Novel hydroxyl tricyclics (e.g., GSK966587) as potent inhibitors of bacterial type IIA topoisomerases

Article abstract of DOI:10.1016/j.bmcl.2013.07.013

During the course of our research to find novel mode of action antibacterials, we discovered a series of hydroxyl tricyclic compounds that showed good potency against Gram-positive and Gram-negative pathogens. These compounds inhibit bacterial type IIA to

Full text of DOI:10.1016/j.bmcl.2013.07.013

Accepted Manuscript
Novel hydroxyl tricyclics (e.g. GSK966587) as potent inhibitors of bacterial
type IIA topoisomerases
Timothy J Miles, Alan J Hennessy, Ben Bax, Gerald Brooks, Barry S Brown,
Pamela Brown, Nathalie Cailleau, Dongzhao Chen, Steven Dabbs, David T
Davies, Joel M Esken, Ilaria Giordano, Jennifer L Hoover, Jianzhong Huang,
Graham E Jones, Senthill K Kusalakumari Sukmar, Claus Spitzfaden, Roger E
Markwell, Elisabeth A Minthorn, Steve Rittenhouse, Michael N Gwynn, Neil
D Pearson
PII:
S0960-894X(13)00847-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.07.013
BMCL 20678
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
2 June 2013
7 July 2013
9 July 2013
Please cite this article as: Miles, T.J., Hennessy, A.J., Bax, B., Brooks, G., Brown, B.S., Brown, P., Cailleau, N.,
Chen, D., Dabbs, S., Davies, D.T., Esken, J.M., Giordano, I., Hoover, J.L., Huang, J., Jones, G.E., Kusalakumari
Sukmar, S.K., Spitzfaden, C., Markwell, R.E., Minthorn, E.A., Rittenhouse, S., Gwynn, M.N., Pearson, N.D., Novel
hydroxyl tricyclics (e.g. GSK966587) as potent inhibitors of bacterial type IIA topoisomerases, Bioorganic &
Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.07.013
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Novel hydroxyl tricyclics (e.g. GSK966587)
as potent inhibitors of bacterial type IIA
topoisomerases
Leave this area blank for abstract info.
Timothy J Miles, Alan J Hennessy, Ben Bax, Gerald Brooks, Barry S Brown, Pamela Brown, Nathalie Cailleau,
Dongzhao Chen, Steven Dabbs, David T Davies, Joel M Esken, Ilaria Giordano, Jennifer L Hoover, Jianzhong
Huang, Graham E Jones, Senthill K Kusalakumari Sukmar, Claus Spitzfaden, Roger E Markwell, Elisabeth A
Minthorn, Steve Rittenhouse, Michael N Gwynn and Neil D Pearson.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Novel hydroxyl tricyclics (e.g. GSK966587) as potent inhibitors of bacterial type
IIA topoisomerases
Timothy J Miles,a, *Alan J Hennessy,b Ben Bax,c Gerald Brooks,d Barry S Brown,e Pamela Brown,d Nathalie
Cailleau,d Dongzhao Chen,e Steven Dabbs,b David T Davies,d Joel M Esken,e Ilaria Giordano,a Jennifer L
Hoover,e Jianzhong Huang,e Graham E Jones,d Senthill K Kusalakumari Sukmar,e Claus Spitzfaden,c Roger
E Markwell,d Elisabeth A Minthorn,f Steve Rittenhouse,e Michael N Gwynn e and Neil D Pearson.e
aDiseases of the Developing World CEDD, GlaxoSmithKline, Calle Severo Ochoa, 2, 28760, Tres Cantos, Madrid, Spain
bInfectious Diseases CEDD, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK
cComputational and Structural Chemistry, GlaxoSmithKline, Gunnels Wood Road, Stevenage, SG1 2NY, UK
dInfectious Diseases CEDD, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW, UK
eInfectious Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
fProtein Dynamics DPU, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
During the course of our research to find novel mode of action antibacterials, we discovered a
series of hydroxyl tricyclic compounds that showed good potency against Gram-positive and
Gram-negative pathogens. These compounds inhibit bacterial type IIA topoisomerases . Herein
we will discuss structure activity relationships in this series and report advanced studies on
compound 1 (GSK966587) which demonstrates good PK and in vivo efficacy properties. X-ray
crystallographic studies were used to provide insight into the structural basis for the difference in
antibacterial potency between enantiomers.
