Novel amino-piperidines as potent antibacterials targeting bacterial type IIA topoisomerases

Article abstract of DOI:10.1016/j.bmcl.2011.09.117

We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We report the investigation of various subunits in this series and advanced studies on compound 8. Compound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral efficacy in a rat lung infection model.

Full text of DOI:10.1016/j.bmcl.2011.09.117

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7489–7495
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel amino-piperidines as potent antibacterials targeting bacterial type IIA
topoisomerases
Timothy J. Miles a, , Jeffrey M. Axten d, Christopher Barfoot b, Gerald Brooks c, Pamela Brown b,
⇑
Dongzhao Chen d, Steven Dabbs b, David T. Davies b, David L. Downie e, Susanne Eyrisch b,
Timothy Gallagher d, Ilaria Giordano a, Michael N. Gwynn d, Alan Hennessy b, Jennifer Hoover d,
Jianzhong Huang d, Graham Jones b, Roger Markwell c, William H. Miller d, Elizabeth A. Minthorn d,
Stephen Rittenhouse d, Mark Seefeld d, Neil Pearson d
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 Infectious Diseases CEDD, GlaxoSmithKline, Third Avenue, Harlow CM19 5AW, UK
d Infectious Diseases CEDD, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, USA
e Biological Reagents & Assay Development, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2011
Revised 26 September 2011
Accepted 27 September 2011
Available online 10 October 2011
We have identified a series of amino-piperidine antibacterials with a good broad spectrum potency. We
report the investigation of various subunits in this series and advanced studies on compound 8. Com-
pound 8 possesses good pharmacokinetics, broad spectrum antibacterial activity and demonstrates oral
efficacy in a rat lung infection model.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterial
Bacterial type IIA topoisomerase
Amino-piperidines
Pharmacokinetics
In vivo efficacy
Novel mechanism antibacterials are needed to combat the glo-
bal spread of diseases caused by increasingly challenging bacterial
pathogens such as methicillin resistant Staphylococcus aureus
(MRSA) and vancomycin resistant enterococci (VRE).1
Mutational and X-ray crystallography studies have shown that
the mode of action and the binding of similar compounds to the
topoisomerases are different to that of the quinolones.4a,b They
can therefore be seen as novel series of antibacterials, which
should address clinically validated targets without the issues of
existing resistance problems. Other compounds with a similar
mechanism but of a distinct chemical series have also been dis-
cussed in the literature.4c–e
In this Letter we describe medicinal chemistry efforts focused
on surveying the potency requirements of the left hand side
(LHS), central unit and right hand side (RHS) portion of amino-
piperidine 2 as shown below in Figure 2.
A typical synthesis of these compounds is described in Scheme
1 (for compound 8) and Scheme 2 (for compound 13). The palla-
dium catalysed vinylation of aryl-bromide 3 gave styrenyl com-
pound 4.5a Asymmetric dihydroxylation gave enantioenriched
5,5b which was transformed into epoxide 6 via activation as the
tosylate and ring closure. Epoxide opening under Lewis acid condi-
tions with amine 7 followed by Boc deprotection under acidic con-
ditions and finally chiral HPLC separation gave enantiopure desired
Bacterial DNA type IIA topoisomerases have been much studied
in the field of antibacterial research, and are the major targets of
quinolones and novobiocin (Fig. 1). However, these drugs suffer
from toxicity issues, which have largely prevented their use in chil-
dren, and increasing clinical resistance may further limit their use.2
In the course of our efforts to develop antibacterials with novel
modes of action and good safety profiles, our group has recently re-
ported a series of cyclohexyl-amide compounds of type 1, Figure 1.
In addition, we also investigated a series of amino-piperidine com-
pounds of general structure 2.3 These inhibitors target both the
GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase
IV,4a and we believe that this dual targeting should delay or pre-
vent emergence of resistance in the clinical setting.
⇑
Corresponding author. Tel.: +34 918 070 507; fax: +34 918 070 550.
E-mail address: tim.j.miles@gsk.com (T.J. Miles).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.117

