Discovery and structure–activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.07.061

There is an urgent and unmet medical need for new antibacterial drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. During the course of our wider efforts to discover and exploit novel mechanism of action antibacterials, we have identified a novel series of isothiazolone based inhibitors of bacterial type II topoisomerase. Compounds from the class displayed excellent activity against both Gram-positive and Gram-negative bacteria with encouraging activity against a panel of MDR clinical Escherichia coli isolates when compared to ciprofloxacin. Representative compounds also displayed a promising in vitro safety profile.

Full text of DOI:10.1016/j.bmcl.2016.07.061

Accepted Manuscript
Discovery and structure-activity relationships of a novel isothiazolone class of
bacterial type II topoisomerase inhibitors
Ian R. Cooper, Andrew J. McCarroll, David McGarry, James Kirkham, Mark
Pichowicz, Rolf Walker, Catherine Warrilow, Anne-Marie Salisbury, Victoria
J. Savage, Emmanuel Moyo, Henry Forward, Jonathan Cheung, Richard
Metzger, Zoe Gault, Gary Nelson, Diarmaid Hughes, Sha Cao, John Maclean,
Cédric Charrier, Mark Craighead, Stuart Best, Neil R. Stokes, Andrew J.
Ratcliffe
PII:
S0960-894X(16)30789-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.061
BMCL 24105
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2016
25 July 2016
26 July 2016
Please cite this article as: Cooper, I.R., McCarroll, A.J., McGarry, D., Kirkham, J., Pichowicz, M., Walker, R.,
Warrilow, C., Salisbury, A-M., Savage, V.J., Moyo, E., Forward, H., Cheung, J., Metzger, R., Gault, Z., Nelson,
G., Hughes, D., Cao, S., Maclean, J., Charrier, C., Craighead, M., Best, S., Stokes, N.R., Ratcliffe, A.J., Discovery
and structure-activity relationships of a novel isothiazolone class of bacterial type II topoisomerase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.061
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Discovery and structure-activity relationships of a novel isothiazolone class of bacterial type II
topoisomerase inhibitors
Ian R. Cooper,a,* Andrew J. McCarrolla, David McGarrya, James Kirkhama, Mark Pichowicza, Rolf
Walkera, Catherine Warrilowa, Anne-Marie Salisburya, Victoria J. Savagea, Emmanuel Moyoa, Henry
Forwarda, Jonathan Cheunga, Richard Metzgera, Zoe Gaulta, Gary Nelsona, Diarmaid Hughesb, Sha
Caob, John Maclean a, Cédric Charriera, Mark Craigheada, Stuart Besta, Neil R. Stokesa, Andrew J.
Ratcliffea.
aRedx Pharma, Alderley Park, Cheshire, SK10 4TG, United Kingdom
bDepartment of Medical Biochemistry and Microbiology, Box 582 Biomedical Center, Uppsala
University, Uppsala, Sweden
*Corresponding author. E-mail: i.cooper@redxpharma.com
Keywords: ESKAPE pathogens; anti-infectives; topoisomerases; DNA gyrase; isothiazolone
Antibiotic resistance is becoming an increasingly urgent threat to public health in both a clinical and
community setting. Failure to combat this crisis is predicted to have catastrophic human and
economic consequences, potentially leading to 10 million extra deaths per year by 2050 and costing
the global economy up to 100 trillion USD.1 The “ESKAPE” group of pathogens (comprising
Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa and Enterobacter species) are of particular concern.2,3
Fluoroquinolones are an important class of bacterial type II topoisomerase (DNA gyrase and
topoisomerase IV) inhibitors that show broad-spectrum activity and are highly effective in the clinic.
In recent years however, the worldwide emergence of fluoroquinolone resistance has raised serious
concerns regarding the future utility of this drug class.4–7 Resistance occurs via a range of
mechanisms including target-site gene mutations, overexpression of multi-drug resistance efflux
pumps, modifying enzymes and target protection proteins.8–10
These factors have increased the need to develop new classes of antibiotics that tackle the issue of
bacterial resistance. One approach is to identify and explore novel targets with no pre-existing
antimicrobial resistance. Issues surrounding target validation along with a lack of physicochemical
diversity within screening collections has hindered progress in this area.11 An alternative approach is
to explore clinically validated targets for new compounds that show limited or no cross-resistance to
existing antibiotics. This avenue removes the risk of target validation and has been employed
effectively within several drug classes.12
Recent reports have described the use of isothiazoloquinolones and isothiazolopyridones as DNA
gyrase inhibitors and some have displayed excellent antimicrobial activity.13–15 Redx Pharma reports
herein the synthesis, structure-activity relationships and in vitro evaluation of a novel class of
isothiazolone inhibitors of bacterial type II topoisomerase. A compound from this series has recently
been reported to display balanced inhibition of both the supercoiling activity of DNA gyrase and the
decatenation function of topoisomerase IV.16

