Synthesis of the amSPCs

Compounds 1950, 1951, 2241, and 2324 were synthesized according to the procedure reported previously [8]. The purity and identity of the compounds was determined using reverse phase Ultra-Performance Liquid Chromatography (UPLC) analysis with Evaporating Light Scattering Detector MS (ELSD/MS) detection and by 1H and 13C NMR spectroscopy. All compounds were confirmed to be >95% pure before being submitted for biological testing. Elemental analysis of select compounds was performed by Atlantic Microlabs to confirm salt composition.

3′-(R)-3′-(N-(4-fluorobenzyl)aminomethyl)dihydrospectinomycin trihydrochloride (1950)

1H NMR (D2O, 400 MHz): δ 7.34 (dd, J = 3.6, 10.3 Hz, 2H), 7.12–7.07 (m, 2H), 4.51 (s, 1H), 4.48 (s, 1H), 3.98 (t, J = 10.5 Hz, 1H), 3.90 (d, J = 13.4 Hz, 1H), 3.86–3.76 (m, 2H), 3.73–3.73 (m, 2H), 2.99 (d, J = 13.5 Hz, 1H), 2.88–2.79 (m, 3H), 2.57 (s, 3H), 2.42 (s, 3H), 1.75–1.60 (m, 2H), 1.13 (d, J = 6.0 Hz, 3H). 13C NMR (126 MHz, D2O): δ 163.20(d), 132.25, 132.18, 125.96, 116.24, 116.06, 93.17, 92.31, 72.16, 69.59, 67.31, 65.87, 65.28, 61.45, 59.46, 58.43, 50.74, 48.88, 40.00, 30.55, 30.29, 19.84. High-resolution electrospray ionization mass spectrometry (HRMS-ESI) calculated for C22H34FN3O7 [M+H]+ 472.2459, found 472.2459. Elemental analysis theoretical composition for empirical formula C22H34FN3O7∙3 HCl∙2.5 H2O: C, 42.46; H, 6.74; N, 6.75; Cl, 17.09; F, 3.05. Found formula for C22H34FN3O7∙3 HCl∙2.5 H2O: C, 42.21; H, 6.86; N, 6.52; Cl, 16.73; F, 2.85.

3′-(R)-3′-(N-(4-ethylbenzyl)aminomethyl)dihydrospectinomycin trihydrochloride (1951)

1H NMR (D2O, 400 MHz): δ 7.44 (d, J = 7.6 Hz, 2H), 7.40 (d, J = 7.6 Hz, 2H), 4.74 (s, 1H), 4.33–4.20 (m, 3H), 4.01 (t, J = 10 Hz, 1H), 3.94 (t, J = 10 Hz, 1H), 3.69 (m, 1H), 3.50 (m, 1H), 3.38 (ABq, J = 14.0 Hz, 1H), 3.17 (ABq, J = 14.0 Hz, 1H), 2.81 (s, 3H), 2.76 (s, 3H), 2.68 (m, 2H), 1.82 (m, 2H), 1.22 (m, 8H). 13C NMR (126 MHz, D2O): δ 146.68, 130.10, 128.77, 127.39, 93.20, 92.35, 72.12, 69.80, 67.27, 66.01, 65.48, 61.58, 59.60, 58.54, 51.11, 48.76, 40.06, 30.54, 30.35, 27.97, 19.84, 14.84. HRMS-ESI calculated for C24H39N3O7 [M+H]+ 482.2788, found 482.2873.

3′-(R)-3′-(N-(3-phenylpropyl)aminomethyl)dihydrospectinomycin trihydrochloride (2241)

1H NMR (D2O, 400 MHz): δ 7.40 (m, 2H), 7.32 (m, 3H), 4.89 (s, 1H), 4.67 (t, J = 4 Hz, 1H), 4.15 (t, J = 12.0 Hz, 1H), 4.01 (t, J= 8.0 Hz, 1H), 3.90 (t, J = 8.0 Hz, 1H), 3.84 (m, 1H), 3.43 (ABq, J = 14.0 Hz, 1H), 3.22 (br, 2H), 3.10 (m, 3H), 2.77 (s, 3H), 2.74 (m, 2H), 2.63 (s, 3H), 2.05 (m, 2H), 1.88 (d, J = 13.2 Hz, 1H), 1.78 (d, J = 13.2 Hz, 1H), 12.5 (d, J= 6.0 Hz, 3H). 13C NMR (126 MHz, D2O): δ 128.80, 128.53, 128.47, 126.50, 96.35, 93.23, 70.02, 67.32, 66.19, 65.72, 61.69, 59.77, 58.64, 50.22, 48.83, 48.15, 42.79, 40.04, 31.84, 30.61, 30.46, 26.59, 23.19, 19.88. HRMS-ESI calculated for C24H39N3O7 [M+H]+ 482.2866, found 482.2860. Elemental analysis theoretical composition for the empirical formula C24H39N3O7∙3 HCl∙2.3 H2O: C, 45.58; H, 7.43; N, 6.64; Cl, 16.82. Found formula for C22H34FN3O7∙3 HCl∙2.3 H2O: C, 45.36; H, 6.96; N, 6.54; Cl, 16.66.

