Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6)

Article abstract of DOI:10.1016/j.bmcl.2015.06.057

Abstract Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED₅₀ 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH₂ is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC₅₀ >30 μM) profile.

Full text of DOI:10.1016/j.bmcl.2015.06.057

Bioorganic & Medicinal Chemistry Letters 25 (2015) 3636–3643
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure activity relationship of pyridoxazinone substituted RHS
analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel
bacterial topoisomerase inhibitors as broad-spectrum antibacterial
agents (Part-6)
a
a
a
a
a
Sheo B. Singh a, , David E. Kaelin , Jin Wu , Lynn Miesel , Christopher M. Tan , Peter T. Meinke ,
David B. Olsen b, Armando Lagrutta b, Changqing Wei c, Yonggang Liao c, Xuanjia Peng c, Xiu Wang c,
Hideyuki Fukuda d, Ryuta Kishii d, Masaya Takei d, Masanobu Yajima d, Taku Shibue d, Takeshi Shibata d,
⇑
Kohei Ohata d, Akinori Nishimura d, Yasumichi Fukuda d,
⇑
a Merck Research Laboratories, Kenilworth, NJ 07033, United States
b Merck Research Laboratories, West Point, PA 19486, United States
c WuXi AppTec, Shanghai, People’s Republic of China
d Kyorin Pharmaceutical Co., Ltd, 1848 Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoi-
somerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than
quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to
combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is
described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and
C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described.
In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-
8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone
moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral effi-
cacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of
Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone
unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker posi-
tion-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained
potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide
NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-
Received 15 May 2015
Revised 12 June 2015
Accepted 15 June 2015
Available online 19 June 2015
Keywords:
Antibacterial
Broad-spectrum
Bacterial topoisomerase inhibitors
Gyrase inhibitors
ParC inhibitors
7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30
profile.
lM)
Ó 2015 Elsevier Ltd. All rights reserved.
Drug resistance bacteria continue to rise and pace of the discov-
ery of new antibacterial agents to combat such bacteria continue to
recede. Recently described bacterial type II topoisomerase inhibi-
tors (NBTIs) are the newest classes of antibacterial agents that
show broad-spectrum of activity without cross-resistance to
known classes of antibiotics and thus has potential to address
unmet need.1–11 The lack of cross-resistance is inherent to their
binding mode to DNA gyrase and topoisomerase IV at a site
different from fluoroquinolones (e.g., ciprofloxacin 1, Fig. 1) and
other known antibiotics. GSK2140944 (2)12 is under phase II
human clinical development for AbSSTI and Gonorrheal diseases
(clinicaltrials.gov) and is the most advanced of the NBTIs. We have
been studying NBTI class of DNA gyrase inhibitors and recently
reported several oxabicyclooctane linked 1,5-naphthyridinyl-pyri-
doxazone NBTIs including AM-8085 (3) and AM-8191 (4)13 as well
as several hydroxy tricyclic series of NBTIs.14 Development of
NBTIs in general and this series in particular has been hampered
due to existence of significant hERG K+ channel activity.
⇑
Corresponding authors at present address: SBS Pharma Consulting LLC, Edison,
NJ 08820, United States (S.B.S.).
AM-8085 and AM-8191 series of oxabicyclooctane linked NBTIs
show broad-spectrum activity covering Gram-positive and Gram-
E-mail addresses: sheo.singh.215@gmail.com (S.B. Singh), yasumichi.fukuda@
gmail.com (Y. Fukuda).
http://dx.doi.org/10.1016/j.bmcl.2015.06.057
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
3637
Figure 1. Chemical structures of NBTIs.
negative pathogens and in vivo efficacy with modest attenuation of
hERG signal.13 The hydroxy tricyclic series of NBTIs showed signif-
icant attenuation of hERG signal unfortunately at the expense of
potency and spectrum against Gram-negative pathogens.14 In
order to maintain Gram-negative activity and spectrum, we
focused our SAR study on the both series. Recently we reported
SAR around the LHS 1,5-naphthyridine15,16 and hydroxy tricyclic
1,5-naphthyridinone,14 as well as a survey of RHS moieties applica-
ble to the hydroxy tricyclic series.17 This Letter focuses on the syn-
thesis and SAR of pyridoxazinone-substituted oxabicyclooctane
linked NBTIs along with the defining an understanding of the role
of the NH at position-7 of the linker.