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T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
NH2
O
O
OH
F
O
O
OH
O
OH
H
N
MeO
OH
N
N
O
HN
O
O
ciprofloxacin
novobiocin
O
OH
HN
N
O
O
O
N
N
H
N
1
Figure 1. Type IIA bacterial DNA toposisomerase inhibitors.
ing acid 17. A Curtius rearrangement followed by Boc deprotection
gave 18. Finally, formation of the diazonium tetrafluoroborate salt
and thermal decomposition gave the fluoronaphthyridine 3.
The synthesis of various amino-piperidine central units is well
described in the patent literature.3 The details of the synthesis of
enantiopure trans hydroxy subunits 24 and 25 are shown in
Scheme 4.
Tetrahydropyridine 19 was protected and epoxidised with
mCPBA to give racemic epoxide 21. Regioselective ring opening
of 21 gave racemic trans azido-alcohol 22. The azide functionality
was reduced under mild conditions and the subsequent primary
amine protected to give racemic 23 which could be separated into
individual enantiomers by chiral HPLC. More forcing hydrogena-
tion conditions then gave the desired amino-piperidine derivatives
24 and 25.5d
Central Unit
HO
N
O
O
O
N
H
N
2
LHS
RHS
Figure 2. Amino-piperidine based inhibitor.
material 8. Amine 7 was made using standard conditions and is de-
scribed in the patent literature.3b
Michael addition of 9 to 4 gave intermediate 10, which was
transformed into the primary amine 11 via Boc deprotection under
acidic conditions. A final reductive alkylation with aldehyde 12
gave the desired compound 13.5c
The synthesis of subunits of type 3 (aryl bromides or triflates) is
well described in the patent literature.3 The synthesis of interme-
diate 3 is shown in Scheme 3 as a representative example.
Reaction of 14 with diethyl ethoxymethylenemalonate and sub-
sequent thermal cyclisation gave quinolone ester 15, which was
transformed to bromo-quinoline ester 16 and then the correspond-
With all of the desired sub-units in hand we proceeded to syn-
thesize and test a range of compounds, a subset of which is shown
in Table 1.
As can be seen from Table 1, we explored a variety of RHS sub-
units, LHS subunits, linkers and amino-piperidine ring substituents
to explore the SAR of this series.
These compounds in Table 1 are well tolerated with activity
seen against Gram-positive (S. aureus WCUH 29, Streptococcus
pneumoniae 1629) and Gram-negative (Haemophilus influenzae
H128) bacteria. Potency was also observed across a more extensive
Br
HO
OH
O
N
F
(a)
(b)
O
N
F
O
N
F
N
N
N
3
4
5
(c)
O
HN
O
O
N
(d)-(f)
O
N
F
+
8
N
O
O
N
7
6
Scheme 1. Reagents and conditions: (a) Aryl bromide 3 (1 equiv), Pd(PPh3)4 (5 mol %), 2,4,6-trivinylcyclotriboroxane–pyridine complex (0.5 equiv), K2CO3 (1 equiv), DME,
H2O, 100 °C, 1.5 h; (b) AD-mix b, t-BuOH, H2O, rt, 48 h; (c) (i) TsCl, n-Bu2SnO, NEt3, 3 h, rt; (ii) K2CO3, MeOH, rt, 1.75 h; (d) LiClO4, MeCN, rt, 48 h; (e) TFA, DCM, rt, 3 h; (f) chiral
HPLC separation on Chiralpak AD column.