Keywords:
Antibacterials;
2009 Elsevier Ltd. All rights reserved.
Bacterial type IIA topoisomerase;
Hydroxyl tricyclics;
Pharmacokinetic;
In vivo efficacy;
There is an unmet medical need for novel antibiotics1 due to
the increasing prevalence of resistant bacteria such as methicillin
resistant Staphylococcus aureus (MRSA), vancomycin resistant
enterococci (VRE) and fluoroquinolone resistant pathogens.
Bacterial type IIA DNA topoisomerases are well validated
targets as they are inhibited by both quinolones and novobiocin.
However both these drug classes suffer from toxicity issues:
novobiocin has very limited use and quinolones are used in
adults only. Moreover resistance to fluoroquinolones is on the
increase making the development of novel antibiotics against this
well-validated target attractive.2
series of hydroxyl tricyclic compounds (e.g. GSK966587, 14)
which have potent antibacterial properties.
This paper will discuss the structure activity relationships
(SAR) of the left hand side (LHS) and central unit portion of this
series of hydroxyl tricyclic compounds (e.g. 14) as shown below
in Figure 1.
It has been established through mutational (Black et al)3a and
X-ray crystallography studies (Bax et al)3b that compounds (such
as GSK2994233b) with a similar mode of action to 14 bind to the
topoisomerases in
a different manner than that of the
quinolones.3c These compounds have a novel mechanism, which
overcomes issues of target-mediated resistance that have
developed in the clinic.2 Compounds with a similar mechanism
but of a distinct chemical series have also been discussed in the
literature.3d-h
Figure 1. Bacterial Type IIA DNA topoisomerase inhibitors
The synthesis of hydroxyl tricyclic compound 14 is described
in Scheme 1. A Suzuki cross coupling of 8-bromo-7-fluoro-2-
methoxy-1,5-napthridine 2,5 with pyridine-tris(1-methylethenyl)
In the course of our efforts to exploit this mode of action to
develop an antibiotic with a good safety profile, we discovered a
———
* Corresponding author. Tel.: +34-650-349-771; fax: +34-918-070-550; e-mail: tim.j.miles@gsk.com

6
boroxin 34,
then chlorination using cerium (III) chloride
binding mode for compound 14 was generally similar to that
described for GSK2994233b, apart from a novel hydrogen
bonding interaction between the bridgehead 4-OH and an NH2
from an adenine base of the DNA (O to N distance of 3.1A as
shown in Figure 2).
heptahydrate7 afforded chloride 5 in an overall yield of 55%.
Subsequent cyclisation using NaI then dihydroxylation afforded
the diol 7 in 96% yield over two steps. Activation of the primary
hydroxyl group as the tosyalte was achieved using dibutyltin
oxide8 and displacement with NBoc piperidine afforded the
NBoc protected intermediate 8 in 90% yield. The free amine 9
was liberated by Boc deprotection and then separated into
enantiomers by prepative chiral HPLC. The desired (4S)-
enantiomer was placed under reductive alkylation conditions
with aldehyde 109 to give the hydroxylated tricyclic 14 in 81%
yield.
Compounds 14 and 114a are enantiomers that have very
different whole cell and molecular target activity against S.
aureus (Table 1). An analytical gel filtration assay3b, 14 was used
to identify a DNA sequence that gave a stable complex with
compound 1 and to investigate the stability of complexes of
compound 14 or compound 114a with eight different 20-base-pair
DNA duplexes and DNA gyrase. The results showed that with
compound 14 all eight DNA duplexes gave stable complexes
(Figure 2), while for compound 114a the stability of the
complexes was variable and four duplexes gave little or no stable
complex. An unfavourable interaction between the chiral centre
at the bridgehead of compound 114a and the surrounding DNA
may partially account for this poor activity.
Cl
Br
O
N
F
B
(b)
(a)
F
O
N
O
N
F
O
B
O
B
+
95%
58%
N
O
N
N
2
3
4
5
36%
(c)
HO
HO
A
B
N
NHBoc
OH
F
O
N
F
(e - f)
O
N
(d)
O
N
F
90% over
2 steps
Quant.
