Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced p K a: Antibacterial agents with an improved safety profile

Article abstract of DOI:10.1021/jm300690s

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. N-L

Full text of DOI:10.1021/jm300690s

Article
pubs.acs.org/jmc
Novel N-Linked Aminopiperidine Inhibitors of Bacterial
Topoisomerase Type II with Reduced pKa: Antibacterial Agents with
an Improved Safety Profile
Folkert Reck,*,† Richard A. Alm,† Patrick Brassil,† Joseph V. Newman,† Paul Ciaccio,‡ John McNulty,‡
Herbert Barthlow,‡ Kosalaram Goteti,† John Breen,† Janelle Comita-Prevoir,† Mark Cronin,†
David E. Ehmann,† Bolin Geng,† Andrew Aydon Godfrey,§ and Stewart L. Fisher†
‡
†Infection Innovative Medicines Unit and Safety Assessment, Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse
Drive, Waltham, Massachusetts 02451, United States
§Pharmaceutical Development, Chemical Sciences, AstraZeneca, Silk Road Business Park, Macclesfield, SK10 4TG Cheshire, England
ABSTRACT: Novel non-fluoroquinolone inhibitors of bacte-
rial type II topoisomerases (DNA gyrase and topoisomerase
IV) are of interest for the development of new antibacterial
agents that are not impacted by target-mediated cross-
resistance with fluoroquinolones. N-Linked amino piperidines,
such as 7a, generally show potent antibacterial activity,
including against quinolone-resistant isolates, but suffer from hERG inhibition (IC50 = 44 μM for 7a) and QT prolongation
in vivo. We now disclose the finding that new analogues of 7a with reduced pKa due to substitution with an electron-withdrawing
substituent in the piperidine moiety, such as R,S-7c, retained the Gram-positive activity of 7a but showed significantly less hERG
inhibition (IC50 = 233 μM for R,S-7c). This compound exhibited moderate clearance in dog, promising efficacy against a MRSA
strain in a mouse infection model, and an improved in vivo QT profile as measured in a guinea pig in vivo model. As a result of its
promising activity, R,S-7c was advanced into phase I clinical studies.
moiety. These compounds are members of the NBTIs (novel
(non-fluoroquinolone) bacterial type II topoisomerase inhib-
itors)6−8 and are not impacted by target mutations that cause
resistance to fluoroquinolones. Members of the NBTI class of
inhibitors form a ternary complex with the topoisomerase and
uncleaved DNA. Structural studies have revealed that the left-
hand side (LHS) of the inhibitors interacts with the DNA
substrate, whereas the right-hand side (RHS) binds to the
topoisomerase protein. Exploration of left-hand side (LHS) and
right-hand side (RHS) modifications led us to focus on two LHS
cores, 7-cyano-2-quinolone and 7-methoxy-2-oxoquinoxalinone,
and two RHS cores, pyrido dioxino and pyrido oxazinone, as
exemplified by 7a and 8b (Figure 1).
N-Linked aminopiperidines such as 7a and 8b exhibit ∼10-
fold less hERG inhibition (44 and 35 μM) compared to earlier C-
linked analogues (hERG IC50 values of <10 μM), likely as a result
of reduced log D.5 However, in our experience, compounds like
7a and 8b still demonstrated QT prolongation in vivo in
preclinical models at less than 20 μM free concentration. For
example, 7a caused >10% MAPD90 prolongation in the guinea
pig at 16 μM free concentration (Figure 2). Our estimates for
effective therapeutic plasma concentrations (ETPCunbound) for
these compounds were typically in the range of 2 to 10 μM free
concentration (data not shown). The resulting ratios for hERG
IC50/ETPCunbound of <22 reflected a high risk for QT
INTRODUCTION
■
The evolution of drug resistance in pathogenic bacteria is of
global concern,1 and research into the development of new
antibacterial agents that lack cross-resistance to commercially
available agents is needed. Gram-positive organisms such as
methicillin-resistant Staphylococcus aureus (MRSA) remain
challenging pathogens capable of producing devastating disease,
such as necrotizing fasciitis, osteomyelitis, and rapidly progessive
pneumonia in the hospital setting.2 Current levels of resistance in
MRSA against the newer agents daptomycin and linezolid are
low, but recent outbreaks of infections due to cfr-mediated
ribosomal methylation that lead to linezolid-resistant MRSA are
concerning because they confer resistance by a single gene on a
transferable plasmid3 and could potentially spread rapidly.
Toxicity concerns associated with the prolonged use of linezolid
often limits its use in longer duration dosing, for example, in the
treatment of osteomyelitis. Inhibition of daptomycin activity by
pulmonary surfactants precludes its use in the treatment of
pneumonia.4 Therefore, there is an urgent need to develop novel
anti-MRSA drugs that are safe and effective to use, especially in a
pneumonia setting, and that are not affected by cross-resistance
with the current agents.
Bacterial type II topoisomerases (DNA gyrase and topo-
isomerase IV) are clinically proven antibacterial targets, with
many important agents of the fluoroquinolone class of drugs
targeting the GyrA and ParC subunits of DNA gyrase and
topoisomerase IV, respectively. We recently reported on novel
N-linked aminopiperidines5 with an unsubstituted piperidine
Received: May 17, 2012
© XXXX American Chemical Society
A
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a
Scheme 1
Figure 1. Unsubstituted N-linked aminopiperidines.
a
Reagents: (a) Molecular sieves 3 Å, CHCl3/MeOH, 70 °C, then
NaBH(OAc)3, 0 °C to room temp.
moiety observed in the characteristic range of 40−45 versus 60−
70 ppm for CH2O in the case of O-alkylation.
The cis- and trans-fluoropiperidine mesylates 2b−d were
synthesized from the known fluoropiperidine derivatives 11−
1313−15 by protection group manipulation and alkylation with
TBDMS-protected bromoethanol as the key synthetic steps
(Scheme 4). For the synthesis of the cis- and trans-hydroxy and
methoxy piperidines, we followed a similar strategy, employing
unprotected bromoethanol for alkylation of the piperidines
(Schemes 7−10).
Figure 2. Effects on cardiac repolarization potential (MAPD90) of the
guinea pig for compound 7a. Following infusion of 7a (closed triangles),
a maximum increase on MAPD90 of 60 10% was observed with 224
μM free plasma exposure for 7a. The calculated EC10 (dashed line) for
7a was 16 μM free concentration. Vehicle controls (open circles) were
performed in separate cohorts of animals and are plotted in alignment
with the compound dosing. Error bars shown are SEM, n = 6.
4b containing a 7-cyano-2-quinolone LHS was initially
synthesized by alkylation of the commercially available 7-
bromoquinolone 1a to give 3a, which was converted to the 7-
cyano derivative by palladium-catalyzed coupling with in situ
generated Bu3SnCN. Subsequently, we developed several
improvements for the synthesis of the compounds that are
worth highlighting. A new efficient synthesis of the 7-cyano-
substituted LHS 1c eliminated the need for a cyanation step later
in the synthesis (Scheme 3). A further improvement to the
synthesis was affected by alkylation of the LHS with 2-bromo-
1,1-diethoxyethane, followed by deprotection of the acetal with
HCl, as described for the aldehyde 34 (Scheme 6), which could
be alkylated reductively with piperidine derivatives such as 31
and 51 (Schemes 5 and 11). This sequence was broadly
applicable to LHS moieties, eliminated the need for the unstable
mesylates, and led to shorter routes for analogues with piperidine
modifications.
prolongation signals in man. A recent analysis of human
thorough QT studies at Pfizer9 demonstrated that a hERG
IC50 within 60-fold of the ETPCunbound results in an 82% chance
of causing a significant signal in a human thorough QT study
(hTQTS).
We therefore aimed to further reduce hERG inhibition within
the N-linked aminopiperidines. To reduce the risk of a positive
signal in the hTQTS, we aimed for a >100 ratio of hERG IC50/
ETPCunbound, which translates approximately into a hERG IC50
target of >200 μM. Since we had previously optimized
antibacterial hERG activities by manipulation of log D,5 our
strategy for this second effort was to reduce pKa.
CHEMISTRY
■
Compounds were assembled through reductive aminations of
amines 4a−l with aldehydes 510or 611 (Scheme 1).12
For the synthesis of the amines 4, LHSs 1a, 1b,5 and 1c were
alkylated with mesylates 2a−g and 39 (Schemes 2 and 7). The
mesylates were unstable under storage and were freshly prepared
for immediate use from the corresponding alcohols (Schemes 4
and 7−10). Alkylations with 2a−g generally afforded the desired
N-alkylated products together with smaller amounts of O-
alkylation products. O-Alkylated products generally had higher
Rf value by TLC than N-alkylated product and were efficiently
removed by chromatography. N-Alkylation in 3 was confirmed
by NMR studies, with carbon chemical shifts for the CH2N
RESULTS AND DISCUSSION
■
We explored reduction of pKa through substitution at the 3-
position of the piperidine moiety with electron-withdrawing
substituents, specifically both cis- and trans-substitution with
hydroxy, methoxy, and fluoro groups (Table 1). In order to
discern small differences in pKa values, these values were
determined by pH metric titration. We discuss the pKa of the
basic secondary amine only because the tertiary piperidine is a
weak base, with pKa values ranging from 3.12 to 5.55 in
B
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a
Scheme 2
a
Reagents: (a) NaH, DMF, 0 °C to room temp; (b) KCN, Sn(Bu)3Cl, Pd2(dba)3, Xantphos, CH3CN, 85 °C, 76%; (c) TBAF, THF, 0 °C to room
temp, 79%; (d) TFA/DCM, 0 °C.
a
Scheme 3
a
Reagents: (a) NaBH4, CH3CN, room temp, then AcOH and Fe, 65 °C, 66%; (b) DBU, CH3CN, 75 °C, 68%.
a
Scheme 4
a
Reagents: (a) BzlOCCl, Na2CO3, dioxane, 0 °C to room temp; (b) HCl, dioxane, 0 °C; (c) TBDMSO(CH2)2Br, Cs2CO3, CH3CN, 60 °C; (d)
Pd(OH)2/C, H2, MeOH, room temp, 98%; (e) Boc2O, THF, room temp, 82%; (f) Pd/C, H2, EtOH, room temp, 93%; (g) TBAF, THF, 0 °C; (h)
Pd(OH)2/C, H2, MeOH, room temp, 76%; (i) MsCl, NEt3, CH2Cl2, 0 °C.
substituted piperidines. Compounds 8b−m with a pyrido
oxazinone RHS showed a third pKa of ∼10.4, which is due to
the weakly acidic nature of the oxazinone RHS NH moiety.