Alkylation of 2-bromo-5-hydroxy-6-amino-pyridine (71) fol-
lowed by reflux produced substituted pyridoxazinone 72. Suzuki
coupling gave 73 and ozonolysis yielded produced 74, which upon
reductive amination afforded the geminal dimethyl NBTI (16) and a
racemic NBTI 17. The chiral HPLC separation of the racemic 17 fur-
nished both enantiomers (+)-17 and (À)-17 (Scheme 8).
Hydrolysis, decarboxylation and ozonolysis of 75 produced alde-
hyde 76 which was reductively aminated with 30a to afford race-
mic 15 which was separated by chiral HPLC to give enantiomers
(+)-15 and (À)-15 (Scheme 8). Methylation of the amide nitrogen
of 77 gave 78, which under standard operations gave NBTI 18.
The amide NBTIs 21, 22, 25 were prepared by EDC couplings of
the acid 7917 with amine 30a, N-methyl-30a and 30b, respectively
(Scheme 9). The linker at position L7 substituted NBTIs 19 and 20
were synthesized by reductive methylation with paraformalde-
hyde and reaction with chloroformate, respectively. All final prod-
ucts were well characterized by NMR and MS. The purity of all
compounds was greater than 95% as assessed by 1H NMR and
HPLC.
The X-ray crystal structure of AM-8191 bound to DNA gyrase-
DNA complex (GyrB27-A56 protein construct and 12–37 bp DNA
duplex, pdb code; 4plb) showed that the 1,5-naphthyridine ring
was sandwiched between two base pairs of the DNA at the top
and RHS moiety pyridoxazinone penetrated into the hydrophobic
pocket created by the two Gyrase A subunits at the bottom portion
of the complex.13 The pyridoxazinone binding was stabilized by
van der Walls interactions of the pyridine ring with Ala68, Val71,
Met75 and Met121. While specific structure-guided modifications
have been not successful in this program nevertheless it was envis-
aged that introduction of various hydrophobic groups on pyridox-
azinone could provide NBTIs with gain of function likely from
additional hydrophobic interactions. Understanding SAR, balancing
potency, spectrum and hERG activity was the goal of this study.
Carefully selected small set of hydrophobic groups was used for
substitution of the hydrogen at the C-3 of AM-8085/AM-8191 to
give chloro (5), fluoro (6, 23), and methyl (8) substituted NBTIs.
C-3 methoxy compounds (7, 24) were prepared to confirm the
minimum tolerance of polarities. With a similar rationale, C-4
methyl (9) and C-3,4-disubstituted analogs with small changes in
the size and shapes of alkyl groups (10–13) at C-3 as well as a C-
3 fluoro group (14) while maintaining a methyl at C-4 were pre-
pared. Likewise to understand substitutions at C-7, enantiomers
of mono-methyl analogs (15), a geminal dimethyl analog (16) were
prepared. In addition, to test whether a polar group was tolerated
at C-7 of pyridoxazinone, both enantiomers of the 7-methyl-7-
ethoxy carbonyl (17) were also prepared. The NH group of the pyri-
doxazinone was methylated (18) to test the role of the NH group.
The X-ray crystal structure suggested that the basic nitrogen at
Chemistry. The chemical synthesis of right-hand-side (RHS)
pyridoxazinone substituted NBTIs (5–25, Tables 1–3) are described
in Schemes 1–9. Chlorination of the intermediate 2613 gave 27
which was transformed to E-styrene 28 by Suzuki coupling and
was oxidized to produce 29. Standard reductive amination of with
30a13 afforded the chloro NBTI 5 (Scheme 1). Fluorination of 31
gave 3,5-difluoro pyridine 32, which was acylated to give N-acyl
derivative 33 (Scheme 2). Refluxing of 33 under base catalysis pro-
duced the cyclic acid 35 (via methyl ester 34), which was reduced
to alcohol 36 and oxidized with MnO2 to afford fluoro-aldehyde 37.
