Synthetic studies with the brevicidine and laterocidine lipopeptide antibiotics including analogues with enhanced properties and in vivo efficacy

Brevicidine and laterocidine are two recently discovered lipopeptide antibiotics with promising antibacterial activity. Possessing a macrocyclic core, multiple positive charges, and a lipidated N-terminus, these lipopeptides exhibit potent and selective activity against Gram-negative pathogens, including polymyxin-resistant isolates. Given the low amounts of brevicidine and laterocidine accessible by fermentation of the producing microorganisms, synthetic routes to these lipopeptides present an attractive alternative. We here report the convenient solid-phase syntheses of both brevicidine and laterocidine and confirm their potent anti-Gram-negative activities. The synthetic routes developed also provide convenient access to novel structural analogues of both brevicidine and laterocidine that display improved hydrolytic stability while maintaining potent antibacterial activity in both in vitro assays and in vivo infection models.

. 1 H NMR (600 MHz, of synthetic brevicidine (top) overlaid with the previously published 1 H NMR (500 MHz, spectrum of natural brevicidine (bottom) isolated from fermentation of the producing organism. Spectra were recorded at room temperature. The spectrum for the synthetic material contains broad signals between 3.30-3.65 ppm due to H 2 0/HDO present in the NMR solvent. The peak at ca. 3.96 ppm in the published spectrum of brevicidine is attributed to an impurity not present in the synthetic material. . Column: Waters Acquity UPLC BEH C18 column (1.7 µm, 130 Å, 2.1 × 150 mm). LC method: The column was maintained at 40˚C and run at a flow rate of 0.2 mL/min, using 0.1% formic acid in H 2 O as solvent A and 0.1% formic acid in acetonitrile as solvent B. A gradient was employed for chromatographic separation starting at 5% B for 2 min, then 5% to 95% B for 15 min, and finally held at 95% for 4 min, The column was reequilibrated to 5% B for 1 min before the next run was started. MS method: The MS system was tuned using standard sodium formate solution. The same solution was used to calibrate the system before starting. All the samples were analyzed in positive polarity, using data-dependent acquisition mode. Detection range: 100-1500 m/z. Figure S6. LPS antagonization assay. A blood agar plate was inoculated with a glycerol stock of E. coli ATCC 25922. The inoculated agar plate was then incubated for 16 h at 37 °C. An individually grown colony was subsequently used to inoculate 3 mL of TSB that was then incubated at 37 °C with shaking at 220 rpm. In parallel, the compounds to be assessed were serially diluted with Mueller-Hinton broth (MHB) in polypropylene 96-well plates (50 µL in each well). Once the OD600 of the bacterial suspensions reached 0.5, the bacteria were diluted with MHB (final concentration 2 × 10 5 CFU mL −1 ). The media were then either supplemented with 1 mg/mL of LPS (lipopolysaccharides from E. coli O55:B5, Sigma-Aldrich) or added directly to the microplates containing the test compounds (50 µL to each well, final volume: 100 µL). The well-plates were sealed with an adhesive membrane and after 16 h of incubation at 37°C with shaking at 220 rpm. The wells were visually inspected for bacterial growth. for 5 min. The peaks were integrated and normalized to the internal standard. Recovery of the peptides at t=0 was compared to control samples without serum and was within the 85%-115% range (data not shown). The t=0 value was then set at 100% for each analogue and all time-points were calculated as a percentage of t=0. Biological duplicates of the experiment were performed. Figure S8. Hemolytic activity of selected analogues against sheep red blood cells. Experiments were performed in triplicate and Triton X-100 used as a positive control. Red blood cells from defibrinated sheep blood obtained from Thermo Fisher were centrifuged (400 g for 15 min at 4°C) and washed with Phosphate-Buffered Saline (PBS) containing 0.002% Tween20 (buffer) for five times. Then, the red blood cells were normalized to obtain a positive control read-out between 2.5 and 3.0 at 415 nm to stay within the linear range with the maximum sensitivity. A serial dilution of the compounds (200 -6.25 µg/mL, 75 µL) was prepared in a 96-well plate. The outer border of the plate was filled with 75 µL buffer. Each plate contained a positive control (0.1% Triton-X final concentration, 75 µL) and a negative control (buffer, 75 µL) in triplicate. The normalized blood cells (75 µL) were added and the plates were incubated at 37 °C for 20 h while shaking at 500 rpm. A flat-bottom plate of polystyrene with 100 µL buffer in each well was prepared. After incubation, the plates were centrifuged (800 g for 5 min at room temperature) and 25 µL of the supernatant was transferred to their respective wells in the flat-bottom plate. The values obtained from a read-out at 415 nm were corrected for background (negative control) and transformed to a percentage relative to the positive control. S10 O r i t a v a n c i n , n

