Novel tetrahydropyran-based bacterial topoisomerase inhibitors with potent anti-gram positive activity and improved safety profile

Article abstract of DOI:10.1021/jm501590q

Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K⁺ channels and hNa_V1.5 Na⁺ channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.

Full text of DOI:10.1021/jm501590q

Article
pubs.acs.org/jmc
Novel Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors
with Potent Anti-Gram Positive Activity and Improved Safety Profile
Jean-Philippe Surivet,* Cornelia Zumbrunn, Georg Rueedi, Daniel Bur, Thierry Bruyere, Hans Locher,
̀
Daniel Ritz, Peter Seiler, Christopher Kohl, Eric A. Ertel, Patrick Hess, Jean-Christophe Gauvin,
Azely Mirre, Verena Kaegi, Marina Dos Santos, Stephanie Kraemer, Mika Gaertner, Jonathan Delers,
́
Michel Enderlin-Paput, Maria Weiss, Romain Sube, Hakim Hadana, Wolfgang Keck,+
and Christian Hubschwerlen+
Actelion Pharmaceuticals Limited, Gewerbestrasse 16, CH-4123 Allschwil, Switzerland
S
* Supporting Information
ABSTRACT: Novel antibacterial drugs that are effective against infections caused by multidrug resistant pathogens are urgently
needed. In a previous report, we have shown that tetrahydropyran-based inhibitors of bacterial type II topoisomerases (DNA
gyrase and topoisomerase IV) display potent antibacterial activity and exhibit no target-mediated cross-resistance with
fluoroquinolones. During the course of our optimization program, lead compound 5 was deprioritized due to adverse findings in
cardiovascular safety studies. In the effort of mitigating these findings and optimizing further the pharmacological profile of this
class of compounds, we have identified a subseries of tetrahydropyran-based molecules that are potent DNA gyrase and
topoisomerase IV inhibitors and display excellent antibacterial activity against Gram positive pathogens, including clinically
relevant resistant isolates. One representative of this class, compound 32d, elicited only weak inhibition of hERG K+ channels
and hNaV1.5 Na+ channels, and no effects were observed on cardiovascular parameters in anesthetized guinea pigs. In vivo
efficacy in animal infection models has been demonstrated against Staphylococcus aureus and Streptococcus pneumoniae strains.
antibacterial drug targets. In this respect, bacterial type II
topoisomerases, DNA gyrase and topoisomerase IV (Topo IV),
have been looked at intensely.7 These two homologous and
essential enzymes are known to regulate the DNA topology
during replication.8 DNA gyrase introduces negative supercoils
into DNA,9 and Topo IV is mainly responsible of the
decatenation of the sister chromatids at the end of the
replication process.10 Topoisomerases are heterotetrameric
A2B2 complexes made of two GyrA/GyrB and ParC/ParE
(GrlA/GrlB in S. aureus) subunits for DNA gyrase and Topo
IV, respectively. Fluoroquinolone11 and coumarin antibiotics,12
two clinically validated classes of antibacterial drugs, block
DNA topoisomerases function. Coumarin antibiotics do so
through binding to the ATP-binding pocket located within
GyrB or ParE subunits.13 Fluoroquinolones function through
binding to the cleavage-ligation site located in GyrA/ParC,
stabilizing double-strand breaks in DNA, eventually leading to a
rapid bacterial killing.14 Resistance to fluoroquinolones is
nowadays widespread.15 The main mechanism providing
INTRODUCTION
■
According to a recent report of the Center for Diseases Control
and Prevention,1 every year in the United States around two
million people are falling ill from antibiotic-resistant infections,
and 23000 people die because of such infections. Antimicrobial
resistance constitutes not only a direct threat, but also put at
risk well-established medical procedures from bone and joint
replacement surgery to dialysis and organ transplants. All over
the world, health authorities are reporting similar threats and
are calling for immediate action to pursue discovery and
development of novel antibacterial drugs.2,3 However, the
number of potential drugs effective against the “ESKAPE”
pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiel-
la pneumoniae, Acinetobacter baumannii, Pseudomonas aerugino-
sa, and Enterobacter species)4 currently in clinical development
remains dramatically low.5 New drugs must also be devoid of
cross-resistance with agents that were formerly of clinical
relevance. To overcome cross-resistance, they can either act on
an untapped drug target or address a novel binding site of a
known drug target. Whereas the identification of novel drug
targets remains of prime importance,6 there are still
considerable efforts dedicated to the clinically validated
Received: October 15, 2014
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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resistance to fluoroquinolone results from amino acid changes
in the quinolone-resistance determining region (QRDR) of
GyrA (Ser84Leu) or GrlA (Ser80Ile).16−18 A few years ago,
GSK19 and Aventis20 had discovered chemical entities, related
either to 1 (GSK299423)21 or 2 (NXL-101)22 (Figure 1), that
infection model against S. aureus; however, NBTI 5 blocked
hKV11.1 (“hERG”) channels slightly. In addition, NBTI 5 also
blocked hNaV1.5 channels slightly and affected various
cardiovascular parameters when administered to anesthetized
guinea pigs (data not shown in our earlier disclosure,29 see
thereafter). We therefore based our lead optimization on
lowering hERG blockade further, while the inhibition of
hNav1.5 and the effects on cardiovascular parameters in vivo
were monitored for selected compounds. Our objective was to
discover THP-based NBTIs with a clean cardiovascular safety
profile before advancing them into preclinical studies.
Structure−activity relationship studies have established the
importance of the two-atom linker −A−B− that unifies the
bicyclic aromatic left-hand side (LHS) to the central THP-core
in modulating hERG K+ channel block (Figure 2).29 To
complete our investigations on hydroxylated −A−B− linkers,
we turned our attention to NBTIs featuring a −CH2−
(CHOH)− linker. We report herein the synthetic routes that
led to the designed hydroxylated linker as well as the fruits of
our investigations devoted to the optimization of the cardiac
safety profile of THP-based NBTIs.30 Key properties of our
preclinical candidate 32d are disclosed and illustrate its
clinically relevant pharmacological profile.
Figure 1. Chemical structures of some NBTIs.
display excellent antibacterial properties and are devoid of
cross-resistance with fluoroquinolones while being potent DNA
gyrase and/or topoisomerase IV inhibitors. In 2010, the
binding mode of compound 1 at the interface between DNA
and gyrase of Staphylococcus aureus (S. aureus) was
unambiguously identified. Intriguingly, this compound binds
at a site different from the one used by fluoroquinolones.23 This
finding immediately triggered intensive research efforts.24
Several pharmaceutical companies have reported their research
programs. Viquidacin (NXL-101) 2, developed at Novexel, was
the first NBTI to enter clinical development but was
discontinued due to QT prolongation.25 More recently, GSK
entered phase 2 dose-ranging study to treat skin infections
caused by S. aureus with 3 (GSK-2140944).26 Astra-Zeneca has
also reported compounds such as 4, which may be suitable for
the treatment of infections caused by Gram-positive bacteria27
or for the treatment of tuberculosis.28
We have previously reported a series of tetrahydropyran
(THP)-based bacterial topoisomerase inhibitors and described
how these compounds act as dual DNA gyrase and Topo IV
inhibitors.29 In those series, we demonstrated that NBTI 5
(Figure 2) displayed an excellent antistaphylococcal activity, no
cross-resistance with fluoroquinolones, low to moderate
resistance frequency, and was efficacious in a murine thigh
CHEMISTRY
■
To rapidly construct the −CH2−(CHOH)− linker and to
examine its influence on biological properties, we first
envisioned the synthesis of amine 14b from ketone 13 obtained
by an hydration reaction of alkyne 10b (R = Bn) (Scheme 1).
Alkyne 10a (R = tBu) was readily built via a Sonogashira-type
coupling31 between known triflate 7a (FG = OSO2CF3) and
alkyne 9, the preparation of which is described in Supporting
Information. Exchange of the NH-protecting group (NH-Boc
to NH-Cbz) was mandatory to perform the Hg(II)-promoted
hydration under harsh acidic conditions.32 Gratifyingly, when
alkyne 10b was treated with mercury oxide in aqueous sulfuric
acid, it afforded a unique product which, after reduction with
sodium borohydride and CBz deprotection, yielded 14b,
isolated as a mixture of epimeric alcohols. Amine 14b was
reductively aminated with aldehyde 15a, leading to compound
16. Its regioisomer 12 was obtained by reductive amination of
amine 11 with aldehyde 15a.29 The construction of amine 11,
featuring the −(CHOH)−CH2− linker, was achieved by
trapping lithio derivatives 7b (FG = Li) with aldehyde 8.
Figure 2. Chemical structures of THP-based NBTIs.
B
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a
Scheme 1
a
Reagents: (a) cat. CuI, cat. PdCl2(PPh3)2, Et3N, DMF, rt; (b) TFA; (c) Cbz-Cl, NaHCO3, aq THF; (d) HgO, aq H2SO4, 55 °C; (e) NaBH4,
MeOH, 0 °C; (f) 15a, MS 3 Å, DCM−MeOH, 50 °C then NaBH4, 0 °C; (g) chiral HPLC separation.
Table 1. Antibacterial, Topoisomerases, and hERG Activities of NBTIs 5, 12a,b, and 16a,b
Topo IV RIA (IC50,
MIC (mg/L)
gyrase SCIA (IC50, μM)
μM)
a
b
c
d
e
f
g
h
i
compds
Sa
SaR
Sp
Ec
Sa
SaR
SaQR
Ec
Sa
Ec
hERG block (%)
5
≤0.03
≤0.03
0.125
≤0.03
≤0.03
0.25
2
1
8
1
4
8
2
0.03
0.125
0.5
8
4
8
4
4
8
4
0.03
0.03
0.125
0.03
0.5
32
>32
>32
>32
>32
>32
>32
0.5
8
8
>8
32
8
0.125
0.125
0.125
0.125
0.125
0.125
0.125
19
99
12
0.03
0.03
2
2
12a
12b
16
100
90
0.125
0.25
0.5
<0.03
0.03
2
8
8
76
16a
16b
0.5
0.125
0.03
32
2
32
2
73
≤0.03
0.125
0.125
76
a
b
c
d
Staphylococcus aureus ATCC 29213. Staphylococcus aureus A-234 gyrA: D83N. Streptococcus pneumoniae ATCC 49619. Escherichia coli ATCC
e
f
g
25922. Isolated from Staphylococcus aureus ATCC 29213. Enzyme containing the D83N mutation isolated from SaR A-234. Quinolone-resistant
h
i
(QR) enzyme containing a gyrA S84L mutation isolated from Staphylococcus aureus A-798. Isolated from Escherichia coli ATCC 25922. % reduction
of hERG K+ current measured at 10 μM.
Compounds 12 and 16, isolated both as mixtures of epimers,
were separated by chiral HPLC, leading to compounds 12a,b
and 16a,b respectively. After characterization of their biological
properties, compound 16b was identified as the most promising
compound (Table 1). The development of an efficient
synthetic access to analogues of NBTI 16b as well as the
assignment of the absolute configuration were prioritized. On
the basis of our previous investigations for which the
−(CHOH)−(CHOH)− linker was preferably (1R,2S)-config-
urated (see compound 5, Figure 2), we inferred that
diastereomer 16b should be (S)-configurated at the level of
−−CH2−(CHOH)− linker.
The stereocontrolled synthesis of 16b was undertaken from
known (1R,2S)-configurated diol 17 as reported in Scheme 2.
After treatment of diol 17 with 1,1′-carbonyl diimidazole
(CDI), the reaction was worked up under basic conditions
(aqueous NaHCO3), giving rise to cyclic carbonate 18.
Subsequent palladium−catalyzed hydrogenolysis of the benzylic
−C−O− bond was readily performed under hydrogen
atmosphere in the presence of palladium on charcoal. However,
some optimization was necessary to minimize the formation of
side products. The use of a mixture of palladium on CaCO3 and
palladium on charcoal in the presence of ammonium formate
gave rise to a much cleaner reaction and afforded, after
C
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Journal of Medicinal Chemistry
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a
Scheme 2
a
Reagents: (a) CDI, 2-butanone, 60 °C then aq NaHCO3; (b) 5% Pd on CaCO3, 10% Pd on C, NH4CO2H, EtOAc−EtOH, 60 °C; (c) TFA; (d)
15a or 15b, MeOH, rt, then NaBH4, 0 °C; (e) 22, 23, 24 or 25, MeOH, 50 °C then NaBH4, 0 °C.
Figure 3. ORTEP representation of the molecular structure of compound 32d as determined by X-ray diffraction. Hydrogen atoms have been
omitted for clarity.35
chromatographic purification, benzylic alcohol 19 in 89% yield.
Treatment of 19 with TFA provided amine 20. Using aldehyde
15a (Q = S), reductive amination of 20 led indeed to derivative
16b, validating thereby our initial hypothesis and ascertaining
the chemical structure. A supplementary confirmation was
achieved by a single molecule X-ray structure of compound 32d
(deposited as CCDC 1022935, Figure 3). A series of analogous
tetrahydropyranyl derivatives 21 and 26a−d, were prepared by
reductive amination of amine 20 with aldehydes 15b33 and 22−
25.
The same synthetic pathway was applied for the preparation
of intermediate amines 30a−c starting from (1R,2S)-configu-
rated diols 27a−c, whose the preparation followed the route
reported previously.34 These resulting amines were eventually
converted by reductive amination to final derivatives 31a,b
using aldehydes 15a,b or to derivatives 32−34(a−d) using
aldehydes 22−25.
RESULTS AND DISCUSSION
■
In Vitro Characterization of NBTIs. Enzyme inhibition
profiles were assessed for novel chemical entities on a panel of
bacterial topoisomerases comprising DNA gyrase and Topo IV
isolated from wild-type S. aureus and Escherichia coli (E. coli)
strains. Inhibition of DNA gyrase isolated either from a
laboratory strain made resistant to 2 (NXL-101) (gyrA:D83N
mutant) or a quinolone-resistant strain (gyrA:S84L mutant,
dubbed QR mutant) were also measured. The antibacterial
activity, expressed as MIC (minimal inhibitory concentration,
[mg/L])36 was measured against a panel of bacterial strains
including one wild-type Streptococcus pneumoniae (S. pneumo-
niae), one wild-type E. coli, and two S. aureus (a wild-type and a
laboratory mutant carrying gyrA:D83N mutation) strains.
Blockade of the hERG K+ channel was also routinely assessed.
As shown in Table 1, compounds 12 and 16, evaluated as
mixture of epimers, showed the same enzymatic profile than
our earlier compound 5, with potent inhibition of wild-type and
QR S84L S. aureus DNA gyrases and E. coli Topo IV (IC50s in
the nanomolar range). Inhibitory activity on E. coli DNA gyrase
and S. aureus Topo IV were weaker. No activity was measured
on gyrA D83N mutated DNA gyrase. Once separated by chiral
(R)-epimers of derivatives 1-epi-32a and 1-epi-32d, featuring
a (R)-configurated −CH2−(CHOH)− two-atom linker, were
also prepared from the corresponding (1S,2R)-configurated
diol. These syntheses are described in Supporting Information.
D
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a
Scheme 3
a
Reagents: (a) CDI, 2-butanone, 60 °C then aq NaHCO3; (b) 5% Pd on CaCO3, 10% Pd on C, NH4CO2H, EtOAc−EtOH, 60 °C; (c) TFA, (d)
15a or 15b, MeOH, rt, then NaBH4, 0 °C; (e) 22, 23, 24 or 25, MeOH, 50 °C then NaBH4, 0 °C.
Table 2. Antibacterial, Topoisomerases, and hERG Activities of NBTIs 21 and 26a−d
Topo IV RIA (IC50,
MIC (mg/L)
gyrase SCIA (IC50, μM)
μM)
a
b
SaR
c
d
e
f
g
h
e
h
i
compd
Sa
Sp
Ec
Sa
SaR
SaQR
Ec
Sa
Ec
hERG block (%)
21
≤0.03
≤0.03
≤0.03
0.25
2
4
0.25
0.125
0.125
1
4
0.125
0.5
>32
>32
8
0.03
0.03
0.03
2
2
0.125
0.5
0.125
8
66
22
44
0
26a
26b
26c
26d
8
>8
>8
>8
32
32
32
32
4
0.12
0.5
>8
8
>32
32
0.125
0.5
>32
>32
>32
>32
≤0.03
0.25
b
0.5
8
14
a
c
d
Staphylococcus aureus ATCC 29213. Staphylococcus aureus A-234 gyrA: D83N. Streptococcus pneumoniae ATCC 49619. Escherichia coli ATCC
e
f
g
25922. Isolated from Staphylococcus aureus ATCC 29213. Enzyme containing the D83N mutation isolated from SaR A-234. Quinolone-resistant
h
i
(QR) enzyme containing a gyrA S84L mutation isolated from Staphylococcus aureus A-798. Isolated from Escherichia coli ATCC 25922. % reduction
of hERG K+ current measured at 10 μM.