The aldehyde 37 was reductively aminated with amines 30a and
30b13 to furnish final fluoro NBTIs
6 and 23, respectively
(Scheme 2).
The nitration of 3-bromo-5-hydroxy-pyridine (38) produced 6-
nitro-pyridine (39) (Scheme 3). Halogen displacement with
methoxide afforded methoxy pyridine (40). Bromination gave 41
and alkylation produced 42 followed by reduction and cyclization
gave 43. Standard protocol using Suzuki coupling (44), oxidation
(45) and reductive amination with appropriate amines (30a, 30b)
afforded NBTIs 7 and 24 (Scheme 3). The 3-methyl substituted
NBTI 8 was synthesized from the aldehyde 4613 via reduction
(47), bromination (48), Suzuki coupling (49) and oxidation (50) fol-
lowed by reductive amination with 30a (Scheme 4). 4-Methyl
NBTIs 9, 10, and 14 were prepared starting from substituted pyri-
dine (51) via N-oxidation (52) followed by acetylation (53), hydrol-
ysis (54), nitration (55), bromination (56) and standard stepwise
operations (57–60) as described in Scheme 5.
The reduction of aldehyde 61 (prepared in Scheme 5) gave alco-
hol 62 which was brominated (63) followed by Suzuki coupling gave
vinyl analog 64. Hydrogenation of 64 to 65 and oxidation to 66 fol-
lowed by reductive amination with 30a gave 3-ethyl-4-methyl
NBTI 11 (Scheme 6). Oxidation of 64 gave 67 followed by reductive
amination produced 4-vinyl-4-methyl NBTI 13 (Scheme 6). Suzuki
coupling of 63 gave 3-isopropenyl-4-methyl pyridine (68), hydro-
genated to afford (69), oxidized to give aldehyde 70 and reductive
amination afforded isopropyl NBTI 12 (Scheme 7).

3638
S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
Table 1
Antibacterial activity and spectrum pyridoxazinone substituted AM-8085 oxabicyclooctane linked NBTIs (MIC,
l
g/mL)a
List
RHS
SaS
Sp
Ef
Ec
Ab
Pa
hERG bind /PX hERG (IC50
2.0/0.6
,
lM)
clogD7.4
3
0.02
0.13
0.5
1
0.5
4
3.2
5
6
7
8
0.02
0.02
4
0.25
0.13
8
1
1
8
1
2
2
8
2
1
2
8
8
8
5.8
1.3
3.98
3.41
3.58
3.56
0.5
8
0.06
1
0.5
1.0/4.3
0.3/2.0
9
0.50
1.06
2
4.0
8.0
32
32
8
8.0
2
8
4
32
16
32
32
32
32
3.61
4
10
11
12
13
14
2
4.0
32
32
32
32
0.5/4.6
0.7
32
32
32
32
32
32
32
32
4.53
4.95
4.37
3.91
8
1.2
0.25
0.5
0.5
4
0.9
(+)-15
(À)-15
0.25
0.06
0.25
0.25
2
2
2
2
2
1
2
2
0.8
1.8
3.61
3.61
16
0.25
16
1
2
2
2
2
3.0
2.4
4.13
3.96
(+)-17
16
16
16
16
16
(À)-17
8
8
8
8
8
8
3.6
3.96

S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
3639
Table 1 (continued)
List
RHS
SaS
2
Sp
2
Ef
Ec
Ab
16
Pa
16
hERG bind /PX hERG (IC50
0.8
,
lM)
clogD7.4
18
16
16
2.59
a
SaS (Staphylococcus aureus Smith, MIC against MRSA OIT971 similar), Sp (Streptococcus pneumoniae IID553), Ef (Enterococcus faecium VanA, VRE), Ec (Escherichia coli ATCC