In the fluoro series, cis-fluoro analogues of 7a, R,S-7c and S,R-
7c, led to a reduction of pKa for the secondary amine
functionality from 8.27 in 7a to 7.03 in 7c. R,S-7c and S,R-7c
inhibited hERG with IC50 values of 233 and 199 μM, respectively,
representing an approximately 5-fold improvement over 7a
(hERG IC50 = 44 μM). The pKa was further reduced in the trans-
fluoro analogue 7e (pKa = 6.66). The same trend was previously
observed for related cis- and trans-3-fluoropiperidines but with
larger shifts in pKa.13 However, 7e was a more potent inhibitor of
hERG with an IC50 of 122 μM. This apparent disconnect in the
SAR can be explained by comparing the log D values of the
parent 7a (log D = 0.68), the cis-fluoro analogue 7c (0.96), and
trans-fluoro analogue 7e (1.53). It is well-established that both
C
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a
Scheme 5
a
Reagents: (a) KOH, EtOH, 90 °C, 43%; (b) 34, THF, 75 °C then NaB(OAc)3H, room temp, 49%; (c) P(Ph)3, CH3CN/H2O, room temp, 90%.
a
Scheme 6
a
Reagents: (a) BrCH2CH(OEt)2, Cs2CO3, NMP, 70 °C, 60%; (b) concd HCl, CH3CN, room temp, 100%.
a
Scheme 7
a
Reagents: (a) Boc2O, KOH, iPrOH/CH2Cl2, 0 °C to room temp, 35%; (b) TBDMSiCl, imidazole, DMF, 0 °C to room temp, 89%; (c) TFA/
CH2Cl2, 0 °C, 98%; (d) Br(CH2)2OH, NEt(iPr)2, CH3CN, 70 °C, 95%; (e) MsCl, NEt3, CH2Cl2, 0 °C; (f) 1b, NaH, DMF then 39, 0 °C to room
temp, 52%; (g) TBAF, THF, room temp, 94%; (h) P(Ph)3, CH3CN/H2O, room temp, 90%.
a
Scheme 8
a
Reagents: (a) Boc2O, NaHCO3, EtOAc/H2O, room temp, quant; (b) TBDMSiCl, imidazole, DMF, room temp, 69%; (c) Pd/C, H2, MeOH, room
temp, quant; (d) Br(CH2)2OH, NEt(iPr)2, CH3CN, 70 °C, 67%; (e) MsCl, NEt3, CH2Cl2, 0 °C.
pKa and log D will have an impact on hERG inhibition.16
Therefore, in the optimization against hERG by lowering pKa,
the concomitant increase of log D needs to be considered. In this
case, a balance was obtained with the cis-fluoro analogue 7c.
Reduction of pKa is expected to potentially reduce hERG
inhibition through reduction of binding affinity. We investigated
binding to the hERG channel with the [3H]-astemizole binding
assay17 on a limited number of compounds, and indeed, both
isomers of 7c showed higher astemizole binding IC50 values
(>200 μM, compared to 39 μM for the parent 7a). Reduced
binding by 7c relative to 7a seems to be a result of the relative
large reduction of pKa for 7c (by 1.24 log units) since the
difference in log D is relatively small (0.28 log units). The trans-
isomer 7e had an astemizole binding IC50 of 57 μM, which is
D
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a
Scheme 9
a
Reagents: (a) Br(CH2)2OH, NEt(iPr)2, CH3CN, 70 °C, 68%; (b) Boc2O, Pd(OH)2/C, H2, MeOH, room temp, 74%; (c) MsCl, NEt3, CH2Cl2, 0
°C.
a
Scheme 10
a
Reagents: (a) NaOH, (Me)2SO4, Bzl(Et)3N+Cl−, toluene/H2O, room temp, 78%; (b) Pd/C, H2, MeOH, room temp, 98%; (c) Br(CH2)2OH,
NEt(iPr)2, CH3CN, 70 °C, 57%; (d) MsCl, NEt3, CH2Cl2, 0 °C.
a
Scheme 11
a
Reagents: (a) 34, THF, 75 °C then NaB(OAc)3H, room temp; (b) TFA/CH2Cl2, 0 °C, quant.
lower than the value for the cis-isomers 7c, despite a further
reduction in pKa for 7e by 0.37 log units. However, the difference
in pKa for 7e and 7c is relatively small and associated with a
relatively large difference in log D by 0.56 log units, indicating
that the higher log D of 7e causes tighter binding to the hERG
channel.
The hERG Ionworks is a cellular assay, and the key binding
sites for small molecule ligands to the hERG channel are facing
the intracellular side.17 Reductions in pKa and resultant increases
in log D are expected to increase functional hERG inhibition in
the Ionworks assay by increasing permeability.
oxazinone RHS; however, the lack of improvement for the cis-
fluoro analogues 8d compared to parent 8b is surprising.
All 3-fluoro-substituted piperidines, especially cis-substituted
analogues (compounds 7c and 8c), showed less potency against
the target topo IV from Escherichia coli than the unsubstituted
parents 7a and 8b. We saw a correlation between the IC50 values
of topo IV from E. coli and the gyrase from Staphylococcus aureus
(S. aureus data not shown). The reduction in target potency with
substituted piperidines was likely due to conformational effects
on the piperidine moiety, which will impact the position of the
LHS and RHS, as well as the position of the basic amine, which
are important for binding. The piperidine moiety itself is
positioned in the target binding site in the solvent-exposed
space.6 Compounds were tested for antibacterial activity against
Gram-positive and Gram-negative organisms. Despite the drop
in target potency with cis-fluoro-substituted piperidines,
antibacterial activity against Gram-positive organisms was largely
maintained (compounds 7c, 8c, and 8d) relative to the
unsubstituted parents 7a and 8b, as apparent by similar MICs
and free fractions (Table 1). This indicated better permeability
into Gram-positive bacteria with the reduced pKa/higher log D
compounds. The higher log D preference for agents targeting
Gram-positive organisms has been noted previously for this
series of compounds5 and as a general trend for antibacterials.18
On the other hand, cellular activity (MICs) against the Gram-
negative organisms Pseudomonas aeruginosa and E. coli was less
potent for all fluoro-substituted piperidines compared to
An optimal balance between pKa and log D will therefore take
into account effects on both binding and permeability.
It is interesting to note that when the same linker
modifications were performed on the parent compound 8b,
bearing a quinoxalinone LHS and a pyrido oxazinone RHS, the
cis-fluoro analogues 8d had higher hERG IC50 values than the
trans-analogue 8f but were not improved over the parent 8b
(hERG IC50 values of 35, 25/32, and 19 μM for parent, cis-fluoro
isomers, and trans-fluoro isomers, respectively). Fluoro-sub-
stituted analogues S,R-8c and 7f that bear either the
quinoxalinone LHS or the pyrido oxazinone RHS are also
slightly more potent hERG inhibitors than the corresponding
analogues with the cyanoquinolone LHS and pyrido dioxino
RHS found in 7a, indicating that hERG inhibition with N-linked
piperidines is more potent with a quinoxalinone LHS or a pyrido
E
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Table 1. SAR of Substituted Piperidines (Minimum Inhibitory Concentration (MIC, μg/mL): Lowest Drug Concentration That
Reduced Growth by 80% or More)
a
b
c
d
e
f
i
S.a. MIC
S.p. MIC
P.ae. MIC
E. coli MIC
fu
E. coli topo IV
IC50 (nM)
log
D
hERG IC50
g
h
compd LHS RHS configuration
(μg/mL)
(μg/mL)
(μg/mL)
(μg/mL)
(%)
pKa
(μM)
7a
8b
1
2
1
2
H
H
0.03
0.03
0.08
0.08
>8
1
0.5
28
26
3.2
2.2
0.68
0.60
8.27/5.75
44
35
0.13
8.42/5.56/10.46
fluoro-substituted analogues (R = F)
R,S-7c
S,R-7c
S,R-8c
R,S-8d
S,R-8d
7e
1
1
1
2
2
1
1
1
2
2
2
1
cis, 3R4S
cis, 3S4R
cis, 3S4R
cis, 3R4S
cis, 3S4R
0.06
0.13
0.06
0.03
0.03
0.13
0.13
0.13
0.25
0.13
0.13
0.25
>8
>8
8
4
25
20
20
ND
25
9
48
0.96
0.97
0.66
ND
0.83
1.53
7.03/4.47
ND
233
199
168
25
4
60
1
11
7.28/4.32/10.28
ND
8
0.5
0.5
8
ND
ND
16
8
ND
32
trans, enan-
tiomer B
>8
6.66/4.07
122
7f
8f
2
2
1
2
trans, enan-
tiomer B
0.06
0.13
0.06
>8
4
8
9
4
26
<9
1.68
1.38
6.46/3.95
75
19
trans, enan-
tiomer B
<0.01
0.5
6.86/3.12/10.40
hydroxy-substituted analogues (R = OH)
R,S-7g
S,R-7g
R,S-8g
S,R-8g
R,S-7h
S,R-7h
R,S-8h
S,R-8h
7i
1
1
1
1
2
2
2
2
2
1
1
2
2
1
1
2
2
1
cis, 3R4S
cis, 3S4R
cis, 3R4S
cis, 3S4R
cis, 3R4S
cis, 3S4R
cis, 3R4S
cis, 3S4R
0.06
0.06
0.25
0.50
0.03
0.06
0.06
0.13
0.06
ND
ND
ND
ND
ND
ND
ND
ND
0.06
>8
>8
2
1
19
24
21
18
19
14
14
5
25
41
17
15
5
0.46
0.45
0.11
0.10
0.77
0.78
0.39
0.37
0.9
8.00/5.55
156
117
305
>333
>33
15
1
0.50
0.50
0.50
1
2
8
>8
2
6
7.97/5.42
7.40/4.33
0.25
0.25
2
17
23
3
172
152
>100
2
trans, enan-
tiomer B
>8
18
methoxy-substituted analogues (R = OMe)
S,R-7j
S,R-8j
S,R-7k
S,R-8k
7l
1
1
2
2
1
1
2
1
2
1
cis, 3S4R
cis, 3S4R
cis, 3S4R
cis, 3S4R
0.13
0.13
0.06
0.06
0.25
0.13
0.50
0.13
0.25
0.50
>8
8
4
1
4
1
8
54
50
33
28
13
55
28
76
76
96
0.63
0.32
0.90
0.51
1.07
7.99/5.15
ND
174
189
75
>8
4
8.01/5.00
8.17/5.10/10.39
7.25/4.34
88
trans, enan-
tiomer 1
>8
176
8l
1
2
2
2
1
2
trans, enan-
tiomer 1
0.13
0.25
0.03
0.25
0.25
0.13
>8
>8
8
2
8
1
27
11
18
35
0.81
1.27
0.97
7.53/4.22/10.46
7.00/4.32
124
93
7m
8m
trans, enan-
tiomer 1
113
trans, enan-
45
7.45/4.34/10.48
27
tiomer 1
a
b
c
d
Methicillin-susceptible Staphylococcus aureus. Streptococcus pneumonia D39 (penicillin-susceptible). Pseudomonas aeruginosa PAO1. Escherichia
e
f
g
h
coli W3110. Fraction unbound, human, % free. Escherichia coli topo IV IC50. Partion coefficient at pH 7.4. First value listed is for secondary
amine. hERG Ionworks IC50.28 ND: Not determined.
i
unsubstituted parents 7a and 8b, indicating that the more basic
7a and 8b may permeate better into Gram-negative bacteria. This
could be due to the nature of the outer membrane in Gram-
negative organisms as a permeability barrier, where porins are
generally more permissive to positively charged compounds.19
Hydroxy-substituted piperidines (compounds 7g−i, 8g−h)
showed a reduction in pKa compared that of parent 7a and 8b
that was more pronounced in the trans series, but to an overall
lesser extent than seen with the more electronegative fluoro-
substituted compounds (pKa of 7.4 and ∼8 for the secondary
amine in the trans- and cis-hydroxy piperidines, respectively). Log
D values for hydroxyl-substituted compounds were lower than
for the corresponding fluoro-substituted analogues. hERG
inhibition was reduced relative to parent 7a and 8b with most
of the analogues, especially with S,R-8g, which had a low log D of
0.1. Compound 7h isomers showed relative potent inhibition of
hERG, which is likely due to the higher log D of ∼0.77 and a
relative high pKa, which is probably similar to 7g at pKa = 8.0. The
F
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trans-isomer 7i has a slightly higher log D of 0.9 but a lower pKa,
which may explain why 7i shows a relatively lower level of hERG
inhibition (IC50 >100 μM) compared to 7h. As with fluoro-
substituted analogues, the balance between reduction in pKa and
the level of lipophilicity seems to be important for the level of
hERG inhibition. With regard to antibacterial activity, cis-
hydroxy substitution led to reduced binding to the target, and
only the trans-analogue 7i showed similar potency on the target
as the parent. Antibacterial activity against the Gram-positive
organism S. aureus was largely maintained relative to parent with
compounds of log D > ∼0.4. The isomers of 8g, which had a low
log D of ∼0.1, were less potent.
