Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents

Article abstract of DOI:10.1021/ml500069w

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibit

Full text of DOI:10.1021/ml500069w

Letter
pubs.acs.org/acsmedchemlett
Oxabicyclooctane-Linked Novel Bacterial Topoisomerase Inhibitors
as Broad Spectrum Antibacterial Agents
Sheo B. Singh,*,†,⊥ David E. Kaelin,† Jin Wu,† Lynn Miesel,† Christopher M. Tan,† Peter T. Meinke,†
David Olsen,‡ Armando Lagrutta,‡ Prudence Bradley,† Jun Lu,‡ Sangita Patel,‡ Keith W. Rickert,‡
Robert F. Smith,‡ Stephen Soisson,‡ Changqing Wei,§ Hideyuki Fukuda,∥ Ryuta Kishii,∥ Masaya Takei,∥
and Yasumichi Fukuda∥
†Merck Research Laboratories, Kenilworth, New Jersey 07033, United States
‡Merck Research Laboratories, West Point, Pennsylvania 19486, United States
§WuXi AppTec, Shanghai, People’s Republic of China
∥Kyorin Pharmaceutical Co., Ltd., 2399-1, Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
S
* Supporting Information
ABSTRACT: Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents.
Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to
inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the
development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors
(NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution
of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC50 > 18 μM) and
improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase
(IC50 = 1.02 μM) and topo IV (IC50 = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg
doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding
interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the
linker.
KEYWORDS: Antibacterial, broad spectrum, novel bacterial topoisomerase inhibitors, NBTI, gyrase inhibitors,
topoisomerase IV inhibitors, oxabicyclooctane
acterial resistance to current antibiotics continues to
bacterial type II topoisomerase inhibitors (NBTIs) have been
recently described.4 These inhibitors bind to different sites of
gyrase A and ParC and generally do not show cross-resistance
to fluoroquinolones.5−15 The alternative-binding site of these
inhibitors was demonstrated by the X-ray crystal structure of
one of the inhibitors (GSK299423, 3) bound to DNA-bound
gyrase complex of S. aureus.5 Several of the NBTIs (e.g.,
NXL101) entered clinical development but were discontinued
due to hERG binding and associated qTC prolongation.8
NBTIs consist of three structural moieties: A left-hand site
(LHS) aromatic heterocycle, a right-hand side (RHS) aromatic
heterocycle, and both connected by a 8-atom central linker.
1,2
Bincrease leaving many antibiotics ineffective.
This
creates a profound global threat and necessitates the discovery
of new antibiotics with novel modes of action as critical.
Bacterial type II topoisomerases (DNA gyrase and topoisomer-
ase IV) are clinically validated targets represented by a series of
clinical agents with different binding sites.3 Fluoroquinolones
(e.g., ciprofloxacin, 1, Chart 1) are highly successful antibiotics
that bind to gyrase A and topoisomerase IV (ParC) subunits,
whereas the coumarin (e.g., novobiocin; the other most
prominent topo II inhibitor, 2) class of antibiotics binds to
gyrase B and topoisomerase IV (ParE), the catalytic subunits.3
Of all the validated bacterial targets, bacterial type II
topoisomerases have been amenable to inhibition by diverse
structural classes of inhibitors without showing significant
cross-resistance to each other due to their action at alternative
and distinct binding sites.3 Several novel (nonfluoroquinolone)
Received: February 12, 2014
Accepted: March 12, 2014
Published: March 12, 2014
© 2014 American Chemical Society
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Letter
diluted HCl to give 13, which was reacted with vinylmagnesium
bromide to provide alcohol 14. Base treatment afforded
oxabicyclooctane 15. The tosylate was quantitatively exchanged
with acetate to give 16, which was then subjected to base
hydrolysis to afford alcohol 17. The primary alcohol was
oxidized with PDC to afford carboxylic acid 18. The carboxylic
acid was converted to the Boc protected amine (19) by Curtius
reaction. The vinyl group was oxidized with osmium tetraoxide
to give diol 20, which was cleaved by sodium periodate to give
the requisite aldehyde 21.