The synthetic route to compounds 7a-o was designed to allow the late stage introduction of
chemical diversity via manipulation of the chloro substituent within compound 6 (Scheme 1).
Starting material 1 was converted to the tert-butyl imine and reacted with triethyl
methanetricarboxylate to afford the pyridone 2. Chlorination and subsequent displacement with
potassium thioacetate afforded thiol 4. Treatment with hydroxylamine-O-sulfonic acid generated the
isothiazolone ring system 5. Trial coupling reactions using intermediate 5 were poor yielding.
Protection of the amide groups with TIPS-Cl to afford 6 allowed the coupling reaction to proceed
with improved yields. The protecting group was removed during the work up procedure.
Scheme 1. General synthesis of isothiazolones. Reagents and conditions: (i) tert-butylamine, Ti(Cl)4,
DCM, rt, 75% (ii) CH(CO2Et)3, (Ph)2O, 160oC, 53% (iii) (COCl)2, DCM, rt, 89% (iv) potassium thioacetate,
DMF, rt, 78% (v) hydroxylamine-O-sulfonic acid, THF/H2O, K3PO4, rt, 79%(vi) triisopropylsilyl
trifluoromethylsulfonate, 2,6-lutidine, rt, 57% (vii) amine, sodium tert-butoxide, Pd2(dba)3, (2-
biphenyl)di-tert-butylphosphine, toluene, 100oC, 4 - 40%.
Regioisomers 8a-c were prepared in a similar fashion from the corresponding isomeric starting
materials.
Figure 1. Ciprofloxacin
The route to prepare derivative 14 involved incorporation of the indazole at an early stage (Scheme
2). Bromo-indazole 9 was lithiated and treated with N-methoxy-N-methyl butanamide to afford
ketone 10. An analogous sequence of steps to Scheme 1 was then followed to afford the final
product 14.

Scheme 2. Synthesis of indazole derivative 14. Reagents and conditions: (i) n-BuLi, N-methyl N-
methoxy-N-methyl butanamide, THF, -78oC, 44% (ii) tert-butylamine, Ti(Cl)4, DCM, rt, 100% (iii)
CH(CO2Et)3, (Ph)2O, 160oC, 43% (iv) (COCl)2, DCM, rt, 82% (v) potassium thioacetate, DMF, rt, 65% (vi)
hydroxylamine-O-sulfonic acid, THF/H2O, K3PO4, rt, 20%.
The antibacterial activity of these compounds was determined against a panel of Gram-positive and
Gram-negative bacterial strains including S. aureus, A. baumannii, K. pneumoniae, P. aeruginosa and
E. coli from the ESKAPE pathogens. Ciprofloxacin, a fluoroquinolone antibiotic, was also included as a
positive control (Figure 1). The MICs (Minimum Inhibitory Concentrations), determined as previously
described, are reported in Table 1 along with data for the highly sensitive and efflux-deficient
(ΔacrA) E. coli N43 strain.16,17
Table 1: In vitro antibacterial activity (MIC, µg/mL) of ciprofloxacin and isothiazolone compoundsa.
List
CIP
5
R
Ab
64
16
2
Kp
0.25
128
4
Pa
1
Sa
0.25
8
Ec
0.03
16
Ec N43
0.004
0.25
>128
1
7a
7b
7c
1
0.25
0.015
0.5
0.03
1
4
1
0.5
2
0.001
0.03
4
8
1
7d
16
2
0.06
0.12
0.008
0.001