HPLC, 12b and 16b proved to be slightly more potent
inhibitors of wild-type S. aureus DNA gyrase and topo IV than
their respective epimers 12a and 16a. The four compounds
12a,b and 16a,b showed good antibacterial activity against wild-
type S. aureus and S. pneumoniae and were also as potent against
a QR S. aureus strain (data not shown). However, we observed
that 12b and 16b demonstrated more pronounced antibacterial
potency against a gyrA D83N S. aureus strain (with MIC = 1
and 2 mg/L, respectively). These results were in agreement
with previous observations for which most potent dual S. aureus
DNA gyrase−Topo IV inhibitors displayed antibacterial activity
against this particular S. aureus mutated strain, most probably
through their action on Topo IV. The antibacterial activity
against E. coli was weak with no notable difference among the
four compounds. High level of hERG block was observed for
these four compounds, with 16a and 16b exhibiting lower
inhibitory activity (73 and 76% at 10 μM compound
concentration) than 12a and 12b (90 and 100%, respectively).
The overall profile of compound 16b emerged as the most
favorable one and therefore our focus was set on establishing
structure−activity relationships of its analogues rather than on
those of 12b. The absolute stereochemistry of 12a and 12b
−(CHOH)−CH2− linker remained undetermined. Analogues
of 16b were produced using to the stereocontrolled syntheses
reported in Schemes 2 and 3 that proved to be versatile enough.
First, analogues of 16b featuring various right-hand side (RHS)
moieties were evaluated. As shown in Table 2, compound 21,
featuring a pyrido-oxazinone RHS, exhibited the same
enzymatic inhibitory and antibacterial activity profile than
compound 16b, with only a slightly lower hERG block (66%
versus 77% at 10 μM compound concentration). Compounds
26a,b containing either a dioxinopyridine or a oxathiinopyr-
idine RHS, retained potent inhibition of wild-type and QR
S84L S. aureus DNA gyrase and E. coli Topo IV. A weaker
inhibitory activity on S. aureus Topo IV was observed for 26a,b
in comparison to either 16b or 21. These enzymatic inhibition
data tracked well with the antibacterial activity observed on
wild-type Gram-positive strains and the weak activity on the
D83N S. aureus strain. Analogues 26c,d featuring a dioxinopyr-
idazine or a oxathiinopyridazine RHS, showed only meaningful
inhibitory activity on S. aureus DNA gyrase. Weak inhibitory
activities on E. coli Topo IV and no activity on E. coli DNA
gyrase or S. aureus Topo IV were measured for either of these
two compounds. Compound 26c showed modest antibacterial
activity on wild-type S. aureus or S. pneumoniae strain.
Nonetheless, compounds 26c,d displayed less block of hERG
E
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Journal of Medicinal Chemistry
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Table 3. Antibacterial, Topoisomerases, and hERG Activities of NBTIs 31,b and 32-34(a−d)
Topo IV RIA (IC50,
μM)
MIC (mg/L)
Gyrase SCIA (IC50, μM)
a
b
c
d
e
f
g
h
e
h
i
compd
31a
Sa
SaR
Sp
Ec
Sa
SaR
SaQR
Ec
Sa
Ec
hERG block (%)
≤0.03
≤0.03
≤0.03
0.06
0.06
0.25
1
≤0.03
≤0.03
0.06
0.25
≤0.06
0.25
0.06
0.5
0.5
0.5
8
0.03
0.125
0.125
0.125
0.03
32
<0.03
0.03
0.03
NT
32
32
2
0.125
2
0.03
NT
83
72
33
37
59
4
31b
>32
>32
>32
2
32a
0.03
8
0.125
2
1-epi-32a
32b
>8
>8
0.125
>0.03
0.03
32
22
32
8
≤0.03
≤0.03
≤0.03
0.5
0.5
4
4
>8
>8
>8
8
0.125
2
32c
0.5
>32
8
>32
32
NT
8
32d
1
0.125
NT
0.03
0.125
NT
10
7
1-epi-32d
33a
>8
NT
8
NT
NT
8
≤0.03
≤0.03
≤0.03
≤0.03
≤0.03
≤0.03
≤0.03
≤0.03
0.5
1
0.12
≤0.03
0.125
0.06
0.06
0.06
0.25
≤0.03
1
0.125
0.03
0.03
0.125
0.125
2
49
72
17
14
58
74
10
20
33b
0.25
2
4
0.5
8
>0.03
0.125
>0.03
0.03
2
8
33c
>8
>8
8
0.125
0.03
32
8
>32
2
33d
1
2
0.5
34a
1
0.125
0.03
>32
2
8
8
0.5
34b
1
8
>0.03
0.125
0.03
2
8
0.125
0.125
0.125
34c
2
>8
>8
≤0.03
0.125
0.125
128
32
8
32
8
32
32
8
34d
1
CIP
0.5
>256
>256
0.5
8
a
b
c
d
Staphylococcus aureus ATCC 29213. Staphylococcus aureus A-234 gyrA: D83N. Streptococcus pneumoniae ATCC 49619. Escherichia coli ATCC
e
f
g
25922. Isolated from Staphylococcus aureus ATCC 29213. Enzyme containing the D83N mutation isolated from SaR A-234. Quinolone-resistant
h
i
(QR) enzyme containing a gyrA S84L mutation isolated from Staphylococcus aureus A-798. Isolated from Escherichia coli ATCC 25922. % reduction
of hERG K+ current measured at 10 μM.
(0 and 14% inhibition at 10 μM compound concentration)
than compounds 26a,b (22 and 44% inhibition, respectively).
Second, the exploration of the structure−activity relationship
of the LHS was based on the knowledge previously gathered on
the diol-linker series (exemplified with compound 5), for which
it was observed that 3-fluoronaphthyridine and 6-fluoroqui-
noxaline LHS provided superior antibacterial activity compared
to 6-methoxyquinoline. Analogous 2-methoxyquinoline deriva-
tives, featuring the nitrogen atom at the preferred position29
were also prepared in this series. As shown in Table 3, 3-
fluoronaphthyridine derivatives 32a−d are consistently more
potent than their des-fluoro analogues 26a−d. Even if
enzymatic inhibitory data collected for 26c and 32c on wild-
type S. aureus DNA gyrase did not show a notable difference,
the antibacterial activity of 32c was significantly improved on S.
aureus and S. pneumoniae, with 4-fold lower MICs than 26c.
There was a trend in favor of improved inhibitory activity on S.
aureus topoisomerases and E. coli Topo IV enzymes for 32a,
32b, and 32d in comparison to their respective des-fluoro
analogues. In agreement with the findings (Table 1), (R)-
epimers 1-epi-32a and 1-epi-32d elicited less potent anti-
bacterial activity than their respective epimers 32a and 32d.
The impact of the −CH2−(CHOH)− linker stereochemistry
on the blockade of hERG was marginal. All fluorinated
compounds presented in Table 3 displayed to a large extent,
excellent antibacterial activity on Gram-positive strains. In
comparison to ciprofloxacin (CIP), a fluoroquinolone known to
target preferentially Topo IV in S. aureus, most of fluorinated
compounds such as 32a,b,d and 33a,b,d inhibited not only S.
aureus Topo IV to a similar extent than CIP but also displayed
excellent inhibitory activity against DNA gyrase. Compounds
32b and 33b, featuring an oxathiinopyridine RHS had good
antibacterial activity on D83N S. aureus strain (MIC < 1 mg/L)
because of either a potent inhibitory activity on S. aureus Topo
IV for 32b or inhibitory activity on the mutant D83N S. aureus
DNA gyrase enzymes for 33b. However, these favorable
antibacterial properties went along with undesired block of
hERG. Similarly, compound 34b, featuring as well an
oxathiinopyridine RHS blocked hERG strongly. Their ana-
logues containing a pyridazine-based RHS, compounds 32d
and 33d exhibited less block of hERG while keeping
satisfactory enzymatic inhibitory and antibacterial properties.
The beneficial effect of the N-2 nitrogen atom of the RHS on
hERG block is a general trend and correlates here with the
decreased log D (from 2.8 for 32b to 1.7 for 32d).
Identification of a few compounds, notably 32d and 33d that
exhibit a better in vitro profile than our former lead NBTI 5
stimulated further efforts to evaluate their in vivo effects.
Cardiovascular Characterization of Selected THP-
Based NBTIs. The effects of compounds 31b, 32a, 32d, and
33d were assessed in more detail on hERG channels as well as
on hNaV1.5 channels. These compounds were also examined
for their actions on cardiovascular function in anesthetized
guinea pigs and compared to the initial compound, NBTI 5.
The four selected compounds exhibited a wide range of hERG
blockade (10−72% and 21−92% at 10 and 30 μM,
respectively) and hNaV1.5 inhibition (0−23% and 0−59% at
10 and 30 μM, respectively). During the in vivo experiment,
three consecutive doses of 3, 10, and 30 mg of compound per
kg of bodyweight were administered intravenously over periods
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of 20 min and mean arterial pressure (MAP), heart rate (HR)
and ECG parameters (PR, RR, QRS, QT intervals) were
recorded continuously. These data, as well as the Bazett-
corrected QT interval (QTcBZ)37 are qualitatively reported in
Table 4 as differences from vehicle-treated animals. Plasma
infection model. When administered subcutaneously at a dose
of 40 mg/kg, compound 32a and 33d (MIC against the S.
aureus in vivo strain = 0.03 and 0.015 mg/L, respectively)
elicited a net 1-log reduction of colony forming units (CFU) in
thighs compared to CFU at treatment start when measured 6 h
postinfection, i.e., 5 h post treatment. For compound 32d
(MIC against the S. aureus in vivo strain = 0.03 mg/L) a net 2-
log reduction of CFU was obtained in similar conditions. In
vivo pharmacodynamic activity of compound 32d was studied
in more depth and it was found efficacious against various S.
pneumoniae and S. aureus strains (including isolates with
demonstrated resistance to beta lactams and quinolones) in
thigh and lung infection models.39 As summarized in Table 5,
Table 4. Effects of Selected THP-Based NBTIs on Human
Cardiac Ion Channels and on MAP, HR, and ECG in
Anesthetized Guinea Pigs
5
31b
32a
32d
33d
a
hERG block
19/46
<15/22
↔
72/92
17/45
↓(--)
↓(--)
↑(++)
↑(++)
↑(++)
↑(+)
↔
33/59
23/59
↓(−)
↓(−)
↑(++)
↑(+)
↑(++)
↑(+)
↔
10/21
<15
↔
14/29
<15
↓(−)
↑(+)
↔
a
hNaV1.5 block
b
MAP
b
HR
↓(−)
↔
↔
Table 5. Mouse, Rat, and Dog Pharmacokinetic Parameters
of Selected THP-Based NBTIs
b
PR
↔
b
RR
↑(+)
↑(+)
↑(+)
↔
↔
↓(−)
↔
↔
b
32a
32d
33d
QRS
↔
↔
b
a
QT
Mouse
b,c
b
QTcBZ
↔
↑(+)
17
AUCn (μg·h/(mL.dose))
Cl/F (mL/min/kg)
β t1/2 (h)
0.35
47.6
0.66
0.65
25.4
0.95
0.18
90.1
0.25
d
[drug] (μM)
67
29
36
39
e
fu
0.235
16
0.168
5
0.11
4
0.201
8
0.274
5
d
c
free [drug] (μM)
Rat
b
a
AUCn (μg·h/(mL.dose))
0.73
23
1.28
13
1.27
13
% reduction of current measured at 10 and 30 μM compound
b
c
Cl (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
concentrations. Compared to vehicle treated animals. QTcBZ =
QT/√RR. At the end of 30 mg/kg/20 min infusion period. (+ or −)
magnitude of the difference from vehicle treatment. Unbound fraction
d
1.1
0.5
33
0.76
5.0
47
0.49
3.1
14
e
measured in guinea pig plasma by rapid equilibrium dialysis.
%F
d
Dog
b
AUCn (μg·h/(mL.dose))
2.56
6.7
1.7
3.9
31
4.14
4.0
4.4
3.8
0.69
2.2
43
drug concentrations were measured at the end of the final
infusion period, and are reported as total and free
concentrations. In the 6-fluoronaphthyridine series, compound
31b induced marked decreases of MAP and HR and it
increased the PR, QT, and QRS intervals. The effects on QT
and QRS can probably be attributed to the blockade of the
guinea pig equivalents of hERG and hNaV1.5 channels by
compound 31b, although Bazett-corrected QT remained
unchanged. Prolongation of the PR-interval suggests that
compound 31b may also block cardiac CaV1.2 channels.38
Compound 32a showed a very similar profile to compound
31b, although effects on MAP, HR, PR, and QT were slightly
less whereas the effect on QRS was somewhat larger. These
effects were consistent with compound 32a blocking hERG less
and NaV1.5 more than compound 31b. The overall profiles of
compounds 31b and 32a are similar to the ones found for our
earlier lead compound 5 (Table 4).
In the case of compound 32d, no effects on MAP, HR, and
ECG parameters were observed above those recorded for
vehicle-treated animals, at the highest drug exposure reached (8
μM free). Compound 32d, that displayed relatively low
inhibition of hERG (21% at 30 μM) and no detectable effect
on hNav1.5; proved to have the cleanest cardiovascular profile
in the 6-fluoronaphthyridine series. Its analogue 33d, featuring
a 3-fluoroquinoxaline LHS, showed as well, relatively weak
hERG inhibition (29% at 30 μM) and was inactive on hNav1.5
but, at the highest drug exposure, slight decreases of MAP and
RR interval were observed and Bazett-corrected QTc interval
was slightly increased. Therefore, from the selected set of
compounds we could identify NBTI 32d with the desired clean
cardiovascular profile in vitro and in the anesthetized guinea
pig.
Cl (mL/min/kg)
Vdss(L/kg)
t1/2 (h)
0.92
3.0
%F
79
a
Mouse dosed sc at 25 mg/kg (compound 32a) or 40 mg/kg
b
(compounds 32d and 33d). Dose-normalized AUC obtained after sc
or iv dosing. Rat dosed iv at 1 mg/kg (compound 32a) or 0.5 mg/kg
c
(compounds 32d and 33d) and orally at 5 mg/kg (compound 32a) or
d
1 mg/kg (compounds 32d and 33d). Beagle dog dosed iv at 0.5 mg/
kg (compounds 32a and 32d) or 1 mg/kg (compound 33d) and orally
at 5 mg/kg (all compounds).
pharmacokinetic properties of compounds 32a,d and 33d were
determined in rodents and dog. In mouse, compound 32d
showed low clearance (Cl = 25.4 mL/min/kg) and a longer
initial half-life (∼1h) when compared to compounds 32a and
33d (0.66 and 0.25 h respectively). The better in vivo efficacy
observed for 32d could be attributed to its superior PK profile
in mice. All compounds showed low volumes of distribution in
rat and dog. Clearance for all compounds was low to medium
in these two species. Oral bioavailability for compound 33d was
rather low in rat (F 14%) but higher in dog (F 43%). The best
overall profile was obtained for compound 32d that showed
good bioavailability in both species (F 47% and 79%
respectively).
No inhibition of the major cytochrome P450 enzymes (IC50s
> 50 μM) and no activation of the pregnane X receptor were
found for compound 32d, suggesting a low potential for drug−
drug interaction.
Microbiological Profile of Compound 32d. Compound
32d was tested against sets of recent clinical isolates of selected
pathogens (Table 6). Compound 32d was highly active against
all S. aureus isolates tested, including MRSA and fluoroquino-
The antistaphylococcal activity of compounds 32a, 32d, and
33d was explored in vivo in a neutropenic murine thigh
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showed in Table 3, these data suggest that 32d is a dual
gyrase and topoisomerase IV inhibitor, with gyrase being the
preferred target in S. aureus.
Table 6. Antibacterial Activity of NBTI 32d against Selected
Pathogens
a
Nb of strains MIC50
MIC90
range
To assess the bactericidal effect of 32d, the minimal
bactericidal concentration (MBC) of the compound against a
panel of eight strains was determined and compared to the
respective MIC. As shown in Table 8, compound 32d acts as
bactericidal drug against all Gram-positive strains tested. MBCs
were generally around 2-fold higher than the MIC (including
MRSA and VRE). Only for one S. aureus strain the MBC was
>16-fold higher, up from ≤0.016 to 0.25. In comparison,
Moxifloxacin (MXF, a fluoroquinolone) displayed a bactericidal
activity against quinolone-susceptible strains, while no MBCs
could be recorded for Linezolid (LZD) that is known to exhibit
a bacteriostatic activity against Gram-positive bacteria (with the
notable exception of S. pneumoniae).
Staphylococcus aureus
MRSA subset
QR subset
48
27
20
24
0.016
0.03
0.03
0.03
0.03
0.03
≤0.008−0.06
≤0.008−0.06
≤0.008−0.03
≤0.016−0.125
0.03
Staphylococcus
epidermidis
0.016
Streptococcus
agalactiae
13
14
31
0.25
0.06
0.125
0.5
0.125−0.5
≤0.016−0.06
0.03−0.25
Streptococcus
pyogenes
0.06
0.25
Streptococcus
pneumoniae
Enterococcus faecalis
Enterococcus faecium
Moraxella catarrhalis
20
21
11
11
0.5
1
0.06−1
0.016−2
0.03−0.25
0.5−8
0.25
0.125
2
1
0.125
8
Haemophilus
influenzae
CONCLUSION
■
Escherichia coli
13
21
16
32
>8
8−32
1−8
We have reported the synthesis of compound 32d, a novel dual
DNA gyrase and Topo IV inhibitor featuring a tetrahydropyran
core and an unprecedented homobenzylic alcohol moiety.40
Our efforts to optimize antibacterial activity while decreasing
the strong blockade of the hERG K+ channel initially observed
for 16b, led to the discovery of a series of promising derivatives.