Chart 1. Chemical Structures of NBTIs
The RHS pyridoxazinecarbaldehyde (Scheme 2) was
prepared by bromination of 2-nitro-3-hydroxypyridine (22) to
a
Scheme 2. Synthesis of RHS Moiety
The hallmark of this linker is the strategic placement of a basic
nitrogen atom at position-7 that shows a salt-bridge interaction
with the Asp83 in the X-ray crystal structure.5 4-Amino-
piperidine (4) and 4-amino-cyclohexyl linkers dominate
NBTIs.5−15 Recently, 3-amino-6-alkyl-pyran linked NBTIs
were reported.16 We embarked on finding alternative linkers
and report herein the discovery of an oxabicyclooctane linker
(e.g., 5−7) with reduced basicity and attenuated hERG activity.
The addition of a hydroxyl group at C-2 (e.g., AM8191) of the
linker reduced hERG activity by more than 30-fold and
improved solubility by over 100-fold with minimum loss of
antibacterial potency and spectrum.
NBTI AM8085 (5) and enantiomers AM8191 (6) and
AM8192 (7) were synthesized via a convergent approach
involving three advanced intermediates representing oxabicy-
clooctane linker, pyridoxazinecarbaldehyde RHS, and 1,5-
naphthyridine LHS moieties. They were then assembled in
two steps to give final products. The synthesis is presented in
Schemes 1−5.
The oxabicyclooctane (Scheme 1) linker was synthesized in
14 steps starting with the reaction of diethyl malonate and ethyl
acrylate to give triethylcarboxycyclohexanone (8), which was
decarboxylated to afford diethylcarboxycyclohexanone (9). The
ketone was protected as its cyclic acetal (10), and the ester
moieties were then reduced with LiAlH4 to furnish the diol
(11) that was subsequently converted to bis tosylate (12). The
ketone group of the tosylate was unmasked by reaction with
a
Reagents: (i) MeONa, MeOH, RT, Br2, 0 °C; (ii) K2CO3,
ethylbromoacetate, acetone, reflux; (iii) Fe, AcOH, 90 °C; (iv)
phenylvinylboronic acid, K2CO3, 1,4-dioxane, water, reflux; (v) O3,
CH2Cl2, MeOH, −78 °C.
produce 6-bromopyridine 23, which was alkylated to produce
ether 24. Reduction of the nitro group at elevated temperatures
produced bromo pyridoxazine 25. Suzuki coupling of phenyl-
vinylboronic acid with 25 yielded E-styrene 26, which was
ozonolyzed to afford desired aldehyde 27.
The synthesis of the LHS 1,5-naphthyridine moiety began by
reaction of ethoxymethyl malonate with 2-methoxy-5-amino-
pyridine (28) to afford 29 (Scheme 3). Heating of 29 in
diphenyl ether produced 8-hydroxy-1,5-naphthyridine 30.
Reaction of 30 with PBr3 afforded bromide 31, which was
hydrolyzed in the presence of base to give the acid 32. Curtius
rearrangement of the acid afforded Boc-protected amine 33.
Boc deprotection afforded the free amine 34, which was
converted to 35 by reaction with nitrosonium tetrafluoroborate.
Cross-coupling of 35 with methylboronic acid produced the
final LHS intermediate 36.
a
Scheme 1. Synthesis of the Linker Moiety
a
Reagents: (i) NaH, THF, 40−45 °C; (ii) NaCl, DMSO, H2O, 160 °C; (iii) ethylene glycol, TsOH, toluene, reflux, Dean−Stark; (iv) LiAlH4, Et2O,
−20 °C; (v) TsCl, pyridine, 0 °C; (vi) 1 N HCl, THF, reflux; (vii) vinylmagnesium bromide, THF, −78 °C; (viii) NaH, DME, 0 °C; (ix) CsOAc,
DMF, 100 °C; (x) K2CO3, MeOH, H2O, RT; (xi) PDC, DMF, 25−40 °C; (xii) (a) DPPA, Et3N, toluene, molecular sieves 4 Å, reflux; (b) t-BuOK,
THF; (xiii) OsO4, NMO, t-BuOH, RT; (xiv) NaIO4, THF, RT.
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ACS Medicinal Chemistry Letters
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a
Scheme 3. Synthesis of LHS Moiety
a
Reagents: (i) ethoxymethylmalonate, EtOH, reflux; (ii) Ph2O, 260 °C; (iii) PBr3, DMF; (iv) NaOH, THF, RT; (v) DPPA, Et3N, t-BuOH, DMF,
100 °C; (vi) TFA, CH2Cl2, −10 °C; (vii) NOBF4, THF, −10 °C-RT; (viii) methylboronic acid, (Ph3P)4Pd, K2CO3, 1,4-dioxane, 100 °C.
