Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

Article abstract of DOI:10.1016/j.bmcl.2003.08.015

Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis β-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-(4′ -benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thiolactomycin.

Full text of DOI:10.1016/j.bmcl.2003.08.015

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3685–3688
Biphenyl-Based Analogues of Thiolactomycin, Active against
Mycobacterium tuberculosis mtFabH Fatty Acid Condensing
Enzyme
Suzanne J. Senior,a,b Petr A. Illarionov,a Sudagar S. Gurcha,a Ian B. Campbell,c
Merrill L. Schaeffer,d David E. Minnikina and Gurdyal S. Besraa,*
aSchool of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
bDepartment of Microbiology &Immunology, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK
cGlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, UK
dGlaxoSmithKline, Collegeville, Pennsylvania, PA 19426, USA
Received 24 June 2003; revised 7 July 2003; accepted 8 August 2003
Abstract—Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro
inhibitory activity against the recombinant Mycobacterium tuberculosis b-ketoacyl-ACP synthase mtFabH condensing enzyme. In
a 4-fold
particular, 5-(40-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately
increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thio-
lactomycin.
# 2003 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis continues to be a primary
cause of morbidity and mortality worldwide; it is cur-
rently estimated that one-third of the world’s popula-
tion is infected with the bacillus.1 The emergence of
bacterial resistance to existing antitubercular agents has
become a significant concern for the effective treatment
of tuberculosis. The determination of the whole-genome
sequence of M. tuberculosis2 has pinpointed targets that
potentially offer improved therapy.3
(CoA):ACP transacylase activities in vivo and in
vitro.9,10 In addition, TLM possesses encouraging anti-
malarial activity, involving inhibition of the type II fatty
acid biosynthetic pathway in apicoplasts.11
TLM inhibits M. tuberculosis FAS-II through inhibition
of both b-ketoacyl-ACP synthase condensing enzymes,
mtFabH and KasA, in vitro and in vivo leading to
inhibition of cell wall mycolic acid biosynthesis and to
cell death.12ꢀ15 Previous studies have shown that a
number of TLM analogues, with aliphatic substituents
at the 5-position of the thiolactone ring, have sig-
nificantly enhanced activity against mycolate synth-
esis.13,16 The best results were obtained with 10-carbon
isoprenoid-based side chains, but the use of more chem-
ically stable aromatic substituents is an attractive alter-
native. It is shown here that a single benzyl 3 group has
little activity against the key condensing enzyme
mtFabH, but addition of para-substituents leads to a
range of encouraging activities (Table 1). In particular,
analogues incorporating a biphenyl unit with a distal
para-oxygen function had excellent activity (Table 1).
These latter analogues were conveniently prepared by
Suzuki coupling protocols.
Thiolactomycin (TLM, 2, Table 1) is a thiolactone
antibiotic isolated from a soil Nocardia spp.4 TLM
exhibits potent in vivo activity against many pathogenic
bacteria, including Gram-negative and Gram-positive
bacteria and M. tuberculosis.5ꢀ7 It is of relevance in the
present context that TLM inhibits bacterial and plant
type II fatty acid synthases (FAS-II) but not mamma-
lian or yeast type I fatty acid synthases (FAS-I).8 For
instance, in Escherichia coli, TLM inhibits both b-
ketoacyl-ACP synthase I to III and acetyl coenzyme A
*Corresponding author. Fax: + 44-121-414-5925; e-mail: g.besra@
bham.ac.uk
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.015

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S. J. Senior et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3685–3688
Table 1. Structures and in vitro mtFabH activity: (a) directly coupled substituents, (b) Suzuki coupled substituents
(a)
R
IC50, mM
R
IC50, mM
52ꢁ8.9
2
3
74.9
8
9
>250
246ꢁ170
263ꢁ86
4
112ꢁ15
10
5
6
7
43ꢁ6.7
155ꢁ26
>500
11
12
13
51ꢁ6.2
215ꢁ77
153ꢁ54
(b)
R0
IC50, mM
52ꢁ8.9
R0
IC50, mM
19ꢁ2.0
14
15
16
17
18
19
25ꢁ3.1
29ꢁ4.8
17ꢁ1.3
119ꢁ17