25922), Ab (Acinetobacter baumannii IID876), Pa (Pseudomonas aeruginosa PAO1), hERG binding (MK499 binding), PX hERG (Patch express, CHO cell),13 Linezolid and
levofloxacin were used as controls for the MIC measurements using the micro broth dilution or agar based methods which yielded the reported the MIC ranges reported by
the Clinical and Laboratory Standards Institute (CLSI M7-A8). AM8085 showed a 4- and 2-fold increase in the MIC under the same test conditions, respectively. All compounds
experienced less than 4 fold MIC shifts against quinolone sensitive and resistant strains of S. aureus MS5935 quinS vs MS5935 quinR). For comparison, the MIC values of
levofloxacin was >64 fold higher with the quinolone resistant (S. aureus MS5935 quinR) as compared to the sensitive (S. aureus MS5935 quinS) strain, (>16 vs 0.25
(not tested). The data for compounds 3 is taken from Singh et al.13
lg/mL), NT
Scheme 1. Synthesis of NBTI 5. Reagents and conditions: (i) Cl2, DMF, (ii) K2CO3, 1,4-dioxane–water, phenylvinylboronic acid, Pd(PPh3)4, (iii) O3, CH2Cl2, MeOH, À71 °C, DMS,
(iv) 30a, DMF, AcOH, NaBH(OAc)3, rt.
Scheme 2. Synthesis of NBTI 6 and 23. Reagents and conditions: (i) F–TEDA–BF4, CH3CN, (ii) ClCOCH2OAc, pyridine, (iii) K2CO3, MeOH, reflux, (iv) NaOH, 1,4-dioxane–water,
(v) (a) i-butyl chloroformate, Et3N, DMF, (b) NaBH4, H2O, (vi) MnO2, THF, (vii) 30a, DMF, AcOH, NaBH(OAc)3, rt, (viii) 30b, DMF, AcOH, NaBH(OAc)3, rt.
Scheme 3. Synthesis of NBTI 7 and 24. Reagents and conditions: (i) HNO3, H2SO4, (ii) NaOMe, Cu, CH3OH, microwave, 150 °C, (iii) benzyltrimethylammonium tribromide,
CH2Cl2, (iv) K2CO3, acetone, BrCH2CO2Et, reflux, (v) Fe, AcOH, 90 °C, (vi) K2CO3, 1,4-dioxane–water, phenylvinylboronic acid, Pd(PPH3)4, (vii) O3, CH2Cl2, MeOH, À71 °C, DMS,
(viii) 30a, DMF, AcOH, NaBH(OAc)3, rt, (ix) 30b, DMF, AcOH, NaBH(OAc)3, rt.
Scheme 4. Synthesis of NBTI 8. Reagents and conditions: (i) NaBH4, MeOH, (ii) Br2, DMF, (iii) MeB(OH)2, 1,4-dioxane, Pd(dppf)2Cl2, K2CO3, (iv) MnO2, THF–CH2Cl2 (1:1), (v)
30a, DMF, AcOH, NaBH(OAc)3, rt.

3640
S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
Scheme 5. Synthesis of NBTI 9, 10, 14. Reagents and conditions: (i) H2O2, AcOH, reflux, (ii) Ac2O, reflux, (iii) NaOH, EtOH, (iv) HNO3, H2SO4, (v) NaOMe, MeOH, Br2, ice bath,
(vi) K2CO3, acetone, BrCH2CO2Et, reflux, (vii) Fe, CaCl2, EtOH, reflux, (viii) K2CO3, 1,4-dioxane, phenylvinylboronic acid, Pd(PPh3)4, (ix) O3, CH2Cl2, MeOH, À71 °C, DMS, (xi) 30a,
DMF, AcOH, NaBH(OAc)3, rt.