MICs against the Gram-negative pathogens P. aeruginosa and
E. coli were consistently higher for all hydroxy-substituted
analogues relative to parent. Although the MIC shift was modest,
it suggests that this substitution, due to its slight reduction in pKa,
may be detrimental to permeability into these Gram-negative
organisms.
Methoxy-substituted piperidines (compounds 7j−m, 8j−m)
showed reductions in pKa very similar to the hydroxy-substituted
analogues for the cis-analogues and a slightly lower pKa for the
trans-isomer (comparing compounds 7m and 7i, pKa of 7.0 and
7.4, respectively). Log D values for methoxy-substituted
compounds were slightly higher than for the hydroxy-substituted
analogues. hERG IC50 values for methoxy-substituted analogues
were, in general, improved over the unsubstituted parents and
similar to the hydroxy analogues. An interesting difference in
hERG inhibition was found between the trans-analogues 7l and
8m, which showed a marked difference in hERG IC50 values (176
vs 27 μM, respectively), with the log D of 8m being only 0.1 units
lower but the pKa by 0.2 units higher than for 7l. While not
conclusive, these results again suggest that relative small
differences in pKa may explain significant differences in hERG
inhibition.
The impact on QT interval was investigated with R,S-7c in
vivo in the guinea pig. The compound did not cause a statistically
significant change in MAPD90 at the highest exposure tested (67
μM total or 30 μM free) (Figure 3). This represents a significant
improvement for R,S-7c over the unsubstituted parent 7a, which
gave an EC10 value of 16 μM free concentration in this model.
Compound R,S-7c also demonstrated reduced ancillary
pharmacology compared to 7a when tested in a secondary
pharmacology panel consisting of 104 targets at 30 μM (results
not shown).
The optimized cis-fluoro analogue R,S-7c showed excellent
activity against hospital- and community-acquired MRSA,
vancomycin-resistant S. aureus (VRSA), as well as against
linezolid-resistant isolates (Table 2), indicating lack of cross-
Table 2. MICs against Resistant Strains of Staphylococcus
a
aureus
phenotype
strain
R,S-7c linezolid vancomycin levofloxacin
b
CA-MRSA
HA-MRSA
ARC3189
ARC1692
ARC3186
ARC3583
0.06
2
1
8
c
0.125
0.015
0.125
2
2
0.125
16
d
VRSA
1
>64
ND
e
LRSA
16
>16
a
Minimum inhibitory concentration (MIC, μg/mL): lowest drug
b
concentration that reduced growth by 80% or more. Community-
acquired methicillin-resistant (USA300). Hospital-acquired methicil-
c
d
lin-resistant (ATCC33591). Vancomycin-resistant, from NARSA
(VRS3). Linezolid-resistant, containing the cfr resistance plasmid.3
e
ND: Not determined.
resistance with anti-MRSA agents. Pharmacokinetic properties of
selected compounds were determined in the rat and dog (Table
3). The fluoro-substituted compounds R,S-7c and S,R-8c showed
lower volumes of distribution in both species compared to
unsubstituted parent compounds 7a and 8b, likely as a result of
reduction in pKa. Clearance for all four compounds was high in
rat and low to medium in dog, correlating roughly with in vitro
intrinsic clearance in hepatocytes. Compounds R,S-7c and S,R-8c
were predicted to show low to medium clearance in man, based
on intrinsic clearance in human hepatocytes. Bioavailability of
compound R,S-7c was low in rat but higher in dog, correlating
with clearance and indicating that first pass metabolism may be a
limiting factor for bioavailability in rodents.
Compound R,S-7c was highly cleared in mouse (results not
shown), and for this reason, it was dosed for efficacy studies in
mice together with the cytochrome P450 inhibitor azabenzo-
triazole (ABT).20 Compound R,S-7c was active against MRSA
ATCC33591 in a neutropenic mouse thigh infection model
(Table 4), with a 1.5 log reduction in colony forming units
(CFU) observed at a dose of 60 mg/kg/day when co-dosed with
ABT. The CFU reduction per dose was similar to that of
levofloxacin in this model; however, the AUC requirement was
higher for R,S-7c.
CONCLUSIONS
■
We report on the reduction of hERG inhibiton in N-linked
aminopiperidine NBTIs by substitution in the 3-position of the
piperidine moiety with electron-withdrawing substituents. The
cis-fluoro-substituted analogues 7c were optimal, resulting in ∼5-
fold increased hERG IC50 values. Reducing pKa, while
minimizing the associated increase in lipophilicity, was important
for successful reduction of hERG inhibition. Small differences in
pKa and log D may explain marked differences in hERG activity.
Compound R,S-7c was tested in the guinea pig model for QT
prolongation to free exposures approximately 2-fold that for 7a
where 10% increased prolongation was observed. We show that
NBTIs with reduced pKa are excellent antibacterial agents against
the Gram-positive bacteria Streptococcus pneumoniae and S.
aureus, including resistant isolates. R,S-7c was evaluated in a
neutropenic mouse thigh infection model against an MRSA
Figure 3. Effects on cardiac repolarization potential (MAPD90) of the
guinea pig for compound R,S-7c. Following infusion of R,S-7c (closed
triangle), MAPD90 was not significantly different (P < 0.05) from vehicle
treated animals (open circles) at any plasma concentration achieved
(highest concentration tested 30 μM free). As there were no biologically
or statistically significant changes, an EC10 (dashed line) could not be
calculated for R,S-7c. Error bars shown are SEM, n = 6.
G
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a
Table 3. Pharmacokinetics in Rat and Dog
compound
Cl int (Hep) (μL/min/106)
clearance (mL/min/kg)
volume (L/kg)
half-life (h)
AUC (iv) (μg·h/mL)
bioavailability (%)
31
rat
7a
42
127
252
54
21
3.0
1.9
0.9
ND
8.4
13
0.37
0.19
3.1
2.5
2.6
6.8
27
8b
26
23
R,S-7c
S,R-8c
7a
13
3.0
3.0
8.0
2.0
1.5
1.7
18
72
60
14
62
dog
man
1.8
<1
4.5
8.6
5.2
2.0
18
8b
8.5
6.2
7.3
R,S-7c
S,R-8c
R,S-7c
S,R-8c
3.4
5.4
25
a
Pharmacokinetic parameters following administration of 3 mg/kg iv bolus injection (compounds 7a, 8b, rat, dog), or 10 mg/kg iv infusion
(compounds R,S-7c, S,R-8c, rat, dog). Oral bioavailability was assessed following a 10 mg/kg oral dose.
treated with methanol (150 μL) containing an internal standard to
Table 4. Exposure and Efficacy of Compound R,S-7c
Compared to Levofloxacin in a Mouse Thigh Model
a
precipitate the protein. Concentration determination was based on a
standard curve (10 nM to 10 μM), and data were processed by the
Analyst version 1.4.1. software.
Log D Determination. The partition coefficient (log D) was
measured by shake flask method, using 10 mM phosphate buffer at pH
7.4 and n-octanol. The samples were allowed to reach equilibrium by
shaking for 1 h at 1200 rpm, and sample analysis was done by LC/UV,
with MS for mass confirmation.
pKa Determination. Values of pKa were determined at Sirius
Analytical Instruments Ltd. (Forest Row Business Park, Station Road,
Forest Row, East Sussex, TH18 5DW) by the Gold Standard pH metric
assay on a Sirius T3 automated system in triplicate. The accuracy of the
measurement was approximately 0.02 log units.
compound R,S-7c
levofloxacin
log reduction in
CFU/g thigh vs
start of treatment
AUC∞
log reduction in
CFU/g thigh vs
start of treatment
AUC∞
(μg/h/
mL)
(μg/h/
mL)
20
mg/kg/day
18
37
53
0.1
1.0
1.5
ND
ND
40
mg/kg/day
4
0.3
b
b
60
mg/kg/day
11
1.4
a
b
In vivo activity against S. aureus ATCC33591 (ARC1692). 80 mg/
kg/day dose.
Animals. Wistar Han rats for pharmacokinetic studies and guinea
pigs for cardiac electrophysiology studies were obtained from Charles
River Laboratories (Raleigh, NC). CD-1 mice were obtained from
Charles River Laboratories (Kingston, NY). All animals were housed
and acclimated in AstraZeneca animal facilities before each study. All
experimental procedures were conducted in accordance with protocols
approved by the Institutional Animal Care and Use Committee.
Pharmacokinetic Studies. Pharmacokinetic properties of selected
compounds were studied in the rat. Groups of three Wistar Han rats
were administered test compound at a dose of 3 or 4 mg/kg, by bolus
injection into a cannulated jugular vein. Oral bioavailability was
determined following a 10 mg/kg dose given by oral gavage. Serial
200 μL samples of whole blood were taken at time intervals.
Concentration of compound in plasma was determined by LC-MS/
MS, and pharmacokinetic parameters were estimated using a non-
compartmental model in WinNonLin (Pharsight). Exposure in CD-1
mice was determined for analysis of the efficacy studies. At timed
intervals, groups of three mice were sacrificed and whole blood samples
collected by cardiac puncture. Plasma samples were prepared and
analyzed as described above. Similarly, plasma pharmacokinetics were
determined from 0 to 24 h in male beagle dogs (n = 3) following 15 min
iv infusions at 3 mg/kg or oral administration at 10 mg/kg.
S. aureus Neutropenic Thigh Infection Model. Compound R,S-
7c was studied in a neutropenic mouse thigh infection model as
described by Mills et al.23 Briefly, mice were rendered neutropenic by
injecting cyclophosphamide (Sigma-Aldrich, St. Louis, MO) intra-
peritoneally 4 days (150 mg/kg of body weight) and 1 day (100 mg/kg)
before experimental infection. Mice were infected with methicillin-
resistant S. aureus ATCC33591 to achieve a target inoculum of 5 × 105
CFU/thigh. Two hours prior to infection, mice received a single
administration of 100 mg/kg aminobenzotriazole (ABT) orally to
inhibit cytochrome P450 activity.20 Groups of five animals each received
an intraperitoneal injection of R,S-7c at the doses specified in Table 4,
prepared in 5% dextrose with lactic acid pH 5.0, on a qd regime starting 2
h after infection. An additional group of five mice received vehicle alone.