S. J. Senior et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3685–3688
3687
Chemical Strategy
of aryl rings enhance activity through an increased
ability to undergo hydrophobic interactions in the
hydrophobic pocket. Previous studies,13,17 using differ-
ent assays, showed enhanced activity for certain TLM
analogues with lipophilic isoprenoid-based side chains.
TLM was synthesized by Wang and Salvino17 using
sodium hydride and n-butyl lithium to deprotonate the
key thiolactone (1, Table 1a) at C-5, followed by reac-
tion with a halide. Previous studies13,17 showed that
other base combinations were possible, with lithium
hexamethyldisilazane (LHMDS) being the most prom-
ising.17 These studies showed that a number of aliphatic
and aromatic analogues had encouraging activity
against fatty acid synthesis.11,13,17 These results demon-
strated the importance of side-chain structure in TLM
analogues, in accordance with previous predictions.9,10
Aromatic substituents are attractive for their stability
and the potential for systematic modification in struc-
ture–activity studies.
The analogues, prepared by Suzuki coupling (Table 1b)
were compared to the unsubstituted analogue 11 (IC50
51 mM) (Table 1a). Introduction of a methyl group 14,
had little effect (IC50, 52 mM), but a para t-butyl group
15 gave an improved IC50 value (IC50, 25 mM). Com-
pounds 16–18, with a para-oxygen functionality, show
an increase in activity (IC50, 17–29 mM). This demon-
strates the possible importance of a hydrogen bond
accepting group, positioned in the active site and inter-
acting with hydrogen bond donating amino acid resi-
dues.22 It can be concluded that the presence of two
aromatic rings, coupled with the substitution of the
second aryl ring at the para position with a hydrogen
bond acceptor group, greatly increases the inhibitory
properties of the analogues compared to TLM.
A range of available arylmethyl bromides (Table 1a)
were linked at position 5 on the thiolactone ring 1. The
optimised conditions involved the addition of three
equivalents of LHMDS to 4-hydroxy-3,5-dimethyl-5H-
thiophen-2-one 1 under nitrogen, followed by cooling to
ꢀ78 ꢂC for 30 min and addition of aryl bromide. Stir-
ring at room temperature for 3 h, gave rise to selective
nucleophilic addition at position-5. All compounds were
completely characterized, as shown for 11.18
In conclusion, new analogues of TLM have been found
to have significantly enhanced activity against mtFabH.
These types of condensing enzymes are of key impor-
tance in the synthesis of fatty acids in a number of
microbial pathogens, as summarised above. It is now
possible, therefore, to use such compounds to probe
important biosynthetic mechanisms and pinpoint effec-
tive drug targets. The synthetic protocols, described,
will readily allow systematic modifications to be made
to effective lead compounds such as 17 (Table 1b).
These stable aromatic compounds have an advantage
over previous active, relatively labile, aliphatic-based
analogues13,17 in that their side chains are con-
formationally more predictable.
Suzuki reactions (Table 1b) were chosen to allow sys-
tematic modification of the substitution pattern, using
aryl-boronic acids according to Miyaura et al.19 Suzuki
coupling reactions were performed by heating the iodo
intermediate, bis(triphenylphosphine) palladium (II)
chloride (5 mol %), dimethoxyethane and aqueous
sodium carbonate under reflux for 6 h before quenching
with acid. Six novel analogues with varying substituents
were isolated and characterized20 and are shown in
Table 1b.
Experimental
Biological Activity
General procedure A: Alkylation of thiolactone 1
TLM 2 and analogues (3–19) were assayed to determine
IC50 values against mtFabH using a well established
assay protocol.21 The aromatic side chain in compound
3, demonstrated poor inhibition towards mtFabH with
an IC50 value of >250 mM. Low activities were evident
with the di-fluoro substituted analogues 9 and 10, and
also with the meta-linked di-aryl analogue 12. Activity
was increased however, by the introduction of a methyl
substituent, 4 (IC50 112 mM). The bulky t-butyl group 5
(IC50 43 mM), demonstrated a further increase in activ-
ity that was comparable to the addition of an aromatic
ring at the para position 11 (51 mM). Inhibitory proper-
ties increase when a methoxy group was introduced at
the para position of the aromatic ring 6 (IC50 155 mM),
compared to 3, but activity decreases when the methoxy
group is attached at the meta position 7 (IC50 >500
mM), indicating the importance of para substitution.
However, introduction of a meta-phenoxy group in 8
increases activity to 52 mM. TLM 2 shows good inhibitory
properties towards mtFabH (IC50 74.9 mM); however,
analogues 8 (IC50 52 mM) and 11 (IC50 51 mM) demon-
strate improved activities, suggesting that the presence
To a stirred solution of 4-hydroxy-3,5-dimethyl-5H-
thiophen-2-one 1 (50 mg, 0.347 mmol, 1 equiv) in
anhydrous tetrahydrofuran (THF) (1 mL) ꢀ78 ꢂC was
added lithium bis(trimethylsilyl)amide, 20% in THF
(LHMDS) (0.98 mL, 1.042 mmol, 3 equiv). The reaction
mixture was stirred for 30 min at ꢀ78 ꢂC before addi-
tion of the respective halide (0.382 mmol, 1.1 equiv).
The mixture was allowed to warm to room temperature
and stirred for a further 3 h, acidified to pH2 with 2 M
aq acetic acid and extracted twice with dichloro-
methane. The organic layers were combined, washed
with satd. brine, dried and reduced in vacuo to yield the
crude product, which was purified by Fisher Matrix
silica gel 60 flash chromatography (0–20% ethyl acetate
in cyclohexane).
General procedure B: Suzuki cross coupling
A solution of 13 (60 mg, 0.167 mmol, 1 equiv), ethyl-
englycol dimethyl ether (DME) (1 mL), aq sodium car-
bonate (0.5 mL, 1 M) and the respective arylboronic