Scheme 6. Synthesis of NBTI 11, 13. Reagents and conditions: (i) NaBH4, MeOH, (ii) Br2, DMF, ice bath, (iii) CH2@CHBF3K, EtOH, Et3N, reflux, (iv) Pd/C, MeOH, H2, (v) MnO2,
THF–CH2Cl2 (1:1), reflux, (vi) 30a, DMF, AcOH, NaBH(OAc)3, rt.
Scheme 7. Synthesis of NBTI 12. Reagents and conditions: (i) 2-isopropenyl-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane, Pd(dppf)2Cl2, Na2CO3, 1,4-dioxane, water, (ii) Pd/C,
MeOH, H2, (iii) MnO2, THF–CH2Cl2 (1:1), reflux, (iv) 30a, DMF, AcOH, NaBH(OAc)3, rt.
Me
Me
OH
O
R
O
R
(i)
(ii)
(iii)
Br
N
71
NH2
Br
N
72
N
H
O
Ph
N
73
N
H
O
Me
O
R
16
(R = Me)
(iv)
(v)
(+)-17 (R = CO2Et)
17
OHC
Ph
N
74
N
H
O
(+)-17 (R = CO2Et)
(-)- (R = CO2Et)
Me
Me
CO2Et
(vi),(iii)
O
H
O
(+)-15
(-)-15
(iv)
(v)
(+)-15
OHC
N
76
N
O
N
75
N
H
O
H
Scheme 8. Synthesis of NBTI 15–17. Reagents and conditions: (i) Br–C(Me,R)CO2Et, K2CO3, acetone, reflux, (ii) K2CO3, 1,4-dioxane–water, phenylvinylboronic acid, Pd(PPh3)4,
(iii) O3, CH2Cl2, MeOH, À71 °C, DMS, (iv) 30a, DMF, AcOH, NaBH(OAc)3, rt, (v) Chiralpak IA, hexane–EtOH (1:4) +0.1% Et2NH, (vi) K2CO3, 1,4-dioxane–water, reflux.
Scheme 9. Synthesis of NBTI 18, 21, 22, 25. Reagents and conditions: (i) MeI, benzyltriethylammonium chloride, K2CO3, CH3CN, (ii) 30a, DMF, AcOH, NaBH(OAc)3, rt, (iii) 30a,
EDC, HOBT, DIEA, CH2Cl2, (iv) N–Me–30a, EDC, HOBT, DIEA, CH2Cl2, (v) 30b, EDC, HOBT, DIEA, CH2Cl2.

S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
3641
Table 2
Antibacterial activity and spectrum of linker (L7) and amide substituted AM-8085 oxabicyclooctane linked NBTIs (MIC,
l
g/mL)a
List
RHS
SaS
Sp
Ef
Ec
Ab
Pa
hERG bind/PX hERG (IC50, uM)
clogD7.4
3
0.02
0.13
0.5
1
0.5
4
2.1/0.6
3.2
19
20
21
22
0.13
16
2
8
16
64
8
8
32
64
8
1.2
3.67
3.92
3.67
64
0.5
4
64
64
6.1
0.02
0.03
1
8
>60.0/>30.0
4
4
4
4
>60.0/>30.0
2.97
a
See footnote of Table 1. The data for compound 3 is taken from Singh et al.13
position-7 (L7) of the linker showed a high affinity polar interac-
tion with the Asp83 side chain. In order to test the role of this
interaction for potency and spectrum we prepared several N-alky-
lated analogs (19 and 20) and amide analogs (21, 22, 25).
NBTIs 5–25 were tested as a freebase or hydrochloride salt(s)
for their potency (MIC) and spectrum against selected Gram-posi-
tive and Gram-negative bacterial strains (Tables 1–3). The activi-
ties of 3 and 4 have been listed for comparison. In our hands,
freebase and the corresponding hydrochloride salt did not show
in vitro MIC activity difference. For in vivo activity, only hydrochlo-
ride salts were used. The Gram-positive strains included
S. pneumoniae activity (MIC 4–32
activity (MIC 32
isopropyl group (12) showed no activity (MIC 32
l
g/mL), and highly diminished
l
g/mL) against other strains (Table 1). The bulky
lg/mL). NBTIs
with mono (15) and dimethyl (16) substitutions at C-7 of pyridox-
azinone exhibited reduction of the potency against S. aureus but
showed flat balanced potency against all other strains and main-
tained the spectrum. No significant stereochemical preference
was observed. NBTIs with C-7 ethyl carboxylate (17) substitution
showed significant diminution of the activity regardless of the
stereochemistry. NH of pyridoxazinone (18) appears to be critical
for the potent antibacterial activities (Table 1).
