J. B. Bremner et al. / Bioorg. Med. Chem. 18 (2010) 2611–2620
2619
(33
l
L, 0.33 mmol). The mixture was stirred overnight at rt, and
2H), 3.72–3.90 (m, 1H), 3.93–4.10 (m, 2H), 4.20–4.54 (m, 4H),
4.94–5.14 (m, 2H), 6.93–6.96 (m, 2H, ArH), 7.03–7.09 (m, 2H,
ArH), 7.14–7.30 (m, 7H, ArH), 7.34 (d, J = 9.1 Hz, 1H, ArH), 7.42
(d, J = 9.1 Hz, 1H, ArH), 7.75–7.93 (m, 4H, ArH); 13C NMR
(75 MHz, CD3OD) d 21.7, 22.5, 22.8, 23.1, 23.3, 25.5, 25.9, 26.2,
27.7, 30.1, 32.1, 39.2, 40.4, 41.0, 41.9, 52.3, 53.6, 54.1, 67.8, 68.9,
69.2, 115.9, 116.9, 120.4, 121.7, 124.8, 125.1, 125.9, 126.3, 127.4,
127.5, 129.0, 129.1, 129.3, 129.5, 129.6, 130.6, 130.8, 130.9,
131.3, 135.0, 135.1, 137.1, 153.9, 155.8, 158.4, 170.9, 173.1,
then the solvent was evaporated to dryness. Purification by flash
column chromatography afforded the desired product (239 mg,
91%) an off white solid. Rf = baseline (5% MeOH/CH2Cl2). 1H NMR
(300 MHz, CDCl3)
d 0.82 (d, J = 5.8 Hz, 3H, CH3), 0.84 (d,
J = 5.8 Hz, 3H, CH3), 1.24–2.02 (m, 13H), 1.27 (s, 6H, 2CH3 (Pmc)),
1.38 (s, 9H, C(CH3)3), 1.76 (t, J = 6.0, 2H, CH2 (Pmc)), 2.07 (s, 3H,
CH3 (Pmc)), 2.52 (s, 3H, CH3 (Pmc)), 2.54 (s, 3H, CH3 (Pmc)),
2.64–2.62 (m, 2H, CH2 (Pmc)), 2.90–3.08 (m, 2H, CH2N (Lys)),
3.09–3.24 (m, 1H, CH2N (Arg, Ha)), 3.25–3.36 (m, 1H, (Arg, Hb)),
4.38–4.61 (m, 2H), 4.84–4.98 (m, NH), 5.03 and 5.09 (ABq,
J = 12.3 Hz, 1H), 6.20–6.50 (m, 3H, NH), 7.24–7.29 (m, 5H, ArH),
7.58 (d, J = 7.9 Hz, NH), 7.95 (d, J = 7.3 Hz, NH); 13C NMR
(75 MHz, CDCl3) d 12.0, 17.3, 18.4, 21.2, 21.4, 22.6, 24.7, 25.4,
26.6, 28.3, 29.6, 29.8, 32.6, 34.6, 40.1, 40.3, 50.8,52.1, 54.8, 66.7,
73.5, 78.8, 117.8, 123.8, 127.8, 128.1, 128.4, 133.3, 134.6, 135.2,
135.3, 153.4, 156.2, 171.8, 172.6, 175.8. MS (ESI +ve) m/z 872
([M+H]+, 100%).
173.6, 174.0. MS (ESI +ve) m/z 902 ([M+H]+, 10%), 452 ([M+H]2+
,
100%). HRMS (ESI +ve) calcd for C52H68N7O7 902.5180, found
902.5189 ([M+H]+, 100%).
3.2. HPLC method
Gradient system comprised of H2O containing 10% CH3CN and
0.1% TFA (A), and CH3CN containing 0.1% TFA (B). The gradient pro-
file was 0–2 min, isocratic 15% B; 3–35 min, linear gradient 15–60%
of B; 35–36 min, isocratic 60% B; 37–42 min, isocratic, 100% B.
tR = 28.7, 99% pure.