In general, these derivatives display strong antibacterial activity
against clinically relevant Gram-positive pathogens and the
required lower hERG inhibition. In comparison to earlier
NBTIs that were identified in the course of our research
program, compound 32d displayed a relatively low inhibition of
the hERG K+ channel with an IC20 ≈ 30 μM and no liabilities
in a cardiovascular safety experiment conducted in guinea pig.
The in vitro properties translated to a useful pharmacological
profile. Indeed, compound 32d was efficacious in vivo in a
murine thigh infection models against S. aureus or S.
pneumoniae, and it showed good oral bioavailability in rat and
dog. Owing to this profile, a preclinical development program
for 32d was initiated. A more efficient synthetic route to this
family of THP-based NBTIs, enabling the preclinical studies
was discovered41 and details will be reported in due course.
Acinetobacter
baumannii
4
a
MIC90 (MIC50) is the MIC in mg/L at which at least 90% (50%) of
the strains were inhibited.
lone-resistant strains (MIC90 ≤ 0.03 mg/L). It also showed
good activity against a variety of other clinically relevant Gram-
positive bacteria, including S. pneumoniae and Enterococcus
faecium (E. faecium), including vancomycin-resistant strains,
with MIC90 ≤ 1 mg/L. While significant activity was observed
against Moraxella catarrhalis, activities against Haemophilus
influenza as well as other Gram-negative pathogens such as E.
coli or Acinetobacter baumannii were weaker with MIC90 ≥ 8
mg/L. In summary, compound 32d has an antibacterial
spectrum well suited for the treatment of Gram-positive
infections.
Spontaneous resistance frequencies of compound 32d,
measured in various representative Gram-positive strains are
summarized in Table 7. In the S. pneumoniae and the E. faecium
Table 7. Spontaneous Resistance Frequencies Measured with
32d on Selected Pathogens
EXPERIMENTAL SECTION
■
resistance frequency
MIC and MBC Testing. Minimal inhibitory concentrations
(MICs) were determined by the broth microdilution method
according to guidelines of the Clinical and Laboratory Standards
Institute (CLSI).36 Stock solutions of compounds were prepared in
DMSO and serially diluted in cation-adjusted Mueller-Hinton broth
(final DMSO concentration, 2.5% (v/v). Reference antibiotics
(ciprofloxacin, linezolid) were tested in parallel and MICs against
quality control strains were within the accepted CLSI ranges. For
determination of the minimal bactericidal concentration (MBC) MICs
were determined as described above. After reading the MICs, the wells
were carefully mixed by pipetting and 10 μL from each well was spread
on Columbia Blood Agar or Mueller-Hinton Agar (for Staphylococcus
aureus strains). Colonies were counted after incubation for 24 h at 37
°C. The MBC was defined as the lowest concentration that reduces
CFU counts by >99.9% compared to the CFU counts of the inoculum
at the beginning of the experiment.
bacterial strains
at 4× the MIC
at 16× the MIC
a
S. aureus ATCC 29213
3.0 × 10−8
6.3 × 10−8
<2.9 × 10−9
<2.7 × 10−9
6.1 × 10−10
9.3 × 10−10
S. aureus BA-1730 (MRSA, QR)
S. pneumoniae A-70 (QR)
E. faecium A-949 (VRE, QR)
a
For CIP: 8.8 × 10−8 (4× the MIC) and <4.6 × 10−10 (16× the MIC)
strains, resistance frequencies of 32d were very low (<10−9 at 4-
fold the MIC), while in S. aureus somewhat higher frequencies
were observed (10−8 at 4-fold the MIC). Nevertheless, very low
frequencies were observed at 16-fold the MIC for S. aureus.
Resistance frequencies of quinolone-resistant strains were not
significantly higher than those of quinolone-susceptible strains.
S. aureus clones spontaneously resistant to 32d were isolated.
All of them showed single mutations in QRDRs of gyrA or gyrB,
the most often encountered mutation occurring at D83 (to N)
of gyrA. Second-step mutants were subsequently generated with
32d and exhibited mutations in the QRDR of grlA,
preferentially at D79. Together with the enzymatic data
Spontaneous Resistance Frequencies. Large numbers of
bacteria (109−1010) were plated on Mueller−Hinton agar plates
containing the antibiotic compound at 4- and 16-fold the MIC
concentration. Resistance frequencies were calculated by dividing the
numbers of colonies growing after 48 h of incubation at 37 °C through
the total numbers of viable bacteria plated (as determined by colony
count on drug-free agar).
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a
Table 8. Minimal Bactericidal Concentration (MBC) of 32d, LZD, and MXF
compounds
LZD
strains
resistance phenotype
32d
MXF
S. aureus ATCC 29213
S. aureus A-798
≤0.016/0.03
≤0.016/0.03
0.03/0.06
0.03/0.06
≤0.016/0.25
0.5/1
2/>32
2/>32
0.06/0.125
8/>8
MRSA, QR
MRSA
S. aureus ATCC 43300
S. aureus H7935 (A-1093)
S. aureus HT203 (A-1160)
E. faecalis ATCC 29212
E. faecium A-949
2/>32
0.06/0.125
8/ND
MRSA, QR
caMRSA
2/>32
2/>32
0.125/0.125
0.25/0.5
>8/ND
0.5/>32
2/>32
VRE, QR
0.25/0.25
0.06/0.06
S. pneumoniae ATCC 49619
0.125/2
0.125/0.25
a
QR, quinolone-resistant; caMRSA, community acquired MRSA; QS, quinolone-sensitive; LZD, linezolid; MXF, moxifloxacin. Values are MIC/
MBC in mg/L.
DNA Gyrase and Topo IV Inhibition Assays. DNA gyrase
supercoiling inhibition assay was performed with relaxed pBR322
(purchased from Inspiralis, UK) as a substrate. For E. coli, the reaction
mixture (20 μL) contained 25 mM Tris HCl pH 7.5, 24 mM KCl, 4
mM MgCl2, 2 mM DTT, 1 mM ATP, 1.6 mM spermidine, 6.5%
glycerol, 50 μg/mL Bovine Serum Albumin (BSA) and 0.1 μg relaxed
plasmid. For S. aureus the reaction mixture (20 μL) contained 50 mM
Tris HCl pH 7.5, 20 mM KCl, 5 mM MgCl2, 5 mM DTT, 3 mM ATP,
700 mM potassium glutamate, 50 μg/mL BSA and 0.1 μg relaxed
plasmid. The reactions were carried out at 37 °C for 1 h. DNA Topo
IV relaxation assay was performed with supercoiled pBR322 as a
substrate. For E. coli, the reaction mixture (20 μL) contained 50 mM
Tris HCl pH 7.8, 20 mM KCl, 6 mM MgCl2, 10 mM DTT, 1 mM
ATP, 1 mM spermidine, 100 μg/mL BSA and 0.1 μg supercoiled
pBR322. For S. aureus, the reaction mixture (20 μL) contained 50 mM
Tris HCl pH 7.5, Twenty mM KCl, 5 mM MgCl2, 5 mM DTT, 1.5
mM ATP, 5 mM spermidine, 100 mM potassium glutamate, 5 μg/mL
BSA, and 0.1 μg supercoiled pBR322. The reaction was carried out at
37 °C for 1 h. Both supercoiling and relaxation reactions were stopped
by adding a mixture of EDTA, bromophenol blue, and glycerol. Ten
μL of each reaction mixture were loaded on a 1% agarose gel in TAE
buffer (40 mM Tris-acetate, 1 mM EDTA pH 8.0) and run at 1.0 V/
cm for 14 to 16 h. Gels were stained in water with ethidium bromide
and visualized under ultraviolet light. For supercoiling inhibition assays
(SCIA), 50% inhibitory concentration (IC50) was determined visually
as being the compound concentration at which the supercoiled band
was reduced by 50%. For relaxation inhibition assays (RIA), IC50 was
defined as the concentration of inhibitor necessary to inhibit the
formation of 50% relaxed DNA from supercoiled. The IC50 was taken
as an average of two inhibitory concentrations if inhibition did not
occur exactly within a particular concentration range (for representa-
tive gels, see Supporting Information). All gels were also subject to
analysis using AlphaEase Stand Alone Software (Alpha Innotech).
hKV11.1 (“hERG”) and hNaV1.5 Assay.42 Compounds were
evaluated for block of hKV11.1 and hNaV1.5 channels using CHO cells
stably expressing the hERG or the SCN5A genes (bSys, Witterswil,
Switzerland) and the QPatch platform (Sophion, Ballerup, Denmark).
For hKV11.1, K+ tail currents were measured at −40 mV following a
500 ms depolarization to +20 mV from a holding voltage of −80 mV.
For hNaV1.5, Na+ peak currents were measured at −20 mV following a
60-s-long, 5-Hz train of 150 ms depolarizations to −20 mV from a
holding voltage of −120 mV. The external solution contained 150 mM
Na+, 4 mM K+, 1 mM Mg2+, and 1.2 mM Ca2+. Compound effects
were quantified 3 min after application to the cells.
the 3Rs and the number of animals was kept to a strict minimum to
allow statistical significance.
On the day of the study, the guinea pigs (400−1000 g of
bodyweight) were anesthetized with a mixture of 60 mg/kg ketamine-
HCl (Ketanarkon, Streuli Pharma AG, Uznach) and 8 mg/kg xylazine
(Xylazin Streuli, Streuli Pharma AG, Uznach) given intramuscularly,
and the animals were placed on a thermostatically controlled heating
table to maintain body temperature at 36−38 °C. A catheter was
inserted into the left jugular vein for infusion of the compound or
vehicle and a derivation lead II was positioned for the ECG recording.
A second catheter was inserted in the right carotid artery to measure
MAP and HR. After the hemodynamic and ECG parameters had
stabilized, they were continuously recorded on a PowerLab data
acquisition system using IOX software (Emka Technologies, Paris,
France). Data were exported to a Dell Optiplex GX620 computer for
analysis (Datanalyst version 2.3.4.6 and ECG-Auto version 2.5.1.18
software, both from Emka Technologies).
The QT interval corrected for HR, QTc Bazett (QTcBZ), was
calculated using the following formula: QTcB Z = QT/√RR. At each
time point, QTcBZ was calculated for 30−60 individual ECG
waveforms and averaged. After baseline measurements, each guinea
pig was infused intravenously with either tested compound or its
vehicle during three consecutive 20 min periods using a Precidor
pump (Infors AG, Bottmingen, Switzerland). The infusion rate was 0.3
mL/kg/20 min for the first 20 min period, 1 mL/kg/20 min for the
second and 3 mL/kg/20 min for the third period. The corresponding
doses of tested compound were 3, 10, and 30 mg/kg.
At the end of the last infusion period, a 250 μL blood sample was
collected in 5% EDTA (K2E EDTA, BD Vacutainer ref 368856) and
centrifuged at 10000g/4 °C. The plasma samples were put in a 96-well
PCR plate and stored at −20 °C until they were analyzed for tested
compounds concentrations using liquid chromatography coupled to
mass spectrometry (LC-MS/MS).
Mice in Life Procedures.43 All animal housing and experiments
were performed in agreement with the Swiss Federal Ordinance for
animal protection, the animal welfare guide lines from the Cantonal
Vetenary Office Basellandschaft, and the Actelion Pharmaceuticals Ltd.
internal animal welfare guidelines. Eight to 12 week-old, specific
pathogen-free, female NMRI mice weighing 27−30 g were used for all
studies (Harlan, The Netherlands). Mice were rendered neutropenic
(neutrophils <100/mm3) by injecting cyclophosphamide (Sigma)
intraperitoneally 4 days (150 mg/kg) and 1 day (100 mg/kg) before
thigh infection. Previous studies have shown that this regimen
produces neutropenia in this model for 5 days. Broth cultures of
freshly plated S. aureus (laboratory descendent of DSM 11823) were
grown to logarithmic phase overnight to an absorbance of 0.3−0.9 at
580 nm. Thigh infections with S. aureus were performed by injection of
0.05 mL of inoculum (107.0−8.0 CFU/ml) intra muscularly into both
thighs of mice 2 h before administration of test compound (formulated
in 5% mannitol in water, administered at 10 mL/kg). Post-mortem,
thigh samples were homogenized (Ultra-Turrax tube drive, IKA),
serially diluted and plated (spiral platter EddyJet (IUL)). Results were
expressed as colony forming units (CFU; CFU-counter Flash & Grow,
Guinea Pig Protocol. Normotensive male Dunkin−Hartley
guinea pigs were delivered from Harlan Laboratories (Horst,
Netherlands) and group-housed during the acclimatization period
(>1 week). All animals were housed under identical conditions in
climate-controlled rooms with a 12-h light/dark cycle and free access
to normal pelleted food, in accordance with the guidelines of the
Basellandschaft Cantonal Veterinary Office (license no. 170). The
experimental design was selected in accordance with the principles of
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IUL), correlating to remaining bacterial load in the thigh at the end of
the study.
proceeded at 60 °C overnight. The reaction mixture was cooled. The
two layers were decanted, and the aqueous layer was extracted with
EtOAc (2 mL/mmol). The combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated to dryness. The
residue was purified by chromatography (heptane−EtOAc) to afford
the corresponding cyclic carbonate.
General Procedure B: Hydrogenolysis of Cyclic Carbonates.
To a suspension of cyclic carbonate (1 equiv) in EtOH (7 mL/mmol)
and EtOAc (0.5 mL/mmol) under nitrogen atmosphere was added
ammonium formate (0.35 g/mmol). The mixture was heated to 70 °C.
At this point, 5% Pd on CaCO3 (0.04 g/mmol) then 10% Pd/C (0.01
g/mmol) were added. The reaction proceeded at 40 °C for 1 h. Water
(3 mL/mmol) and EtOAc (3 mL/mmol): The mixture was filtered
and the solids were washed with water (10 mL/mmol) and EtOAc (30
mL/mmol). The two layers were separated, and the aqueous layer was
extracted with EtOAc (10 mL/mmol). The combined organic layers
were dried over Na2SO4, filtered, and concentrated to dryness. The
residue was purified by chromatography (heptane−EtOAc) to afford
the corresponding alcohol.
General Procedure C: Boc Deprotection. A solution of the Boc-
protected amine (1 equiv) in TFA (2 mL/mmol) was stirred at rt for 1
h. The solvent was evaporated in vacuo, and the residue was
partitioned between DCM−MeOH mixture (9:1, 10 mL/mmol) and
saturated NaHCO3 solution (5 mL/mmol). The pH was adjusted to
10 by adding 32% aqueous NaOH. The two layers were separated, and
the aqueous layer was extracted three times with the same mixture (3
× 10 mL/mmol) and the combined organic layers were dried over
Na2SO4, filtered, and concentrated to dryness. The residue was
purified by chromatography (DCM−MeOH 19:1, containing 1% aq
NH4OH) to afford the corresponding amine.
General Procedure D: Reductive Amination Using Alde-
hydes 15a,b. To a solution of amine (1 equiv) in DCE (10 mL/
mmol) and MeOH (4 mL/mmol) were added 3 Å molecular sieves (3
g/mmol) and 15a or 15b (1.1 equiv). The reaction was heated at 50
°C overnight. After cooling to 0 °C, NaBH4 (400 mol %) was added
and the reaction was stirred for 2 h. The reaction mixture was filtered,
and the filtrate was diluted with DCM−MeOH (9:1, 10 mL/mmol)
and saturated NaHCO3 solution (5 mL/mmol). The two layers were
separated, and the organic layer was dried over dried over Na2SO4,
filtered, and concentrated to dryness. The residue was purified by
chromatography (DCM−MeOH containing 1% NH4OH) to afford
the alkylated product.
Pharmacokinetics in the Mouse. The pharmacokinetic profile
was determined in female NMRI mice (n = 18). For this purpose, each
compound was formulated as aqueous isotonic 5% D-mannitol solution
for subcutaneous administration at a dose of 25, 40, and 40 mg/kg for
32a, 32d, and 33d, respectively. Blood samples (two per mouse) were
taken at regular time intervals to cover a 24 h period, and plasma was
generated by centrifugation at 4000 rpm at 4 °C. Plasma
concentrations were determined using a specific and sensitive
LCMS/MS method with a limit of quantification of 1.5 ng/mL.
Pharmacokinetics in the Rat. The pharmacokinetic profile was
determined in the male Wistar rat (n = 3). For this purpose,
compound 32a was formulated as an aqueous solution at pH 4.5 for
intravenous administration at a dose of 1 mg/kg and as aqueous
suspension pH 5.6 for oral dosing at 5 mg/kg; compound 32d was
formulated as a solution in 5% DMSO in pure water for intravenous
administration at a dose of 0.5 mg/kg, and as a microsuspension in
7.5% aqueous modified gelatin for oral dosing at 1 mg/kg; compound
33d was formulated as an aqueous solution at pH 4.74 for intravenous
administration at a dose of 0.5 mg/kg and as as a microsuspension in
7.5% aqueous modified gelatin for oral dosing at 1 mg/kg. Blood
samples were taken at regular time intervals over a period of 24 h from
a preimplanted catheter, and plasma was generated by centrifugation at
4000 rpm at 4 °C. Plasma concentrations were determined using a
specific and sensitive LCMS/MS method with a limit of quantification
of 1.5 ng/mL.
