Novel tricyclics (e.g., GSK945237) as potent inhibitors of bacterial type IIA topoisomerases

Article abstract of DOI:10.1016/j.bmcl.2016.03.106

During the course of our research on the lead optimisation of the NBTI (Novel Bacterial Type II Topoisomerase Inhibitors) class of antibacterials, we discovered a series of tricyclic compounds that showed good Gram-positive and Gram-negative potency. Herein we will discuss the various subunits that were investigated in this series and report advanced studies on compound 1 (GSK945237) which demonstrates good PK and in vivo efficacy properties.

Full text of DOI:10.1016/j.bmcl.2016.03.106

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel tricyclics (e.g., GSK945237) as potent inhibitors of bacterial
type IIA topoisomerases
b
c
d
e
b
Timothy J. Miles a, , Alan J. Hennessy , Ben Bax , Gerald Brooks , Barry S. Brown , Pamela Brown ,
Nathalie Cailleau d, Dongzhao Chen e, Steven Dabbs b, David T. Davies d, Joel M. Esken e, Ilaria Giordano a,
Jennifer L. Hoover e, Graham E. Jones d, Senthill K. Kusalakumari Sukmar e, Roger E. Markwell d,
Elisabeth A. Minthorn f, Steve Rittenhouse e, Michael N. Gwynn e, Neil D. Pearson e
⇑
a Diseases of the Developing World CEDD, GlaxoSmithKline, Calle Severo Ochoa, 2, 28760 Tres Cantos, Madrid, Spain
b Infectious Diseases CEDD, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK
c Platform Technology & Science, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK
d Infectious Diseases CEDD, GlaxoSmithKline, Third Avenue, Harlow CM19 5AW, UK
e Infectious Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
f Oncology TA, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
During the course of our research on the lead optimisation of the NBTI (Novel Bacterial Type II
Topoisomerase Inhibitors) class of antibacterials, we discovered a series of tricyclic compounds that
showed good Gram-positive and Gram-negative potency. Herein we will discuss the various subunits
that were investigated in this series and report advanced studies on compound 1 (GSK945237) which
demonstrates good PK and in vivo efficacy properties.
Received 3 March 2016
Revised 28 March 2016
Accepted 29 March 2016
Available online xxxx
Ó 2016 Published by Elsevier Ltd.
Keywords:
Antibacterials
Bacterial type IIA topoisomerase
NBTI
GSK945237
Tricyclics
Pharmacokinetic
In vivo efficacy
Since infection due to resistant bacteria such as methicillin
resistant Staphylococcus aureus (MRSA) and vancomycin resistant
enterococci (VRE) are on the increase there is an unmet medical
need for novel antibiotics.1
GSK2994233b) with a similar mode of action to 14 (Fig. 1) bind to
the topoisomerases in a different manner than the quinolones.3c
These compounds have a novel mechanism, which overcomes
issues of target-mediated resistance for fluoroquinolones that
have developed in the clinic.2 Compounds with a similar mecha-
nism but of a distinct chemical series have also been discussed in
the literature.3d–z Frequency of resistance of these compounds,
like 14, is describe in the literature as being low.3m,r,s
Bacterial DNA type IIA topoisomerases are inhibited by quino-
lones and novobiocin, which have been used in the clinic to treat
bacterial infections. However, both these drugs classes suffer from
toxicity issues: novobiocin has very limited use and quinolones can
cause pathologic lesions in tendon tissue (tendinopathy) restrict-
ing their use in females and in adolescent children.2a,d,e Moreover
target-mediated resistance to fluoroquinolones2f is on the
increase so the development of novel antibiotics against this
target is attractive.2
In the course of our efforts to develop antibacterials with novel
modes of action, good safety profile and based on our previous
published material,3b,f,g,i we discovered
a series of tricyclic
compounds (e.g., 14) which have potent antibacterial properties.
They target both DNA gyrase and topoisomerase IV and this dual
targeting should confer resistance benefits in a clinical setting.
This Letter will discuss the structure activity relationship (SAR)
of the left hand side (LHS) and central unit portion of this series of
tricyclic compounds (e.g., 14) as shown in Figure 1. The fit of the
right hand side (RHS) in the largely hydrophobic pocket between
the two GyrA subunits,5 plus the nitrogen of the RHS forming a
It has been established through mutational (Black et al.)3a and
our X-ray crystallography studies (Bax et al.)3b that NBTI (Novel
Bacterial Type II Topoisomerase Inhibitors) compounds (such as
⇑
Corresponding author. Tel.: +34 650 349 771; fax: +34 918 070 550.