Hydroboration of vinyl oxabicyclooctane 19 followed by
Suzuki coupling with 8-bromo-1,5-naphthyridine (35) led to 37
(Scheme 4). Boc-deprotection followed by reductive amination
42, which were reductively aminated with aldehyde 27 to afford
AM8191 (6) and AM8192 (7), respectively.
The hydrochloride salts of oxabicyclooctane-linked NBTIs
5−7 showed broad-spectrum antibacterial activities (Table 1)
a
Scheme 4. Synthesis of AM8085
Table 1. Antibacterial, Gyrase, and hERG Activities of
a
NBTIs
AM8085
AM8191
AM8192
Bacterial Strains
S. aureus MSSA
S. aureus MRSA
S. pneumoniae
E. faecalis
MIC, μg/mL
0.016
0.008
0.064
1
0.02
0.06
0.05
0.5
0.06
0.5
0.25
1
E. faecium VRE
H. influenzae
E. coli
0.5
0.5
2
1
0.5
NT
4
1
2
a
Reagents: (i) 9-BBN, THF; (ii) 35, Pd(PPh3)4, K3PO4, EtOH/H2O;
A. baumannii
M. catarrhalis
P. aeruginosa
0.5
0.5
1
(iii) TFA, CH2Cl2; (iv) 27, NaBH(OAc)3, DMF, AcOH, 2−6, RT.
NT
0.015
8
NT
8
4
of 38 using pyridoxazinecarbaldehyde 27 afforded AM8085 (5),
which was converted to the corresponding hydrochloride salt
by treatment with HCl in dioxane.
Aldol reaction of 36 with aldehyde 21 afforded an
enantiomeric mixture of alcohols 39 and 40 (Scheme 5).
b
S. aureus quinS
0.016
0.064
IC50, μM
0.22
4.4
0.06
0.25
0.06
0.5
b
S. aureus quinR
Bacterial Enzymes
S. aureus gyrase
1.02
10.4
0.78
0.5
NT
NT
NT
NT
S. aureus topo IV
E. coli gyrase
0.18
0.2
a
Scheme 5. Synthesis of AM8191 and AM8192
E. coli topo IV
Off target
IC50, μM
0.6
hERG patch express (CHO cell)
18
NT
a
Linezolid and levofloxacin were used as controls for the MIC
measurements using the micro broth dilution or agar based methods,
which yielded the MIC ranges reported by the Clinical and Laboratory
Standards Institute (CLSI M7-A8). AM8191 and AM8192 did not
show any alteration of the S. aureus MIC in the presence of 50%
human serum or 500 μg/mL DNA. AM8085 showed a 4- and 2-fold
increase in the MIC under the same test conditions, respectively. NT
b
indicates not tested. For comparison, the MIC values of levofloxacin
was >64-fold higher with the quinolone resistant (quinR) as compared
to the sensitive (quinS) strain, (>16 vs 0.25 μg/mL).
a
Reagents: (i) LDA, THF, −78 °C, 21, chiral SFC separation; (ii)
against most ESKAPE pathogens, as well as MRSA and Gram-
negative pathogens, A. baumannii and E. coli. They showed
weaker activities against P. aeruginosa. AM8191, the S
enantiomer at C-2 of the linker was slightly more potent
than the R enantiomer AM8192. These compounds retained
activity against quinolone-resistant strains of S. aureus and S.
pneumoniae. S. aureus MIC values were not significantly altered
in the presence of 50% human serum or sheared DNA (500
μg/mL) indicating that they did not bind significantly to serum
TFA, CH2Cl2; (iii) 27, NaBH(OAc)3, DMF, AcOH, 2−6, RT.
Chiral SFC separation provided enantiomerically pure 39
(faster eluting) and 40. The absolute configuration of the chiral
center was elucidated independently by calculated and
experimental vibrational circular dichroism (VCD) and Mosher
methods. The Boc-group of the two pure enantiomers 39 (2S)
and 40 (2R) were deprotected separately to give amines 41 and
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or DNA. AM8085 and AM8191 showed potent activity against
S. aureus and E. coli gyrase with IC50 values of 1.0 μM or less.
Both compounds showed better activity against E. coli topo IV
(IC50 0.2 and 0.5 μM) compared to S. aureus topo IV (4.4 and
10.4 μM) when compared to respective gyrase IC50s (Table 1).