3688
S. J. Senior et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3685–3688
acid (0.183 mmol, 1.2 equiv) was degassed in a ‘green-
house’ parallel synthesis reaction vessel for 10 min.
Bis(triphenylphosphine) palladium (II) chloride (8 mg,
7ꢃ10ꢀ3 mmol, 5 mol %) was added and the mixture
was heated under reflux for 6 h. The mixture was parti-
tioned between water (10 mL) and ethyl acetate (10 mL)
and separated. The aqueous layer was acidified to pH 2
with dilute hydrochloric acid (2 M) and the product was
extracted with ethyl acetate (2ꢃ10 mL). The organic
layers were combined, washed with satd. brine (3ꢃ10
mL), dried and reduced to give the crude product, which
was purified as above.
Krogh, A.; McLean, J.; Moule, S.; Murphy, L.; Oliver, K.;
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MtFabH assay: determination of IC50 values
Direct, end-point scintillation proximity assays (SPA)
were performed according to an established SPA
assay.21 The assay contained a mixture of recombinant
E. coli (ec) produced mtFabH12 (1.0 nM), biotinylated-
malonyl-ecACP (2.5 mM),21 and myristoyl-CoA (0.4
mM) in 100 mM sodium phosphate buffer with 0.01%
CHAPS and 1 mM DTT. Compounds in DMSO were
added to the assay plate to provide concentrations ran-
ging from 500 to 0.98 mM, followed by recombinant
mtFabH and biotinylated-malonyl-ecACP generated
from malonyl-ecACP and NHS-biotin as described pre-
viously.21 Following a 5-min preincubation at 30 ꢂC,
[3H] myristoyl-CoA and unlabeled myristoyl-CoA were
added to reach a final concentration of 0.4 mM con-
taining approximately 30,000 cpm per well. Reactions
were incubated at 30 ꢂC and 100 mL of ethanol was
added to quench the reaction after 20 min. Streptavidin-
coated beads (40 mL) (10 mg/mL in DPBS) (Amersham)
were added to each well, and the plates sealed with
Topseal-A press-on, transparent adhesive sealing film
(Packard) and shaken for 30 min. The plates were cen-
trifuged for 1 min at 1700g to pellet the beads in each
well and the incorporated radioactivity was counted
using a 1450 Microbeta Trilux liquid scintillation coun-
ter (Wallac).
10. Tsay, J. T.; Rock, C. O.; Jackowski, S. J. Bacteriology
1992, 174, 508.
