Analogues of SB-203207 as inhibitors of tRNA synthetases

Article abstract of DOI:10.1016/S0960-894X(00)00456-X

SB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl, leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00456-X

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2263±2266
Analogues of SB-203207 as Inhibitors of tRNA Synthetases
Martin G. Banwell,a,* Curtis F. Crasto,a Christopher J. Easton,a Andrew K. Forrest,b
Tomislav Karoli,a Darren R. March,a Lucy Mensah,b Michael R. Nairn,a
Peter J. O'Hanlon,b Mark D. Oldhama and Weimin Yuea
aResearch School of Chemistry, Australian National University, Canberra, ACT 0200, Australia
bSmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
Received 2 June 2000; accepted 28 July 2000
AbstractÐSB-203207 and 10 analogues have been prepared, by elaboration of altemicidin, and evaluated as inhibitors of isoleucyl,.
leucyl and valyl tRNA synthetases (IRS, LRS, and VRS, respectively). Substituting the isoleucine residue of SB-203207 with leucine
and valine increased the potency of inhibition of LRS and VRS, respectively. The leucine derivative showed low level antibacterial
activity, while several of the compounds inhibited IRS from Staphylococcus aureus WCUH29 more strongly than rat liver IRS.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
as a starting material in the preparation of analogues.
Instead, we used altemicidin (12).5 While this compound
is available via total synthesis,6 for our purposes it was
produced through fermentation.7 The methods used to
synthesise compounds 1±3 and 6 from 12 are shown in
Scheme 1.8 Compound 10 corresponds to the inter-
mediate 13 in the case when (S)-3-methylpentanoic acid
was used as the acylating agent. Hydrogenolysis of 10
a€orded 8. Compound 5 was prepared using tBoc-(S)-
isoleucine instead of the Cbz-derivative, and in that case
the intermediate corresponding to 13 was deprotected
through base hydrolysis, then acid hydrolysis. Com-
pounds 4, 7, 9, and 11 were prepared from altemicidin
methyl ester9 by applying similar methodology.
SB-203207 (1) was isolated from Streptomyces NCIMB
40513 and shown to inhibit isoleucyl tRNA synthetase
(IRS) from Staphylococcus aureus Oxford and rat liver,
with IC50 values of 1.7 and <2 nM, respectively.1,2 The
inhibition of IRS by SB-203207 (1) implies a structural
resemblance between this compound and the enzyme's
natural substrate. The natural substrates of other tRNA
synthetases are the same as that of IRS, except that they
have di€erent amino acid side chains. Therefore, it
seemed likely that replacing the isoleucine residue of SB-
203207 (1) with, for example, leucine and valine would
produce selective inhibitors of LRS and VRS, respec-
tively. By analogy, Creppy et al.3,4 reported that
whereas ochratoxin A is an inhibitor of a phenylalanyl
tRNA synthetase, substituting the phenylalanine residue
of this compound with valine a€orded a VRS inhibitor. To
examine this hypothesis and carry out related structure±
activity studies, SB-203207 (1) and the analogues 2±11
have been prepared from altemicidin (12), and eval-
uated as inhibitors of IRS, LRS, and VRS.