Efficacy was determined 24 h after the start of treatment. Thigh tissue
was homogenized with an Omni TH homogenizer (Omni International,
strain and found to be efficacious with a 1.5 log reduction in CFU
at a dose of 60 mg/kg/day in the presense of ABT. The
pharmacokinetic properties of selected compounds were studied
in rat and dog. A good correlation was seen for clearance to in
vitro hepatocyte values for intrinsic clearance. Human
hepatocyte values predicted low to medium clearance in man
for cis-fluoro-substituted analogues. The cis-fluoro analogue R,S-
7c had the overall best profile of the compounds investigated for
this study and was advanced into phase I studies.
EXPERIMENTAL SECTION
■
Minimum Inhibitory Concentration Testing. Minimum inhib-
itory concentrations were determined by broth microdilution according
to the Clinical and Laboratory Standards Institute guidelines.21
Inoculants were incubated at 35 °C on blood agar plates (Remel
#01202) for 18 to 24 h. Compounds were dissolved in 100% DMSO and
diluted to 2% DMSO (v/v) in culture medium to 11 doubling dilutions
from 64 to 0.06 μg/mL. Specific culture media: For S. aureus, P.
aeruginosa, and E. coli, Mueller Hinton Broth 2 (Difco). For S.
pneumoniae, Mueller Hinton Broth 2 plus 2.5% (v/v) lysed horse blood
(Hema Resource and Supply #15-14-0100-28). Plates were read by
spectrophotometry at 620 nm.
Topoisomerase IV Assay. The assay for the ATPase activity E. coli
topo IV, employing the malachite green phosphate detection reagent,22
was performed as described previously.5
Plasma Protein Binding. Plasma protein binding was determined
using the Dianorm equilibrium dialysis chamber. Compound (10 μM
concentration) was spiked in the plasma chamber (donor side), and
phosphate buffer was placed in the receiver side. The unit was rotated at
37 °C for 16 h. Drug concentration was determined for the plasma
sample that represents the bound fraction and the buffer sample that
represents the free fraction. LC/MS-MS quantitative sample analysis
was achieved using an Ace C18 50 × 4.6 mm column (MacMod, PA)
and electrospray ionization MRM detection (PE Sciex API 4000 mass
spectrometer, Applied Biosystems CA). Plasma samples (50 μL) were
H
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Warranton, VA), plated onto tryptic soy agar plates, and incubated at 37
°C overnight for CFU/gram thigh determination.
chloride (0.71 mL, 9.15 mmol) were reacted following the procedure for
2b. The crude product was used directly in the next step without further
purification.
Effects on Cardiac Repolarization Potential of the Guinea Pig
in Vivo. Cardiac electrophysiological characterization of compounds
was performed by the method described by Duker et al.29 Male guinea
pigs were anaesthetized with Nembutal and tracheotomized for
mechanical ventilation. The carotid artery and jugular vein were
cannulated, and to eliminate autonomic influence on the heart, a
bilateral vagotomy was performed and propranolol (0.5 mg/kg) given
intravenously. Needle electrodes were positioned for recording of lead II
electrocardiogram. The chest was opened and a bipolar electrode was
clipped to the left atrial appendage for cardiac pacing. A suction
electrode for recording of the monophasic action potential (MAPD) was
positioned on the left ventricular epicardial wall. Delay of cardiac
repolarization is reflected in an electrocardiogram as an increase in the
QT interval and can be indirectly measured as a prolongation of the
ventricular monophasic action potential (MAP) duration. Compounds
were infused intravenously with two 17 min infusion periods, and
monophasic action potential was continuously recorded during cardiac
pacing. During the first infusion period for 7a (vehicle was water pH
adjusted to 5 with lactic acid), 11.7 mg/kg was administered, while 29.4
mg/kg was delivered during the second infusion period. During first
infusion period for R,S-7c (20% SBECD vehicle), 11.7 mg/kg was
administered, while 14.7 mg/kg was delivered during the second
infusion period. Blood samples were collected at each dose level for
determination of plasma concentration.
2-(trans( )-4-[(tert-Butoxycarbonyl)amino]-3-{[tert-butyl-
(dimethyl)silyl]oxy}piperidin-1-yl)ethyl methanesulfonate (2e).
46 (1.0 g, 2.7 mmol), triethylamine (0.52 mL, 3.74 mmol), and
methanesulfonyl chloride (0.25 mL, 3.21 mmol) were reacted using the
general procedure for 2. The crude product was used directly in the next
step without further purification.
2-{(3S,4R)-4-[(tert-Butoxycarbonyl)amino]-3-methoxypiperi-
din-1-yl}ethyl methanesulfonate (2f). 49 (540 mg, 1.97 mmol),
triethylamine (0.38 mL, 2.76 mmol), and methanesulfonyl chloride
(0.18 mL, 2.36 mmol) were reacted as described for 2b. The crude
preparation of the mesylate product in DMF was used without delay for
the next step.
2-{trans( )-4-[(tert-Butoxycarbonyl)amino]-3-methoxypi-
peridin-1-yl}ethyl methanesulfonate (2g). 52 (0.74 g, 2.7 mmol),
triethylamine (0.53 mL, 3.78 mmol), and methanesulfonyl chloride
(0.25 mL, 3.24 mmol) were reacted as described for 2b. The crude
preparation of the mesylate product in DMF was used without delay for
the next step.
General Procedure for 3 by Alkylation of 1 with 2. A solution of
1 (2 mmol) in dry dimethylformamide (10 mL) was treated at 0 °C
under stirring with sodium hydride (60% in oil, 2 mmol). The cooling
bath was removed, and the mixture was stirred for 30 min at room
temperature. Freshly prepared 2 in a solution with DMF (0.58 mmol/
mL, 2 mmol) was added, and the resulting mixture was stirred overnight
at room temperature. DMF was removed under reduced pressure, and
the residue was taken up in ethyl acetate (100 mL) and saturated
aqueous sodium hydrogencarbonate solution (30 mL). The aqueous
phase was back-extracted once with ethyl acetate (50 mL). The
combined organic phases were dried over sodium sulfate and
concentrated under reduced pressure. Generally, O-alkylation was
observed as a minor product and removed by chromatography.
Chromatography conditions are given below for individual compounds.
tert-Butyl-{1-[2-(7-bromo-2-oxoquinolin-1(2H)-yl)ethyl]-
piperidin-4-yl}carbamate (3a). Prepared from commercially avail-
able 1a and 2a5 according to the general procedure for 3.
Chromatography on silica gel with hexanes/acetone (5:2) gave 9.87 g
(66%) of the product as a colorless solid: mp 155 °C; MS (ESP) m/z
450/452 (MH+); 1H NMR (DMSO-d6) δ 1.32 (m, 2H); 1.36 (s, 9H);
1.65 (m, 2H); 2.01 (t, 2H); 2.46 (m, 2H); 2.90 (m, 2H); 3.19 (m, 1H);
4.29 (t, 2H); 6.61 (d, 1H); 6.75 (d, 1H); 7.41 (d, 1H); 7.65 (d, 1H); 7.73
(br s, 1H); 7.89 (d, 1H).
tert-Butyl-{1-[2-(7-cyano-2-oxoquinolin-1(2H)-yl)ethyl]-
piperidin-4-yl}carbamate (3b). A mixture of 3a (9.85 g, 21.9 mmol)
and potassium cyanide (2.14 g, 32.8 mmol) in dry acetonitrile (60 mL)
was degassed and flushed with nitrogen three times. Tributyltinchloride
(0.059 mmol, 1.13 mL of a 51.6 mM solution in heptane) was added,
followed by 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (63 mg,
0.11 mmol) and tris(dibenzylideneacetone)dipalladium(0) (100 mg,
0.11 mmol), and it was degassed and flushed with nitrogen like above.
The mixture was stirred for 30 min at room temperature and then
degassed and flushed with nitrogen again. It was heated at 85 °C for 20 h.
The solvent was removed under reduced pressure and the residue taken
up in dichloromethane (500 mL) and washed with water (200 mL). The
aqueous phase was back-extracted once with dichloromethane (200
mL), and combined organic phases were dried over sodium sulfate.
Solvent was removed under reduced pressure, and the residue was
crystallized from acetonitrile (∼60 mL) to give the product as a colorless
solid: 6.57 g (76%), mp 202 °C; MS (ESP) m/z 397 (MH+); 1H NMR
(DMSO-d6) δ 1.30 (m, 2H); 1.36 (s, 9H); 1.64 (m, 2H); 2.02 (m, 2H);
2.50 (m, 2H, under solvent peak); 2.90 (m, 2H); 3.15 (m, 1H); 4.34 (t,
2H); 6.74−6.78 (m, 2H); 7.63 (m, 1H); 7.89 (d, 1H); 7.99 (d, 1H); 8.05
(s, 1H).
General Chemical Methods. All commercially available solvents
and reagents were used without further purification. All moisture-
sensitive reactions were carried out under a nitrogen atmosphere in
commercially available anhydrous solvents. Column chromatography
was performed on 230−400 mesh silica gel 60. Aluminum-backed sheets
of silica gel 60 F254 (EM Science) were used for TLC. Melting points
were obtained with a Mel-TempII melting point apparatus from
1
Laboratory Devices, Inc. and are uncorrected. H NMR spectra were
recorded at 300 or 400 MHz. Chemical shifts are reported in parts per
million (δ) relative to solvent. The purity of tested compounds was
assessed by LC-MS. Reverse phase HPLC was carried out using YMC
Pack ODS-AQ (100 × 20 mm ID, S-5 μ particle size, 12 nm pore size)
on Agilent instruments. Mass spectroscopy was performed using a
Micromass Quattro Micro mass spectrometer (for ESP) and an Agilent
1100 MSD instrument (for APCI). All compounds tested possessed a
purity of ≥95%.
2-Oxo-1,2-dihydroquinoline-7-carbonitrile (1c). A mixture of
10 (15.51 g, 82.42 mmol) and 1,8-diazabicyclo[5.4.0]undec-7ene
(DBU) in acetonitrile (155 mL) was heated at 75 °C for 2.5 h. The
reaction mixture was cooled to room temperature, and a precipitate was
collected by filtration, washed with water (77 mL) and with methanol
(77 mL), and dried under reduced pressure to give the product as an off-
white solid, 9.71 g (68%): mp > 250 °C MS (ESP) m/z 171 (MH+); 1H
NMR (DMSO-d6) δ 6.67 (d, 1H); 7.48−7.68 (m, 2H); 7.85 (d, 1H);
7.98 (d, 1H); 12.01 (s, 1H).
cis( )-2-{4-[(tert-Butoxycarbonyl)amino]-3-fluoropiperidin-
1-yl}ethyl methanesulfonate (2b). A mixture of 26 (314 mg, 1.2
mmol) in dry dichloromethane (5 mL) and triethylamine (0.236 mL, 1.7
mmol) was treated at 0 °C with methanesulfonyl chloride (0.111 mL,
1.44 mmol). After 90 min, potassium phosphate buffer (pH 7, 1 M, 5
mL) was added, dichloromethane was removed under reduced pressure,
and it was extracted with ice-cold ethyl acetate (20 mL). The aqueous
phase was back-extracted once with ethyl acetate (10 mL), and the
combined organic phases were dried over sodium sulfate. The solvent
was removed under reduced pressure, and the residue was taken up in
DMF (2 mL). This crude preparation of the mesylate was used without
delay for the next step.