11. Waller, R. F.; Ralph, S. A.; Reed, M. B.; Su, V.; Douglas,
J. D.; Minnikin, D. E.; Cowman, A. F.; Besra, G. S.; McFad-
den, G. I. Antimicrob. Agents. Chemother. 2003, 47, 297.
12. Choi, K. H.; Kremer, L.; Besra, G. S.; Rock, C. O. J. Biol.
Chem. 2000, 275, 28201.
13. Kremer, L.; Douglas, J. D.; Baulard, A. R.; Morehouse,
C.; Guy, M. R.; Alland, D.; Dover, L. G.; Lakey, J. H.;
Jacobs, W. R.; Brennan, P. J.; Minnikin, D. E.; Besra, G. S. J.
Biol. Chem. 2000, 275, 16857.
14. Schaeffer, M. L.; Agnihotri, G.; Volker, C.; Kallender,
H.; Brennan, P. J.; Lonsdale, J. T. J. Biol. Chem. 2001, 276,
47029.
15. Kremer, L.; Dover, L. G.; Carrere, S.; Nampoothiri,
K. M.; Lesjean, S.; Brown, A. K.; Brennan, P. J.; Minnikin,
D. E.; Locht, C.; Besra, G. S. Biochem. J. 2002, 364, 423.
16. Douglas, J. D.; Senior, S. J.; Morehouse, C.; Phetsukiri,
B.; Campbell, I. B.; Besra, G. S.; Minnikin, D. E. Micro-
biology 2002, 148, 3101.
17. Wang, C. L. J.; Salvino, J. M. Tetrahedron Lett. 1984, 25,
5243.
18. Analytical data for compound 11: 1H NMR (D3COD,
400 MHz) d 1.54 (3H, s), 1.65 (3H, s), 3.00–3.15 (2H, m), 7.25
(2H, d, J=5 Hz), 7.28 (1H, m), 7.35 (2H, t, J=8 Hz), 7.42
(2H, d, J=8 Hz), 7.57 (2H, d, J=8 Hz); 13C NMR (D3COD,
100.6 MHz) d 8.0 (C-6), 26.0 (C-7), 44.0, 59.0 (C-5), 110.0 (C-
3), 126.0, 127.0, 128.0, 130.0, 132.0, 136.0, 140.0, 141.0, 181.0
(C-4), 194.0 (C-2); HRMS calcd for C17H22O2 S (MH+):
311.1098, found 311.1106 (ꢀ2.5 ppm).
Acknowledgements
This work was supported by The MRC, National
Cooperative Drug Discovery Groups for the Treatment
of Opportunistic Infections (NIH), GSK ActionTB.
G.S.B. is a Lister Institute Jenner Research Fellow.
19. Miyaura, N.; Yanagi, T.; Suzuki, A. Synth. Commun.
1981, 11, 513.
20. Analytical data for compound 17: 1H NMR (D3COD,
400 MHz) d 1.43 (9H, s), 1.52 (3H, s), 1.69 (3H, s), 2.97–3.08
(2H, m), 4.47 (2H, s), 7.13 (2H, d, J=8 Hz), 7.24 (2H, d, J=8
Hz), 7.34 (2H, d, J=8 Hz), 7.41 (2H, d, J=8 Hz); 13C NMR
(D3COD, 100.6 MHz) d 7.9 (C-6), 26.6 (C-7), 45.4, 59.8 (C-5),
65.3, 111.6 (C-3), 127.5, 128.2, 18.9, 132.5, 137.2, 141.2, 141.5,
142.1, 182.8 (C-4), 197.8 (C-2); HRMS calcd for C21H19O3S
(MH+): 341.1211, found 341.1225 (4.0 ppm).
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