3.1.2.5. Benzyl (2S,5R,8R)-3,6,9-triaza-5-{3-[(3,4-dihydro-2,2,5,
7,8-pentamethyl-2H-1-benzopyran-6-yl)sulfonyl]guanidinopro-
pyl}-11-[(S)-20-(3-methylbutoxy)-1,10-binaphthalen-2-yloxy]-8-
[4-(1,1-dimethylethoxycarbonylamino)butyl]-2-(2-methylpro-
pyl)-4,7,10-trioxoundecanoate (protected 96). To a solution of
8714 (50 mg, 0.121 mmol) and 50 (110 mg, 0.126 mmol) in acetoni-
trile (10 mL) was added HOBt (20 mg, 0.15 mmol) and EDCI
(28 mg, 0.15 mmol). The mixture was stirred at rt for 3 h before
the solvent being evaporated to dryness. Purification by flash col-
umn chromatography (3% MeOH/CH2Cl2) afforded the desired
product (114 mg, 74%) as an off white solid. Rf = 0.16 (5% MeOH/
CH2Cl2). 1H NMR (300 MHz, CDCl3) d 0.46 (d, J = 6.2 Hz, 3H), 0.52
(d, J = 6.2 Hz, 3H), 0.70–1.00 (m, 9H), 1.20–1.35 (m, 5H), 1.28 (s,
6H, CH3 (Pmc)), 1.38–1.54 (m, 2H), 1.41 (s, 9H, C(CH3)3), 1.57–
1.87 (m, 7H), 2.09 (s, 3H, CH3 (Pmc)), 2.40–2.48 (m, 2H, CH2
(Pmc)), 2.55 (s, 3H, CH3 (Pmc)), 2.57 (s, 3H, CH3 (Pmc)), 2.80–
2.98 (m, 2H, CH2N (Lys)), 3.00–3.30 (m, 2H, CH2N (Arg)), 3.78–
3.92 (m, 1H), 3.99–4.15 (m, 2H), 4.34–4.60 (m, 4H), 4.78–4.90
(m, 1H, NH), 5.07 and 5.16 (ABq, J = 12.6 Hz, 1H), 6.08 (s, br, NH),
6.18 (d, J = 7.0 Hz, NH), 6.29 (br s, NH), 6.48 (br s, NH), 7.09–7.21
(m, 11H, ArH), 7.45 (d, J = 9.1 Hz, 2H, ArH), 7.69–7.90 (m, 2H,
ArH), 7.91–8.01 (m, 2H, ArH), 8.06 (d, J = 8.8 Hz, NH); 13C NMR
(75 MHz, CDCl3) d 12.1, 17.5, 18.6, 21.4, 21.6, 22.0, 22.3, 22.9,
24.5, 24.9, 25.2, 26.8, 28.5, 29.2, 32.8, 38.0, 39.9, 40.1, 40.4, 40.6,
51.1, 52.2, 53.3, 67.0, 68.0, 68.7, 73.6, 79.2, 114.1, 116.2, 117.9,
119.8, 120.2, 124.0, 124.1, 124.3, 125.1, 125.5, 126.7, 126.8,
128.0, 128.1, 128.3, 128.5, 128.6, 129.3, 129.6, 129.8, 130.4,
133.5, 133.6, 133.9, 134.9, 135.47, 135.54, 152.1, 153.5, 154.4,
156.2, 169.5, 171.4, 173.1. MS (ESI +ve) m/z 1291 ([M+Na]+, 70%),
3.3. Determination of minimum inhibitory concentration (MIC)
MIC studies were performed on S. aureus wild type (ATCC
6538P), Mu50 (ATCC 700699) and MRSA (ATCC 43300) in Luria
Broth. MIC determinations for wild type and clinical isolates of
Enterococcus faecium were conducted by growth in Enterococcosal
broth (Becton Dickinson Microbiology Systems). Briefly, overnight
stationary phase cultures were diluted 1:1000 into fresh media and
then incubated with two fold dilutions of compound in media, typ-
ically with a highest concentration of 128 lg/ml, in a 96 well plate.
Plates were incubated overnight at 37 °C and the MIC recorded as
the highest concentration at which bacterial growth was observed.
Compound 96 was tested at ImQuest BioSciences on S. aureus
wild types (ATCC 6538P), Mu50 (ATCC 700699) and MRSA (ATCC
43300), S. epidermidis (ATCC 700562) and E. faecium (ATCC
700221). MICs of compounds were determined using micro-broth
dilution analysis grown in Mueller Hinton II broth (cation ad-
justed) to a concentration of 1 ꢀ 106 colony forming units per
mL. 100 mL was placed into triplicate wells containing 100 mL of
test compound serially diluted twofold in Mueller Hinton II broth.
The plates were incubated for 24 h at 37 °C and the MIC deter-
mined at the lowest compound dilution that completely inhibited
microbial growth.
Acknowledgments
We thank the former Amrad Operations Pty Ltd and Avexa Lim-
ited, the University of Wollongong and the National Health and
Medical Research Council (Development Grants 303415 and
404528) for their support, Dr. Susan Cox for her support in the ini-
tial development of this project and R. W. Buckheit, Jr. and K. M.
Watson from ImQuest BioSciences, 7340 Executive Way, Suite R,
Frederick, MD 21704, USA for providing some assays.
1268 ([M+H]+, 100%). HRMS (ESI +ve) calcd for C71H94N7O12
1268.6681, found 1268.6687 ([M+H]+, 100%).
S
3.1.2.6. Benzyl (2S,5R,8R)-3,6,9-triaza-8-butylamino-5-(3-gua-
nidinopropyl)-11-[(S)-20-(3-methylbutoxy)-1,10-binaphthalen-
2-yloxy]-8-(4-(1,1-dimethylethoxycarbonylamino)butyl)-2-(2-
methylpropyl)-4,7,10-trioxoundecanoate
dihydrochloride
(96). To a solution of protected 96 (104 mg, 0.082 mmol) in
CH2Cl2 (5 mL) was added TFA (5 mL). The mixture was stirred for
16 h at rt. The solvent was evaporated under reduced pressure.
The crude residue was redissolved in CH2Cl2 (3 mL). 1 M HCl–
Et2O (excess) was added, and the mixture was stirred for a minute
before the solvent being removed. This step was repeated twice
more. The crude product was purified by precipitation from CH2Cl2
by additional of diethyl ether to yield the desired product (78 mg,
98%) as a white solid. 1H NMR (300 MHz, CD3OD) d 0.38 (d,
J = 6.2 Hz, 3H), 0.43 (d, J = 6.2 Hz, 3H), 0.64–0.93 (m, 9H), 0.94–
1.20 (m, 3H), 1.1.24–1.82 (m, 10H), 2.69 (s, br, 2H), 3.05 (s, br,
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
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