T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
7491
O
O
N
F
HN
O
O
N
F
O
N
(a)
+
O
N
H
N
N
10
N
H
9
4
(b)
O
N
F
O
O
O
N
(c)
H
+
NH2
13
N
N
12
11
Scheme 2. Reagents and conditions: (a) DMF, 105–110 °C, 29 h; (b) TFA, rt, 0.5 h; (c) (i) CHCl3, MeOH, 3 Å molecular sieves, 70 °C, 4 h; (ii) NaBH(OAc)3, rt, 18 h.
O
O
Br
N
O
O
N
O
N
O
N
O
(a), (b)
(c)
O
NH2
N
H
14
15
16
(d)
Br
Br
Br
O
O
N
F
O
N
NH2
O
N
(g), (h)
(e), (f)
OH
N
N
N
3
18
17
Scheme 3. Reagents and conditions: (a) diethyl ethoxymethylenemalonate, EtOH, reflux, 4 h; (b) Dowtherm AÒ, reflux, 15 min; (c) PBr3, DMF; (d) NaOH, THF, H2O, rt, 18 h; (e)
DPPA, t-BuOH, NEt3, DMF, 100 °C, 1 h; (f) TFA, DCM, rt, 3 h; (g) NOBF4, THF, À10 °C, 0.5 h; (h) Decalin, ꢀ170 °C, 2 min.
O
N
(a)
N
(b)
N
H
O
O
O
O
20
21
19
(c)
O
O
N3
N
O
O
HN
N
O
OH
OH
HN
O
HN
O
(f), (g)
(d), (e)
OH
OH
+
O
O
N
H
N
H
O
23 +
25
24
22 +
enantiomer
enantiomer
Scheme 4. Reagents and conditions: (a) CbzCl, Na2CO3, H2O, rt, 18 h; (b) mCPBA, DCM, rt, 4 h; (c) NaN3, NH4Cl, MeOH, H2O, 65 °C, 18 h; (d) Pd/C, EtOAc, 1 atm H2, rt, 22 h; (e)
Boc2O, DCM, rt, 18 h; (f) chiral HPLC separation on Chiralpak AD column; (g) Pd(OH)2, MeOH, 1 atm H2, rt, 18 h.
panel of organisms. The apparent trend that this series of com-
pounds is more potent towards Gram-positive than Gram-negative
bacteria could be attributed to reduced cell membrane penetration
and/or efflux pump differences.
This data tracks well with the good fit obtained by modelling
studies, where 8 was docked into the X-ray crystal structure previ-
ously obtained with compound 35 (GSK299423).3b,9
We measured target potency using a gyrase-dependent DNA
replication assay reconfigured in an unpublished format at GSK
using Scintillation Proximity Assay (SPA) technology.7 For this ser-
ies of compounds, this assay tracked well with whole cell antibac-
terial potency.
Table 1 shows it is possible to alter each subunit of compound 2.
From the benzodioxin RHS unit of 2 nitrogens can be introduced to

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T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
Table 1
In vitro activities of a set of amino-piperidine compounds6,7,8a,b
Compound Structure
H. influenzae DNA
gyrase-dependent
DNA replication IC50
MIC (
l
g/mL) H. hERG inhibition hERG
LogD
pH7.4
influenzae/S.
aureus/S.
whole cell patch inhibition
clamp IC50
dofetilide
(
l
g/mL)7
pneumoniae6
(l
M)8a
binding IC50
(l
M)8b
HO
N
O
O
O
0.5, 0.125,
0.125
2
0.008*
nd
39.8
25.1
0.87
0.62
N
H
N
HO
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
26
0.056*, 0.016
2, 0.06, 0.125
4
N
H
N
N
N
N
N
N
N
N
N
N
N
F
27
13
28
29
0.031
0.051
0.027
0.144
0.25, 0.01, 0.03
6
5.0
2.03
1.81
2.40
1.48
N
H
N
F
N
N
N
0.125, 0.01,
0.01
nd
15.1
2.0
N
H
N
Cl
N
0.25, 0.01, 0.01
0.5, 0.01, 0.03
2
N
H
nd
7.9
N
H
N
HO
HO
N
F
O
O
O
O
O
O
N
N
N
N
8
30
31
32
0.078
0.098
0.072
0.051
0.5, 0.03, 0.06
1, 0.06, 0.125
0.5, 0.03, 0.06
0.5, 0.01, 0.06
5
79.4
63.1
7.9
0.59
N
H
N
N
N
N
N
N
N
N
N
F
O
O
nd
nd
N
H
OH
N
F
O
O
3
1.52
1.38
N
H
OH
N
F
O
O
nd
4.9
N
H