Staphylococcus
aureus,
methicillin
resistant
S.
aureus,
The substitution of the hydrogen of basic NH at the position-7
of the linker (L7) with a methyl group (19) reduced activity
against all strains by 6–16 folds (Table 2). On the other hand sub-
stitution with a carboxy methyl group (20) significantly dimin-
Streptococcus pneumoniae, Enterococcus faecium, and Gram-nega-
tive strains Escherichia coli, Acinetobacter baumannii, and
Pseudomonas aeruginosa. Since no MIC difference between S. aureus
Smith and MRSA strains was observed only S. aureus Smith MIC
data has been reported in Tables 1–3.
The C-3 chloro (5) and fluoro (6) pyridoxazinone analogs
showed similar potency and spectrum as AM-8085 (Table 1) unfor-
tunately without significantly impacting hERG signal. The methyl
substitution at C-3 (8) had marginal impact on the activity with
the exception of S. pneumoniae, which showed 8-fold reduction
of the MIC. On the other hand, a C-4 methyl (9) substitution had
negative effect on the potency and spectrum (Table 1). Even a mod-
estly polar group such as a methoxy group (7) was not tolerated at
C-3. These observations were consistent with the observed results
ished activity (MIC 16–32
with or without NH (21 and 22) retained potent S. aureus activity
(MIC 0.02–0.03 g/mL) while maintaining spectrum with a 2–4
lg/mL). The amide linked compounds
l
folds loss of the potency against other strains. These data suggest
that a hydrogen-bond donor basic NH appears to be important for
potent S. aureus cellular activity. However, when the position-7
nitrogen was attached to a keto group (linker position-8) forming
an amide, the requirement of the hydrogen-bond donor group for
the cellular activity was not essential particularly for S. aureus
activity. Both NH and N-methyl amides and the parent 3 exhibited
identical potency against S. aureus and maintained spectrum. This
would suggest that basicity of the nitrogen or free NH at position-
7 of the linker is not essential for the cellular activity in S. aureus
for the amides not only in AM8085 series but also tricyclic
series.17 Whether this is due to altered target binding or altered
cellular accumulation of 21 and 22 need further exploration. The
amides (21 and 22) showed significant attenuation of the hERG
of C-3 pyridoxazinone substituted hydroxy tricyclic series.17
methyl substitution at C-4 with a methyl at C-3 (10) lost some
potency but maintained susceptibility against most strains except
S. pneumoniae (MIC 8 lg/mL) and P. aeruginosa (MIC 16 lg/mL).
NBTIs with a slightly larger alkyl groups (e.g., ethyl, 11, and vinyl,
13) and a fluoro group (14) at C-3 along with a methyl at C-4
A
showed good S. aureus activity (MIC 0.5–2
lg/mL), modest to weak
binding (IC50 >60 lM) and functional (IC50 >30 lM) activities.

3642
S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
Table 3
Antibacterial activity and spectrum of pyridoxazinone substituted AM-8191 oxabicyclooctane linked NBTIs (MIC,
l
g/mL)a
List
RHS
SaS
Sp
Ef
Ec
Ab
Pa
hERG bind/PX hERG (IC50
,
l
M)
clogD7.4
4
0.02
0.05
0.5
2
0.5
8
26.0/18.0
1.9
23
24
0.03
0.25
2
4
1
16
16
2.4
NT
2.13
2.31
16
16
16
16
16
25
0.03
2
2
16
2
32
NT
2.4
a
See footnote of Table 1. The data for compound 4 was taken from Singh et al.13
Table 4
and hydrogen donating properties of NH of the linker at position-7
is important for the potency and spectrum but compounds in
which NH is tied in the form of an amide bond neither basicity
nor NH is critical for the potency and spectrum. Importantly,
amides showed significant improvement of the functional hERG
activity.