Macromolecular labeling studies demonstrated that AM8085
selectively inhibited DNA synthesis over RNA, protein, cell
wall, and lipid (Figure 1).17 AM8191 exhibited >3 log10
AM8085 were less than 2%. All three compounds showed
good (30−60 min) stability in human, mouse, and dog liver
microsomal incubations. To assess for in vivo efficacy, all
compounds were evaluated in a S. aureus murine survival model
of bacteremia. In this model, AM8085 showed ED50 values of
5.5 mg/kg by intravenous (i.v.) dosing and 17 mg/kg by oral
(p.o.) dosing. AM8191 showed ED50 values of 2.5 mg/kg (i.v.)
and 2.2 mg/kg (p.o.). AM8192 showed good in vivo efficacy as
well in this model, providing ED50 values of 3.5 mg/kg (i.v.)
and 4.5 mg/kg (p.o.). AM8191 also showed efficacy in a similar
E. coli murine survival model exhibiting ED50 values of 23 mg/
kg (i.v.) and 85 mg/kg (p.o.). Mouse PK data (Table 2)
generally supported the observed efficacy.
Table 2. Mouse and Dog Pharmacokinetics Parameters of
NBTIs
AM8085
AM8191
AM8192
a
Mouse
AUCn (μM·h/mpk)
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
0.98
34.6
2.22
2.24
71.6
0.76
43.3
1.61
1.70
30.1
0.68
48.7
4.94
2.15
25.3
%F
Figure 1. Macromolecular synthesis inhibition of AM8085 in S. aureus.
b
Dog
AUCn (μM·h/mpk)
Clp (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
0.43
39.8
5.36
1.85
>100
3.57
9.16
2.60
4.49
40.4
NT
NT
NT
NT
NT
reduction of colony forming units (CFU) of S. aureus in an
in vitro time kill kinetic experiment (>105 CFU/mL inoculum)
at >4× of MIC (0.06−0.12 μg/mL) confirming bactericidal
effect of this compound (Figure 2).
%F
a
C57BL/6 mouse (n = 2) dosed at 1 mg/kg (i.v.) and 2 mg/kg (p.o.).
Beagle dog (n = 2) dosed at 1 mg/kg (i.v.) and 2 mg/kg (p.o.).
b
AM8191 was cocrystallized with the DNA−gyrase complex
(Figure 3) in a manner similar to that reported by Bax et al.5
Figure 2. Time kill kinetics of AM8191 in S. aureus. The MIC in the
experiment was 0.06−0.12 μg/mL.
Significant attenuation of undesirable hERG activity is critical
to the future development of these compounds as antibacterial
agents. We evaluated hERG activity in a functional automated
patch clamp assay (see Supporting Information for Methods).
In this assay, AM8085 showed an IC50 of 0.6 μM. The 2S-
hydroxy group of AM8191 improved the polarity and
attenuated the hERG activity (IC50 = 18 μM). However,
more than an order of magnitude attenuation of hERG activity
may be required for a clinical development compound.
AM8191 HCl salt showed excellent (>50 mg/mL)
solubilities in various acceptable intravenous and oral vehicles.
It showed PBS solubility of ∼154 μM (pH 7) and ∼200 μM
(pH 2), whereas AM8085 showed significantly lower PBS
solubility of <0.89 μM (pH 7) and 20.3 μM (pH 2). AM8191
and AM8192 showed >25% free fractions in human, dog, or
mouse plasma, whereas corresponding free fractions for
Figure 3. The 2.7 Å cocrystal structure of AM8191 bound to a DNA−
gyrase complex. Gyrases A and B are shown in green and cyan.
AM8191 is colored in yellow and DNA duplex in magenta. Upper left:
the electron density map of AM8191.
The binding mode of AM8191 resembles that of GSK299423.5
The 1,5-naphthyridine (LHS) ring binds between the two
central base pairs of the stretched DNA through van der Waals
interactions between the DNA base pairs and the aromatic 1,5-
naphthyridine ring. The pyridoxazine (RHS) moiety penetrates
into the hydrophobic pocket between the two gyrase A subunits
at the bottom portion of the complex. van der Waals
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Letter
(4) Mayer, C.; Janin, Y. L. Non-quinolone inhibitors of bacterial type
IIA topoisomerases: a feat of bioisosterism. Chem. Rev. 2014, 114,
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interactions of Ala68, Val 71, Met75, and Met121 with the
pyridine ring further stabilize the ternary complex. The linker
moiety of AM8191 is located in the donut-hole and generally
exposed to solvent other than the position-7 basic amine, which
showed a polar interaction with the Asp83 side chain and
accounts at least partially for the high affinity of the compound.