2-((3S,4R)-4-(tert-Butoxycarbonylamino)-3-fluoropiperidin-
1-yl)ethylmethanesulfonate (2c). The compound was prepared
from 24 using the same sequence of reactions as for the converion of
racemic 21 to 2b: MS (ESP) m/z 341 (MH+).
2-{4-[(tert-Butoxycarbonyl)amino]-3-fluoropiperidin-1-yl}-
ethyl methanesulfonate, trans-Enantiomer B (2d). 29 (2.0 g, 7.62
mmol), triethylamine (1.5 mL, 10.7 mmol), and methanesulfonyl
cis( )-tert-Butyl-{1-[2-(7-cyano-2-oxoquinolin-1(2H)-yl)-
ethyl]-3-fluoropiperidin-4-yl}carbamate (3c). Prepared from 1c
and 2b according to the general procedure for 3. Chromatography on
silica gel with hexanes/ethyl acetate (2:3) gave the product as a colorless
solid: 397 mg (73%); MS (ESP) m/z 415 (MH+); 1H NMR (DMSO-
I
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d6) δ 1.37 (s, 9H); 1.48 (m, 1H); 1.67 (m, 1H); 2.27 (m, 2H); 2.56 (m,
2H); 2.96 (m, 1H); 3.15 (m, 1H); 3.46 (m, 1H); 4.34 (m, 2H); 4.60 (m,
1H); 6.77 (m, 1H); 6.91 (m, 1H); 7.64 (m, 1H); 7.90 (m, 1H); 7.99 (m,
1H); 8.07 (s, 1H).
tert-Butyl-(3S,4R)-1-(2-(7-cyano-2-oxoquinolin-1(2H)-yl)-
ethyl)-3-fluoropiperidin-4-ylcarbamate (3c, Single Enan-
tiomer). Prepared from 1c and 2c according to the procedure for
racemic 3c: [α]D = +0.063 (c = 0.2, DMSO).
(dd, 1H); 3.92 (s, 3H); 4.32 (t, 2H); 4.67 (d, 1H); 6.62 (d, 1H); 6.94−
7.06 (m, 2H); 7.69−7.79 (m, 1H); 8.04 (s, 1H).
The mixture of enantiomers was separated by supercritical fluid
chromatography on a Chiralpak AD-H column (250 × 21 mm, 5 μm)
eluting with an isocratic gradient of 25% isopropyl alcohol/0.1%
dimethylethylamine at a flow rate of 60 mL/min. This gave 130 mg of
3h, isomer 1 (first eluting enantiomer) and 130 mg of 3h, isomer 2
(second eluting enantiomer).
cis( )-tert-Butyl-{3-fluoro-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}carbamate (3d). Prepared from 1b5
and 2b according to the general procedure for 3. Chromatography on
silica gel with hexanes/ethyl acetate (2:3) gave 222 mg (51%) product as
a solid: MS (ESP) m/z 421 (MH+); 1H NMR (CDCl3-d) δ 1.44 (s, 9H);
1.86 (m, 2H); 2.40 (m, 2H); 2.80 (m, 2H); 3.15 (m, 1H); 3.41 (m, 1H);
3.70 (m, 1H); 3.94 (s, 3H); 4.42 (m, 2H); 4.70 (m, 2H); 6.93 (m, 2H);
7.77 (m, 1H); 8.11 (s, 1H).
tert-Butyl-{1-[2-(7-cyano-2-oxoquinolin-1(2H)-yl)ethyl]-3-flu-
oropiperidin-4-yl}carbamate, trans-Enantiomer B (3e). Prepared
from 1c (0.5 g, 2.94 mmol) and 2d (3.82 mmol) according to the general
procedure for 3. Chromatography on silica gel with a gradient of 10−
50% acetone in hexanes gave 0.64 g (53%) of the product as an off-white
solid: MS (ESP) m/z 415 (MH+); 1H NMR (DMSO-d6) δ 1.25−1.43
(m, 11H); 1.67−1.78 (m, 1H); 2.04−2.17 (m, 2H); 2.57−2.68 (m, 2H);
2.80−2.89 (m, 1H); 3.25−3.32 (m, 1H); 4.27−4.47 (m, 2H); 4.30 (m,
1H); 6.78 (d, 1H); 6.99 (d, 1H); 7.66 (dd, 1H); 7.91 (d, 1H); 8.01 (d,
1H); 8.09 (s, 1H).
tert-Butyl-{3-fluoro-1-[2-(7-methoxy-2-oxoquinoxalin-1(2H)-
yl)ethyl]piperidin-4-yl}carbamate, trans-Enantiomer B (3f).
Prepared from 1b (0.52 g, 2.95 mmol) and 2d (3.82 mmol) according
to the procedure for 3e, to give 0.93 g (78%) of the product as an off-
white solid: MS (ESP) m/z 421 (MH+); 1H NMR (DMSO-d6) δ 1.23−
1.45 (m, 11H); 1.64−1.80 (m, 1H); 2.04−2.19 (m, 2H); 2.61−2.71 (m,
2H); 2.84 (d, 1H); 3.25−3.33 (m, 1H); 3.92 (s, 3H); 4.27−4.43 (m,
2H); 4.28 (m, 1H); 16.94−7.05 (m, 3H); 7.75 (d, 1H); 8.04 (s, 1H).
tert-Butyl-{trans( )-3-{[tert-butyl(dimethyl)silyl]oxy}-1-[2-(7-
methoxy-2-oxoquinoxalin-1(2H)-yl)ethyl]piperidin-4-yl}-
carbamate (3g). 1b (430 mg, 2.43 mmol), 2e in DMF (∼0.27 mmol/
mL, 2.70 mmol), and sodium hydride (60% in oil, 110 mg, 2.70 mmol)
were reacted using the general procedure for 3. The crude product was
purified by chromatography on silica gel, eluting with a gradient of 10−
25% acetone in hexanes to give 600 mg (46%) of the product as an off-
white solid: MS (ESP) m/z 533 (MH+); 1H NMR (DMSO-d6) δ 0.00 (s,
6H); 0.79 (s, 9H); 1.33 (s, 9H); 1.37−1.47 (m, 1H); 1.49−1.59 (m,
1H); 1.87 (t, 1H); 1.95−2.07 (m, 1H); 2.55−2.66 (m, 2H); 2.77−2.89
(m, 1H); 2.92−3.02 (m, 1H); 3.11 (s, 1H); 3.30−3.40 (m, 1H); 3.89 (s,
3H); 4.17−4.41 (m, 2H); 6.57 (d, 1H); 6.91−7.04 (m, 2H); 7.72 (d,
1H); 8.01 (s, 1H).
tert-Butyl-{(3S,4R)-1-[2-(7-cyano-2-oxoquinolin-1(2H)-yl)-
ethyl]-3-methoxypiperidin-4-yl}carbamate (3i). 1c (370 mg, 2.20
mmol), 2f in DMF (∼0.24 mmol/mL, 2.40 mmol), and sodium hydride
(60% in oil, 110 mg, 2.60 mmol) were reacted following the general
procedure for 3. Chromatography on silica gel with 25−35% acetone in
hexanes gave 370 mg (39%) of the product as an off-white solid: MS
1
(ESP) m/z 427 (MH+); H NMR (DMSO-d6) δ 1.38 (s, 9H); 1.42−
1.51 (m, 1H); 1.57−1.71 (m, 1H); 2.18−2.40 (m, 2H); 2.56 (t, 2H);
2.65−2.76 (m, 1H); 2.78−2.90 (m, 1H); 3.18 (s, 3H); 3.27 (s, 1H); 3.58
(s, 1H); 4.30−4.46 (m, 2H); 6.37 (d, 1H); 6.78 (d, 1H); 7.66 (dd, 1H);
7.91 (d, 1H); 8.01 (d, 1H); 8.09 (s, 1H).
tert-Butyl-{(3S,4R)-3-methoxy-1-[2-(7-methoxy-2-oxoqui-
noxalin-1(2H)-yl)ethyl]piperidin-4-yl}carbamate (3j). 1b (320
mg, 1.79 mmol), 2f in DMF (∼0.20 mmol/mL, 1.97 mmol), and
sodium hydride (60% in oil, 86 mg, 2.15 mmol) were reacted following
the general procedure for 3. Chromatography on silica gel with 15−25%
acetone in hexanes gave 420 mg (55%) of the product as a colorless
solid: MS (ESP) m/z 433 (MH+); 1H NMR (DMSO-d6) δ 1.38 (s, 9H);
1.43−1.51 (m, 1H); 1.57−1.72 (m, 1H); 2.20−2.40 (m, 2H); 2.55−2.66
(m, 2H); 2.67−2.78 (m, 1H); 2.80−2.93 (m, 1H); 3.18 (s, 3H); 3.29 (s,
1H); 3.51−3.65 (m, 1H); 3.92 (s, 3H); 4.24−4.43 (m, 2H); 6.40 (d,
1H); 6.96−7.05 (m, 2H); 7.75 (d, 1H); 8.04 (s, 1H).
tert-Butyl-{1-[2-(7-cyano-2-oxoquinolin-1(2H)-yl)ethyl]-3-
methoxypiperidin-4-yl}carbamate, trans-Enantiomer 1 (3k). A
mixture of 51 (0.63 g, 2.7 mmol), 34 (0.57 g, 2.7 mmol), and sodium
triacetoxyborohydride (1.7 g, 8.1 mmol) were reacted following the
procedure for 32 to give 0.74 g (62%) of the racemic mixture of the
1
product: MS (ESP) m/z 427 (MH+); H NMR (DMSO-d6) δ 1.19−
1.33 (m, 1H); 1.37 (s, 9H); 1.64−1.73 (m, 1H); 1.77 (m, 1H); 1.99 (m,
1H); 2.59 (m, 2H); 2.79−2.87 (m, 1H); 2.93−3.04 (m, 1H); 3.05−3.15
(m, 1H); 3.23−3.30 (m, 1H); 3.28 (s, 3H); 4.30−4.47 (m, 2H); 6.79 (d,
2H); 7.66 (dd, 1H); 7.91 (d, 1H); 8.01 (d, 1H); 8.09 (s, 1H).