Pharmacokinetics in the Dog. The pharmacokinetic profile was
determined in male Beagle dogs (n = 3). For this purpose, compounds
32a, 32d, and 33d were formulated as aqueous isotonic 5% D-mannitol
solutions intravenous administration at a dose of 0.5, 0.5, and 1 mg/kg
respectively and as dispersion in gelatin 7.5% via 5% DMSO aqueous
suspension for oral dosing at 5 mg/kg. Blood samples were taken at
regular time intervals over a period of 24 h from a preimplanted
catheter, and plasma was generated by centrifugation at 4000 rpm at 4
°C. Plasma concentrations were determined using a specific and
sensitive LCMS/MS method with a limit of quantification of 1.5 ng/
mL.
General Chemical Methods. Starting materials, reagents, and
solvents were obtained from commercial sources and used as received.
All reactions were carried out with continuous stirring under
1
atmosphere of dry nitrogen. The resonance frequency for H (13C)
on a Varian Gemini 300 is 300 MHz (75 MHz) or on a Bruker Ascend
500 is 500 MHz (125 MHz). Chemical shifts (δ) are reported in parts
per million (ppm) relative to deuterated solvent as the internal
standard (δH: CDCl3 7.26 ppm, DMSO-d6 2.50 ppm), coupling
constants (J) are in hertz (Hz). Peak multiplicities are expressed as
follows: singlet (s), doublet (d), doublet of doublets (dd), triplet (t),
quartet (q), multiplet (m), and broad singlet (br s). Melting points
General Procedure E: Reductive Amination Reaction Using
Aldehydes 22−25. To a solution of amine (1equiv) in MeOH (5
mL/mmol) was added the appropriate aldehyde (1 equiv). The
reaction mixture was stirred at rt overnight. The reaction mixture was
cooled to 0 °C, and NaBH4 (400 mol %) was added. After stirring for
40 min, DCM−MeOH (9:1, 6 mL/mmol) and saturated NaHCO3 (2
mL/mmol) were added. The two layers were decanted, and the
aqueous layer was extracted with a DCM−MeOH mixture (9:1, 4 × 5
mL/mmol). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated to dryness. The residue
was purified by chromatography (DCM−MeOH containing 1% aq
NH4OH) to afford the corresponding alkylated product.
tert-Butyl ((3R,6S)-6-((6-Methoxy-1,5-naphthyridin-4-yl)-
ethynyl)tetrahydro-2H-pyran-3-yl)carbamate (10a). CuI (0.2 g,
0.9 mmol), PdCl2(PPh3)2 (0.32 g, 0.45 mmol), 9 (1 g, 4.4 mmol), and
7a (1.37 g, 4.4 mmol), DMF (5 mL) and Et3N (3 mL, 21.6 mmol)
were successively introduced in a flask. The reaction proceeded 6 h at
rt. After the solvent was evaporated, the residue was purified by
chromatography (heptane−EtOAc) to afford 10a (1.07 g, 63% yield).
Orange solid; mp = 125 °C. 1H NMR (500 MHz, CDCl3) δ: 8.74 (d, J
= 4.5 Hz, 1 H), 8.22 (d, J = 9.0 Hz, 1 H), 7.63 (d, J = 4.5 Hz, 1 H),
7.18 (d, J = 9.0 Hz, 1 H), 4.99 (m, 1 H), 4.84 (m, 1 H), 4.39 (m, 1 H),
3.76 (m, 1 H), 3.53 (m, 1 H), 2.30 (m, 1 H), 2.22 (m, 1 H), 1.90 (m, 1
H), 1.76 (m, 1 H), 1.48 (s, 9H). 13C NMR (125 MHz, CDCl3) δ:
162.6, 155.2, 147.2, 142.1, 142.0, 140.4, 128.5, 126.7, 117.2, 82.0, 79.6,
67.8, 66.4, 53.9, 45.4, 28.4 (3C), 27.8, 25.9. ESI-MS (M + H)+ m/z
384.0. HR ESI-MS (M + H)+ m/z = 384.1927 (calcd for C21H26N3O4:
384.1923); tR =1.28.
were recorded with a Buchi 520 apparatus and are uncorrected.
̈
Purification of intermediates and final products was carried out on
normal phase using an ISCO CombiFlash system and prepacked SiO2
cartridges eluted with optimized gradients of either heptane−ethyl
acetate mixture or dichloromethane−methanol (doped with 1% v/v of
aq NH4OH for basic compounds). Progress of the reactions was
monitored by thin-layer chromatography (TLC) analysis (Merck, 0.2
mm silica gel 60 F254 on glass plates) or by LC-MS (methods and
equipment are described in Supporting Information). All target
compounds had purity >95%, established on a Waters Atlantis T3, 5
μm, 4.6 mm × 30 mm, eluting with a gradient of 5−95% of acetonitrile
in water containing 0.04% of trifluoroacetic acid or on a Waters
XBridge C18, OBD, 5 μm, 4.6 mm × 50 mm (Waters, Switzerland),
eluting with a gradient of 5−95% of acetonitrile in water containing 13
mM of NH4OH UV at 230 and 254 nm. Purity and identity were
further confirmed by NMR spectroscopy. Retention times (tR) are
expressed in minutes.
General Procedure A: Cyclic Carbonates Formation. To a
mixture of diol (1 equiv) in 2-butanone (4 mL/mmol), heated to 60
°C, was added CDI (1.5 equiv). The mixture was stirred 1 h and
Na2CO3 (5% aqueous, 1.5 mL/mmol) was added. The reaction
J
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(3R,6S)-[6-(6-Methoxy-[1,5]naphthyridin-4-ylethynyl)-tetra-
hydro-pyran-3-yl]-carbamic Acid Benzyl Ester (10b). A solution
of 10a (0.75 g, 1.94 mmol) in TFA (12 mL) was stirred at rt for 10
min. After evaporation to dryness, the residue was partitioned between
aqueous NaOH (1M, 7 mL) and a DCM−MeOH mixture (9:1, 50
mL). The aqueous layer was extracted with the same mixture (2 × 50
mL). The combined extracts were washed with brine, dried over
Na2SO4, filtered, and concentrated to dryness. The residue was taken
up in EtOAc (9 mL) and water (9 mL). NaHCO3 (0.5 g) and benzyl
chloroformate (0.32 mL, 2.2 mmol) were added. The reaction
proceeded for 30 min. The two layers were decanted, and the aqueous
layer was extracted once with EtOAc. The combined organic layers
were concentrated to dryness, and the residue was purified by
chromatography (heptane−EtOAc) to afford 10b (R = Bn) (0.24g,
J = 9.1 Hz, 0.5 H), 7.49 (d, J = 4.4 Hz, 0.5 H), 7.47 (d, J = 4.4 Hz, 0.5
H), 7.15 (d, J = 9.1 Hz, 0.5 H), 7.14 (d, J = 9.1 Hz, 0.5 H), 5.00 (br s,
1 H), 4.09 (s, 3 × 0.5 H), 4.08 (s, 3 × 0.5 H), 3.99−4.04 (m, 2 × 0.5
H), 3.89−3.94 (m, 2 × 0.5 H), 3.57 (dd, J = 2.8, 13.7 Hz, 0.5 H), 3.43
(dd, J = 3.5, 13.4 Hz, 0.5 H), 3.33 (dd, J = 6.8, 7.2 Hz, 0.5 H), 3.30
(dd, J = 6.8, 7.2 Hz, 0.5 H), 3.25 (ddd, J = 2.1, 5.0, 11.3 Hz, 0.5 H),
3.03 (t, J = 10.7 Hz, 0.5 H), 3.03 (overlaid m, 0.5 H), 2.97 (t, J = 10.7
Hz, 0.5 H), 2.81−2.88 (m, 1 H), 2.05−2.13 (m, 1 H), 1.99 (m, 0.5 H),
1.83 (m, 0.5 H), 1.66 (m, 0.5 H), 1.50 (m, 0.5 H), 1.37 (br s, 2 H),
1.26 (m, 0.5 H), 1.17 (m, 0.5 H). ESI-MS (M + H)+ 304.4.
6-((((3R,6S)-6-((R and S)-1-/hydroxy-2-(6-methoxy-1,5-naph-
thyridin-4-yl)ethyl)tetrahydro-2H-pyran-3-yl)amino)methyl)-
2H-pyrido[3,2-b][1,4]thiazin-3(4H)-one (16a). Starting from of
14b (0.029 g, 0.095 mmol) and 15 (0.018g, 0.095 mmol), 16 (0.021 g,
47% yield) was prepared using the general procedure D. The
diastereomeric mixture was separated on a Chiracel OZ-H column
(250 mm × 20 mm, 5 μm) with EtOH−heptane (9:1), containing
0.1% diethylamine. Isomer 16a eluted first (tR = 10.0 min), followed
1
30% yield). White solid; mp = 133 °C. H NMR (500 MHz, CDCl3)
δ: 8.74 (d, J = 4.5 Hz, 1 H), 8.22 (d, J = 9.0 Hz, 1 H), 7.63 (d, J = 4.5
Hz, 1 H), 7.33−7.41 (m, 5 H), 7.18 (d, J = 9.0 Hz, 1 H), 5.23 (d, J =
7.6 Hz, 1 H), 5.14 (s, 2 H), 4.87 (br s, 1 H), 4.44 (dd, J = 2.0, 11.7 Hz,
1 H), 4.14 (s, 3 H), 3.84 (m, 1 H), 3.57 (dd, J = 3.7, 11.5 Hz, 1 H),
2.32 (m, 1 H), 2.22 (m, 1 H), 1.90 (m, 1 H), 1.81 (m, 1 H). 13C NMR
(125 MHz, CDCl3) δ: 162.6, 155.7, 147.2, 142.1, 141.8, 140.4, 136.4,
128.6 (2C), 128.4, 128.3 (2C), 128.2, 126.7, 117.2, 96.3, 82.2, 67.5,
66.8, 66.4, 53.9, 46.0, 27.6, 25.8. ESI-MS (M + H)+ m/z 418.0. HR
ESI-MS (M + H)+ m/z = 418.1772 (calcd for C24H24N3O4:
418.1767); tR = 1.28.
1
by isomer 16b (tR = 13.5 min). For isomer 16a: yellowish solid. H
NMR (500 MHz, DMSO-d6) δ: 10.88 (s, 1 H), 8.66 (d, J = 4.4 Hz, 1
H), 8.24 (d, J = 9.0 Hz, 1 H), 7.74 (d, J = 7.8 Hz, 1 H), 7.54 (d, J = 4.5
Hz, 1 H), 7.24 (d, J = 9.0 Hz, 1 H), 7.09 (d, J = 7.9 Hz, 1 H), 4.71 (d, J
= 6.4 Hz, 1 H), 4.01 (s, 3 H), 3.96 (m, 1 H), 3.84 (m, 1 H), 3.69−3.77
(m, 2 H), 3.60 (dd, J = 3.4, 13.2 Hz, 1 H), 3.53 (s, 2 H), 3.12 (ddd, J =
1.6, 5.1, 11.9 Hz, 1 H); 2.95 (t, J = 10.5 Hz, 1 H), 2.88 (dd, J = 9.2,
13.3 Hz, 1 H), 2.50 (overlaid m, 1 H), 2.05 (m, 1 H), 1.86 (m, 1 H),
1.58 (m, 1 H), 1.24 (br s, 1 H), 1.19 (m, 1 H). ESI-MS (M + H)+
482.4. HR ESI-MS (M + H)+ m/z = 482.1879 (calcd for
C24H28N5O4S: 482.1862); tR = 0.59. For isomer 16b: off-white solid;
mp = 172 °C. 1H NMR (300 MHz, DMSO-d6) δ: 10.93 (s, 1 H), 8.67
(d, J = 4.4 Hz, 1 H), 8.25 (d, J = 9.0 Hz, 1 H), 7.97 (d, J = 7.8 Hz, 1
H), 7.93 (d, J = 8.5 Hz, 1 H), 7.60 (d, J = 7.8 Hz, 1 H), 7.55 (d, J = 4.4
Hz, 1 H), 7.25 (d, J = 9.0 Hz, 1 H), 4.69 (d, J = 6.4 Hz, 1 H), 4.10 (s, 3
H), 3.84−4.1 (overlaid m, 3 H), 3.64 (s, 2 H), 3.50 (dd, J = 3.7, 12.6
Hz, 1 H), 3.21 (m, 1 H), 3.13 (t, J = 10.4 Hz, 1 H), 3.03 (dd, J = 8.9,
12.8 Hz, 1 H), 2.03 (m, 1 H), 1.79−1.50 (m, 3 H). 13C NMR (125
MHz, DMSO-d6) δ: 166.6, 161.2, 158.3, 149.2, 148.0, 145.8, 141.4,
141.1, 141.0, 136.4, 126.2, 117.1, 116.4, 113.2, 80.6, 72.4, 72.1, 53.9,
53.6, 51.6, 34.8, 30.8, 29.2. ESI-MS (M + H)+ m/z 482.2. HR ESI-MS
(M + H)+ m/z = 482.1859 (calcd for C24H28N5O4S: 482.1862); tR =
0.58.
tert-Butyl ((3R,6S)-6-((4R,5R)-5-(6-Methoxy-1,5-naphthyri-
din-4-yl)-2-oxo-1,3-dioxolan-4-yl)tetrahydro-2H-pyran-3-yl)-
carbamate (18). The compound 18 was prepared in 68% yield from
the diol 1729 according to general procedure A. White solid; mp = 206
°C. 1H NMR (500 MHz, CDCl3) δ: 8.84 (d, J = 4.5 Hz, 1 H), 8.29 (d,
J = 9.1 Hz, 1 H), 7.61 (d, J = 4.5 Hz, 1 H), 7.22 (d, J = 9.1 Hz, 1 H),
7.19 (d, J = 0.8 Hz, 1 H), 6.22 (s, 1 H), 4.71 (d, J = 1.9 Hz, 1 H), 4.29
(ddd, J = 2.0, 4.7, 10.7 Hz, 1 H), 4.09 (s, 3 H), 3.80 (dt, J = 2.2, 11.7, 1
H), 3.69 (m, 1 H), 3.13 (m, 1 H), 2.24 (m, 1 H), 1.91 (m, 1 H), 1.72
(m, 1 H), 1.48 (s, 9 H), 1.35 (m, 1 H). 13C NMR (125 MHz, CDCl3)
δ: 162.2, 154.8, 147.9, 142.1, 141.1, 140.7, 138.8, 135.0, 121.9, 117.5,
82.7, 79.7, 76.5, 76.0, 71.6, 54.2, 46.1, 30.0, 28.6 (3C), 25.7. ESI-MS
(M + H)+ m/z 446.2. HR ESI-MS (M + H)+ m/z = 446.1924 (calcd
for C22H28N3O7: 446.1927); tR = 1.2.
(3R,6S)-(6-[2-(6-Methoxy-[1,5]naphthyridin-4-yl)-acetyl]-tet-
rahydro-pyran-3-yl)-carbamic Acid Benzyl Ester (13). To a
solution of 10b (0.24 g, 0.57 mmol) in MeOH (2 mL) and THF (2
mL) was added a solution of mercury oxide (5.1 mL, prepared from
0.075 g of mercury oxide in 12 mL of a 4% (wt/wt) H2SO4 solution in
water). The reaction mixture was heated at 55 °C for 4 h. After cooling
to rt, NaHCO3 was added until pH 8 was reached. The aqueous layer
was extracted with EtOAc (2 × 100 mL). The combined extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by chromatography (DCM−MeOH
19:1) to afford 13 (0.17 g, 67% yield). White solid; mp = 173 °C. 1H
NMR (500 MHz, DMSO-d6) δ: 8.72 (d, J = 4.4 Hz, 1 H), 8.26 (d, J =
9.0 Hz, 1 H), 7.56 (d, J = 4.4 Hz, 1 H), 7.30−7.41 (m, 6 H), 7.25 (d, J
= 9.0 Hz, 1 H), 5.00−5.06 (m, 2 H), 4.35−4.47 (m, 2 H), 4.02 (m, 1
H), 3.95 (s, 3 H), 3.50 (m, 1 H), 3.15 (m, 1 H), 2.54 (overlaid m, 1
H), 1.96−2.03 (m, 2 H), 1.47−1.64 (m, 2 H). 13C NMR (125 MHz,
CDCl3) δ: 206.1, 161.6, 155.6, 147.5, 141.5, 140.8, 140.3, 136.2, 128.6
(2C), 128.3 (2C), 128.2, 128.1, 125.7, 166.6, 81.8, 70.8, 66.9, 53.7,
46.6, 40.3, 29.7, 27.0. ESI-MS (M + H)+ m/z 436.4. HR ESI-MS (M +
H)+ m/z = 436.1873 (calcd for C24H26N3O5: 436.1872); tR = 1.15.
(3R,6S)-(6-((1RS)-1-Hydroxy-2-(6-methoxy-[1,5]-
naphthyridin-4-yl)-ethyl)-tetrahydro-pyran-3-yl)-carbamic
Acid Benzyl Ester (14a). To a solution of 13 (0.17 g, 0.39 mmol) in
THF (2 mL) and MeOH (2 mL) was added NaBH4 (0.077 g). The
reaction was stirred 30 min, and water (5 mL) was added. The
volatiles were removed under reduced pressure, and the residue was
extracted twice with EtOAc (2 × 25 mL). The combined extracts were
concentrated to dryness, and the residue was purified chromatography
(DCM−MeOH 19:1) to afford 14a (0.045 g, 27% yield). White solid.