Inhibition Assays
The results of studies of the interactions of compounds
1±11 with IRS, LRS, and VRS are summarised in Table
1. The esters 4 and 5 are less potent than 1 as inhibitors
of all the synthetases studied, and the des-amino deri-
vative 6 of SB-203207 (1) is even less active. The more
substantially modi®ed analogues 7±11 show only low
activity. SB-203207 (1) inhibits bacterial and mammalian
IRS more strongly than LRS and VRS, and this beha-
viour is re¯ected with compounds 4±11, presumably
because each contains the sec-butyl side chain of iso-
leucine. In contrast, the leucine derivative 2 selectively
inhibits LRS. The valine derivative 3 is more potent as
an inhibitor of VRS than either SB-203207 (1) or the
Synthesis
Compound 1 was not available from natural sources in
sucient quantity to allow for further testing or for use
*Corresponding author. Tel.: +61-2-6279-8201; fax: +61-2-6279-
8114; e-mail: easton@rsc.anu.edu.au
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00456-X

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M. G. Banwell et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2263±2266
Table 1. Inhibition of tRNA synthetases by SB-203207 (1) and related compounds
Synthetase IC50 values (mM) or percentage inhibition10
Compound
IRSa
IRSb
LRSa
VRSa
1
2
3
4
5
6
7
8
0.014
0.91
0.037
0.06
0.12
0.0042
0.068
0.0029
0.017
0.025
1.55
0.016
2.3
0.126
0.29
0.03
186
40% @ 100 mM
NI @ 10 mM
NI @ 100 mM
NI @ 100 mM
65% @ 100 mM
NI @ 100 mM
6% @ 100 mM
NI @ 100 mM
NI @ 10 mM
NI @ 100 mM
13% @ 100 mM
NI @ 100 mM
NI @ 100 mM
NI @ 100 mM
NI @ 100 mM
3.27
23% @ 100 mM
14.6
18% @ 100 mM
19% @ 100 mM
46% @ 100 mM
22.3
25% @ 100 mM
6.1
9% @ 100 mM
20% @ 100 mM
0.5
9
10
11
aFrom Staphylococcus aureus WCUH29.
bFrom rat liver.
Scheme 1.

M. G. Banwell et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2263±2266
2265
Table 2. Antibacterial activity of SB-203207 (1) and the leucine ana-
logue 2
7. Streptomyces A5941 was grown according to the litera-
ture.12 The extraction procedure at 300 L scale was as follows.
The harvested broth was centrifuged and the supernatant was
loaded onto a PK 208 cation exchange column. Altemicidin
(12) was eluted with 0.5 M NH4OH. The eluate was con-
centrated by reverse osmosis to low volume, and acidi®ed to
pH 2. The precipitate which formed was separated by cen-
trifugation and discarded. The supernatant was loaded onto a
Biotage HP2Oss cartridge at pH 2, the column was washed
with water, and altemicidin (12) was eluted with 2% acetoni-
trile. The eluate was evaporated and freeze-dried. The product
was further puri®ed on a Sephadex G10 size exclusion column,
then by crystallisation from water, to give pure altemicidin
(12).
MIC value (mg/mL)11
Bacterial strain
1
2
Staphylococcus aureus Oxford
Staphylococcus aureus WCUH29
Enterococcus faecalis 1
Enterococcus faecalis 7
Haemophilus in¯uenzae Q1
>64
>64
>64
>64
64
>64
>64
>64
>64
64
Haemophilus in¯uenzae NEMC1
Moraxella catarrhalis 1502
64
64
>64
32
64
16
>64
>6 4
>64
>64
>64
64
>64
>6 4
Streptococcus pneumoniae 1629
Streptococcus pneumoniae N1387
Streptococcus pneumoniae ERY2
Escherichia coli 7623 AcrABEFD+
Escherichia coli 120 AcrAB
8. Data for compound 1: mp 203±204 ꢀC (decomp.); FTIR
(KBr disk) 3440 (sh), 3357, 3229, 1718 (sh), 1633, 1510, 1393,
1282, 1114, 849 cm 1; 1H NMR (300 MHz, CD3OD: 0.04 equiv
AcOH) d 0.96 (3H, t, J=7.5 Hz), 1.05 (3H, d, J=7.0 Hz),
1.17±1.33 (2H, m), 1.55±1.70 (1H, m), 1.94±2.09 (1H, m),
2.59±2.76 (1H, m), 2.81±3.05 (4H, m), 2.97 (3H, s), 3.57 (1H,
d, J=4.3 Hz), 4.10 (1H, J=13.9 Hz), 4.30 (1H, J=13.9 Hz),
4.24±4.34 (lH, m), 7.34 (1H, s); 13C NMR (75 MHz, CD3OD)
d 12.1, 15.6, 25.5, 32.6, 38.0, 41.5, 43.1 (2 signals), 45.9, 58.5,
61.6, 67.9, 76.4, 99.5, 146.3, 165.9, 174.0, 175.5, 176.5;
ESMS( ) 488 (M H, 100); LSIMS-HRMS calcd for
C19H32N5O8S (M+H): 490.1973. Found: 490.1955. Anal.
leucine analogue 2. To this extent, the data for com-
pounds 1±3 indicate that replacing the isoleucine moiety
of SB-203207 (1) with other amino acid residues pro-
duces inhibitors of the corresponding synthetases.