HPLC purification of synthetic peptides
Brevicidine and analogues were purified using a Perkin
The resin was then capped by adding a solution of methanol, DIPEA and DCM (3 mL, 10 : 5 : 85) and bubbled with argon for 1 h. The solution was discharged and the resin was washed with DCM (3 x 5 mL) before being dried under a stream of argon. A small portion of resin was then used to ascertain the loading. Estimation of loading level of first residue onto resin (0.15 mmol g -1 ) was calculated via an Fmoc loading test, as described by Gude et al. [6] Standard Fmoc SPPS protocol was used to extend the peptide to the linear Fmoc-Thr-Ile-Gly-Ser stage. Specifically, resin (670 mg, 0.1 mmol) was added to a manual SPPS vessel and bubbled in DMF (3 mL) to swell. The solvent was discharged and the resin was bubbled in an Fmoc deprotection solution of 20% piperidine in DMF (3 x 3 mL, 2 x 1 min then 1 x 5 min) with argon.
The resin was washed with DMF (3 x 3 mL) and a coupling solution of amino acid (6 equiv), HATU (6 equiv) and DIPEA (12 equiv) in DMF (3 mL) was added. The solution was then bubbled with argon for 1 h, before the solution was discharged and the resin washed with DMF (3 x 3 mL).
This process was repeated to obtain on-resin linear Fmoc-tetrapeptide. At this stage the resin was S20 split an a portion of this on-resin allyl-protected tetrapeptide (78.0 mg. 0.01 mmol) was added to a manual SPPS vessel and bubbled in DCM (3 mL) with argon for 15 min. The solvent was discharged and an allyl deprotection solution of tetrakis(triphenylphosphine) palladium (231 mg, 0.200 mmol) and phenylsilane (123 µL, 0.998 mmol) in DCM and DMF (1:1, 2 mL) was added.
The solution was bubbled with argon for 2 h in darkness, after which the deprotection solution was discharged and the resin was washed with DMF (3 x 3

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The resin was then capped and loading level of the first residue onto resin (0.25 mmol g -1 ) was calculated as per the synthesis of brevicidine.

Synthesis of Ser9-Brevicidine (3)
The desired Fmoc-tetrapeptide was synthesized from Fmoc-Ser-OAll as described above, with Fmoc-Ser used in place of Fmoc-Thr. This resin-bound tetrapeptide (0.065 mmol, 0.14 mmol/g) was added to a manual SPPS vessel and bubbled with DMF ( (1 mL

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The desired Fmoc-tetrapeptide was synthesized from Fmoc-Ser-OAll as described above, with  (1 mL

Synthesis of MeDap9-Laterocidine (8)
Rink amide MBHA resin loaded with Fmoc-Asp-OAll (680 mg, 0.25 mmol) was was treated with Pd(PPh 3 ) 4 (75 mg, 0.075 mmol) and PhSiH 3 (0.75 mL, 7.5 mmol) in DCM (ca. 15 mL) under nitrogen for 2 h before being washed with DCM (5 × 10 mL), followed by a solution of diethyldithiocarbamic acid trihydrate sodium salt (5 mg mL −1 in DMF, 5 × 10 mL), and DMF (5 × 10 mL). TFA·H 2 N-Gly-OAll (115 mg, 0.5 mmol, 2 eq.) was then coupled using BOP (221 mg, 0.5 mmol, 2 eq.) and DiPEA (  Once the OD 600 of the bacterial suspensions reached 0.5, the bacteria were diluted with MHB (final concentration 2 × 10 5 CFU mL −1 ) and added to the microplates containing the test compounds (50 µL to each well, final volume: 100 µL). The well-plates were sealed with an adhesive membrane and after 16 h of incubation at 37°C with shaking at 220 rpm the wells were visually inspected for bacterial growth. MIC values reported are based on three technical replicates and defined as the lowest concentration of the compound that prevented visible growth of bacteria.

In vivo tolerability and efficacy studies
Ethical Issues. Animal experiments were performed under UK Home Office Licences P89653310 (tolerability and PK) and PA67E0BAA (thigh efficacy), with local ethical committee clearance.
Animal Strain. Mice used in these studies were supplied by Charles River (Margate UK) and were specific pathogen free. The strain of mice used was ICR (also known as CD1 Mice) which is a well characterized outbred murine strain. Mice (male) were 11-15 g on receipt and were allowed to acclimatise for at least 7 days.

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Animal Housing. Mice were housed in sterilised individual ventilated cages exposing the mice at all times to HEPA filtered sterile air. Mice had free access to food and water and had aspen chip bedding (changed at least once weekly). The room temperature was 22°C +/-1°C, with a relative humidity of 60% and maximum background noise of 56 dB. Mice were exposed to 12 h light/dark cycles.
Test compounds. Compound 6 was dissolved in water for injection in which it formed a clear colourless solution. Polymyxin B was dissolved in saline for injection to produce a clear colourless solution .
Tolerability study. The tolerability of compound 6 was assessed in the same mouse strain used for the efficacy studies. Compound 6 was administered via subcutaneous administration route at 3 8-h intervals indicating good tolerability up to 40 mg/kg. The mice used in the tolerability study were naïve and were not immunosuppressed or infected.
Efficacy study. The in vivo efficacy of compound 6 was assessed in a mouse thigh abscess model where both thighs of each mouse were infected with E. coli ATCC 25922 Immunosuppression. Mice were rendered neutropenic with subcutaneous injections of cyclophosphamide at 150 mg/kg 4 days before infection and 100 mg/kg 1 day before infection. The immunosuppression regime leads to neutropenia starting 24 h post administration of the first injection, which continues throughout the study.
Infection. The bacterial strain used was E. coli ATCC 25922. An aliquot of a previously prepared frozen stock of the strain was thawed and diluted in sterile PBS to the desired inoculum just prior to infection. Mice were infected with 0.05 mL of the bacterial strain suspensions by intramuscular (IM) injection under temporary inhaled anaesthesia (2.5% isofluorane for 3-4 min) into both thighs.

Analgesia.
At the time of thigh infection, buprenorphine analgesia was administered at 0.03mg/kg subcutaneously using a 0.015mg/mL solution delivered at 2 mL/kg. The same dose was administered again 9 and 17 h post-infection.