No interaction partners were identified for the hydroxyl group
at C-2 of the linker, indicative of no direct binding contribution.
This finding is consistent with the observation that the two
enantiomers demonstrated identical biological activity.
In summary, this letter describes the discovery, synthesis, and
characterization of new NBTIs with an oxabicyclooctane linker
and attenuated hERG activity with potent broad-spectrum
antibacterial activity, including efficacy in mouse models of S.
aureus and E. coli infections. Hydroxylation of the linker chain
provided compounds with improved physical properties leading
to improved oral efficacy. X-ray crystallographic studies
provided binding insights consistent with amino-piperidine
linked NBTIs.
ASSOCIATED CONTENT
* Supporting Information
■
S
(9) Gomez, L.; Hack, M. D.; Wu, J.; Wiener, J. J.; Venkatesan, H.;
Santillan, A., Jr.; Pippel, D. J.; Mani, N.; Morrow, B. J.; Motley, S. T.;
Shaw, K. J.; Wolin, R.; Grice, C. A.; Jones, T. K. Novel pyrazole
derivatives as potent inhibitors of type II topoisomerases. Part 1:
synthesis and preliminary SAR analysis. Bioorg. Med. Chem. Lett. 2007,
17, 2723−2727.
Experimental procedures of synthesis of 5−42, Mosher ester
analysis, materials and methods for MIC, systemic infection
model, and hERG assay. This material is available free of charge
via the Internet at http://pubs.acs.org.
(10) Miles, T. J.; Axten, J. M.; Barfoot, C.; Brooks, G.; Brown, P.;
Chen, D.; Dabbs, S.; Davies, D. T.; Downie, D. L.; Eyrisch, S.;
Gallagher, T.; Giordano, I.; Gwynn, M. N.; Hennessy, A.; Hoover, J.;
Huang, J.; Jones, G.; Markwell, R.; Miller, W. H.; Minthorn, E. A.;
Rittenhouse, S.; Seefeld, M.; Pearson, N. Novel amino-piperidines as
potent antibacterials targeting bacterial type IIA topoisomerases.
Bioorg. Med. Chem. Lett. 2011, 21, 7489−7495.
AUTHOR INFORMATION
Corresponding Author
*(S.B.S.) E-mail: sheo.singh.215@gmail.com.
■
Present Address
⊥(S.B.S.) Pharma Consulting, Edison, New Jersey 08820,
United States.
(11) 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, J.; Huang, J.; Jones, G.;
Markwell, R.; Rittenhouse, S.; Xiang, H.; Pearson, N. Novel
cyclohexyl-amides as potent antibacterials targeting bacterial type IIA
topoisomerases. Bioorg. Med. Chem. Lett. 2011, 21, 7483−7488.
(12) Mitton-Fry, M. J.; Brickner, S. J.; Hamel, J. C.; Brennan, L.;
Casavant, J. M.; Chen, M.; Chen, T.; Ding, X.; Driscoll, J.; Hardink, J.;
Hoang, T.; Hua, E.; Huband, M. D.; Maloney, M.; Marfat, A.;
McCurdy, S. P.; McLeod, D.; Plotkin, M.; Reilly, U.; Robinson, S.;
Schafer, J.; Shepard, R. M.; Smith, J. F.; Stone, G. G.; Subramanyam,
C.; Yoon, K.; Yuan, W.; Zaniewski, R. P.; Zook, C. Novel quinoline
derivatives as inhibitors of bacterial DNA gyrase and topoisomerase
IV. Bioorg. Med. Chem. Lett. 2013, 23, 2955−2961.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors acknowledge technical assistance from WuXi chem-
istry, DMPK, and pharmaceutical sciences teams. The authors
thank Drs. Chris Pillar and Dean Shinabarger of Micromyx for
providing a portion of the MIC, MML, and Kill kinetics data.
(13) 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.; 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.
Bioorg. Med. Chem. Lett. 2013, 23, 5437−5441.
(14) Reck, F.; Alm, R.; Brassil, P.; Newman, J.; Dejonge, B.;
Eyermann, C. J.; Breault, G.; Breen, J.; Comita-Prevoir, J.; Cronin, M.;
Davis, H.; Ehmann, D.; Galullo, V.; Geng, B.; Grebe, T.; Morningstar,
M.; Walker, P.; Hayter, B.; Fisher, S. Novel N-linked aminopiperidine
inhibitors of bacterial topoisomerase type II: broad-spectrum
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