However, the leucine and valine derivatives 2 and 3 are
also both potent inhibitors of IRS, more so than of VRS
in the case of 3 and mammalian IRS. This lack of ®d-
elity displayed by the synthetases is surprising.
.
calcd for C19H31N5O8S 3H2O: C, 41.98; H, 6.86; N, 12.88.
Found: C, 42.12; H, 6.69; N, 12.99%. Data for compound 2:
mp 195 ꢀC (deform), 201 ꢀC (melt); FTIR (KBr disk) 3435,
1
1717 (sh), 1634, 1510, 1384, 1340, 1282, 1112, 844 cm
;
1H
Compounds 2, 3, and 11 show a marked preference for
inhibition of the rat IRS over the bacterial protein,
whereas compound 6 selectively binds to bacterial IRS.
This indicates that there are di€erences between bacterial
and mammalian tRNA synthetases that may allow
development of antibiotics through selective inhibition
of the bacterial enzymes. Compounds 3±11 did not
show antibacterial activity against any of the organisms
tested, but the leucine derivative 2 of SB-203207 (1)
displayed low level activity (Table 2) and was more
potent than 1 against three strains of Streptococcus
pneumoniae.
NMR (300 MHz, CD3OD: 0.13 equiv AcOH) d 0.97 (3H, d,
J=6.3 Hz), 0.99 (3H, d, J=6.4 Hz), 1.27±1.32 (1H, m), 1.57±
1.70 (1H, m), 1.74±1.87 (2H, m), 2.61±2.75 (1H, m), 2.82±3.04
(4H, m), 2.97 (3H, s), 3.66 (lH, dd, J=5.0, 8.7 Hz), 4.12 (1H,
J=14.0 Hz), 4.30 (1H, J=14.0 Hz), 4.24±4.36 (1H, m), 7.34
(1H, s); 13C NMR (75 MHz, CD3OD) d 22.4, 23.6, 25.9, 32.9,
41.7, 42.0, 43.5 (2 signals), 46.2, 55.9, 58.7, 68.2, 76.7, 99.8,
146.6, 166.2, 174.3, 176.9, 177.1; ESMS( ) 488 (M H, 100);
LSIMS-HRMS calcd for C19H32N5O8S (M+H): 490.1973.
.
Found: 490.1979. Anal. calcd for C19H31N5O8S 2H2O: C,
43.42; H, 6.71 N, 13.33. Found: C, 43.67; H, 6.47; N, l3.27%.
Data for compound 3: mp 199±202 ꢀC (decomp.); FTIR (KBr
disk) 3437, 1717 (sh), 1634, 1509, 1399, 1384, 1342, 1284, 1112,
1
851 cm 1; H NMR (300 MHz, CD3OD: 0.12 equiv AcOH) d
1.02 (3H, d, J=6.9 Hz), 1.08 (3H, d, J=7.0 Hz), 1.17±1.32
(1H, m), 2.36±2.41 (1H, m), 2.62±2.74 (1H, m), 2.78±3.03 (4H,
m), 2.97 (3H, s), 3.51 (1H, d, J=4.6 Hz), 4.12 (1H,
J=13.9 Hz), 4.31 (1H, J=13.9 Hz), 4.24±4.36 (1H, m), 7.34
(1H, s); 13C NMR (75 MHz, CD3OD) d 17.8, 19.6, 31.4, 32.9,
41.8, 43.4 (2 signals), 46.2, 58.8, 62.7, 68.2, 76.7, 99.8, 146.6,
166.2, 174.3, 176.0, 176.9; ESMS( ) 474 (M H, 100); LSIMS-
HRMS calcd for C18H30N5O8S (M+H): 476.1815. Found:
Acknowledgements
The authors acknowledge the assistance of Anna Stefanska
in supplying altemicidin (12) and Stephen Rittenhouse in
providing the antibacterial pro®ling data, as well as
Brian Metcalf (SmithKline Beecham Pharmaceuticals,
USA) for supporting this collaboration.