3′-(R)-3′-(N-(4-chlorobenzyl)aminomethyl)dihydrospectinomycin trihydrobromide (2324)

1H NMR (D2O, 400 MHz): δ 7.48–7.37 (m, 4H), 4.30–4.16 (m, 3H), 3.97 (t, J = 9.9 Hz, 1H), 3.88 (t, J = 10.0 Hz, 1H), 3.73–3.62 (m,1H), 3.50–3.45 (m, 1H), 3.33 (d, J = 13.6 Hz, 1H), 3.26 (s, 2H), 3.21–3.12 (m, 2H), 2.75 (s, 6H), 1.83–1.67 (m, 2H), 1.15 (d, J = 6.0 Hz, 3H). 13C NMR (126 MHz, D2O): δ 135.28, 131.57, 129.34, 128.53, 93.16, 92.32, 72.18, 69.56, 67.33, 65.86, 65.28, 61.43, 59.45, 58.43, 50.76, 49.03, 40.02, 30.60, 30.37, 19.87. HRMS-ESI calculated for C22H34ClN3O7 [M+H]+ 488.2164, found 488.2169.


MIC assay

The MICs were determined as recommended by CLSI in 96-well plates with compound at an initial concentration of 200 µg ml−1 and serially diluted twofold. Approximately 5 × 104 CFU of bacteria in CAMHB (or CAMHB supplemented with 2% IsoVitalex for F. tularensis) were added to each well and plates were incubated at 37°C for 24–48 h. The MIC was determined as the lowest concentration of the compound that prevented growth of the bacterial strain. The MIC50 and MIC90 values were determined as the concentration of the amSPCs at which growth was inhibited in 50 and 90%, respectively, of the strains that were tested.

The MICs of spectinomycin and compound 1950 were also tested in combination with other antibiotics or polymyxin B nonapeptide (PMBN). For the dual antibiotic studies, a checkerboard serial dilution of antibiotics was completed in 96-well plates so that, when possible, the MIC of an individual antibiotic was in the middle of the dilutions (i.e., there were at least two dilutions above and below the MIC in the checkerboard). The bacteria were applied to the plates and incubated as described above. The fractional inhibitory concentration (FIC) index was calculated with the following formula: FIC = (A/MICA) + (B/MICB), where MICA and MICB are the MIC of each drug individually and A and B are the MICs of each antibiotic in combination. Effects were determined to be synergistic (FIC ≤ 0.5), additive (0.5 < FIC ≤ 1.0) indifferent (1.0 < FIC ≤ 4.0), or antagonistic (FIC > 4.0).

For the PMBN combination studies, a checkerboard serial dilution of spectinomycin or compound 1950 (initial concentration 64 µg ml−1) and PMBN (initial concentration 32 µg ml−1) were completed in a 96-well plate. F. tularensis LVS or B. pseudomallei strain Bp82 were applied to the plate as described above and incubated at 37°C with 5% CO2 for 40–48 h. The MIC of spectinomycin or 1950 was recorded for each concentration of PMBN.


Time-kill assay

An overnight culture of B. mallei strain FMH in CAMHB was diluted to an OD600 of 0.01 and incubated, with shaking, at 37 °C for 6 h to allow the culture to reach the mid-exponential phase of growth. The culture was then diluted to a concentration of 5 × 105 CFU ml−1. Compound 1950, spectinomycin, or an equal volume of DMSO/diluent was added such that the final concentration of compound/antibiotic was 50 µg ml−1 and DMSO was 0.5%. The samples were incubated at 37°C with aeration. A sample was removed from the culture at 0, 1.25, 2.5, 4, 6, and 24 h for serial dilution and enumeration of the bacteria on sheep blood agar (SBA) plates. Significant differences between the groups at each time point were determined by multiple t tests with the Holm-Šídák method to correct for multiple comparisons in GraphPad Prism 7.


Mouse infection and colonization

Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. Six- to eight-week-old BALB/c mice (Charles River Laboratories/NCI, Frederick, MD) were used for all experiments.

Mice received an intranasal (i.n.) instillation of 3 × 104 CFU B. mallei strain FMH in 20 µl. Treatment with amSPC 1950 (25 or 50 mg kg−1) or saline was initiated at 1 h post-infection. Treatments were administered twice daily (BID) via s.c. injection in 200 µl for 14 days. Mice were closely monitored for morbidity and mortality. On day 40, five mice were removed from the 1950-treated groups (25 and 50 mg kg−1) for necropsy and were marked as censored subjects for the survival curve. Mice that survived until day 60 were euthanized and necropsy was completed. The lungs, livers, and spleens of mice were removed, homogenized, and CFU g−1 of tissue was determined.

To determine if a delay in initiation of treatment had an effect on survival of mice post-infection, mice received an i.n. inoculum of 2 × 104 CFU B. mallei strain FMH in 20 µl. Treatment with 1950 (50 mg kg−1, BID), administered s.c. in 200 µl, was initiated at 1, 6, 24, or 48 h post infection and continued for 14 days. Control mice were administered saline or spectinomycin (50 mg kg−1, BID) starting at 1 h post infection. Mice were closely monitored for morbidity and mortality for 42 days.

Mice were infected i.n. with 1 × 103 CFU F. tularensis strain SchuS4 in 20 µl. Treatment with compound 1950 (50 or 75 mg kg−1) or spectinomycin (50 mg kg−1) in 200 µl was initiated at 1 h post-infection and continued BID for 14 days. Mice were closely monitored for morbidity and mortality for 55 days. In a second experiment, mice were infected with 900 CFU F. tularensis SchuS4 and s.c. administration of saline, spectinomycin (50 mg kg−1), or 1950 (50 mg kg−1) in 200 µl was initiated at 1 h post-infection and continued three times daily (TID) for 14 days. Mice were closely monitored for morbidity and mortality for 37 days.

Significant differences in survival between groups were determined by log-rank (Mantel–Cox) test in GraphPad Prism 7. Significant differences in bacterial load were determined by Mann–Whitney test in GraphPad Prism 7.

Source