E-mail address: tim.j.miles@gsk.com (T.J. Miles).
http://dx.doi.org/10.1016/j.bmcl.2016.03.106
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Miles, T. J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.106

2
T. J. Miles et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
NH2
O
O
OH
F
O
O
OH
OH
H
N
MeO
OH
N
N
O
HN
O
O
O
novobiocin
ciprofloxacin
O
S
N
N
F
N
H
N
H
O
O
N
N
O
CN
N
O
N
Central Unit
RHS
N
GSK299423
LHS
GSK945237, 1
Figure 1. Type IIA bacterial DNA topoisomerase inhibitors.
hydrogen bond interaction to the GyrA through a solvent water
(Fig. 2) adds to the number of possible interactions that could be
exploited to alter potency within this series.
The synthesis of aldehyde 107 is described in Scheme 2.
Commercially available bromomaleic anhydride 11 was displaced
by hydrazine (slight variation on the method of Kasnar et al.)8
followed by chlorination to give trichloropyridazine 4 in an overall
yield of 87% (+20–30% bromide isomer which was carried over the
next 3 steps). The following two steps involved alkylation using
ethylene glycol and cyclisation to afford intermediate 14 in 64%
yield. The palladium catalysed vinylation of intermediate 14 gave
styrenyl compound 15,9 which was oxidatively cleaved to give
aldehyde 107 in 32% yield over 2 steps.
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 TPS3 [quinolone-
resistant]) and Gram-negative (Haemophilus influenzae H128)
bacteria.
The synthesis of tricyclic compound 14 is described in Scheme 1.
Commercially available 3-fluoro-2-methylaniline 2, underwent an
alkylation with cinnamyl chloride 3 then cyclisation using alu-
minium (III) chloride6 to give quinolinone 4 in overall yield of
79%. The next steps involved alkylation using MeI, bromination
then cyanide displacement to give cyano 5 in 71% yield over three
steps. Hydrolysis of the cyano group to the acid followed by ester-
ification then vinylation gave acrylate 6 in 57% overall yield.
Michael addition using NBoc piperidine afforded the ester 7 in
92% yield. This intermediate 7 was reduced to the alcohol which
was activated and cyclised using MsCl to give NBoc protected
intermediate 8. The free amine 9 was liberated by Boc deprotection
and then separated into enantiomers by preparative chiral HPLC.
The desired (1R)-enantiomer was placed under reductive alkyla-
tion conditions with aldehyde 107 to give the tricyclic compound
14 in 73% yield.
We 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 this assay showed good correlation with the
whole-cell antibacterial potency.
Within these tricyclic examples and particular bacterial strains
shown in Table 1 the following observations can be made (please
note MICs of 4
lg/mL or more was deemed no further interest):
enantiomer 14 is better tolerated than 164. Quinoxaline (1711
)
shows that variations in LHS subunits are tolerated. It is possible
to replace the 9-F in 14 with either H (18) or CN (204) although
for Me (19) and OMe (214) we observed reduced activity. Alterna-
tive central units such as 22 and 23 are tolerated although not as
potent as the parent compound 14 within these bacterial strains.
Finally substitution of a methyl group in 7-position (24) was
tolerated.
Within the literature it has been reported that hERG inhibition
is related to their lipophilicity and basicity,12 for our series we too
observed a similar relationship. Two compounds (14 and 1711
)
were progressed to hERG profiling (IC50 238 and 42 lM, respec-
tively), where example 14 demonstrated a desirable profile for pro-
gression into PK and in vivo efficacy studies. We will now discuss
each of these studies in turn.
As can be seen in Table 2,14 the blood clearance was high in the
rat (ꢀ100% of hepatic blood flow) but moderate in dog and monkey
(40–50% of hepatic blood flow). The renal clearance in dog and
monkey was moderate (ꢀ74% and 45% of the total clearance in
dog and monkey, respectively), and volume of distribution was
high (greater than total body water). Reasonable oral bioavailabil-
ity (FPO) was achieved in all species. Plasma Protein Binding and
Figure 2. A 2.05 Å structure of compound 1 in complex with DNA (green carbons)
and S. aureus DNA gyrase (blue carbons). Waters are shown as small red spheres and
hydrogen bonds as dotted black lines.