.
476.1824. Anal. calcd for C18H29N5O8S l.5H2O: C, 43.02; H,
6.42; N, 13.94. Found: C, 42.88; H, 6.39; N, 13.77%. Data for
compound 6: mp 165.5±168.0 ꢀC; FTIR (KBr disk) 3457 (sh),
3358, 3237 (sh), 1712, 1670 (sh), 1638, 1507, 1463, 1404, 1342,
References and Notes
1
1. Stefanska, A. L.; Cassels, R.; Ready, S. J.; Warr, S. R. J.
Antibiot. 2000, 53, 357.
2. Houge-Frydrych, C. S. V.; Gilpin, M. L.; Skett, P. W.;
Tyler, J. W. J. Antibiot. 2000, 53, 364.
3. Creppy, E. E.; Mayer, M.; Kern, D.; Schlegel, M.; Steyn, P.
S.; Vleggaar, R.; Dirheimer, G. Biochim. Biophys. Acta. 1981,
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schenthaler, R.; Dirheimer, G. Toxicol. Lett. 1983, 19, 217.
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Takeuchi, T. J. Antibiot. 1989, 42, 1556.
1290, 1157, 1143, 1080, 906, 865 cm
;
1H NMR (300 MHz,
CD3OD: 0.15 equiv AcOH) d 0.91 (3H, t, J=7.5 Hz), 0.95
(3H, d, J=6.7 Hz), 1.16±1.32 (2H, m), 1.34±1.47 (1H, m),
1.84±1.97 (1H, m), 2.13 (1H, dd, J=8.1, 14.8 Hz), 2.35 (1H,
dd, J=6.3, 14.8 Hz), 2.60±2.72 (1H, m), 2.80±3.02 (4H, m),
2.96 (3H, s), 4.25±4.30 (1H, m), 4.42 (1H, J=14.5 Hz), 4.56
(1H, J=14.5 Hz), 7.34 (1H, s); 13C NMR (75 MHz, CD3OD) d
11.6, 19.4, 30.3, 32.5, 33.0, 41.4, 43.l, 43.2, 44.4, 45.8, 57.9,
67.9, 76.2, 99.6, 146.2, 163.5, 174.0, 175.0, 176.1; ESMS( )
473 (M H, 100); LSIMS-HRMS calcd for C19H31N4O8S
(M+H): 475.1864. Found: 475.1843. Anal. calcd for
.
6. Kende, A. S.; Liu, K.; Jos Brands, K. M. J. Am. Chem.
Soc. 1995, 117, 10597.
C19H30N4O8S 1.5H2O: C, 45.50; H, 6.63; N, 11.17. Found: C,
45.82; H, 6.52; N, 11.15%.

2266
M. G. Banwell et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2263±2266
9. Takahashi, A.; Ikeda, D.; Nakamura, H.; Naganawa, H.;
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11. Whole-cell antibacterial activity was determined by broth
microdilution. Compounds were dissolved in DMSO and diluted
1:10 in water to produce a 256 mg mL 1 stock solution. Using a
96-well microtitre plate, a Microlab AT Plus 2 (Hamilton Co.,
Reno, NV) serially diluted 50 mL of the stock solution into
supplemented cation adjusted Mueller Hinton broth (Beckton
Dickinson, Cockeysville, MD). After the compounds were
1
diluted, a 50 mL aliquot of the test isolate (ꢁ1Â106 cfu mL
)
was added to each well of the microtitre plate using the
Microlab AT Plus 2. The ®nal test concentrations ranged from
0.06 to 64 mg mL 1. Inoculated plates were incubated at 35 ꢀC
in ambient air for 18±24 h. Following incubation, a microtitre
mirror reader (Cooke Instruments Ltd, UK) was used to assist
in reading the minimum inhibitory concentration (MIC). The
MIC was determined as the lowest concentration of com-
pound that inhibited visible growth of the test isolates.
12. Isaac, B. G.; Ayer, S. W.; Stonard, R. J. Environ. Sci. Res.
1992, 44, 157.