In vivo activity of NBTI’s in S. aureus murine survival model of bacteremia by
intravenous (iv) and oral (po) dosinga
List
iv (ED50, mpk)
po (ED50, mpk)
3
4
6
5.5
2.5
2.5
14
17
2.2
3.9
28
(À)-15
Acknowledgment
23
3.2
6.7
a
Dosing vehicle: iv [(2 mg/mL in EtOH/PEG400/water (10:25:65), clear solution],
The authors thank technical assistance from WuXi AppTec tech-
nical staff for completion of some of the experiments and com-
pound synthesis.
po [(tween 80:0.5% methocel (10:90)].
The C-3 fluoro (23) and C-3 methoxy (24) and the amide 25 of
AM-8191 series showed similar effect on the corresponding MIC
values as observed for AM-8085 series and thus confirmed cross-
series SAR (Table 3).
References and notes
1. Bax, B. D.; Chan, P. F.; Eggleston, D. S.; Fosberry, A.; Gentry, D. R.; Gorrec, F.;
Giordano, I.; Hann, M. M.; Hennessy, A.; Hibbs, M.; Huang, J.; Jones, E.; Jones, J.;
Brown, K. K.; Lewis, C. J.; May, E. W.; Saunders, M. R.; Singh, O.; Spitzfaden, C.
E.; Shen, C.; Shillings, A.; Theobald, A. J.; Wohlkonig, A.; Pearson, N. D.; Gwynn,
M. N. Nature 2010, 466, 935.
The hydrochloride salts of 6, (À)-15, and 23 representing C-3
fluoro analogs of AM-8085 and AM-8191 and the C-7 methyl ana-
log of AM-8085 with potent S. aureus MIC (0.02–0.06 lg/mL) were
tested in the murine bacteremia survival model of S. aureus infec-
tion.13 Their efficacy was compared with the reported efficacy of
AM-8085 and AM-8191 (Table 4). 50 mg/kg was used as the
highest starting dose for all compounds. The fluoro analog (6) of
AM-8085 showed about two-fold (ED50 2.5 mg/kg) improved intra-
venous efficacy and over four-fold (ED50 3.9 mg/kg) improved oral
efficacy compared to AM-8085 likely due to improved metabolic
stability. The efficacy of the hydroxy fluoro analog 23 was some-
what inferior to the parent AM-8191 (Table 4). The efficacy of
the C-7 methyl analog (À)-15 was significantly decreased. The PK
data of these compounds was not collected.
2. Wiener, J. J.; Gomez, L.; Venkatesan, H.; Santillan, A., Jr.; Allison, B. D.; Schwarz,
K. L.; Shinde, S.; Tang, L.; Hack, M. D.; Morrow, B. J.; Motley, S. T.; Goldschmidt,
R. M.; Shaw, K. J.; Jones, T. K.; Grice, C. A. Bioorg. Med. Chem. Lett. 2007, 17, 2718.
3. Black, M. T.; Stachyra, T.; Platel, D.; Girard, A. M.; Claudon, M.; Bruneau, J. M.;
Miossec, C. Antimicrob. Agents Chemother. 2008, 52, 3339.
4. Black, M. T.; Coleman, K. Curr. Opin. Investig. Drugs 2009, 10, 804.
5. 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. Bioorg. Med. Chem. Lett. 2007, 17, 2723.
6. 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. Bioorg. Med. Chem. Lett.
2011, 21, 7489.