N
N
N
8
7
6
68% over
two steps
(g - h)
O
O
O
N
HO
O
O
HO
N
NH2
N
N
H
10
N
O
N
F
O
N
F
(i)
81%
N
N
9
GSK966587, 1
Scheme 1. Reagents and conditions: (a) 25 (1 equiv), Pd(P(Ph)3)4 (5 mol%),
dimethoxy ethane, RT, 30 min then 34, 6 (0.4 equiv), K2CO3 (1 equiv), H2O,
reflux, 10 h; (b) CeCl3.7H2O (1 equiv), NaClO (2 equiv), tBuOH, RT, 10 min;
Figure 2. A The 2.6Å crystal structure14 of compound 1, GSK966587
(yellow carbons) in a complex with DNA (green carbons) and DNA gyrase
(cyan carbons). B An analytical gel filtration assay3b was used to assay the
stability of complexes of compound 14 (red) or it’s enantiomer compound
114a (blue) with eight different DNA duplexes. The bases surrounding the
compound binding site were varied, as indicated (top line 5’to 3’; bottom line
complementary strand sequence is shown 3’to 5’).
t
(c) NaI (10 equiv), acetone, reflux, 18 h; (d) AD-mix , BuOH, H2O, RT, 16
h; (e) TsCl (1 equiv), nBu2SnO (5 mol%), Et3N (1.5 equiv), DCM, THF,
DMF, RT, 18 h; (f) 1,1-dimethylethyl 4-piperidinylcarbamate (1.33 equiv),
NaHCO3, DMF, EtOH, RT, 2.5 d; (g) HCl in 1,4-dioxane (4M), DCM,
MeOH, RT, 1.5 h; (h) Chiral Separation using Chiralpak AD column; (i) 109
(1 equiv), Na(OAc)3BH (2 equiv), DCM, MeOH, 0oC RT, 2.5 h.
The hERG inhibition of the compounds described was
generally related to their lipophilicity and basicity.15a-c Several
compounds within this series were identified with hERG
inhibition that was suitable for progression.
To further profile this series of compounds we decided to
examine compound 14 in PK and in-vivo efficacy studies. We
will now discuss each of these studies in turn.
From Table 1 it can be seen that this series of compounds has
broad spectrum activity against Gram-positive (Staphylococcus.
aureus WCUH 29, Streptococcus pneumoniae 1629) and Gram-
negative (Haemophilus influenzae H128) bacteria. These are just
representative strains and the potency was maintained against a
more extensive panel of organisms.
We indirectly measured target potency by using a gyrase-
dependent DNA replication assay reconfigured in an unpublished
format at GSK using Scintillation Proximity Assay (SPA)
technology.10 For this series of compounds the IC50 in this assay
tracked the whole cell antibacterial potency well.
Table 2. Summary of in vivo pharmacokinetics and in vitro study results for
compound 1.16
Parameters (Units)
Rat
Dog
Monkey
Human
IV DMPK
Within these hydroxyl tricyclic examples and for the
bacterial strains shown in Table 1 the following observations can
be made: enantiomer 14 is better tolerated for antibacterial
potency than 114a. It is possible to alter the bridgehead 4-OH in
14 with either F (124a) or H (1311). Variations of alternative LHS
Dose (mg/kg)
CLb (mL/min/kg)
Vss (L/kg)
3
3
3
nd
nd
nd
nd
62.2
8.6
3.1
18.1
6.4
7.1
25.1
3.4
2.3
Half-life (h)
Oral DMPK
subunits are tolerated; examples shown in Table
1 are
naphthyridine (14), quinoline (1412), quinoxaline (1513) and a
further substituted naphthyridine (164a). Finally, alternative
central units such as 174a and 184a are tolerated, although not as
potent as the parent compound 14 within these bacterial strains.
As described below, structural studies have shown that these
central units would be largely solvent exposed, without target or
DNA interaction.
Dose (mg/kg)
5
0.46
34
42.29 ±
9.60
5
2.82
63
17.74 ±
8.68
5
1.92
55
21.91 ±
7.35
nd
nd
nd
22.25 ±
5.97
AUC (ug.h/mL)
Oral Bioavailability (%)
Plasma Protein Binding
(% bound)
CLi microsomes
(mL/min/g liver)
nd: not determined.