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T. J. Miles et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
CN
O
H
N
F
H2
N
O
F
(c - e)
71%
(a - b)
F
O
N
Cl
+
79% over
over
3 steps
Ph
2 steps
2
3
4
5
NHBoc
57% over
(f - h)
3 steps
N
MeO2C
NHBoc
N
O
N
F
(j - k)
(i)
92%
MeO2
N
C
F
O
N
71% over
F
O
2 steps
8
6
51% over
7
(l - m)
O
two steps
O
O
N
O
N
N
F
N
NH2
10
N
H
O
N
O
N
O
N
F
N
(n)
73%
9
GSK945237, 1
Scheme 1. Reagents and conditions: (a) 2 (1.0 equiv), 3 (1.0 equiv), EtOAc, NaHCO3, H2O, 0 °C, 0.25 h; (b) AlCl3 (0.4 mol %), chlorobenzene, 65 °C, 1 h then 75 °C, 30 min;
(c) KOtBu (1.1 equiv), DMSO, 35 ? 45 °C, 15 min then MeI (1.3 equiv), 60 °C, 30 min; (d) NBS (1.1 equiv), benzoyl peroxide (0.5 mol %), trifluoromethylbenzene, hv, 70 °C, 1 h;
(e) KCN (4.6 equiv), DMF, 70 °C, 18 h; (f) TMSCl (3.4 equiv), MeOH, 79 °C, 2.25 h; (g) TMSCH2N2 (1.3 equiv), Et3N (1.2 equiv), MeOH, CH3CN, rt, 3 h; (h) paraformaldehyde
(7.1 equiv), K2CO3 (1.54 equiv), BnNEt3Cl (1.6 equiv), cyclohexane, 86 °C, 5 h; (i) 1,1-dimethylethyl 4-piperidinylcarbamate (1.1 equiv), 1,1,3,3-tetramethylguanidine, DMF,
80 °C, 2 h; (j) LiAlH4 (1.2 equiv), THF, À70 °C, 1 h then 0 ? 10 °C, 1 h; (k) MsCl (1.1 equiv), iPr2NEt (2.2 equiv), CHCl3, 65 °C, 3 h; (l) Conc. HCl, H2O, 50 °C, 3 h; (m) chiral
separation using Chiralpak AD column; (n) 107 (1.1 equiv), Na(OAc)3BH (2.5 equiv), DCM, MeOH, 0 °C ? rt, 3 h then 107 (0.33 equiv), Na(OAc)3BH (0.2 equiv), rt, 18 h.
O
Br
Cl
Cl
N
Cl
N
HO
(c)
(a - b)
N
N
O
87% over
83%
Cl
O
Cl
2 steps
O
12
13
11
+ 20-30% Br isomer
+ ~15% Br isomer
(d) 77%
O
N
O
O
N
N
N
O
O
N
(e)
50%
(f)
N
O
O
64%
Cl
10
15
14
+ ~15% Br isomer
Scheme 2. Reagents and conditions: (a) N2H4 (0.75 equiv), H2O, reflux, 4 h; (b) reaction was split into two batches: POCl3 (200 mL), reflux, 3.5 h; (c) ethylene glycol
(6.7 equiv), NaH (1.0 equiv), THF, 0 °C, 40 min then 12 (1.0 equiv), THF, 0 °C, 1 h followed by 0 °C ? rt, o/n; (d) LiH (4.0 equiv), dioxane, rt, 1 h then 110 °C, o/n; (e) Pd(PPh3)4
(2.2 mol %), K2CO3 (0.95 equiv), 2,4,6-trivinylcyclotriboroxane pyridine complex (0.6 equiv), H2O, DME, 95 °C, 48 h; (f) Aq. sol. osmium tetroxide (4% w/v), sodium periodate
(2.8 equiv), dioxane, H2O, 0 °C then 0 °C ? rt followed by rt, 7 h.
microsomal intrinsic clearance of 14 were not high and similar
across species.
compared to those at the start of therapy (1 h NTC). Compound
14 showed excellent efficacy at 50 and 100 mg/kg b.i.d., with bacte-
rial numbers reduced below the limit of detection in 3/5 and 5/5
animals, respectively. The comparator moxifloxacin was ineffective
Compound 14 was progressed into in vivo efficacy studies,
including
a rat respiratory tract infection model caused by
the quinolone-resistant S. pneumoniae TPS3, Figure 3.15 Rats
were infected in the lungs via intrabronchial instillation and
received either 14 or moxifloxacin via oral gavage at 1, 7, 24
and 31 h post infection. We measured the exposure in blood
associated with these doses to determine the potential thera-
peutic window.
in this in vivo model (MIC is 4 lg/mL against S. pneumoniae TPS3
[quinolone-resistant], which is considered resistant by current
susceptibility criteria).16
The AUC associated with a twice daily 100 mg/kg oral dose in
rats was approximately 30
lg h/mL per day with an associated
Cmax of approximately 3 g/mL. This demonstrates the excellent
l
After 48 h, bacterial numbers in non-treated control animals
(NTC) increased by more than 1 log10 colony forming units (cfu)
in vivo efficacy for a compound from this class in a clinically
relevant animal infection model.
