L. C. Axford et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6598–6603
6599
O
O
HO
N
N
N
N
HO
O
O
O
N
N
H
N
H
N
H
N
S
N
S
N
S
5
2
N
H
N
H
N
H
7
N
N
N
1
2
3
IC50a (µg/mL)
0.014
0.12
6.25
0.001
0.12
50
0.010
0.015
>800
E. fs MIC (µg/mL)
Solubility (µg/mL)
Figure 1. Progression of early hits to isonipecotic acid containing analogues. aDNA gyrase ATPase (S. aureus).
explored that would not interfere with the promising antimicrobial
profile. It was discovered that the addition of a carboxylic acid moi-
ety appended to the C-5 aryl group, 2, indeed improved the solubil-
(CF3) or methanesulfonyl chloride (SO2Me). Some analogues were
prepared via a modified synthetic route or from alternative starting
materials as described elsewhere.9
ity8 at physiological pH from 6.25
lg/mL to 50
lg/mL, whilst
Compounds were tested in a DNA gyrase ATPase activity
assay.10 The antimicrobial activity of the compounds was mea-
sured by susceptibility testing using the broth microdilution
method11 and MICs were determined against Staphylococcus aureus
(ATCC 29213), E. faecalis (ATCC 29212), Streptococcus pyogenes
(ATCC 51339) and Haemophilus influenzae (ATCC 49247).
Table 1 indicates the structure activity relationship (SAR) with-
in the series. All analogues tested displayed potent activity in the
DNA gyrase ATPase assay. Compounds 3 and 18 have IC50 values
maintaining antibacterial activity against the target pathogens
(minimal inhibitory concentration (MIC) data for Enterococcus fae-
calis shown as an example).
By linking the carboxylate to the C-5 aryl group by a cyclic
amine, for example the isonipecotic acid appendage, 3, both the
antibacterial activity and the solubility increased. It was discov-
ered that the addition of a substituent
a to the carboxylic acid im-
proved the PK profile of the series without loss of potency or
solubility. Compounds of this class possessed enhanced solubilities
at physiologically acceptable pH, enabling IV administration, along
with excellent oral bioavailability, lower clearance, and higher
for S. aureus topoisomerase IV (0.012 and 0.008 lg/mL respec-
tively)10 comparable to their S. aureus DNA gyrase ATPase IC50 val-
ues. Compounds 3 and 18 also showed specificity for bacterial
topoisomerases, with no inhibition of human topoisomerase II
being observed.7 A dual targeting intracellular inhibitor of GyrB
and ParE would be expected to show a low frequency of resistance
(FoR).13 The FoR to compound 3 and 18 in S. aureus (ATCC 29213)
was determined.7 At concentrations equivalent to four- and eight-
fold the MICs no spontaneous mutants were isolated, giving mea-
sured FoRs of 610À10. Taken together this suggests the compounds
are potent and specific inhibitors of both DNA gyrase and topoiso-
merase IV. Small alkyl groups gave the best antibacterial profile as
well as excellent solubility and stability in mouse microsomes
(intrinsic clearance, CLint). For example, methyl 18 and ethyl 19
groups were shown to have the most favourable activity across
the panel. Aryl containing groups such as phenyl 20 lost activity
against S. pyogenes (S. py) and H. influenzae (H. in) and demon-
strated poor solubility. Addition of a fluorine-containing group
such as CF3 21 led to a marked decrease in activity, particularly
against S. aureus (S. au). Other electron withdrawing groups such
as SO2Me 22 led to a further decrease in activity across the species
tested. Electron rich groups such as OH 23 and NH2 24 generally
showed poor activity, particularly against S. aureus, E. faecalis (E.
exposure compared to compounds without an
a substituent. The
discovery and preparation of this class of compounds are now
described.
During the exploration of C-5, a 2-pyridyl group was main-
tained at the C-7 position of the benzothiazole core. Boronic acid
intermediate 4 was prepared as an advanced precursor, according
to Scheme 1.
Synthesis commenced with bromination of 2-amino-5-nitro-
phenol 5 in acetonitrile followed by deamination with sodium ni-
trite and sulfuric acid in ethanol. Benzylation of phenol 6 and
reduction of the nitro group afforded 7. Addition of benzoyl isothi-
ocyanate to 7 furnished N-benzoylthiourea 8, which was readily
cleaved to the thiourea 9 with sodium hydroxide. Bromine-medi-
ated cyclisation to the benzothiazole core followed, with subse-
quent conversion to the ethyl urea 10. Stille coupling installed
the C-7 pyridyl group. Treatment of 11 with methane sulfonic acid
yielded alcohol 12, which upon conversion to triflate 13 and a
Miyaura borylation reaction yielded the key intermediate 4 for fur-
ther derivatisation.
Initially, the introduction of an
a substituent to the carboxylic
acid was investigated. As shown in Scheme 2, commercially avail-
able ethyl N-Boc-piperidine-4-carboxylate 14 was treated with
lithium diisopropylamide at À78 °C, quenched with an alkyl halide,
then warmed to room temperature to furnish 15. Removal of the
Boc group with hydrogen chloride in 1,4-dioxane gave crude 16
which was then used in an aromatic nucleophilic substitution reac-
tion with 5-bromo-2-chloropyrimidine to yield bromide 17. Suzuki
cross-coupling with boronic acid 4, followed by saponification,
yielded the target products.
fs) and H. influenzae. The nature of the a-substituent coupled with
the carboxylate was key to fine tuning the ideal profile of the ser-
ies. With this information in mind, subsequent analogues were
prepared with an
SAR.
a-methyl substituent in order to obtain further
Next, the effect of the heterocycle directly attached to the C-5
position of the core was investigated. A set of analogues was
prepared9 using the
a-methyl isonipecotic acid group as the opti-
mal group from the earlier exploration. Selected compounds are
described in Table 2. The 3,5-pyrimidyl group 18 (data in Table 1)
gave the best overall profile, followed by the 3-pyridyl 25. The
3,5-pyrimidyl and 3-pyridyl groups showed an improvement in
Alternative
a-substituents were prepared by quenching the
lithiated species (step a) with alternative electrophiles that is, S-
(trifluoromethyl)dibenzothiophenium trifluoromethanesulfonate