1H NMR (500 MHz, CDCl3) mixture of diastereomers δ: 8.69−8.75
tert-Butyl ((3R,6S)-6-((S)-1-Hydroxy-2-(6-methoxy-1,5-naph-
thyridin-4-yl)ethyl)tetrahydro-2H-pyran-3-yl)carbamate (19).
The compound 19 was prepared in 89% yield from carbonate 18
according to general procedure B. White solid; mp = 168 °C. 1H NMR
(500 MHz, CDCl3) δ: 8.71 (d, J = 4.4 Hz, 1 H), 8.25 (d, J = 9.0 Hz, 1
H), 7.50 (d, J = 4.5 Hz, 1 H), 7.15 (d, J = 9.0 Hz, 1 H), 4.28 (m, 1 H),
4.18 (ddd, J = 2.1, 4.8, 10.6 Hz, 1 H), 4.09 (s, 3 H), 4.02 (m, 1 H),
3.62−3.74 (m, 2 H), 3.41 (dd, J = 3.5, 13.3 Hz, 1 H), 3.32 (dd, J = 8.7,
13.3 Hz, 1 H), 3.25 (ddd, J = 2.1, 5.0, 11.3 Hz, 1 H), 3.04 (t, J = 10.7
Hz, 1 H), 2.17 (m, 1 H), 1.87 (m, 1 H), 1.71 (m, 1 H), 1.47 (s, 9 H),
1.31 (m, 1 H). 13C NMR (125 MHz, CDCl3) δ: 161.7, 155.1, 147.8,
145.1, 141.5, 141.1, 140.7, 125.6, 116.5, 79.6, 79.5, 73.6, 71.4, 53.9,
46.6, 35.2, 30.3, 28.4 (3C), 26.3. ESI-MS (M + H)+ m/z 404.0. HR
ESI-MS (M + H)+ m/z = 404.2191 (calcd for C21H30N3O5:
404.2185); tR = 1.01.
(two overlaid d, 2 × 0.5 H), 8.27 (d, J = 9.0 Hz, 0.5 H), 8.24 (d, J = 9.0
Hz, 0.5 H), 7.46−7.50 (m, 1 H), 7.33−7.41 (m, 5 H), 7.16 (d, J = 6.8
Hz, 0.5 H), 7.15 (d, J = 6.8 Hz, 0.5 H), 5.02−5.21 (m, 2 H), 4.46−4.55
(m, 1 H), 4.18−4.29 (m, 1 H), 4.10−4.12 (two overlaid s, 3 H), 4.02
(m, 0.5 H), 3.92 (m, 0.5 H), 3.65−3.83 (m, 1.5 H), 3.57 (m, 0.5 H),
3.21−3.43 (m, 3 H), 2.95−3.11 (m, 2 H), 2.10−2.24 (m, 1 H), 2.03
(m, 0.5 H), 1.87 (m, 0.5 H), 1.72 (m, 0.5 H), 1.56 (m, 0.5 H). ESI-MS
(M + H)+ 438.4. HR ESI-MS (M + H)+ m/z = 438.2032 (calcd for
C24H28N3O5: 438.2028); tR = 1.04.
(1RS)-1-((2S,5R)-5-Amino-tetrahydro-pyran-2-yl)-2-(6-
methoxy[1,5]naphthyridin-4 yl)-ethanol (14b). The compound
14b (0.029 g) was prepared in 92% yield starting from 14a (0.045 g,
0.10 mmol) according to general procedure C. Colorless oil. 1H NMR
(500 MHz, CDCl3) mixture of diastereomers δ: 8.72 (d, J = 4.4 Hz,
0.5 H), 8.70 (d, J = 4.4 Hz, 0.5 H), 8.26 (d, J = 9.1 Hz, 0.5 H), 8.23 (d,
K
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(S)-1-((2S,5R)-5-Aminotetrahydro-2H-pyran-2-yl)-2-(6-me-
thoxy-1,5-naphthyridin-4-yl)ethan-1-ol (20). The compound 20
was prepared in 98% yield from 19 according to general procedure B.
White solid; mp = 103 °C. 1H NMR (500 MHz, CDCl3) δ: 8.71 (d, J
= 4.4 Hz, 1 H), 8.24 (d, J = 9.0 Hz, 1 H), 7.50 (d, J = 4.5 Hz, 1 H),
7.15 (d, J = 9.0 Hz, 1 H), 4.09 (s, 3 H), 3.98−4.05 (m, 2 H), 3.43 (dd,
J = 3.5, 13.4 Hz, 1 H), 3.32 (dd, J = 8.7, 13.4 Hz, 1 H), 3.25 (ddd, J =
2.2, 5.0, 11.3 Hz, 1 H), 3.03 (t, J = 10.6 Hz, 1 H), 2.86 (m, 1 H), 2.11
(m, 1 H), 1.83 (m, 1 H), 1.66 (m, 1 H), 1.54 (br s, 3 H), 1.26 (m, 1
H). 13C NMR (125 MHz, CDCl3) δ: 161.7, 147.8, 145.1, 141.6, 141.1,
140.8, 125.6, 116.5, 79.6, 75.0, 73.8, 53.9, 47.5, 35.5, 33.4, 26.6. ESI-
MS (M + H)+ m/z 304.0. HR ESI-MS (M + H)+ m/z = 304.1657
(calcd for C16H21N3O3: 304.1661); tR = 0.46.
6-((((3R,6S)-6-((S)-1-Hydroxy-2-(6-methoxy-1,5-naphthyri-
din-4-yl)ethyl)tetrahydro-2H-pyran-3-yl)amino)methyl)-2H-
pyrido[3,2-b][1,4]thiazin-3(4H)-one (16b). Starting from 20 (0.59
g, 1.97 mmol) and 15a (0.42 g, 2.1 mmol), 16b (0.45 g, 47% yield)
was prepared using the general procedure D. Analytical data recorded
for this sample matched those recorded previously (vide infra).
6-((((3R,6S)-6-((S)-1-Hydroxy-2-(6-methoxy-1,5-naphthyri-
din-4-yl)ethyl)tetrahydro-2H-pyran-3-yl)amino)methyl)-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one (21). Starting from 20 (0.709
g, 2.34 mmol) and 15b (0.42 g, 2.34 mmol), 21 (0.19 g, 18% yield)
was prepared according to the general procedure D. Off-white solid;
mp = 188 °C. 1H NMR (300 MHz, DMSO-d6) δ: 11.14 (s, 1 H), 8.63
(d, J = 4.4 Hz, 1 H), 8.21 (d, J = 9.1 Hz, 1 H), 7.50 (d, J = 4.5 Hz, 1
H), 7.28 (d, J = 8.1 Hz, 1 H), 7.21 (d, J = 9.1 Hz, 1 H), 6.99 (d, J = 8.1
Hz, 1 H), 4.59 (s, 2 H), 4.53 (d, J = 6.4 Hz, 1 H), 3.98 (s, 3 H), 3.98
(overlaid m, 1 H), 3.85 (m, 1 H), 3.62−3.73 (m, 2 H), 3.42 (dd, J =
3.8, 12.8 Hz, 1 H), 3.10 (m, 1 H), 2.88−3.00 (m, 2 H), 1.95−2.07 (m,
2 H), 1.63 (m, 1 H), 1.48 (m, 1 H), 1.19 (m, 1 H). ESI-MS (M + H)+
m/z 466.3.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]dioxino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-methoxy-1,5-
naphthyridin-4-yl)ethan-1-ol (26a). Starting from 20 (0.08 g, 0.26
mmol) and 22 (0.044 g, 0.26 mmol), 26a (0.044 g, 47% yield) was
prepared according to the general procedure E. Off-white solid; mp =
116 °C. 1H NMR (500 MHz, CDCl3) δ 8.71 (d, J = 4.4 Hz, 1 H), 8.23
(d, J = 9.0 Hz, 1 H), 8.13 (s, 1 H), 7.49 (d, J = 4.4 Hz, 1 H), 7.14 (d, J
= 9.0 Hz, 1 H), 6.82 (s, 1 H), 4.33−4.36 (m, 2 H), 4.28−4.31 (m, 2
H), 4.16 (m, 1 H), 4.08 (s, 3 H), 3.99 (m, 1 H), 3.78−3.85 (m, 2 H),
3.70 (m, 1 H), 3.42 (dd, J = 3.4, 13.4 Hz, 1 H), 3.25−3.32 (m, 2 H),
3.15 (t, J = 10.6 Hz, 1 H), 2.71 (m, 1 H), 2.17 (m, 1 H), 1.83 (m, 1
H), 1.59−1.71 (m, 2 H), 1.34 (m, 1 H). 13C NMR (125 MHz, CDCl3)
δ: 161.7, 153.4, 150.2, 147.9, 145.1, 141.7, 141.1, 140.8, 140.1, 138.9,
125.6, 116.4, 110.7, 80.0, 73.8, 72.8, 65.0, 64.1, 53.9, 53.3, 52.1, 35.5,
30.8, 26.5. ESI-MS (M + H)+ m/z 453.0. HR ESI-MS (M + H)+ m/z =
453.2142 (calcd for C24H29N4O5: 453.2137); tR = 0.58.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]oxathiino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-methoxy-1,5-
naphthyridin-4-yl)ethan-1-ol (26b). Starting from 20 (0.1 g, 0.33
mmol) and 23 (0.06 g, 0.33 mmol), 26b (0.14 g, 90% yield) was
prepared according to the general procedure E. White solid; mp = 50
°C. 1H NMR (500 MHz, CDCl3) δ: 8.71 (d, J = 4.4 Hz, 1 H), 8.24 (d,
J = 9.0 Hz, 1 H), 8.05 (s, 1 H), 7.49 (d, J = 4.5 Hz, 1 H), 7.14 (d, J =
9.0 Hz, 1 H), 7.00 (s, 1 H), 4.41−4.45 (m, 2 H), 4.17 (m, 1 H), 4.08
(s, 3 H), 3.99 (m, 1 H), 3.78−3.84 (m, 2 H), 3.42 (dd, J = 3.4, 13.4
Hz, 1 H), 3.26−3.33 (m, 2 H), 3.18−3.31 (m, 2 H), 3.15 (t, J = 10.5
Hz, 1 H), 2.72 (m, 1 H), 2.58 (br s, 2 H), 2.17 (m, 1 H), 1.84 (m, 1
H), 1.65 (m, 1 H), 1.36 (m, 1 H). 13C NMR (125 MHz, CDCl3) δ:
161.7, 151.3, 147.8, 147.5, 145.2, 141.6, 141.1, 140.7, 139.1, 129.4,
125.6, 120.0, 116.5, 80.0, 73.8, 72.6, 64.8, 54.0, 53.3, 51.7, 35.6, 30.7,
26.5, 25.7. ESI-MS (M + H)+ m/z 469.0. HR ESI-MS (M + H)+ m/z =
469.191 (calcd for C24H29N4O4S: 469.1909); tR = 0.61.
Hz, 1 H), 7.02 (s, 1 H), 4.52−4.55 (m, 2 H), 4.37−4.41 (m, 2 H), 4.16
(ddd, J = 2.2, 4.4, 10.8 Hz, 1 H), 4.08 (s, 3 H), 4.03 (apparent d, J =
5.4 Hz, 2 H), 3.99 (m, 1 H), 3.72 (br s, 1 H), 3.42 (dd, J = 3.5, 13.4
Hz, 1 H), 3.26−3.33 (m, 2 H), 3.13 (t, J = 10.6 Hz, 1 H), 2.73 (m, 1
H), 2.20 (m, 1 H), 1.84 (m, 1 H), 1.59−1.72 (m, 2 H), 1.32 (m, 1 H).
13C NMR (125 MHz, CDCl3) δ: 161.8, 159.3, 155.5, 147.8, 145.1,
144.3, 141.6, 141.1, 140.8, 125.6, 116.5, 113.4, 80.0, 73.8, 72.6, 64.8
(2C), 54.0, 53.4, 50.4, 35.5, 30.8, 26.5. ESI-MS (M + H)+ m/z 454.1.
HR ESI-MS (M + H)+ m/z = 454.2091 (calcd for C23H28N5O5:
454.2090); tR = 0.52.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]oxathiino[2,3-c]-
pyridazin-3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-
methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (26d). Starting from
18d (0.072 g, 0.23 mmol) and 25 (0.046 g, 0.25 mmol), 26d (0.070 g,
63% yield) was prepared according to the general procedure E. Off-
white foam. 1H NMR (300 MHz, CDCl3) δ: 8.63 (d, J = 4.4 Hz, 1 H),
8.21 (d, J = 9.0 Hz, 1 H), 7.53 (s, 1 H), 7.50 (d, J = 4.5 Hz, 1 H), 7.21
(d, J = 9.0 Hz, 1 H), 4.54−4.58 (m, 2 H), 4.52 (d, J = 6.4 Hz, 1 H),
3.98 (s, 3 H), 3.95 (m, 1 H), 3.80−3.89 (m, 3 H), 3.42 (dd, J = 3.9,
12.9 Hz, 1 H), 3.35−3.30 (m, 2 H), 3.09 (m, 1 H), 2.95 (dd, J = 9.2,
11.8 Hz, 1 H), 2.90 (t, J = 10.7 Hz, 1 H), 2.43 (overlaid m, 1 H), 2.21
(m, 1 H), 2.01 (m, 1 H), 1.62 (m, 1 H); 1.47 (m, 1 H), 1.16 (m, 1 H).
ESI-MS (M + H)+ m/z 470.2; .HR ESI-MS (M + H)+ m/z = 470.1861
(calcd for C23H28N5O4S: 470.1862); tR = 0.54.
tert-Butyl ((3R,6S)-6-((4R,5R)-5-(3-Fluoro-6-methoxy-1,5-
naphthyridin-4-yl)-2-oxo-1,3-dioxolan-4-yl)tetrahydro-2H-
pyran-3-yl)carbamate (28a). The compound 28a was prepared in
74% yield from diol 27a29 using the general procedure A. White solid;
mp = 135 °C. 1H NMR (500 MHz, CDCl3) δ: 8.72 (s, 1 H), 8.24 (d, J
= 9.1 Hz, 1 H), 7.15 (d, J = 9.1 Hz, 1 H), 6.34 (d, J = 5.8 Hz, 1 H),
4.86 (t, J = 5.5 Hz, 1 H), 4.29 (m, 1 H), 4.18 (m, 1 H), 4.11 (s, 3 H),
3.59−3.69 (m, 2 H), 3.11 (t, J = 10.8 Hz, 1 H), 2.17 (m, 1 H), 1.82
(m, 1 H), 1.45 (s, 9 H), 1.43 (overlaid m, 1 H), 1.39 (m, 1 H). 13C
NMR (125 MHz, CDCl3) δ: 163.3, 156.2 (d, J = 263 Hz), 155.0,
154.9, 140.6, 140.3 (d, J = 4 Hz), 139.4 (d, J = 3 Hz), 138.1 (d, J = 28
Hz), 123.8 (d, J = 8 Hz), 116.6 (d, J = 2 Hz), 81.6, 79.9, 76.3, 71.1,
70.5 (d, J = 6 Hz), 54.6, 46.3, 29.6, 28.4 (3C), 26.6. ESI-MS (M + H)+
m/z 464.3. HR ESI-MS (M + H)+ m/z = 464.1833 (calcd for
C22H27N3O7F: 464.1833); tR = 1.27.
tert-Butyl ((3R,6S)-6-((4R,5R)-5-(6-Fluoro-3-methoxyquinox-
alin-5-yl)-2-oxo-1,3-dioxolan-4-yl)tetrahydro-2H-pyran-3-yl)-
carbamate (28b). The compound 28b was prepared in 66% yield
from the diol 27b29 according to general procedure A. White solid; mp
1
= 183.8 °C. H NMR (300 MHz, DMSO-d6) δ: 8.65 (s, 1 H), 8.17
(dd, J = 5.9, 9.2 Hz, 1 H), 7.62 (t, J = 9.5 Hz, 1 H), 6.78 (d, J = 7.7 Hz,
1 H), 6.28 (d, J = 6.0 Hz, 1 H), 5.01 (dd, J = 3.6, 5.9 Hz, 1 H), 4.03 (s,
3 H), 3.92 (m, 1 H), 3.51 (m, 1 H), 3.34 (m, 1 H), 3.07 (t, J = 10.5
Hz, 1 H), 1.89 (m, 1 H), 1.64 (m, 1 H), 1.33−1.50 (m, 2 H), 1.36 (s,
9H). 13C NMR (125 MHz, CDCl3) δ: 161.3 (d, J = 255 Hz), 157.8,
155.2, 155.0 (d, J = 5 Hz), 140.0 (d, J = 7 Hz), 139.6 (d, J = 3 Hz),
136.0, 132.9 (d, J = 11 Hz), 116.5 (d, J = 12 Hz), 115.7 (d, J = 26 Hz),
81.8, 79.8, 75.8, 71.4, 70.7 (d, J = 7 Hz), 54.6, 46.1, 28.3 (3C), 25.3.