7e
7f
64
8
4
8
0.06
8
0.12
4
0.002
0.5
≤0.0001
0.06
7g
64
64
32
4
4
1
7h
7i
8
4
1
2
1
2
0.5
1
0.008
0.008
>128
32
7j
7k
7l
>64
>64
>64
>64
>64
64
64
64
16
16
16
8
32
16
4
1
0.5
0.5
7m
16
2
1
0.015
0.12
0.008
7n
7o
14
32
>64
2
4
0.5
4
0.12
0.12
0.12
0.25
0.5
0.03
0.12
>64
16
4
0.5
0.004
8a
8b
8c
64
>128
>128
>64
>128
>128
>64
32
32
64
>64
N.D
>64
8
N.D
8
>128
>64
a Sa (Staphylococcus aureus ATCC 29213), Ab (Acinetobacter baumannii NCTC 13420), Kp (Klebsiella
pneumoniae ATCC 700603), Pa (Pseudomonas aeruginosa ATCC 27853), Ec (Escherichia coli W4573),
Ec N43 (Escherichia coli N43). CIP (ciprofloxacin). N.D (Not determined).

Racemic 7a displayed broad-spectrum activity across most strains tested. The stereochemistry of
enantiomers 7b and 7c had a limited effect on the activity.
Removal of one or both methyl groups had a pronounced effect on activity as shown by 7d and 7e.
Both compounds demonstrated increased potency against E. coli and P. aeruginosa in particular but
also suffered a corresponding loss of potency against A. baumannii. This could be attributed to the
increased polarity (Table 3) relative to the dimethyl parent compound 7c causing an increased
susceptibility to the efflux pump mechanisms of A. baumannii.18
Bicyclic amine analogues gave varying results with both 7f and 7g showing reduced activity. Potency
for the 5,6-bicyclic analogue was restored by removal of the methyl group 7h.
In comparison to 7d, homologated analogue 7i displayed reduced activity against all strains except
for the E. coli N43 efflux-deficient strain. This suggests 7i may retain potency at the enzyme level but
suffer from an increased efflux liability.
Switching from a pyrrolidine ring to a 6-membered piperidine or piperazine was detrimental to
activity as shown for 7j, 7k and 7l. The reduced activity against the E. coli N43 strain was considered
to be indicative of reduced enzyme activity.
A series of non-basic compounds were prepared and showed retention of activity in many Gram-
negative strains. Hydroxyl analogues 7m and 7n retained good activity against S. aureus, E. coli and
P. aeruginosa. Difluoro analogue 7o retained reasonable potency against several strains and showed
a low efflux ratio between E. coli N43 and its isogenic parent E. coli W4573. Indazole analogue 14
retained broad-spectrum activity.
Meta substitution was detrimental to activity as shown by examples 8a, 8b and 8c. Again, this was
attributed to reduced enzyme activity as indicated by the relatively elevated MICs against the efflux-
deficient E. coli N43 strain.
Point mutations within the QRDR (quinolone-resistance determining region) of gyrA, gyrB, parC
and/or parE are a common source of fluoroquinolone resistance with mutations at S83 and D87 of
GyrA being particularly prevalent.19 Representative compounds, 7a, 7g and 14, were tested against a
panel of isogenic laboratory strains of E. coli bearing multiple target specific mutations (e.g. LM693)
and efflux mutations (e.g. LM367).20 All compounds, including ciprofloxacin, displayed a similar fold
change reduction in activity against LM625 and LM367 compared to the isogenic parent strain E. coli
MG1655 (LM179). However, 7a was observed to show a much less significant decrease in activity
against isogenic strains bearing a greater level of mutations (LM693 and LM705) compared to
ciprofloxacin. The compounds were further evaluated against a panel of characterised MDR (multi-
drug resistant) clinical E. coli UTI (urinary tract infection) isolates (CH440, CH460, CH418 and CH448)
which also included resistance obtained via horizontal gene transfer. 7a, 7g and 14 all showed a
significantly reduced susceptibility to a range of key fluoroquinolone mutations in comparison to
ciprofloxacin (Table 2). However, the elevated MIC values for the tested isothiazolones across both
panels exposed an underlying level of fluoroquinolone cross-resistance.