In summary, this Letter describes the SAR of C-3, C-4 and C-7
substituted pyridoxazinone oxabicyclooctane linked 1,5-naph-
thyridinyl NBTIs. The study concludes that small hydrophobic
groups are tolerated at C-3 but not at C-4 or C-3 and C-4. A C-3
Fluoro analog (6) showed four-fold improvement of in vivo oral
efficacy. Methyl substitution at C-7 showed slightly diminished
but flat potency and spectrum including activity against P. aerugi-
nosa and would benefit from additional studies. In general basicity
7. 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. Bioorg.
Med. Chem. Lett. 2011, 21, 7483.
8. 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. Bioorg. Med. Chem. Lett. 2013,
23, 2955.

S. B. Singh et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3636–3643
3643
9. 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. Bioorg. Med. Chem. Lett. 2013,
23, 5437.
14. Singh, S. B.; Kaelin, D. E.; Wu, J.; Miesel, L.; Tan, C. M.; Black, T.; Nargund, R.;
Meinke, P. T.; Olsen, D. B.; Lagrutta, A.; Lu, J.; Patel, S.; Rickert, K. W.; Smith, R.
F.; Soisson, S.; Sherer, E.; Joyce, L. O.; Wei, C.; Peng, X.; Wang, X.; Fukuda, H.;
Kishii, R.; Takei, M.; Takano, H.; Shibasaki, M.; Yajima, M.; Nishimura, A.;
Shibata, T.; Fukuda, Y. Bioorg. Med. Chem. Lett. 2015, 25, 1831.
10. 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. J. Med.
Chem. 2011, 54, 7834.
15. Singh, S. B.; Kaelin, D. E.; Wu, J.; Miesel, L.; Tan, C. M.; Meinke, P. T.; Olsen, D. B.;
Lagrutta, A.; Wei, C.; Peng, X.; Wang, X.; Fukuda, H.; Kishii, R.; Takei, M.;
Shibata, T.; Ohata, K.; Takano, H.; Kurasaki, H.; Takeuchi, T.; Nishimura, A.;
Fukuda, Y. Bioorg. Med. Chem. Lett. 2015, 25, 2409.
11. Reck, F.; Alm, R. A.; Brassil, P.; Newman, J. V.; Ciaccio, P.; McNulty, J.; Barthlow,
H.; Goteti, K.; Breen, J.; Comita-Prevoir, J.; Cronin, M.; Ehmann, D. E.; Geng, B.;
Godfrey, A. A.; Fisher, S. L. J. Med. Chem. 2012, 55, 6916.
12. Ross, J. E.; Scangarella-Oman, N. E.; Flamm, R. K.; Jones, R. N. J. Clin. Microbiol.
2014, 52, 2629.
13. Singh, S. B.; Kaelin, D. E.; Wu, J.; Miesel, L.; Tan, C. M.; Meinke, P. T.; Olsen, D.;
Lagrutta, A.; Bradley, P.; Lu, J.; Patel, S.; Rickert, K. W.; Smith, R. F.; Soisson, S.;
Wei, C.; Fukuda, H.; Kishii, R.; Takei, M.; Fukuda, Y. ACS Med. Chem. Lett. 2014, 5,
609.
16. Singh, S. B.; Kaelin, D. E.; Meinke, P. T.; Wu, J.; Miesel, L.; Tan, C. M.; Olsen, D. B.;
Lagrutta, A.; Fukuda, H.; Kishii, R.; Takei, M.; Takeuchi, T.; Takano, H.; Ohata, K.;
Kurasaki, H.; Nishimura, A.; Shibata, T.; Fukuda, Y. Bioorg. Med. Chem. Lett.
2015, 25, 3630.
17. Singh, S. B.; Kaelin, D. E.; Wu, J.; Miesel, L.; Tan, C. M.; Gill, C.; Black, T.;
Nargund, R.; Meinke, P. T.; Olsen, D. B.; Lagrutta, A.; Wei, C.; Peng, X.; Wang, X.;
Fukuda, H.; Kishii, R.; Takei, M.; Takeuchi, T.; Shibue, T.; Ohata, K.; Takano, H.;
Ban, S.; Nishimura, A.; Fukuda, Y. Bioorg. Med. Chem. Lett. 2015, 25, 2473.