ESI-MS (M + H)+ m/z 464.6. HR ESI-MS (M + H)+ m/z = 464.1826
(calcd for C22H27N3O7F: 464.1833); tR = 1.26.
tert-Butyl ((3R,6S)-6-((4R,5R)-5-(7-Fluoro-2-methoxyquino-
lin-8-yl)-2-oxo-1,3-dioxolan-4-yl)tetrahydro-2H-pyran-3-yl)-
carbamate (28c). The compound 28c was prepared in 63% yield
from the diol 27c29 according to general procedure A. White solid; mp
1
= 93 °C. H NMR (500 MHz, CDCl3) δ: 8.00 (d, J = 8.9 Hz, 1 H),
7.79 (dd, J = 6.1, 8.9 Hz, 1 H), 7.19 (t, J = 9.3 Hz, 1 H), 6.94 (d, J =
8.9 Hz, 1 H), 6.33 (m, 1 H), 5.05 (m, 1 H), 4.29 (m, 1 H), 4.23 (m, 1
H), 4.10 (s, 3 H) 3.67 (m, 1 H), 3.49 (m, 1 H), 3.12 (t, J = 10.7 Hz, 1
H), 2.19 (m, 1 H); 1.65−1.78 (m, 2 H), 1.46 (s, 9H), 1.34 (m, 1 H).
13C NMR (125 MHz, CDCl3) δ: 163.3, 161.8 (d, J = 252 Hz), 155.4,
155.0, 145.9 (d, J = 7 Hz), 139.1, 131.4 (d, J = 12 Hz), 122.4, 116.0 (d,
J = 10 Hz), 113.3 (d, J = 9 Hz), 113.2 (J = 14 Hz), 81.7, 79.7, 76.0,
71.4, 71.1 (d, J = 7 Hz), 54.1, 46.1; 29.9, 28.3 (3C), 26.6. ESI-MS (M
+ H)+ m/z 463.0. HR ESI-MS (M + H)+ m/z = 463.1888 (calcd for
C23H28N2O7F: 463.1880) ; tR = 1.33.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]dioxino[2,3-c]pyridazin-
3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-methoxy-
1,5-naphthyridin-4-yl)ethan-1-ol (26c). Starting from 18d (0.08 g,
0.26 mmol) and 24 (0.046 g, 0.26 mmol), 26c (0.082 g, 67% yield)
was prepared according to the general procedure E. Off-white solid;
mp = 45 °C. H NMR (500 MHz, CDCl3) δ: 8.70 (d, J = 4.4 Hz, 1
H), 8.23 (d, J = 9.0 Hz, 1 H), 7.49 (d, J = 4.5 Hz, 1 H), 7.14 (d, J = 9.0
1
tert-Butyl ((3R,6S)-6-((S)-2-(3-Fluoro-6-methoxy-1,5-naph-
thyridin-4-yl)-1-hydroxyethyl)tetrahydro-2H-pyran-3-yl)-
L
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
carbamate (29a). The compound 29a was prepared in 78% yield
from carbonate 28a according to general procedure B. White solid; mp
= 170 °C. 1H NMR (500 MHz, CDCl3) δ: 8.67 (s, 1 H), 8.22 (d, J =
9.0 Hz, 1 H), 7.10 (d, J = 9.0 Hz, 1 H), 4.28 (m, 1 H), 4.18 (ddd, J =
2.0, 4.7, 10.7 Hz, 1 H), 4.09 (s, 3 H), 4.00 (m, 1 H), 3.63 (m, 1 H),
3.52 (m, 1 H), 3.38−3.46 (m, 2 H), 3.28 (ddd, J = 2.5, 4.4, 11.0 Hz, 1
H), 3.06 (t, J = 10.7 Hz, 1 H), 2.18 (d, J = 12.2 Hz, 1 H), 1.71−1.87
(m, 2 H), 1.47 (s, 9 H), 1.33 (m, 1 H). 13C NMR (125 MHz, CDCl3)
δ: 162.7, 157.6 (d, J = 256 Hz), 155.1, 141.9 (d, J = 6 Hz), 140.6, 138.6
(d, J = 2 Hz), 138.2 (d, J = 29 Hz), 129.1 (d, J = 13 Hz), 115.4 (d, J =
3 Hz), 79.6, 79.5, 73.3, 71.4, 54.1, 46.5, 30.3, 28.4 (3C), 27.8, 26.6.
ESI-MS (M + H)+ m/z 422.2. HR ESI-MS (M + H)+ m/z = 422.2094
(calcd for C21H29N3O5F: 422.2091); tR = 1.21.
tert-Butyl ((3R,6S)-6-((S)-2-(6-Fluoro-3-methoxyquinoxalin-
5-yl)-1-hydroxyethyl)tetrahydro-2H-pyran-3-yl)carbamate
(29b). The compound 29b was prepared in 73% yield from carbonate
28b according to general procedure B. Colorless foam. 1H NMR (300
MHz, CDCl3) δ: 8.54 (s, 1 H), 7.88 (dd, J = 5.9, 9.1 Hz, 1 H), 7.47 (t,
J = 9.2 Hz, 1 H), 4.38 (d, J = 6.1 Hz, 1 H), 4.04 (s, 4 H), 3.74−3.84
(m, 2 H), 3.24−3.33 (m, 2 H), 3.00−3.13 (m, 2 H), 2.79 (t, J = 10.5
Hz, 1 H), 2.53 (overlaid m, 1 H), 1.90 (m, 1 H), 1.44−1.65 (m, 2 H),
1.27 (br s, 2 H), 1.10 (m, 1 H). ESI-MS (M + H)+ m/z 422.5. HR ESI-
MS (M + H)+ m/z = 422.2094 (calcd for C21H29N3O5F: 422.2091); tR
= 1.21.
tert-Butyl ((3R,6S)-6-((S)-2-(7-Fluoro-2-methoxyquinolin-8-
yl)-1-hydroxyethyl)tetrahydro-2H-pyran-3-yl)carbamate (29c).
The compound 29c was prepared in 70% yield from carbonate 28c
according to general procedure B. White solid; mp = 67 °C. 1H NMR
(500 MHz, CDCl3) δ: 7.99 (d, J = 8.8 Hz, 1 H), 7.61 (dd, J = 6.1, 8.8
Hz, 1 H), 7.19 (t, J = 9.0 Hz, 1 H), 6.89 (d, J = 8.8 Hz, 1 H), 4.27 (br
s, 1 H), 4.19 (ddd, J = 2.1, 4.8, 10.7 Hz, 1 H), 4.08 (s, 3 H), 4.02 (br s,
1 H), 3.97 (m, 1 H), 3.67 (br s, 1 H), 3.45 (ddd, J = 0.8, 2.1, 11.0 Hz,
1 H), 3.38 (ddd, J = 1.8, 8.4, 11.0 Hz, 1 H), 3.29 (ddd, J = 2.1, 4.7,
11.2 Hz, 1 H), 3.06 (t, J = 10.7 Hz, 1 H) 2.17 (m, 1 H), 1.86 (m, 1 H),
1.73 (m, 1 H), 1.47 (m, 9 H), 1.32 (m, 1 H). 13C NMR (125 MHz,
CDCl3) δ: 162.8, 161.7 (d, J = 247 Hz), 155.1, 146.5 (d, J = 9 Hz),
139.4, 127.2 (d, J = 11 Hz), 121.9, 120.6 (d, J = 16 Hz), 113.8 (d, J =
27 Hz), 111.9 (d, J = 2 Hz), 79.8, 79.5, 74.2, 71.5, 53.7, 46.6, 30.5, 28.4
(3C), 27.7, 26.4. ESI-MS (M + H)+ m/z 421.0. HR ESI-MS (M + H)+
m/z = 421.2139 (calcd for C22H30N2O5F: 421.2138); tR =1.35.
(S)-1-((2S,5R)-5-Aminotetrahydro-2H-pyran-2-yl)-2-(3-fluo-
ro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (30a). The com-
pound 30a was prepared in 90% yield from 29a according to general
procedure B. White solid; mp = 118 °C. 1H NMR (500 MHz, CDCl3)
δ:. 8.67 (s, 1 H), 8.22 (d, J = 9.0 Hz, 1 H), 7.10 (d, J = 9.0 Hz, 1 H),
4.10 (s, 3 H), 3.97−4.04 (m, 2 H), 3.56 (br s, 1 H), 3.46 (ddd, J = 1.5,
4.0, 13.0 Hz, 1 H), 3.40 (ddd, J = 1.4, 8.4, 13.0 Hz, 1 H), 3.29 (ddd, J
= 2.3, 4.5, 11.2 Hz, 1 H), 3.05 (t, J = 10.6 Hz, 1 H), 2.86 (m, 1 H),
2.12 (m, 1 H), 1.79 (m, 1 H), 1.70 (m, 1 H), 1.28 (m, 1 H), 1.10 (br s,
2 H). 13C NMR (125 MHz, CDCl3) δ: 162.7, 157.6 (d, J = 256 Hz),
141.9 (d, J = 6 Hz), 138.6, 138.5, 138.2 (d, J = 29 Hz), 129.2 (d, J = 13
Hz), 115.4 (d, J = 3 Hz), 79.6, 75.0, 73.4, 54.0, 47.4, 33.5, 28.0, 26.9.
ESI-MS (M + H)+ m/z 322.2. HR ESI-MS (M + H)+ m/z = 322.1567
(calcd for C16H21N3O3F: 322.1567); tR = 0.57.
(S)-1-((2S,5R)-5-Aminotetrahydro-2H-pyran-2-yl)-2-(7-fluo-
ro-2-methoxyquinolin-8-yl)ethan-1-ol (30c). The compound 30c
was prepared in quantitative yield from 29c according to general
1
procedure B. White solid; mp = 65 °C. H NMR (500 MHz, CDCl3)
δ: 7.99 (d, J = 8.9 Hz, 1 H), 7.61 (dd, J = 6.0, 8.8 Hz, 1 H), 7.19 (t, J =
9.0 Hz, 1 H), 6.89 (d, J = 8.8 Hz, 1 H), 4.08 (s, 3 H), 4.08 (overlaid1
m, 1 H), 3.95 (m, 1 H), 3.45 (ddd, J = 1.6, 3.8, 13.4 Hz, 1 H), 3.38
(ddd, J = 1.8, 8.1, 13.4 Hz, 1 H), 3.30 (ddd, J = 2.0, 5.0, 11.2 Hz, 1 H),
3.12 (t, J = 10.7 Hz, 1 H), 2.94 (m, 1 H), 2.82 (br s, 3 H), 2.13 (m, 1
H), 1.85 (m, 1 H), 1.68 (m, 1 H), 1.35 (m, 1 H). 13C NMR (125
MHz, CDCl3) δ: 162.8, 161.7 (d, J = 246 Hz), 146.5 (d, J = 9 Hz),
139.5, 127.3 (d, J = 11 Hz), 121.9, 120.4 (d, J = 16 Hz), 113.8 (d, J =
27 Hz), 112.0 (d, J = 2 Hz), 79.9, 74.3, 73.6, 53.8, 47.4, 32.3, 27.8,
26.5. ESI-MS (M + H)+ m/z 321.0. HR ESI-MS (M + H)+ m/z =
321.1615 (calcd for C17H22N2O3F: 321.1614); tR = 0.67.
6-((((3R,6S)-6-((S)-2-(3-Fluoro-6-methoxy-1,5-naphthyridin-
4-yl)-1-hydroxyethyl)tetrahydro-2H-pyran-3-yl)amino)-
methyl)-2H-pyrido[3,2-b][1,4]thiazin-3(4H)-one (31a). Starting
from 30a (0.092 g, 0.28 mmol) and 15a (0.055 g, 0.28 mmol), 31a
(0.113 g, 79% yield) was prepared according to the general procedure
D. Yellowish solid; mp = 93 °C. 1H NMR (500 MHz, CDCl3) δ: 8.67
(s, 1 H), 8.29 (br s, 1 H), 8.22 (d, J = 9.0 Hz, 1 H), 7.60 (d, J = 7.8 Hz,
1 H), 7.10 (d, J = 9.0 Hz, 1 H), 6.98 (d, J = 7.8 Hz, 1 H), 4.17 (ddd, J
= 2.1, 4.3, 10.7 Hz, 1 H), 4.09 (s, 3 H), 3.99 (m, 1 H), 3.83−3.90 (m,
2 H), 3.50 (s, 2 H), 3.46 (ddd, J = 1.2, 4.0, 13.4 Hz, 1 H), 3.40 (ddd, J
= 1.2, 8.5, 13.4 Hz, 1 H), 3.33 (ddd, J = 2.2, 4.3, 11.2 Hz, 1 H), 3.18 (t,
J = 10.6 Hz, 1 H), 2.73 (m, 1 H), 2.18 (m, 1 H), 1.80 (m, 1 H), 1.74
(br s, 2H), 1 0.69 (m, 1 H), 1.40 (m, 1 H). 13C NMR (125 MHz,
CDCl3) δ: 165.6, 162.7, 157.5 (d, J = 256 Hz), 156.8, 148.2, 141.8 (d, J
= 6 Hz), 140.6, 138.6 (d, J = 2 Hz), 138.1 (d, J = 9 Hz), 136.2, 129.2
(d, J = 13 Hz), 117.9, 115.4 (d, J = 3 Hz), 113.6, 80.1, 73.4, 72.5, 54.1,
53.3, 51.5, 30.7, 29.7, 28.0, 26.7. ESI-MS (M + H)+ m/z 499.9.
6-((((3R,6S)-6-((S)-2-(3-Fluoro-6-methoxy-1,5-naphthyridin-
4-yl)-1-hydroxyethyl)tetrahydro-2H-pyran-3-yl)amino)-
methyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (31b). Starting
from 30a (0.092 g, 0.28 mmol) and 15b (0.055 g, 0.28 mmol), 31b
(0.119 g, 79% yield) was prepared according to the general procedure
1
D. Yellowish solid; mp = 187−188 °C. H NMR (300 MHz, CDCl3)
δ: 8.67 (s, 1 H), 8.52 (br s, 1 H), 8.22 (d, J = 9.0 Hz, 1 H), 7.23 (d, J =
8.0 Hz, 1 H), 7.10 (d, J = 9.0 Hz, 1 H), 6.94 (d, J = 8.0 Hz, 1 H), 4.67
(s, 2 H), 4.17 (m, 1 H), 4.09 (s, 3 H), 3.99 (m, 1 H), 3.80−3.87 (m, 2
H), 3.45 (ddd, J = 1.2, 4.0, 13.4 Hz, 1 H), 3.40 (ddd, J = 1.2, 8.5, 13.4
Hz, 1 H), 3.33 (ddd, J = 2.2, 4.3, 11.2 Hz, 1 H), 3.16 (t, J = 10.6 Hz, 1
H), 2.72 (m, 1 H), 2.16 (m, 1 H), 1.80 (m, 1 H), 1.59−1.73 (m, 3 H),
1.38 (m, 1 H). 13C NMR (125 MHz, CDCl3) δ: 165.3, 162.7, 157.4 (d,
J = 256 Hz), 151.9, 141.8 (d, J = 6 Hz), 140.6, 140.0, 138.6 (d, J = 2
Hz), 138.3, 138.1 (d, J = 9 Hz), 129.2 (d, J = 13 Hz), 124.1, 118.1,
115.4 (d, J = 2 Hz), 80.1, 73.4, 72.7, 67.3, 54.1, 53.3, 51.4, 30.8, 28.1,
26.7. ESI-MS (M + H)+ m/z 484.3. HR ESI-MS (M + H)+ m/z =
484.1996 (calcd for C24H27N5O5F: 484.1996); tR = 0.64.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]dioxino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(3-fluoro-6-me-
thoxy-1,5-naphthyridin-4-yl)ethan-1-ol (32a). Starting from 30a
(1.47 g, 4.6 mmol) and 22 (0.758 g, 4.6 mmol), 32a (1.31 g, 61%
yield) was prepared according to the general procedure E. Off-white
1
solid. H NMR (300 MHz, CDCl3) δ: 8.72 (s, 1 H), 8.24 (d, J = 9.0
(S)-1-((2S,5R)-5-Aminotetrahydro-2H-pyran-2-yl)-2-(6-fluo-
ro-3-methoxyquinoxalin-5-yl)ethan-1-ol (30b). The compound
30b was prepared in quantitative yield from 29b according to general
Hz, 1 H), 7.99 (s, 1 H), 7.19 (d, J = 9.0 Hz, 1 H), 6.91 (s, 1 H), 4.53
(d, J = 6.1 Hz, 1 H), 4.30−4.37 (m, 2 H), 4.23−4.27 (m, 2 H), 4.00 (s,
3 H), 3.85−3.96 (m, 2 H), 3.59−3.70 (m, 2 H), 3.31 (m, 1 H), 3.07−
3.10 (m, 2 H), 2.90 (t, J = 10.5 Hz, 1 H), 2.41 (m, 1 H), 2.09 (m, 1
H), 2.00 (m, 1 H), 1.64 (m, 1 H), 1.47 (m, 1 H), 1.19 (m, 1 H). 13C
NMR (125 MHz, CDCl3) δ: 162.6, 157.6 (d, J = 256 Hz), 153.3,
150.2, 141.9 (d, J = 6 Hz), 140.6, 140.1, 138.9, 138.5 (d, J = 2 Hz),
138.2 (d, J = 28 Hz), 129.2 (d, J = 13 Hz), 115.3 (d, J = 2 Hz), 110.7,
80.0, 73.4, 72.7, 65.0, 64.1, 54.0, 53.2, 52.0, 30.7, 28.0, 26.7. ESI-MS
(M + H)+ m/z 471.2. HR ESI-MS (M + H)+ m/z = 471.2054 (calcd
for C24H28N4O5F: 471.2043); tR = 0.67.