The mixture of enantiomers was separated by HPLC on a Chiralpak
AD column (20 × 250 mm, 10 μm) with an isocratic gradient of 80%
hexanes, 20% 1:1 ethanol/methanol, 0.1% diethylamine at a flow rate of
20 mL/min. This gave 0.28 g of 3k (trans-enantiomer 1) (second eluting
peak, (+) isomer) and 0.32 g of tert-butyl-{1-[2-(7-cyano-2-
oxoquinolin-1(2H)-yl)ethyl]-3-methoxypiperidin-4-yl}carbamate (first
eluting peak, (−) isomer).
tert-Butyl-{3-hydroxy-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}carbamate trans-Enantiomers (3h,
Isomers 1 and 2). A solution of 3g (0.60 g, 1.13 mmol) in THF (20
mL) was treated at 0 °C with a solution of tetrabutylammonium fluoride
in THF (1M, 2.2 mL). The reaction was stirred at room temperature for
2 h, then concentrated to dryness under reduced pressure. The crude
residue was taken up in ethyl acetate and washed with water. The
aqueous phase was re-extracted three times with ethyl acetate. The
combined organic phases were dried over sodium sulfate and
concentrated under reduced pressure. Chromatography on silica gel
with 0−5% methanol in dichloromethane gave 0.27 g of the desired
product and 0.12 g of an O-acetylated side product. The side product
was taken up in methanol and treated with a catalytic amount of
potassium carbonate. This was stirred at room temperature for 1 h,
resulting in complete conversion to the alcohol. The reaction mixture
was concentrated to dryness. The residue was partitioned between
aqueous potassium phosphate buffer (pH = 7) and ethyl acetate. The
aqueous phase was re-extracted 2× with ethyl acetate. The combined
organic phases were dried over sodium sulfate, filtered, and concentrated
to dryness giving an additional 100 mg of the desired (79% total) of
tert-Butyl-{3-methoxy-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}carbamate, (+) trans-Enantiomer
1 (3l). 1b (430 mg, 2.45 mmol), 2g (∼0.27 mmol/mL, 2.70 mmol), and
sodium hydride (60% in oil, 110 mg, 2.70 mmol) were reacted using a
the general procedure for 3. The crude product was purified by flash
chromatography eluting with a gradient of 15−35% acetone in hexanes
to give 490 mg (45%) of the racemic mixture of the products as an off-
white solid: MS (ESP) m/z 433 (MH+); 1H NMR (DMSO-d6) δ 1.21−
1.33 (m, 1H); 1.37 (s, 9H); 1.63−1.74 (m, 1H); 1.78 (t, 1H); 2.01 (t,
1H); 2.62 (t, 2H); 2.80−2.90 (m, 1H); 2.96−3.06 (m, 1H); 3.07−3.18
(m, 1H); 3.22−3.29 (m, 4H); 3.93 (s, 3H); 4.27−4.43 (m, 2H); 6.78 (d,
1H); 6.96−7.06 (m, 2H); 7.75 (d, 1H); 8.05 (s, 1H).
The mixture of enantiomers was separated by supercritical fluid
chromatography on a Chiralpak AD-H column (250 × 21 mm, 5 μm)
eluting with an isocratic gradient of 20% isopropyl alcohol/0.1%
dimethylethylamine at a flow rate of 60 mL/min. This gave 190 mg of 3l
(second eluting compound, (+) trans-enantiomer 1) and 190 mg of tert-
butyl-{3-methoxy-1-[2-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)ethyl]-
piperidin-4-yl}carbamate (first eluting peak, (−) isomer).
General Procedure for 4 by Deprotection of 3. A solution of 3
(16.6 mmol) in dichloromethane (100 mL) was treated with
trifluoroacetic acid (40 mL) at 0 °C for 30 min. The solvent was
removed under reduced pressure and the residue co-distilled once with
1
product: MS (ESP) m/z 419 (MH+); H NMR (DMSO-d6) δ 1.18−
1.33 (m, 1H); 1.38 (s, 9H); 1.63−1.77 (m, 1H); 1.86 (t, 1H); 1.97 (t,
1H); 2.54−2.64 (m, 2H); 2.80−2.93 (m, 1H); 2.96−3.09 (m, 2H); 3.23
J
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dichloromethane, then taken up in dichloromethane (200 mL) and
washed with saturated sodium hydrogencarbonate solution (50 mL, pH
adjusted to 10 with sodium hydroxide). The aqueous phase was back-
extracted with dichloromethane (3 × 100 mL) and dried over sodium
sulfate. The combined organic phases were concentrated under reduced
pressure to give the products.
1-[2-(4-Aminopiperidin-1-yl)ethyl]-2-oxo-1,2-dihydroquino-
line-7-carbonitrile (4a). Prepared from 3b (6.57 g, 16.57 mmol)
according to the general procedure for 4, in quantitative yield: off-white
solid, mp 138 °C; MS (ESP) m/z 297 (MH+); 1H NMR (DMSO-d6) δ
1.13 (m, 2H); 1.48 (m, 1H); 1.62 (m, 2H); 2.01 (t, 2H); 2.50 (m, 2H,
under solvent peak); 2.86 (m, 2H); 4.35 (t, 2H); 6.76 (d, 1H); 7.63 (d,
1H); 7.90 (d, 1H); 7.98 (d, 1H); 8.07 (s, 1H).
(1:1), containing 0.1% diethyl amine. Isomer 2 was eluting first, [α]D =
+45.5, followed by isomer 1, [α]D = −44.7 (in methanol/chloroform
1:1, c = 1).
1-{2-[4-Amino-3-hydroxypiperidin-1-yl]ethyl}-7-methoxy-
quinoxalin-2(1H)-one, trans-Isomer 2 (4h). 3h, isomer 2 (130 mg,
0.31 mmol) was reacted with trifluoroacetic acid following the general
procedure for 4 to give 84 mg (85%) of the crude product as an off-white
foam: MS (ESP) m/z 319 (MH+).
1-{2-[(3S,4R)-4-Amino-3-methoxypiperidin-1-yl]ethyl}-2-
oxo-1,2-dihydroquinoline-7-carbonitrile (4i). 3i (370 mg, 0.87
mmol) was reacted with trifluoroacetic acid according to the general
procedure for 4 give 300 mg (quant) of the crude product as an oil: MS
(ESP) m/z 327 (MH+).
cis( )-1-[2-(4-Amino-3-fluoropiperidin-1-yl)ethyl]-2-oxo-
1,2-dihydroquinoline-7-carbonitrile (4b). Prepared from 3c (397
mg, 0.95 mmol) according to the general procedure for 4, except
chloroform was used as the solvent and aqueous workup was omitted. 4c
was obtained as the trifluoroacetate salt in quantitative yield: MS (ESP)
m/z 315 (MH+).
1-(2-((3S,4R)-4-Amino-3-fluoropiperidin-1-yl)ethyl)-2-oxo-
1,2-dihydroquinoline-7-carbonitrile (4b, Single Isomer). Pre-
pared from chiral 3c, according to the procedure for racemic 4b: MS
(ESP) m/z 315 (MH+).
1-{2-[(3S,4R)-4-Amino-3-methoxypiperidin-1-yl]ethyl}-7-me-
thoxyquinoxalin-2(1H)-one (4j). 3j (420 mg, 0.97 mmol) was
reacted with trifluoroacetic acid according to the general procedure for 4
to give 310 mg (97%) of the crude product as an oil: MS (ESP) m/z 333
(MH+).
1-{2-[4-Amino-3-methoxypiperidin-1-yl]ethyl}-2-oxo-1,2-di-
hydroquinoline-7-carbonitrile, trans-Enantiomer 1 (4k). 3k (280
mg, 0.66 mmol) was reacted with trifluoroacetic acid according to the
general procedure for 4 to give 240 mg of the crude product as an oil: MS
(ESP) m/z 327 (MH+).
cis( )-1-[2-(4-Amino-3-fluoropiperidin-1-yl)ethyl]-7-me-
thoxyquinoxalin-2(1H)-one, Trifluoroacetic Acid Salt (4c). 3d
(222 mg, 0.53 mmol) was reacted following the procedure for 4b. The
title compound was obtained in the form of a bis-trifluoroacetic acid salt
in quantitative yield: MS (ESP) m/z 315 (MH+).
1-{2-[4-Amino-3-methoxypiperidin-1-yl]ethyl}-7-methoxy-
quinoxalin-2(1H)-one, trans-Enantiomer 1 (4l). 3l (190 mg, 0.44
mmol) was reacted with trifluoroacetic acid according to the general
procedure for 4 to give 150 mg (quant) of the crude product as an oil:
MS (ESP) m/z 333 (MH+).
1-{2-[4-Amino-3-fluoropiperidin-1-yl]ethyl}-2-oxo-1,2-dihy-
droquinoline-7-carbonitrile, trans-Enantiomer B (4d). Prepared
from 3e (300 mg, 0.72 mmol) according to the general procedure for 4
to give 0.25 g of the crude product as an oil: MS (ESP) m/z 315 (MH+).
1-{2-[4-Amino-3-fluoropiperidin-1-yl]ethyl}-7-methoxyqui-
noxalin-2(1H)-one, trans-Enantiomer B (4e). Prepared from 3f
(330 mg, 0.78 mmol) according to the general procedure for 4 to give
0.27 g of the crude product as an oil: MS (ESP) m/z 321 (MH+).
1-{2-[(3R,4S)-4-Amino-3-hydroxypiperidin-1-yl]ethyl}-2-oxo-
1,2-dihydroquinoline-7-carbonitrile (4f, Isomer 1) and 1-{2-
[(3S,4R)-4-Amino-3-hydroxypiperidin-1-yl]ethyl}-2-oxo-1,2-di-
hydroquinoline-7-carbonitrile (4f, Isomer 2). A mixture of 32
(0.545 g, 1.61 mmol) and triphenylphophine (0.507 g, 1.93 mmol) in
acetonitrile/water (9:1, 50 mL) was stirred at room temperature for 6
days. The reaction mixture was concentrated to dryness under reduced
pressure. Chromatography on silica gel with dichloromethane/
methanol (6:1, containing 0.2% ammonium hydroxide) gave the
racemic mixture of 4f as a colorless hard foam: 0.452 g (90%); MS (ESP)
m/z 313 (MH+); 1H NMR (DMSO-d6) δ 1.48 (m, 2H); 2.20−2.40 (m,
2H); 2.45−2.61 (m, 4H); 2.72 (m, 1H); 3.44 (m, 1H); 4.35 (dd, 2H);
6.78 (d, 1H); 7.64 (d, 1H); 7.90 (d, 1H); 8.00 (d, 1H); 8.08 (d, 1H)
(OH and NH2 protons were exchanged with methanol).
General Procedure for 7 and 8 by Reductive Amination of 4. A
solution of 4 (0.20 mmol) and 1 equiv of aldehyde, either (2,3-
dihydro[1,4]dioxino[2,3-c]pyridine-7-carbaldehyde 510 for 7 or 3-oxo-
3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde 611 for 8, in
dry chloroform/methanol (5 mL, 1:1) was heated over freshly activated
3 Å molecular sieves (pearled) at 70 °C for 3 h. The reaction mixture was
cooled to 0 °C, and sodium triacetoxyborohydride (0.6 mmol) was
added. The reaction mixture was stirred at room temperature for 30 min,
then filtered. The filtrate was concentrated to dryness under reduced
pressure. The residue was taken up in dichloromethane (50 mL) and
saturated aqueous sodium hydrogencarbonate solution (5 mL). The pH
of the aqueous phase was adjusted to a pH of 10 with 1 M aqueous
sodium hydroxide solution. The aqueous phase was back-extracted twice
with dichloromethane (2 × 20 mL), and the combined organic phases
were dried over sodium sulfate and concentrated under reduced
pressure.