<0.5
<0.5
1.6
<0.5
The 2.6Å crystal structure of compound 14 in complex with
DNA and S. aureus DNA gyrase was determined using a similar
14
technique as previously described in the literature.3b,
The

As can be seen in Table 2,16 the blood clearance was high in
the rat (~100% of hepatic blood flow) but moderate in dog and
monkey (50-60% of hepatic blood flow). The renal clearance in
dog and monkey was low to moderate (~27% and 15% of the
total clearance in dog and monkey, respectively), and volume of
distribution was high (greater than total body water). Reasonable
oral bioavailability (FPO) was achieved in all species. Plasma
protein binding and microsomal intrinsic clearance of 14 were
similar across species.
Table 1: Antibacterial activity of a set of hydroxyl tricyclic compounds10, 15d-e, 17
hERG
inhibition
hERG
inhibition
whole cell
patch (IC50,
µM)15e
H. influenzae. DNA
MIC (µg/ml) H.
influenzae / S.
aureus / S.
gyrase-dependent
DNA replication
IC50 (µg/ml)10
Compound
Structure
PatchXpress
(IC50, µM)15d
pneumoniae17
14
0.035
>1.324
0.04
1, 0.125, 0.125
239
nd
310
nd
(GSK966587)
HO
N
114a
(enantiomer of
GSK966587)
O
O
O
O
O
N
N
N
F
F
F
>16, 8, 4
N
H
N
N
N
N
F
N
124a
1311
1412
1513
164a
174a
184a
0.5, 0.03, 0.06
0.25, 0.03, 0.06
1, 0.06, 0.125
0.5, 0.125, 0.125
0.5, 0.25, 0.25
16, 4, 4
16
nd
O
O
N
H
N
H
N
O
O
0.035
0.071
0.058
0.037
1.213
0.361
58
122
nd
N
H
N
nd
HO
N
O
O
O
N
N
F
101
289
nd
206
nd
N
H
N
nd
4, >16, 4
nd
nd
Haemophilus influenzae H128, Staphylococcus aureus WCUH 29, Streptococcus pneumoniae 1629, nd: not determined.

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Figure 3. In vivo efficacy of compound 1 (GSK966587)4 against S.
pneumoniae 1629. Each circle represents bacterial counts recovered from the
lungs of one animal. Filled circles represent animals sacrificed prior to the
end of the study.
Compound 14 (GSK966587) was progressed into in vivo
efficacy studies, including
a S. pneumoniae 1629 mouse
respiratory tract infection model , Figure 3.18 Non-neutropenic
mice were infected in the lungs via intranasal inhalation and
received either 14 or a positive control (moxifloxacin) via oral
gavage at 1, 7, 24 and 31 hours post infection. Pharmacokinetics
were evaluated at these doses to determine the exposure in blood
required for efficacy.
4.
5.
After 48 hours, bacterial numbers in non-treated control
animals (NTC) increased by more than 2 log10 colony forming
units (cfu) compared to those at the start of therapy (1 hour
NTC), indicating a robust infection was achieved. Compound 14
showed excellent efficacy in this model. At 50 mg/kg BID, a
mean reduction in bacterial counts of approximately 2 log10 cfu
was obtained compared with 1hour NTC. The higher dose
reduced bacterial burden below the limit of detection (1.7 log10
cfu/lungs) in 4/5 animals.
The AUC measured for the 50 mg/kg BID dose was
approximately 50 µg.h/ml per day with an associated Cmax of
approximately 8 µg/ml. This data demonstrates that excellent in
vivo efficacy can be obtained for a compound from this class in a
clinically relevant animal infection model.
In conclusion, we achieved our goal of demonstrating
efficacy and promising PK attributes with a tool compound 14.
Progression of compound 14 towards the clinic was discontinued
due to hepatic portal tract lesions observed in the 14-day dog
GLP safety toxicology study across all dose levels. This type of
toxicity has not been observed for other compounds within this
class, therefore we continue to evaluate the class for clinical
development.
6.
7.
8.
9.
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Bosworth, N.; Towers, P. Nature, 1989, 341, 167; The published
toluenized cell DNA replication assay was configured to a microtiter-
based Scintillation Proximity (SPA) format. H. influenzae was rendered
permeable to nucleotide substrates and cofactors by treatment with 1%
toluene for 10 min. DNA gyrase activity was determined by measuring
gyrase-dependent DNA replication in the toluenized cells by the ATP-
dependent incorporation of 33P-TTP. Final concentrations (per well):
20% toluenized H. influenzae cells, 0.05 µCi 33P-TTP, 33 µM dATP, 33
µM dGTP, 33 µM dCTP,13 µM dTTP, 2 mM ATP, 70 mM KPO4, 5 mM
DTT, 13 mM MgSO4, 0.01% BSA, 0.01% CHAPS, pH 7.4. Reactions,
incubated at 37 oC for approximately 30 min, were stopped with 10%
TCA, and SPA beads added for scintillation counting.