1
procedure B. White solid; mp = 139−140 °C. H NMR (500 MHz,
CDCl3) δ: 8.46 (s, 1 H), 7.92 (dd, J = 5.8, 9.1 Hz, 1 H), 7.36 (t, J = 9.1
Hz, 1 H), 4.12 (s, 3 H), 4.04 (ddd, J = 2.2, 4.5, 10.7 Hz, 1 H), 3.93 (m,
1 H), 3.42 (ddd, J = 1.5, 4.0, 13.0 Hz, 1 H), 3.36 (ddd, J = 1.4, 8.4,
13.0 Hz, 1 H), 3.26 (ddd, J = 2.3, 4.5, 11.1 Hz, 1 H), 3.06 (t, J = 10.6
Hz, 1 H), 2.86 (m, 1 H), 2.11 (m, 1 H), 1.77 (m, 1 H), 1.69 (m, 1 H),
1.29 (m, 1 H), 1.25−2.20 (br s, 3 H). 13C NMR (125 MHz, CDCl3) δ:
161.7 (d, J = 248 Hz), 157.4, 140.3 (d, J = 10 Hz), 138.2 (d, J = 3 Hz),
136.0, 128.7 (d, J = 11 Hz), 120.7 (d, J = 17 Hz), 116.1 (d, J = 27 Hz),
79.5, 74.9, 73.8, 53.9, 47.4, 33.4, 28.0, 27.0. ESI-MS (M + H)+ m/z
322.0. HR ESI-MS (M + H)+ m/z = 322.1567 (calcd for
C16H21N3O3F: 322.1567); tR = 0.59.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]oxathiino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(3-fluoro-6-me-
thoxy-1,5-naphthyridin-4-yl)ethan-1-ol (32b). Starting from 30a
(5.0 g, 15.7 mmol) and 23 (2.87 g, 15.7 mmol), 32b (5.47 g, 71%
M
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
yield) was prepared according to the general procedure E. Off-white
solid. 1H NMR (300 MHz, CDCl3) δ: 8.72 (d, J = 0.6 Hz, 1 H), 8.25
(d, J = 9.0 Hz, 1 H), 7.92 (s, 1 H), 7.19 (d, J = 9.0 Hz, 1 H), 7.13 (s, 1
H), 4.53 (d, J = 6.1 Hz, 1 H) 4.33−4.37 (m, 2 H), 4.00 (s, 3 H), 3.85−
3.96 (m, 2 H), 3.59−3.70 (m, 2 H), 3.31 (m, 1 H), 3.21−3.25 (m, 2
H), 3.06−3.16 (m, 2 H), 2.90 (t, J = 10.5 Hz, 1 H), 2.41 (m, 1 H),
1.95−2.05 (m, 2 H), 1.65 (m, 1 H), 1.48 (m, 1 H), 1.17 (m, 1 H). 13C
NMR (125 MHz, CDCl3) δ: 162.6, 157.5 (d, J = 256 Hz), 151.5,
147.4, 141.8 (d, J = 6 Hz), 140.5, 139.2, 138.5 (d, J = 2 Hz), 138.2 (d, J
= 29 Hz), 129.3, 129.2 (d, J = 13 Hz), 120.0, 115.3 (d, J = 2 Hz), 80.0,
73.4, 72.7, 64.8, 54.1, 53.2, 51.8, 30.7, 28.0, 26.7, 25.7. ESI-MS (M +
H)+ m/z 487.3. HR ESI-MS (M + H)+ m/z = 487.1821 (calcd for
C24H28N4O4FS: 487.1815); tR = 0.71.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]dioxino[2,3-c]pyridazin-
3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(3-fluoro-6-
methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (32c). Starting from
30a (0.053 g, 0.165 mmol) and 24 (0.027 g, 0.17 mmol), 32c (0.02 g,
25% yield) was prepared according to the general procedure E. Off-
white solid. 1H NMR (300 MHz, CDCl3) δ: 8.73 (s, 1 H), 8.25 (d, J =
9.0 Hz, 1 H), 7.20 (d, J = 9.1 Hz, 1 H), 7.17 (s, 1 H), 4.56 (d, J = 6.0
Hz, 1 H) 4.47−4.51 (m, 2 H), 4.36−4.40 (m, 2 H),), 4.00 (s, 3 H),
3.85−3.96 (m, 2 H), 3.81−3.84 (m, 2 H), 3.31 (m, 1 H), 3.07−3.17
(m, 2 H), 2.90 (t, J = 10.5 Hz, 1 H), 2.41 (m, 1 H), 2.26 (m, 1 H),
2.00 (m, 1 H), 1.64 (m, 1 H), 1.49 (m, 1 H), 1.17 (m, 1 H). ESI-MS
(M + H)+ m/z 472.2. HR ESI-MS (M + H)+ m/z = 472.1993 (calcd
for C23H27N5O5F: 472.1996); tR = 0.61.
151.4, 147.4, 140.3 (d, J = 10 Hz), 139.2, 138.2 (d, J = 3 Hz), 136.0,
129.3, 128.7 (d, J = 11 Hz), 120.7 (d, J = 17 Hz), 120.0, 116.1 (d, J =
27 Hz), 79.9, 73.8, 72.6, 64.8, 53.9, 53.3, 51.8, 30.7, 28.0, 26.9, 25.7.
ESI-MS (M + H)+ m/z 487.2.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]dioxino[2,3-c]pyridazin-
3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-fluoro-3-
methoxyquinoxalin-5-yl)ethan-1-ol (33c). Starting from 30b
(0.297 g, 0.92 mmol) and 24 (0.153 g, 0.92 mmol), 33c (0.124 g,
28% yield) was prepared according to the general procedure E. Off-
1
white solid. H NMR (300 MHz, DMSO-d6) δ: 8.53 (s, 1 H), 7.88
(dd, J = 5.9, 9.1 Hz, 1 H), 7.46 (t, J = 9.2 Hz, 1 H), 7.16 (s, 1 H),
4.45−4.51 (m, 2 H), 4.33−4.40 (m, 3 H), 4.03 (s, 3 H), 3.92 (m, 1 H),
3.75−3.84 (m, 3 H), 3.30 (m, 1 H), 3.03−3.12 (m, 2 H), 2.88 (t, J =
10.5 Hz, 1 H), 2.43 (overlaid m, 1 H), 2.25 (br s, 1 H), 1.99 (m, 1 H),
1.61 (m, 1 H), 1.47 (m, 1 H), 1.16 (m, 1 H). ESI-MS (M + H)+ m/z
472.2. HR ESI-MS (M + H)+ m/z = 472.1990 (calcd for
C23H27N5O5F: 472.1996); tR = 0.64.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]oxathiino[2,3-c]-
pyridazin-3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-
fluoro-3-methoxyquinoxalin-5-yl)ethan-1-ol (33d). Starting
from 30b (1.8 g, 5.6 mmol) and 25 (1.0 g, 5.6 mmol), 33d (1.99 g,
73% yield) was prepared using the general procedure E. White solid.
1H NMR (300 MHz, DMSO-d6) δ: 8.53 (s, 1 H), 7.88 (dd, J = 5.9, 9.1
Hz, 1 H), 7.53 (s, 1 H), 7.47 (t, J = 9.2 Hz, 1 H), 4.54−4.59 (m, 2 H),
4.39 (d, J = 6.1 Hz, 1 H), 4.02 (s, 4 H), 3.92 (m, 1 H), 3.74−3.83 (m,
3 H), 3.24−3.32 (m, 3 H), 3.02−3.13 (m, 2 H), 2.88 (t, J = 10.4 Hz, 1
H), 2.42 (overlaid m, 1 H), 2.18 (m, 1 H), 1.62 (m, 1 H), 1.48 (m, 1
H). 13C NMR (125 MHz, CDCl3) δ: 161.6 (d, J = 249 Hz), 159.9,
157.4, 156.8, 140.3 (d, J = 10 Hz), 138.3 (d, J = 3 Hz), 136.0, 128.7 (d,
J = 11 Hz), 125.7, 125.2, 120.6 (d, J = 17 Hz), 116.1 (d, J = 27 Hz),
79.9, 73.8, 72.6, 66.2, 53.9, 53.4, 50.2, 30.8, 28.0, 26.8, 25.6. ESI-MS
(M + H)+ m/z 488.5. HR ESI-MS (M + H)+ m/z = 488.1776 (calcd
for C23H27N5O4FS: 488.1767); tR = 0.66.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]oxathiino[2,3-c]-
pyridazin-3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(3-
fluoro-6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol (32d).
Starting from 30a (2.0 g, 6.27 mmol) and 25 (1.34 g, 6.27 mmol),
32d (2.06 g, 67% yield) was prepared according to the general
1
procedure E. Off-white solid; mp = 129 °C. H NMR (300 MHz,
DMSO-d6) δ: 8.72 (s, 1 H), 8.24 (d, J = 9.0 Hz, 1 H), 7.53 (s, 1 H),
7.19 (d, J = 9.0 Hz, 1 H), 4.50−4.61 (m, 3 H), 3.98 (s, 3 H), 3.85−
3.95 (m, 2 H), 3.77−3.83 (m, 2 H), 3.31 (overlaid m, 1 H), 3.25−3.30
(m, 2 H), 3.05−3.15 (m, 2 H), 2.90 (t, J = 10.7 Hz, 1 H), 2.41 (m, 1
H), 2.20 (m, 1 H), 2.02 (m, 1 H), 1.65 (m, 1 H); 1.50 (m, 1 H), 1.17
(m, 1 H). 13C NMR (125 MHz, CDCl3) δ: 162.7, 159.9, 157.5 (d, J =
256 Hz), 156.7, 141.9 (d, J = 6 Hz), 140.6, 138.6 (d, J = 2 Hz), 138.2
(d, J = 29 Hz), 129.2 (d, J = 13 Hz), 125.8, 125.2, 115.4 (d, J = 3 Hz),
80.0, 73.4, 72.6, 66.2, 54.1, 53.4, 50.2, 30.8, 28.0, 26.7, 25.6. ESI-MS
(M + H)+ m/z 488.3. HR ESI-MS (M + H)+ m/z = 488.1771 (calcd
for C23H27N5O4FS: 488.1767); tR = 0.63.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]dioxino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(7-fluoro-2-me-
thoxyquinolin-8-yl)ethan-1-ol (34a). Starting from 30c (0.73 g, 5.6
mmol) and 22 (0.38 g, 2.3 mmol), 34a (0.275 g, 26% yield) was
1
prepared according to the general procedure E. Off-white solid. H
NMR (300 MHz, DMSO-d6) δ: 8.20 (d, J = 8.9 Hz, 1 H), 7.99 (s, 1
H), 7.76 (dd, J = 6.3, 8.9 Hz, 1 H), 7.25 (t, J = 9.1 Hz, 1 H), 6.95 (d, J
= 8.8 Hz, 1 H), 6.91 (s, 1 H), 4.29−4.34 (m, 3 H), 4.23−4.27 (m, 2
H), 3.96 (s, 3 H), 3.95 (overlaid m, 1 H), 3.82 (m, 1 H), 3.60−3.70
(m, 2 H), 3.31 (overlaid m, 1 H), 3.03−3.12 (m, 2 H), 2.90 (t, J = 10.5
Hz, 1 H), 2.43 (overlaid m, 1 H), 1.93−2.07 (m, 2 H), 1.61 (m, 1 H),
1.47 (m, 1 H), 1.16 (m, 1 H). 13C NMR (125 MHz, CDCl3) δ: 162.7,
161.6 (d, J = 246 Hz), 153.5, 150.2, 146.5 (d, J = 9 Hz), 140.1, 139.4,
138.9, 127.2 (d, J = 11 Hz), 121.9, 120.7 (d, J = 16 Hz), 113.8 (d, J =
27 Hz), 111.9 (d, J = 3 Hz), 110.7, 80.3, 74.4, 72.8, 65.0, 64.1, 53.8,
53.3, 52.1, 30.9, 28.0 (d, J = 2 Hz), 26.6. ESI-MS (M + H)+ m/z 470.1.
HR ESI-MS (M + H)+ m/z = 470.2093 (calcd for C25H29N3O5F:
470.2091); tR = 0.76.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]oxathiino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(7-fluoro-2-me-
thoxyquinolin-8-yl)ethan-1-ol (34b). Starting from 30c (0.072 g,
0.23 mmol) and 23 (0.046 g, 0.25 mmol), 34b (0.082 g, 67% yield)
was prepared according to the general procedure E. Off-white solid. 1H
NMR (300 MHz, DMSO-d6) δ: 8.20 (d, J = 8.9 Hz, 1 H), 7.92 (s, 1
H), 7.76 (dd, J = 6.2, 8.9 Hz, 1 H), 7.25 (t, J = 9.2 Hz, 1 H), 7.13 (s, 1
H), 6.95 (d, J = 8.8 Hz, 1 H), 4.29−4.32 (m, 3 H), 3.97 (s, 3 H), 3.95
(overlaid m, 1 H), 3.84 (m, 1 H), 3.59−3.70 (m, 2 H), 3.31 (overlaid
m, 1 H), 3.20−3.25 (m, 2 H), 3.02−3.11 (m, 2 H), 2.89 (t, J = 10.5
Hz, 1 H), 2.44 (overlaid m, 1 H), 1.94−2.08 (m, 2 H), 1.61 (m, 1 H),
1.49 (m, 1 H), 1.17 (m, 1 H). ESI-MS (M + H)+ m/z 486.1. HR ESI-
MS (M + H)+ m/z = 486.1863 (calcd for C25H29N3O4FS: 486.1862);
tR = 0.79.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]dioxino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-fluoro-3-me-
thoxyquinoxalin-5-yl)ethan-1-ol (33a). Starting from 30b (0.344
g, 1.0 mmol) and 22 (0.177 g, 1.0 mmol), 33a (0.334 g, 66% yield)
was prepared according to the general procedure E. Off-white foam.
1H NMR (300 MHz, DMSO-d6) δ: 8.53 (s, 1 H), 7.99 (s, 1 H), 7.88
(dd, J = 5.8, 9.1 Hz, 1 H), 7.46 (t, J = 9.2 Hz, 1 H), 6.91 (s, 1 H), 4.38
(d, J = 6.1 Hz, 1 H), 4.29−4.34 (m, 2 H), 4.23−4.28 (m, 2 H), 4.02 (s,
3 H), 3.93 (m, 1 H), 3.79 (m, 1 H), 3.60−3.70 (m, 2 H), 3.28 (m, 1
H), 3.03−3.12 (m, 2 H), 2.88 (t, J = 10.5 Hz, 1 H), 2.43 (overlaid m, 1
H), 2.06 (br s, 1 H), 1.98 (m, 1 H), 1.61 (m, 1 H), 1.47 (m, 1 H), 1.17
(m, 1 H). 13C NMR (125 MHz, CDCl3) δ: 161.6 (d, J = 249 Hz),
157.4, 153.4, 150.2, 140.3 (d, J = 10 Hz), 140.1, 138.9, 138.2 (d, J = 3
Hz), 136.0, 128.7 (d, J = 11 Hz), 120.7 (d, J = 17 Hz), 116.1 (d, J = 27
Hz), 110.7, 79.9, 73.8, 72.7, 65.0, 64.1, 53.9, 53.2, 52.1, 30.8, 28.0, 26.9.
ESI-MS (M + H)+ m/z 471.2. HR ESI-MS (M + H)+ m/z = 471.2040
(calcd for C24H28N4O5F: 471.2043); tR = 0.69.