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]piperidin-1-yl}ethyl)-2-oxo-1,2-dihydroquinoline-7-car-
bonitrile (7a). 4a (70 mg, 0.24 mmol), 510 (40 mg, 0.24 mmol), and
sodium triacetoxyborohydride (150 mg, 0.75 mmol) were reacted as
described in the general procedure for 7. Chromatography on silica gel
with dichloromethane/methanol (6:1) and crystallization from
dichloromethane/ether/hexanes gave the monoacetate salt of the
product as a colorless solid: 69 mg (58%), mp 130−135 °C; MS (ESP)
m/z 446 (MH+); 1H NMR (CDCl3-d) δ 1.19 (m, 2H); 1.73 (m, 2H);
1.89 (s, 3H); 2.00 (t, 2H); 2.34 (m, 1H); 2.51 (m, 2H, under solvent
peak); 2.88 (m, 2H); 3.65 (s, 2H); 4.24−4.37 (m, 6H); 6.76 (d, 1H);
6.92 (s, 1H); 7.63 (dd, 1H); 7.90 (d, 1H); 7.97−8.00 (m, 2H); 8.07 (br
s, 1H).
1-(2-{(3R,4S)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-fluoropiperidin-1-yl}ethyl)-2-oxo-1,2-dihy-
droquinoline-7-carbonitrile (R,S-7c) and 1-(2-{(3S,4R)-4-[(2,3-
Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)amino]-3-fluoro-
piperidin-1-yl}ethyl)-2-oxo-1,2-dihydroquinoline-7-carboni-
trile (S,R-7c). 4b (0.95 mmol), 510 (160 mg, 0.95 mmol), and sodium
triacetoxyborohydride (600 mg, 2.8 mmol) were reacted as described in
the general procedure for 7, except N,N-diisopropylethylamine (15
equiv) was added together with 4b. Reverse phase chromatography with
water/acetonitrile/ammonium acetate afforded the product as a tan
foam: 276 mg (62%); MS (ESP) m/z 464 (MH+); 1H NMR (CDCl3-d)
δ 1.86 (m, 2H); 2.34 (m, 3H); 2.71 (m, 2H); 2.82 (m, 1H); 3.03 (m,
1H); 3.30 (m, 1H); 3.93 (m, 2H); 4.30 (m, 4H); 4.42 (m, 2H); 4.90 (m,
The racemic mixture was separated on a Chiralpak AD column (250
× 20 mm, 10 μm) with 60% hexanes and 40% ethanol/methanol (1:1),
containing 0.1% diethyl amine. Isomer 2 was eluting first, [α]D = +45.5,
followed by isomer 1, [α]D = −45.9 (in methanol/chloroform 1:1, c =
1).
1-{2-[(3R,4S)-4-Amino-3-hydroxypiperidin-1-yl]ethyl}-7-me-
thoxyquinoxalin-2(1H)-one (4g, Isomer 1) and 1-{2-[(3S,4R)-4-
Amino-3-hydroxypiperidin-1-yl]ethyl}-7-methoxyquinoxalin-
2(1H)-one (4g, Isomer 2). A mixture of 41 (0.507 g, 1.47 mmol) and
triphenylphophine (0.463 g, 1.77 mmol) in acetonitrile/water (9:1, 20
mL) was stirred at room temperature for 5 days. The reaction mixture
was concentrated to dryness under reduced pressure. The residue was
taken up in dichloromethane (5 mL) and chromatographed on silica gel
with dichloromethane/methanol (6:1, containing 0.2% ammonium
hydroxide) to give the racemic mixture of 4g as a colorless hard foam
1
(0.422 g, 90%): MS (ESP) m/z 319 (MH+); H NMR (DMSO-d6) δ
1.48 (m, 2H); 1.89 (m, 1H); 2.28 (m, 1H); 2.37 (dd, 1H); 2.54−2.62
(m, 3H); 2.71 (m, 1H); 3.45 (m, 1H); 3.91 (s, 3H); 4.30 (dd, 2H);
6.96−7.00 (m, 2H); 7.73 (d, 1H); 8.03 (s, 1H) (OH and NH2 protons
were exchanged with methanol). The racemic mixture was separated on
a Chiralpak AD column (250 × 20 mm, 10 μm) with ethanol/methanol
K
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Journal of Medicinal Chemistry
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1H); 6.80 (d, 1H); 6.90 (s, 1H); 7.45 (d, 1H); 7.65 (m, 2H); 7.78 (s,
1H); 8.09 (s, 1H).
The racemic mixture was separated on a Chiralpak AD, 250 × 20 mm,
10 μ column (50% methanol, 50% ethanol, 0.1% diethylamine). R,S-7c
eluted first, [α]D = +14.3 (c = 0.3, methanol) (89 mg), followed by S,R-
7c, [α]D = −11.6 (c = 0.328, methanol) (80 mg).
2(1H)-one, trans-Enantiomer B, Bis-hydrochloride Salt (7i). 4h
(42 mg, 0.13 mmol) and 510 (22 mg, 0.13 mmol) were reacted with
sodium triacetoxyborohydride (83 mg, 0.39 mmol) as described in the
general procedure for 7g to give 45 mg of the product as a yellow solid:
1
MS (ESP) m/z 467 (MH+); H NMR (D2O) δ 1.82−1.99 (m, 1H);
2.42−2.53 (m, 1H); 3.01 (t, 1H); 3.08−3.20 (m, 1H); 3.32−3.44 (m,
1H); 3.58 (t, 2H); 3.80−3.95 (m, 5H); 3.96−4.09 (m, 1H); 4.32−4.50
(m, 6H); 4.65 (t, 2H); 6.87 (d, 1H); 7.07 (dd, 1H); 7.29 (s, 1H); 7.76
(d, 1H); 8.00 (s, 1H); 8.21 (s, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]-3-fluoropiperidin-1-yl}ethyl)-2-oxo-1,2-dihydroquino-
line-7-carbonitrile, trans-Enantiomer B, Bis-hydrochloride Salt
(7e). 4d, 5,10 and sodium triacetoxyborohydride were reacted as
described in the general procedure for 7. Chromatography on silica gel
with a gradient of 0−5% methanol in dichloromethane gave the free base
of the title composition. This was taken up in 1:1 dichloromethane/
diethyl ether (5 mL) and treated with 1.0 M HCl in ether (∼2 equiv),
resulting in a precipitate. This mixture was concentrated to dryness, and
the resulting solid was reconstituted in water and lyophilized to give the
title compound: MS (ESP) m/z 464 (MH+); 1H NMR (D2O) δ 1.81−
1.94 (m, 1H); 2.30−2.43 (m, 1H); 3.13−3.26 (m, 1H); 3.32−3.42 (m,
1H); 3.42−3.50 (m, 1H); 3.53 (t, 2H); 3.57−3.66 (m, 1H); 3.79−3.93
(m, 1H); 4.11−4.27 (m, 2H); 4.38−4.43 (m, 2H); 4.50−4.56 (m, 2H);
4.62−4.75 (m, 1H); 4.80−4.88 (m, 1H); 4.85 (m, 1H); 6.85 (d, 1H);
7.25 (s, 1H); 7.68 (dd, 1H); 7.89 (d, 1H); 7.98 (s, 1H); 8.03 (d, 1H);
8.20 (s, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]-3-fluoropiperidin-1-yl}ethyl)-7-methoxyquinoxalin-
2(1H)-one, trans-Enantiomer B, Bis-hydrochloride Salt (7f). 4e,
5,10 and sodium triacetoxyborohydride were reacted as described in the
procedure for 7e to give the title compound as the bis-hydrochloride
salt: MS (ESP) m/z 470 (MH+); 1H NMR (D2O) δ 1.81−1.97 (m, 1H);
2.32−2.45 (m, 1H); 3.20−3.32 (m, 1H); 3.35−3.44 (m, 1H); 3.52−3.72
(m, 4H); 3.91 (s, 3H); 3.85−3.95 (m, 1H); 4.13−4.28 (m, 2H); 4.37−
4.43 (m, 2H); 4.51−4.56 (m, 2H); 4.57−4.73 (m, 2H); 4.94 (m, 1H);
6.90 (d, 1H); 7.10 (dd, 1H); 7.30 (s, 1H); 7.78 (d, 1H); 8.03 (s, 1H);
8.22 (s, 1H).
1-(2-{(3S,4R)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-methoxypiperidin-1-yl}ethyl)-2-oxo-1,2-di-
hydroquinoline-7-carbonitrile, Bis-hydrochloride Salt (S,R-7j).
4i (93 mg, 0.29 mmol), 510 (47 mg, 0.29 mmol), and sodium
triacetoxyborohydride (180 mg, 0.86 mmol) were reacted according to
the procedure for 7g to give 95 mg of the product as a colorless solid: MS
(ESP) m/z 476 (MH+); 1H NMR (D2O) δ 2.14−2.33 (m, 2H); 3.12−
3.27 (m, 2H); 3.42−3.49 (m, 3H); 3.54−3.62 (m, 2H); 3.63−3.76 (m,
2H); 4.09 (s, 1H); 4.25 (d, 1H); 4.32−4.39 (m, 4H); 4.43−4.50 (m,
2H); 4.50−4.65 (m, 1H); 4.83 (d, 1H); 6.80 (d, 1H); 7.29 (s, 1H); 7.61
(dd, 1H); 7.82 (d, 1H); 7.91 (s, 1H); 7.97 (d, 1H); 8.22 (s, 1H).