T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
7493
Table 1 (continued)
Compound Structure
H. influenzae DNA
gyrase-dependent
DNA replication IC50
MIC (
l
g/mL) H. hERG inhibition hERG
LogD
pH7.4
influenzae/S.
aureus/S.
whole cell patch inhibition
clamp IC50
dofetilide
(
l
g/mL)7
pneumoniae6
(l
M)8a
binding IC50
(l
M)8b
OH
N
F
O
O
O
N
N
33
0.058
0.5, 0.01, 0.03
nd
nd
2.10
N
H
N
N
OH
N
F
O
O
O
34
0.058
0.06
0.5, 0.01, 0.03
0.25, 0.01, 0.01
nd
29.5
0.5
2.16
2.32
N
H
N
N
N
N
N
O
S
35
2
N
H
N
O
Haemophilus influenzae H128, Staphylococcus aureus WCUH 29, Streptococcus pneumoniae 1629, nd: not determined.
*
E. coli DNA gyrase-dependent DNA replication assay used.
Figure 3. In vivo efficacy of amino-piperidine compounds in a rat respiratory tract infection model as measured by mean log10 change compared to controls at the start of
dosing (1 h NTC). The non-treated control (NTC) rats showed an increase in S. pneumoniae 1629 colony forming units (cfu) of greater than 2log10 at 48 h post infection.
Compounds 13, 28, 8, 31 and 32 all showed excellent oral efficacy at 50 mg/kg (approx. 2log10 decrease in cfu/lungs compared to 1 h NTC), equivalent to or better than the
comparator moxifloxacin.
The stereochemistry of the benzylic hydroxyl group has some
influence on activity (8 vs 30) but was less important in the central
unit (31–34). A variety of quinolines and naphthyridine LHS units
were potent with substitution in the 3-position with H, F, Cl and
CN groups all well tolerated.
Table 2
Summary of in vivo and in vitro DMPK of compound 811
Parameters (Units)
Rat
Dog
Monkey
Human
IV DMPK
Intravenous dose (mg/kg)
CLb (mL/min/kg)
Vss (L/kg)
Half-life (h)
MRT (h)
3.5
67.2
9.8
2.5
2.5
3.5
3.1
15.2
4.1
5.6
4.6
—
There was a general issue of hERG inhibition with this series.8c–e
We used a dofetilide binding assay to screen our compounds and
took whole cell patch clamp measurements for selected examples.
We found that the dofetilide binding assay could underestimate
the levels of hERG inhibition so we used this as an early filter in
our compound progression cascade.
16.7
16.5
12.4
16.5
nd
nd
nd
nd
Oral DMPK
Oral dose (mg/kg)
AUC (
Oral bioavailability (%)
5.6
0.72
51
4.0
2.51
76
5.2
0.72
12
nd
nd
nd
l
g h/mL)
The patch clamp measurements indicated that hERG inhibition
was generally <10 lM for this series despite achieving LogD values
Plasma protein binding (% bound)
CLi microsomes (mL/min/g liver)
41.1
<0.6
nd
0.7
64.6
4.8
69.3
<0.6
from 2.40 (compound 28) to as low as 0.59 (compound 8). Addition
of more polarity led to a loss of antibacterial activity in this series.
As the MIC values were very low, we wished to determine the min-
imum efficacious doses of our more potent compounds in an
in vivo rat respiratory tract infection model to assess the potential
for a therapeutic window (Fig. 3).10
nd = not determined.
give pyridyl analogues 8, 13 and 26–35. Hydroxylation of the linker
and central unit is also possible (compounds 2, 8, 26 and 30–34).