(S)-1-((2S,5R)-5-(((2,3-Dihydro-[1,4]oxathiino[2,3-c]pyridin-7-
yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(6-fluoro-3-me-
thoxyquinoxalin-5-yl)ethan-1-ol (33b). Starting from 30b (0.057
g, 0.177 mmol) and 23 (0.031 g, 0.18 mmol), 33b (0.027 g, 31% yield)
1
was prepared according to the general procedure E. White foam. H
NMR (300 MHz, DMSO-d6) δ: 8.54 (s, 1 H), 7.92 (s, 1 H), 7.89 (dd,
J = 5.8, 9.1 Hz, 1 H), 4.32−4.39 (m, 3 H), 4.03 (s, 3 H), 3.93 (m, 1
H), 3.79 (m, 1 H), 3.59−3.70 (m, 2 H), 3.30 (m, 1 H), 3.21−3.26 (m,
2 H), 3.03−3.12 (m, 2 H), 2.89 (t, J = 10.4 Hz, 1 H), 2.43 (overlaid m,
1 H), 1.94−2.07 (m, 2 H), 1.62 (m, 1 H), 1.48 (m, 1 H), 1.18 (m, 1
H). 13C NMR (125 MHz, CDCl3) δ: 161.7 (d, J = 249 Hz), 157.4,
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]dioxino[2,3-c]pyridazin-
3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(7-fluoro-2-
methoxyquinolin-8-yl)ethan-1-ol (34c). Starting from 30c (0.083
g, 0.26 mmol) and 25 (0.043 g, 0.26 mmol), 34c (0.061 g, 50% yield)
was prepared according to the general procedure E. Off-white solid;
1
mp = 161 °C. H NMR (500 MHz, CDCl3) δ: 7.99 (d, J = 8.8 Hz, 1
N
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
H), 7.61 (dd, J = 6.1, 8.8 Hz, 1 H), 7.19 (t, J = 9.0 Hz, 1 H), 6.88 (d, J
= 8.8 Hz, 1 H), 4.51−4.55 (m, 2 H), 4.37−4.40 (m, 2 H),), 4.17 (ddd,
J = 2.2, 4.4, 10.8 Hz, 1 H), 4.08 (s, 3 H), 4.00−4.06 (m, 2 H), 3.95 (m,
2 H), 3.45 (ddd, J = 1.7, 3.7, 14.4 Hz, 1 H), 3.37 (ddd, J = 1.9, 8.5,
14.4 Hz, 1 H), 3.32 (ddd, J = 2.1, 4.8, 11.5 Hz, 1 H), 3.15 (t, J = 10.5
Hz, 1 H), 2.74 (tt, J = 4.1, 11.1 Hz, 1 H), 2.20 (m, 1 H), 1.84 (m, 1
H), 1.74 br s, 2 H), 1.67 (m, 1 H), 1.34 (m, 1 H). 13C NMR (125
MHz, CDCl3) δ: 162.7, 161.6 (d, J = 246 Hz), 159.4, 155.5, 146.5 (d, J
= 9 Hz), 144.2, 139.4, 127.2 (d, J = 11 Hz), 121.9, 120.7 (d, J = 16
Hz), 113.8 (d, J = 27 Hz), 113.4, 111.9 (d, J = 3 Hz), 80.3, 74.3, 72.7,
64.7, 64.6, 53.8, 53.4, 50.4, 30.9, 27.9 (d, J = 3 Hz), 26.6. ESI-MS (M +
H)+ m/z 471.0. HR ESI-MS (M + H)+ m/z = 471.2046 (calcd for
C24H28N4O5F: 471.2043); tR = 0.71.
(S)-1-((2S,5R)-5-(((6,7-Dihydro-[1,4]oxathiino[2,3-c]-
pyridazin-3-yl)methyl)amino)tetrahydro-2H-pyran-2-yl)-2-(7-
fluoro-2-methoxyquinolin-8-yl)ethan-1-ol (34d). Starting from
30c (0.053 g, 0.16 mmol) and 25 (0.030 g, 0.16 mmol), 34d (0.03 g,
37% yield) was prepared according to the general procedure E. Off-
white solid; mp = 165 °C. 1H NMR (500 MHz, CDCl3) δ: 7.99 (d, J =
8.8 Hz, 1 H), 7.61 (dd, J = 6.1, 8.9 Hz, 1 H), 7.33 (s, 1 H), 7.19 (t, J =
9.0 Hz, 1 H), 6.88 (d, J = 8.8 Hz, 1 H), 4.64−4.69 (m, 2 H), 4.17
(ddd, J = 2.2, 4.4, 10.9 Hz, 1 H), 4.08 (s, 3 H), 3.98−4.05 (m, 2 H),
3.46 (ddd, J = 1.6, 3.6, 13.8 Hz, 1 H), 3.38 (ddd, J = 1.8, 8.5, 13.8 Hz,
1 H), 3.32 (ddd, J = 2.0, 4.8, 11.4 Hz, 1 H), 3.21−3.25 (m, 2 H), 3.15
(t, J = 10.5 Hz, 1 H), 2.74 (tt, J = 4.1, 11.1 Hz, 1 H), 2.20 (m, 1 H),
1.84 (m, 1 H), 1.74 br s, 2 H), 1.67 (m, 1 H), 1.34 (m, 1 H). 13C
NMR (125 MHz, CDCl3) δ: 162.7, 161.7 (d, J = 246 Hz), 159.9,
156.8, 146.5 (d, J = 9 Hz), 139.4, 127.2 (d, J = 11 Hz), 125.7, 125.2,
121.9, 120.7 (d, J = 16 Hz), 113.8 (d, J = 27 Hz), 111.9 (d, J = 3 Hz),
80.3, 74.3, 72.6, 66.2, 53.8, 53.5, 50.2, 30.9, 27.9 (d, J = 2 Hz), 26.6,
25.6. ESI-MS (M + H)+ m/z 487.0. HR ESI-MS (M + H)+ m/z =
487.1815 (calcd for C24H28N4O4FS: 487.1815); tR = 0.73.
SCIA, supercoiling inhibitory activity; MeOH, methanol;
EtOH, ethanol; EtOAc, ethyl acetate
REFERENCES
■
(1) Antibiotic Resistance Threats in the United States; Centers for
Disease Control and Prevention (CDC):Atlanta, GA, 2013; http://
www.cdc.gov/drugresistance/threat-report-2013/.
(2) Infectious Diseases Society of America. The 10 x ’20 Initiative:
pursuing a global commitment to develop 10 new antibacterial drugs
by 2020. Clin. Infect. Dis. 2010, 50, 1081−3.
(3) Spellberg, B.; Blaser, M.; Guidos, R. J.; Boucher, H. W.; Bradley,
J. S.; Eisenstein, B. I.; Gerding, D.; Lynfield, R.; Reller, L. B.; Rex, J.;
Schwartz, D.; Septimus, E.; Tenover, F. C.; Gilbert, D. N. Combating
antimicrobial resistance: policy recommendations to save lives. Clin.
Infect. Dis. 2011, 52 (Suppl 5), S397−S428.
(4) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E.;
Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad bugs,
no drugs: no ESKAPE! An update from the Infectious Diseases Society
of America. Clin. Infect. Dis. 2009, 48, 1−12.
(5) Boucher, H. W.; Talbot, G. H.; Benjamin, D. K., Jr.; Bradley, J.;
Guidos, R. J.; Jones, R. N.; Murray, B. E.; Bonomo, R. A.; Gilbert, D.
10 x ’20 Progressdevelopment of new drugs active against Gram-
negative bacilli: an update from the Infectious Diseases Society of
America. Clin. Infect. Dis. 2013, 56, 1685−1694.
(6) East, S. P.; Silver, L. L. Multitarget ligands in antibacterial
research: progress and opportunities. Expert Opin. Drug Discovery
2013, 8, 143−156.
(7) Mayer, C.; Janin, Y. L. Non-quinolone inhibitors of bacterial type
IIA topoisomerases: a feat of bioisosterism. Chem. Rev. 2014, 114,
2313−2342.
(8) Sanyal, G.; Doig, P. Bacterial DNA replication enzymes as targets
for antibacterial drug discovery. Expert Opin. Drug Discovery 2012, 7,
327−339.
ASSOCIATED CONTENT
* Supporting Information
■
S
(9) Gellert, M.; Mizuuchi, K.; O’Dea, M. H.; Nash, H. A. DNA
gyrase: an enzyme that introduces superhelical turns into DNA. Proc.
Natl. Acad. Sci. U. S. A. 1976, 73, 3872−3876.
(10) Kato, J.; Nishimura, Y.; Imamura, R.; Niki, H.; Hiraga, S.;
Suzuki, H. New topoisomerase essential for chromosome segregation
in E. coli. Cell 1990, 63, 393−404.
Experimental details on synthesis and characterization of
compounds 9, 12a and 12b, 27c, 1-epi-32a, and 1-epi-32d.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(11) Mitscher, L. A. Bacterial topoisomerase inhibitors: quinolone
and pyridone antibacterial agents. Chem. Rev. 2005, 105, 559−592.
(12) Hoeksema, H.; Caron, E. L.; Hinman, J. W. Novobiocin. III.
The structure of novobiocin. J. Am. Chem. Soc. 1956, 78.
(13) Smith, D. H.; Davis, B. D. Mode of action of novobiocin in
Escherichia coli. J. Bacteriol. 1967, 93, 71−79.
(14) Shen, L. L.; Mitscher, L. A.; Sharma, P. N.; O’Donnell, T. J.;
Chu, D. W.; Cooper, C. S.; Rosen, T.; Pernet, A. G. Mechanism of
inhibition of DNA gyrase by quinolone antibacterials: a cooperative
drug−DNA binding model. Biochemistry 1989, 28, 3886−3894.
(15) Jacoby, G. A. Mechanisms of resistance to quinolones. Clin.
Infect. Dis. 2005, 41, S120−S126.
(16) S. aureus numbering was employed throughout the document.
(17) Tanaka, M.; Wang, T.; Onodera, Y.; Uchida, Y.; Sato, K.
Mechanism of quinolone resistance in Staphylococcus aureus. J. Infect.
Chemother. 2000, 6, 131−139.
(18) Drlica, K.; Zhao, X. DNA gyrase, topoisomerase IV, and the 4-
quinolones. Microbiol. Mol. Biol. Rev. 1997, 61, 377−392.
(19) Coates, W. J.; Gwynn, M. N.; Hatton, I. K.; Masters, P. J.;
Pearson, N. D.; Rahman, S. S.; Slocombe, B.; Warrack, J. D. Quinoline
derivatives as antibacterials. WO 99376635, 1999.
AUTHOR INFORMATION
Corresponding Author
*Phone: +41 61 565 65 65. Fax: + 41 61 565 65 00. E-mail:
jean-philippe.surivet@actelion.com.
■
Notes
The authors declare no competing financial interest.
+W.K. and C.H. retired on December 31, 2012.
ACKNOWLEDGMENTS
■
We gratefully acknowledge our colleagues from Process
Research, Dr. Stefan Abele, Ste
Schindelholz, and Dr. Jacques-Alexis Funel (who realized the
crystallization of compound 32d) for the excellent support they
provided. Kevin Groshaeny, Daniel Angst, and Franco̧ is Le Goff
́
are warmly thanked for the technical support they provided
during the preparation of the manuscript.
phanie Combes, Ivan
ABBREVIATIONS USED
■
(20) Malleron, J.-L.; Tabart, M.; Carry, J. C.; Evers, M.; El Ahmad, Y.;
Mignani, S.; Viviani, F. Quinolyl propyl piperidine derivatives and their
use as antibacterial agents. WO 200125227, 2001.
(21) 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
NBTI, novel (nonfluoroquinolone) bacterial type II topo-
isomerase inhibitor; QR, quinolone-resistant; QS, quinolone-
sensitive; QTcBZ, Bazett-corrected QT interval (= QT/√RR);
CIP, ciprofloxacin; LZD, linezolid; MXF, moxifloxacin; LHS,
bicyclic aromatic left-hand side; RHS, aromatic right-hand side
(as positioned in Figure 2); CDI, 1,1′-carbonyl diimidazole;
CFU, colony forming unit; RIA, relaxation inhibitory activity;
O
dx.doi.org/10.1021/jm501590q | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
potent antibacterials targeting bacterial type IIA topoisomerases.
Bioorg. Med. Chem. Lett. 2011, 21, 7489−7495.
(35) The diffraction data was deposited into the Cambridge
Crystallographic Data Centre (CCDC 1022935). Information
concerning the X-ray structure determination of compound 32d can
be retrieved from http://www.ccdc.cam.ac.uk/.
(36) Clinical Laboratory and Standards Institut. Methods for Dilution
Susceptibility Test for Bacteria That Grow Aerobically, 7thed.; Approved
Standard, document. M7-A7; Clinical Laboratory and Standards
Institut, Wayne, PA, 2006.
(37) Salvi, V.; Karnad, D. R.; Panicker, G. K.; Kothari, S. Update on
the evaluation of a new drug for effects on cardiac repolarization in
humans: issues in early drug development. Br. J. Pharmacol. 2010, 159,
34−48.
(22) Black, M. T.; Stachyra, T.; Platel, D.; Girard, A. M.; Claudon,
M.; Bruneau, J. M.; Miossec, C. Mechanism of action of the antibiotic
NXL101, a novel nonfluoroquinolone inhibitor of bacterial type II
topoisomerases. Antimicrob. Agents Chemother. 2008, 52, 3339−3349.
(23) 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. Type
IIA topoisomerase inhibition by a new class of antibacterial agents.
Nature 2010, 466, 935−940.
(38) Morissette, P.; Nishida, M.; Trepakova, E.; Imredy, J.; Lagrutta,
A.; Chaves, A.; Hoagland, K.; Hoe, C. M.; Zrada, M. M.; Travis, J. J.;
Zingaro, G. J.; Gerenser, P.; Friedrichs, G.; Salata, J. J. The
anesthetized guinea pig: an effective early cardiovascular derisking
and lead optimization model. J. Pharmacol. Toxicol. Methods 2013, 68,
137−149.
(24) 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.
(25) Black, M. T.; Coleman, K. New inhibitors of bacterial
topoisomerase GyrA/ParC subunits. Curr. Opin. Invest. Drugs 2009,
10, 804−810.
(26) Bouchillon, S. K.; Hackel, M.; Miller, L. A.; Scangarella-Oman,
N. E. In vitro activity of GSK2140944, a Novel topoisomerase
inhibitor, against isolates associated with lower respiratory tract and
skin infections. In 53rd Interscience Conference on Antimicrobial
Agents and Chemotherapy (ICAAC), Denver, CO, September 10−13,
2013, Poster F1−1216.
(27) 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.
Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase
type II with reduced pKa: antibacterial agents with an improved safety
profile. J. Med. Chem. 2012, 55, 6916−6933.
(39) Andes, D. unpublished results.
(40) 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. Oxabicyclooctane-linked novel bacterial
topoisomerase inhibitors as broad spectrum antibacterial agents. ACS
Med. Chem. Lett. 2014, 5, 609−14.
(41) Abele, S.; Funel, J. A.; Schmidt, G.; Surivet, J. P. New Synthetic
Intermediates for Manufacturing Antibiotics. WO 2012164498, 2012.
(42) Kutchinsky, J.; Friis, S.; Asmild, M.; Taboryski, R.; Pedersen, S.;
Vestergaard, R. K.; Jacobsen, R. B.; Krzywkowski, K.; Schroder, R. L.;
Ljungstrom, T.; Helix, N.; Sorensen, C. B.; Bech, M.; Willumsen, N. J.
Characterization of potassium channel modulators with QPatch
automated patch-clamp technology: system characteristics and
performance. Assay Drug Dev. Technol. 2003, 1, 685−693.
(43) Andes, D.; Craig, W. A. Animal model pharmacokinetics and
pharmacodynamics: a critical review. Int. J. Antimicrob. Agents 2002, 19,
261−268.
(28) Hameed, P. S.; Patil, V.; Solapure, S.; Sharma, U.;
Madhavapeddi, P.; Raichurkar, A.; Chinnapattu, M.; Manjrekar, P.;
Shanbhag, G.; Puttur, J.; Shinde, V.; Menasinakai, S.; Rudrapatana, S.;
Achar, V.; Awasthy, D.; Nandishaiah, R.; Humnabadkar, V.; Ghosh, A.;
Narayan, C.; Ramya, V. K.; Kaur, P.; Sharma, S.; Werngren, J.;
Hoffner, S.; Panduga, V.; Kumar, C. N.; Reddy, J.; Mahesh Kumar, K.
N.; Ganguly, S.; Bharath, S.; Bheemarao, U.; Mukherjee, K.; Arora, U.;
Gaonkar, S.; Coulson, M.; Waterson, D.; Sambandamurthy, V. K.; de
Sousa, S. M. Novel N-Linked Aminopiperidine-Based Gyrase
Inhibitors with Improved hERG and in Vivo Efficacy against
Mycobacterium tuberculosis. J. Med. Chem. 2014, 57, 4889−4905.
(29) Surivet, J. P.; Zumbrunn, C.; Rueedi, G.; Hubschwerlen, C.; Bur,
D.; Bruyere, T.; Locher, H.; Ritz, D.; Keck, W.; Seiler, P.; Kohl, C.;
Gauvin, J. C.; Mirre, A.; Kaegi, V.; Dos Santos, M.; Gaertner, M.;
Delers, J.; Enderlin-Paput, M.; Boehme, M. Design, synthesis, and
characterization of novel tetrahydropyran-based bacterial topoisomer-
ase inhibitors with potent anti-gram-positive activity. J. Med. Chem.
2013, 56, 7396−7415.
(30) Hubschwerlen, C.; Rueedi, G.; Surivet, J. P.; Zumbrunn Acklin,
C. 5-Amino-2-(2-heteroaryl-1-hydroxyethyl)tetrahydropyran deriva-
tives. WO 2010067332, 2010.
(31) Chinchilla, R.; Najera, C. The Sonogashira Reaction: A
́
Booming Methodology in Synthetic Organic Chemistry. Chem. Rev.
2007, 107, 874−922.
(32) Thomas, R. J.; Campbell, K. N.; Hennion, G. F. Catalytic
Hydration of Alkylacetylenes. J. Am. Chem. Soc. 1938, 60, 718−720.
(33) Brooks, G.; Dabbs, S.; Davies, D. T.; Hennessy, A. J.; Jones, G.
E.; Markwell, R. E.; Miles, T. J.; Owston, N. A.; Pearson, N. D.; Peng,
T. W. The design of efficient and selective routes to pyridyl analogues
of 3-oxo-3,4-dihydro-2H-1,4-(benzothiazine or benzoxazine)-6-carbal-
dehydes. Tetrahedron Lett. 2010, 51, 5035−5037.
(34) See ref 29. The preparation of compound 27c is described in
Supporting Information.
P
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