Table 2: Antibacterial activity (MIC, µg/mL) of ciprofloxacin and selected compounds against E. coli
mutant strains
CIP
7a
MIC fold x
WT
7g
MIC fold x
WT
14
MIC fold
x WT
Strain
genotype
MIC
fold x
WT
LM179a Wild-type
0.016
0.25
0.12
32
0.5
4
0.5
LM625a GyrA S83L D87N
LM367a ∆marR, ∆acrR
16
8
4
2
8
8
4
16
16
4
4
8
4
16
8
LM693a GyrA S83L D87N, ParC
S80I
2000
16
>64
>16
16
32
LM705a GyrA S83L D87N, ParC
S80I, ∆marR, ∆acrR
64
4000
64
16
128
32
>64
>64
>16
>16
>64 >128
CH440b GyrA S83L D87N, ParC
S80I E84V, aac(6’)-Ib-
crc
>64 >4000
>64 >4000
16
32
CH460b GyrA S83L D87N, ParC
16
16
16
32
32
32
64
64
32
16
16
8
16
16
16
32
32
32
S80I E84V, qepAc
CH418b GyrA S83L D87N, ParC
64
4000
S80I E84G, qnrAc
CH448b GyrA S83L, qnrSc
32
2000
a isogenic laboratory strain b MDR clinical UTI isolate c relevant genotype
Representative compounds were evaluated for in vitro toxicity as shown in Table 3. No toxic effects
were observed in a Hep G2 mammalian cytotoxicity assay for all tested compounds. 7a was
measured for hERG inhibition and displayed 84% inhibition at a concentration of 100 µM, with an
IC50 of 20 µM. In line with previous literature reports describing the effects of logD and pKa on hERG
inhibition, significant reductions in hERG inhibition were measured for both the more polar analogue
7d and the less basic compound 7n, although subtle structural changes could also be playing a
role.21–23
Table 3: In vitro safety profiles of representative isothiazolones and ciprofloxacin
b
Compound
HepG2
(IC50,
logD7.4
hERG (% block
at 100µM)c
µg/mL)a
CIP
>128
N.D
28
7a
7d
7n
>128
>128
>16
1.4
1.2
1.5
84 (20)
34
22
a Hep G2 cells incubated for 24 h at 37 oC in 5 % CO2 and viability determined using CellTiter-Glo®
(Promega, WI, USA) b Partition coefficient (LogD) determined by shake-flask method, using 10 mM
phosphate buffer at pH 7.4 and n-octanol c Percent block of hERG K+ channel measured via IonWorks
at 100 µM. Value in parentheses indicates IC50 (µM)

In summary, this paper describes the SAR and in vitro evaluation of a novel isothiazolone-based
series of bacterial topoisomerase II inhibitors. Broad-spectrum activity was observed for many
compounds and representative examples showed a promising in vitro safety profile. Examples from
the series showed encouraging activity against a panel of MDR clinical E. coli UTI isolates in
comparison to ciprofloxacin. Further work is required to understand the binding mode of the series
and the impact this has on cross-resistance with fluoroquinolones.
Acknowledgements
The research leading to these results was conducted in part with the ND4BB ENABLE Consortium and
has received support from the Innovative Medicines Joint Undertaking under Grant Agreement n°
115583, resources which are composed of financial contribution from the European Union’s seventh
framework programme (FP7/2007-2013) and EFPIA companies in kind contribution.
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Graphical abstract