 x 3 mL), CH 2 Cl 2 (5 x 3 mL) and DMF (5 x 3 mL).

General procedure 7: Cleavage from 2-chlorotrityl chloride resin and work-up
A solution of 30 vol.% hexafluoroisopropanol (HFIP) in CH 2 Cl 2 (5-10 mL) was added to the resin and shaken for 30 min at rt. The resin was subsequently washed with CH 2 Cl 2 (6´ 10 mL) and the combined cleavage and washing solutions were concentrated in vacuo. The resulting residue was dissolved in 9:1 v/v MeCN: H 2 O and purified by reverse phase HPLC.
Following cleavage from the resin (general procedure 7), the peptide was purified by

IR (ATR
Carbamate S12 (75 mg, 140 µmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was
Carbamate S12 (73 mg, 138 µmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was
Carbamate S12 (73.6 mg, 140 µmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was provided as a 9:1 mixture of diastereomers, which were readily separable by reversephase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S49 as a single diastereomer as a fluffy white solid (14.5 mg, 25%).
Carbamate S12 (73.6 mg, 140 µmol) was subsequently coupled according to general procedure 4. Following cleavage from the resin (general procedure 7), the peptide was provided as a 9:1 mixture of diastereomers which were readily separable by reversephase HPLC (50 to 100% MeCN over 40 min) to afford depsipeptide S50 as a single diastereomer as a fluffy white solid (18.8 mg, 27%).