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T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
7. (a) Moses, R. E.; Richardson, C. C. Proc. Natl. Acad. Sci. U.S.A. 1970, 67, 674; (b)
In this model, rat lungs were infected with S. pneumoniae 1629
Winston, S.; Masushita, T. J. Bacteriol. 1975, 123, 921; (c) Bosworth, N.; Towers,
P. Nature 1989, 341, 167.
and animals were dosed orally with either a test compound or a
positive control (moxifloxacin) at 50 mg/kg (1, 7, 24 and 31 h post
infection).
8. (a) Zhou, Z.; Gong, Q.; Ye, B.; Fan, Z.; Makielski, J. C.; Robertson, G. A.; January, C.
T. Biophys. J. 1998, 74, 230; (b) Chadwick, C. C.; Ezrin, A. M.; O’Connor, B.;
Volberg, W. A.; Smith, D. I.; Wedge, K. J.; Hill, R. J.; Briggs, G. M.; Pagani, E. D.;
Silver, P. J.; Krafte, D. S. Circ. Res. 1993, 72, 707; hERG inhibition reviews: (c)
Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishert, G. J. Med. Chem. 2006, 49, 4029;
(d) Hancox, J. C.; McPate, M. J.; El Harchi, A.; Zhang, Y. H. Pharmacol. Ther. 2008,
119, 118; (e) Recanatini, M.; Cavalli, A.; Masetti, M. ChemMedChem 2008, 3,
523.
9. Docking of 8 into X-ray crystal structure (Ref. 4b) in S. aureus gyrase can help to
explain the antibacterial potency observed. Compound 8 binds very similarly to
compound 35 (GSK299423, Fig. 4). The key interactions are shown below,
Figure 5.
Excellent in vivo efficacy was obtained with compounds 13, 28,
8, 31 and 32 following oral dosing at 50 mg/kg, and final mean bac-
terial burdens were 103 cfu/lungs or less (approximately 2log10
reduction compared with 1 h NTC and P4log10 reduction com-
pared with 48 h NTC). Complete eradication of the infection was
observed in multiple animals among these groups. Overall, this
demonstrated the excellent in vivo oral efficacy for compounds
of this class in a relevant animal infection model.
Figure 4. X-ray binding mode of compound 35 (GSK299423) in S. aureus gyrase
(pdb code: 2XCS).4b
We then fully profiled the DMPK characteristics of compounds
with the best efficacy profiles. The DMPK attributes of compound
8 are shown in Table 2.11
As can be seen in Table 2, the blood clearance of 8 was high in
the rat (approximately 100% of hepatic blood flow) but moderate in
dog and monkey (35–55% of hepatic blood flow). Moderate to high
oral bioavailability (FPO) can be achieved in the rat and the dog, but
oral exposure is relatively low in the monkey. Protein binding of
compound 8 was moderate to low in all species tested. Intrinsic
clearance in microsomes was very low to low across species.
In conclusion, we achieved our goal of demonstrating efficacy
and achieving promising PK attributes with a tool compound 8.
Further efforts are ongoing in this area to optimize all of the re-
quired properties for clinical development.
Acknowledgment
Acknowledgment is given to all the chemists and biologists who
have been involved in this work.
References and notes
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Figure 5. Compound 8 was manually placed in the binding site of compound 35
(GSK299423) in S. aureus gyrase and then optimised using Embrace (Maestro)
with the OPLS2005 force field and GB/SA solvation model.
3. (a) Miles, T. J.; Barfoot, C.; Brooks, G.; Brown, P.; Chen, D.; Dabbs, S.; Davies, D.
T.; Downie, D. L.; Eyrisch, S.; Giordano, I.; Gwynn, M. N.; Hennessy, A.; Hoover,
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j.bmcl.2011.09.114.; (b) For the syntheses of these compounds and
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6. 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 Â 105 CFU/mL.

T. J. Miles et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7489–7495
7495
10. Infection model details: S. pneumoniae 1629 was grown overnight on trypticase
soy agar plates supplemented with 5% horse blood. The inoculum was prepared
by harvesting bacterial growth from the plates into phosphate-buffered saline
with a further 1/10 dilution into cooled molten nutrient agar (42 °C). Rats were
kinetics of compound 8 was 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 LCMS/MS analysis and the concentration-time data were analyzed
by non-compartmental methods (WinNonlin, v3.2). 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.
infected with S. pneumoniae 1629 by intrabronchial instillation of 50 ll of the
inoculum via non surgical intratracheal intubation. Animals received
approximately 6log10 cfu/rat. At 48 h post infection, the animals were
euthanized and the lungs excised for the enumeration of viable bacteria.
11. All animal experiment protocols were approved by the Animal Care and Use
Committee at GlaxoSmithKline Pharmaceuticals (PA, USA). The pharmaco-