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4
T. J. Miles et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Antibacterial activity of a set of tricyclic compounds10,13
Compound
Structure
H. influenzae DNA gyrase-dependent DNA
MIC H. influenzae/S. aureus/S.
pneumoniae13
(lg/mL)
10
replication IC50
(lg/mL)
O
O
O
N
N
H
14 (GSK945237)
0.034
1, 0.06, 0.25
O
O
O
N
N
N
O
O
O
N
F
N
N
N
N
N
H
164 (enantiomer of
GSK945237)
0.121
0.036
>16, 2, 8
N
F
N
N
H
1711
N
N
F
0.5, 0.06, nd
O
O
O
O
N
N
N
N
H
O
O
O
O
N
N
N
N
18
nd
2, 0.5, 1
O
O
O
O
N
N
N
N
N
N
N
N
N
H
19
0.076
0.062
0.276
4, 0.25, 1
2, 0.25, 0.5
16, 2, 8
N
H
204
CN
N
N
H
214
O
O
O
N
N
H
22
23
24
0.166
0.666
0.099
>16, 16, 16
O
N
N
F
O
N
O
N
H
O
N
N
N
F
>16, 1, 4
O
N
O
N
F
N
H
O
N
O
N
N
2, 0.06, 0.5
H. aemophilus influenzae H128, S. taphylococcus aureus WCUH 29, S. treptococcus pneumoniae TPS3 (quinolone-resistant), nd: not determined.
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T. J. Miles et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
5
Table 2
References and notes
Summary of in vivo pharmacokinetics and in vitro ADME study results for compound
14
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Parameters (units)
Rat
Dog
Monkey
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IV DMPK
Dose (mg/kg)
CLb (mL/min/kg)
Vss (L/kg)
3
3
3
60.8
7.2
1.9
11.9
6.1
8.8
22.6
8.2
7.6
b-Half-life (h)
Oral DMPK
Dose (mg/kg)
5
5
5
Cmax
(
lg/mL)
0.04
0.29
22
1.07
6.05
ꢀ100
0.21
1.19
34
AUC (
l
g h/mL)
Oral bioavailability (%)
Plasma protein binding (%
bound)
63.25 6.44 47.03 5.34 42.45 4.04
CLi microsomes (mL/min/g liver) 0.7
<0.6
1.5
Figure 3. In vivo efficacy of compound 14 against Streptococcus pneumoniae TPS3
(quinolone-resistant). Each circle represents the bacterial counts recovered from
the lungs of one animal.
In conclusion, we achieved our goal of demonstrating efficacy
and promising PK attributes with compound 14, as such this asset
was progressed to phase I (escalating single oral dose studies in
healthy volunteers). Actual human PK parameters were predicted
by allometric scaling and a linear dose to exposure relationship
was seen in the predicted therapeutic range for efficacy. A species
specific ocular toxicity issue in pigmented rats and testicular
effects in 3 month dog studies led to a decision to discontinue pro-
gression of compound 14 in favour of gepotidacin (GSK2140944).17
Acknowledgments
Acknowledgment is given to all the chemists and biologists who
have been involved in this work.
We would also like to make a special acknowledgement to Mick
Gwynn, who died on 16th December 2013. He was highly
respected friend, scientist, mentor and colleague who accom-
plished so much in his incredible 36 year career at GSK. Mick spent
most of his career working in antibacterials and worked with the
team that discovered and developed Augmentin. On moving to
the US Mick worked primarily on bacterial gyrase. Notably this
included a key role in the discovery of a new class of antibacterial
agents and obtaining structural data for enzyme-DNA-inhibitor
complexes that allowed us to drive this project to its conclusion.
More recently, Mick led GSK initiatives to develop antibacterials
by public private partnerships and became a beacon of expertise
on government contracts, where he was greatly trusted and
respected by both these organizations. He leaves us with wonder-
ful memories but with very heavy hearts at his loss.
Please cite this article in press as: Miles, T. J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.106

6
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Please cite this article in press as: Miles, T. J.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.106