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WO2007122258; Chem. Abstr. 2007, 147, 502337.
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Sukumar, S. K.; Markwell, R. E.; Miles, T. J.; Pearson, N. D.
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Acknowledgment
Acknowledgment is given to all the chemists and biologists
who have been involved in this work.
13. Jones, G. E.; Miles, T. J.; Pearson, N. D. WO2007115947; Chem. Abstr.
2007, 147, 469374.
14.
A
complex of
1
(GSK966587) with S. aureus GyrB27-
A56(GKdel/Tyr123Phe) (ref 3b) and a 20 base-pair DNA heteroduplex
(sequence below) was purified by size exclusion chromatography in
100mM Na2SO4, 20mM HEPES, 5mM MnCl2, pH 7.0. The purified
complex, 6.4mg/ml in 33mM Na2SO4, 20mM HEPES, 13mM NaCl,
1.7mM MnCl2, pH 7.0 was crystallised by the hanging drop method
against 11% PEG 5000MME, 100mM BisTris pH6.2. A crystal was
transferred into 15% glycerol + well solution and frozen in liquid
nitrogen. A 2.6Å dataset was collected from a single crystal at the ESRF
on beamline ID23-1 (cell P61 a=b=93.9Å, c=416.4Å). The structure was
refined starting from the complex with GSK299423 (PDB code: 2XCS -
cell P61 a=b=93.3Å, c=412.8Å – ref 3b), and coordinates have been
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5' TGTGCGGTGTACCTACGGCT 3'
3' ACACGCCACATGGATGCCGA 5'
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5’-TGTGCGGTGXXCCTACGGCT – 3’
3’-ACACGCCACXXGGATGCCGA – 5’
were varied as described in the legend to figure 2.
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Guoliang, T.; Zozulya, Z.; Saya, S.; Phimmachack, R.; Sie, C.; Yuan, J.;
Wu, L.; Xu, J.; Ghetti, A. Assay Drug Dev.Technol. 2004, 2:497; (e)
Zhou, Z.; Gong, Q.; Ye, B.; Fan, Z.; Makielski, J. C.; Robertson, G. A.;
January, C. T. Biophys. J. 1998, 74, 230.
16. All animal experiment protocols were approved by the Animal Care and
Use Committee at GlaxoSmithKline Pharmaceuticals (PA, US). The
pharmacokinetics of compound 1 were studied in male Sprague-Dawley
rats, male beagle dogs and male Cynomolgus monkeys following single
intravenous and oral administration. Absolute oral bioavailability was
estimated using a cross-over study design (n=3). Blood samples were
assayed using protein precipitation followed by LC/MS/MS analysis and
the concentration-time data were analyzed by non-compartmental
methods (WinNonlin, v4.1). The in vitro plasma protein binding and
microsomal intrinsic clearance were determined as described in Xiang,
H.; McSurdy-Freed, J.; Moorthy, G. S.; Hugger, E.; Bambal, R.; Han, C.;
Ferrer, S.; Gargallo, D.; Davis, C. B. J. Pharm. Sci. 2006, 95, 2657.
17. Minimum inhibitory concentrations: MICs were determined by broth
microdilution methods according to Clinical and Laboratory Standards
Institute guidelines 7. The MIC was the lowest concentration of an
antibacterial that showed no visible growth after incubation at 37°C for
18-24 h, with a starting inoculum of ~5.5 X 105 cfu/ml
18. Infection model details: S. pneumoniae 1629 was grown overnight on
trypticase soy agar plates supplemented with 5% sheep blood. The
inoculum was prepared by harvesting bacterial growth from the plates
into phosphate-buffered saline. Mice were infected with S. pneumoniae
1629 by intranasal inhalation of
a 50 µl inoculum, receiving
approximately 7.6 log10 cfu/mouse. At 48 h post infection, the animals
were euthanized and the lungs excised for the enumeration of viable
bacteria. PK was performed separately using infected mice.

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19.
Novel hydroxyl tricyclics (e.g. GSK966587)
as potent inhibitors of bacterial type IIA
topoisomerases
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