1-(2-{(3S,4R)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-methoxypiperidin-1-yl}ethyl)-7-methoxy-
quinoxalin-2(1H)-one, Bis-hydrochloride Salt (S,R-7k). 4j (160
mg crude, 0.48 mmol), 510 (80 mg, 0.48 mmol), and sodium
triacetoxyborohydride (310 mg, 1.44 mmol) were reacted following
the general procedure for 7. Chromatography on silica gel with a
gradient of 2−5% methanol in dichloromethane containing 0.25%
ammonium hydroxide gave 160 mg (70%) of the free base of the title
composition as an oil. This was taken up in dichloromethane/diethyl
ether (1:1, 5 mL) and treated with 2.0 M HCl in ether (∼2 equiv). The
resulting precipitate was collected by filtration, reconstituted in water,
and lyophilized to give 148 mg of the of the bis-hydrochloride salt of the
product as a solid: MS (ESP) m/z 482 (MH+); 1H NMR (D2O) δ 2.04−
2.33 (m, 2H); 3.05−3.25 (m, 2H); 3.44 (s, 3H); 3.50−3.71 (m, 3H);
3.87 (s, 3H); 4.04 (s, 1H); 4.21 (d, 3H); 4.26−4.32 (m, 3H); 4.33−4.40
(m, 2H); 4.45−4.58 (m, 1H); 4.74−4.87 (m, 1H); 6.82−6.92 (m, 1H);
7.02−7.12 (m, 2H); 7.75 (d, 1H); 8.01 (s, 1H); 8.09 (s, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]-3-methoxypiperidin-1-yl}ethyl)-2-oxo-1,2-dihydroqui-
noline-7-carbonitrile, trans-Enantiomer 1, Bis-hydrochloride
Salt (7l). 4k (105 mg crude, 0.32 mmol), 510 (53 mg, 0.32 mmol), and
sodium triacetoxyborohydride (205 mg, 0.97 mmol) were reacted
following the procedure for S,R-7k to give 63 mg of the title composition
as a solid: MS (ESP) m/z 476 (MH+); 1H NMR (D2O) δ 1.84−2.00 (m,
1H); 2.37−2.49 (m, 1H); 2.93−3.06 (m, 1H); 3.10−3.23 (m, 1H);
3.33−3.44 (m, 4H); 3.53−3.80 (m, 4H); 4.19 (d, 1H); 4.27−4.40 (m,
4H); 4.40−4.48 (m, 2H); 4.62−4.76 (m, 2H); 6.79 (d, 1H); 7.22 (s,
1H); 7.62 (dd, 1H); 7.83 (d, 1H); 7.91 (s, 1H); 7.97 (d, 1H); 8.14−8.20
(m, 1H).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]-3-methoxypiperidin-1-yl}ethyl)-7-methoxyquinoxalin-
2(1H)-one, trans-Enantiomer 1, Bis-hydrochloride Salt (7m). 4l
(75 mg, 0.23 mmol), 510 (37 mg, 0.23 mmol), and sodium
triacetoxyborohydride (150 mg, 0.69 mmol) were reacted following
the procedure for 7g to give 63 mg of the product as a yellow solid: MS
(ESP) m/z 482 (MH+); 1H NMR (D2O) δ 1.81−2.02 (m, 1H); 2.34−
2.52 (m, 1H); 2.92−3.05 (m, 1H); 3.10−3.23 (m, 1H); 3.37−3.49 (m,
4H); 3.54−3.72 (m, 3H); 3.73−3.82 (m, 1H); 3.85−3.90 (m, 3H); 4.22
(d, 1H); 4.29−4.41 (m, 4H); 4.43−4.51 (m, 2H); 4.60−4.80 (m, 2H);
6.83−6.91 (m, 1H); 7.07 (dd, 1H); 7.26−7.31 (m, 1H); 7.74 (d, 1H);
7.97−8.03 (m, 1H); 8.17−8.26 (m, 1H).
6-[({1-[2-(7-Methoxy-2-oxoquinoxalin-1(2H)-yl)ethyl]-
piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]oxazin-
3(4H)-one (8b). 1-[2-(4-Aminopiperidin-1-yl)ethyl]-7-methoxyqui-
noxalin-2(1H)-one (60 mg, 0.20 mmol),5 611 (36 mg, 0.20 mmol),
and sodium triacetoxyborohydride (130 mg, 0.60 mmol) were reacted as
described in the general procedure for 8. Chromatography on silica gel
with 5% methanol in dichloromethane containing 0.25% ammonium
hydroxide and crystallization from dichloromethane/ethyl acetate gave
the free base of the product as a colorless solid: 45 mg (50%); MS (ESP)
m/z 465 (MH+); 1H NMR (CDCl3-d) δ 1.06−1.39 (m, 2H); 1.66−1.87
1-(2-{(3R,4S)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-hydroxypiperidin-1-yl}ethyl)-2-oxo-1,2-di-
hydroquinoline-7-carbonitrile, Bis-hydrochloride Salt (R,S-7g).
4f (3R,4S isomer) (74 mg, 0.24 mmol), 510 (39 mg, 0.24 mmol), and
sodium triacetoxyborohydride (150 mg, 0.75 mmol) were reacted as
described in the general procedure for 7. Chromatography was done on
silica gel with dichloromethane/methanol (8:1 to 4:1). Fractions
containing product were pooled and concentrated to dryness. The
residue was taken up in dichloromethane/diethyl ether (1:2, 10 mL),
and HCl in diethyl ether (2 M, ∼0.15 mL) was added. The mixture was
concentrated to dryness under reduced pressure, co-distilled two times
with dichloromethane (2 × 15 mL), and titurated from ether to give the
title composition as a colorless solid: 91 mg (72%), mp >210 °C; MS
(ESP) m/z 462 (MH+); 1H NMR (DMSO-d6) δ 2.18 (m, 2H); 3.15 (m,
1H); 3.25−3.36 (m, 4H); 3.69 (m, 2H); 4.10−4.49 (m, 7H); 4.61 (dd,
2H); 6.64 (br s, 1H); 6.83 (d, 1H); 7.30 (s, 1H); 7.72 (d, 1H); 7.97 (d,
1H); 8.08 (d, 1H); 8.22 (m, 2H); 9.45 (br s, 2H); 10.00 (br s, 1H).
1-(2-{(3S,4R)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-hydroxypiperidin-1-yl}ethyl)-2-oxo-1,2-di-
hydroquinoline-7-carbonitrile, Bis-hydrochloride Salt (S,R-7g).
5
10 and 4f (3S,4R isomer) were reacted using the procedure for R,S-7g:
MS (ESP) m/z 462 (MH+).
1-(2-{(3R,4S)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-hydroxypiperidin-1-yl}ethyl)-7-methoxy-
quinoxalin-2(1H)-one, Bis-hydrochloride Salt (R,S-7h). 510 and 4g
(3R,4S isomer) were reacted following the procedure for 7g: MS (ESP)
m/z 468 (MH+); 1H NMR (D2O) δ 2.28 (m, 2H); 3.20 (ddd, 1H); 3.31
(m, 1H); 3.45−3.76 (m, 4H); 3.92 (s, 3H); 3.99 (m, 1H); 4.29−4.62
(m, 8H); 4.83 (m, 1H); 6.93 (m, 1H); 7.12 (m, 1H); 7.22 (d, 1H); 7.81
(dd, 1H); 8.06 (d, 1H); 8.19 (d, 1H).
1-(2-{(3S,4R)-4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-
ylmethyl)amino]-3-hydroxypiperidin-1-yl}ethyl)-7-methoxy-
quinoxalin-2(1H)-one, Bis-hydrochloride Salt (S,R-7h). 510 and 4g
(3S,4R isomer) were reacted following the procedure for 7g: MS (ESP)
m/z 468 (MH+).
1-(2-{4-[(2,3-Dihydro[1,4]dioxino[2,3-c]pyridin-7-ylmethyl)-
amino]-3-hydroxypiperidin-1-yl}ethyl)-7-methoxyquinoxalin-
L
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(m, 2H); 2.04 (t, 2H); 2.33−2.49 (m, 1H); 2.56 (t, 2H); 2.92 (d, 2H);
3.70 (s, 2H); 3.92 (s, 3H); 4.32 (t, 2H); 4.61 (s, 2H); 6.86−7.09 (m,
3H); 7.30 (d, 1H); 7.75 (d, 1H); 8.04 (s, 1H); 11.18 (s, 1 H).
for 7g to give the title compound: MS (ESP) m/z 475 (MH+); 1H NMR
(D2O) δ 2.28 (m, 2H); 3.15 (ddd, 1H); 3.26 (m, 1H); 3.40−3.72 (m,
4H); 3.91 (m, 1H); 4.28 (s, 2H); 4.52 (m, 1H); 4.61 (m, 1H); 4.71 (s,
2H); 4.80 (m, 1H); 6.65 (d, 1H); 7.08 (d, 1H); 7.35 (d, 1H); 7.66 (d,
1H); 7.87 (d, 1H); 7.96 (s, 1H); 8.02 (d, 1H).
1-[2-((3S,4R)-3-Hydroxy-4-{[(3-oxo-3,4-dihydro-2H-pyrido-
[3,2-b][1,4]oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-2-
oxo-1,2-dihydroquinoline-7-carbonitrile, Bis-hydrochloride
Salt (S,R-8g). 611 and 4f (3S4R isomer) were reacted as described for
7g to give the title compound: MS (ESP) m/z 475 (MH+).
1-[2-((3S,4R)-3-Fluoro-4-{[(3-oxo-3,4-dihydro-2H-pyrido[3,2-
b][1,4]oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-2-oxo-
1,2-dihydroquinoline-7-carbonitrile (8c). 1-{2-[(3S,4R)-4-Amino-
3-fluoropiperidin-1-yl]ethyl}-2-oxo-1,2-dihydroquinoline-7-carbonitrile
trifluoroacetate, 4b (3S4R isomer) (1.2 mmol), prepared following the
procedure for racemic 4b, except instead of racemic 2b, the single
enantiomer 2c was used, was suspended in chloroform/methanol (1:2,
30 mL) and neutralized by dropwise addition of N,N-diisopropylethyl-
amine. To this solution were added 611 (258 mg, 1.45 mmol) and
sodium triacetoxyborohydride (512 mg, 2.42 mmol), and the reaction
was preformed as described in the general procedure for 8. Reverse
phase chromatography with water/acetonitrile/ammonium acetate
afforded the product as an off-white solid after lyophilization. The
hydrochloride salt was prepared by dissolving the lyopholization
product in dichloromethane (5 mL) and adding 2.2 equiv of 4 N HCl
in dioxane, which gave a colorless solid: 154 mg (26%); MS (ESP) m/z
477 (MH+); 1H NMR (DMSO-d6) δ 1.61 (m, 2H); 2.20 (m, 2H); 2.52
(m, 4H); 3.04 (m, 2H); 3.72 (s, 2H); 4.34 (m, 2H); 4.59 (s, 2H); 4.82
(m, 1H); 6.67 (d, 1H); 7.01 (d, 1H); 7.28 (d, 1H); 7.64 (d, 1H); 7.89 (d,
1H); 8.00 (d, 1H); 8.09 (s, 1H); 11.19 (s, 1H).
6-[({(3R,4S)-3-Hydroxy-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b]-
[1,4]oxazin-3(4H)-one, Bis-hydrochloride Salt (R,S-8h). 611 and
4g (3R,4S isomer) were reacted as described for 7g to give the title
compound: MS (ESP) m/z 481 (MH+); 1H NMR (DMSO-d6) δ 2.19
(m, 2H); 3.19 (m, 1H); 3.30−3.48 (m, 4H); 3.62−3.83 (m, 2H); 3.96
(s, 3H); 4.18 (m, 2H); 4.54−4.76 (m, 3H); 4.69 (s, 2H); 6.54 (m, 1H);
7.04 (dd, 1H); 7.21 (d, 1H); 7.26 (d, 1H); 7.44 (d, 1H); 7.79 (d, 1H);
8.09 (s, 1H); 9.28 (m, 1H); 9.61 (m, 1H); 10.26 (m, 1H); 11.41 (s, 1H).
6-[({(3S,4R)-3-Hydroxy-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b]-
[1,4]oxazin-3(4H)-one, Bis-hydrochloride Salt (S,R-8h). 611 and
4g (3S,4R isomer) were reacted as described for 7g to give the title
1
compound: MS (ESP) m/z 481 (MH+); H NMR (D2O) δ 2.28 (m,
6-[({(3R,4S)-3-Fluoro-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b]-
[1,4]oxazin-3(4H)-one, Bis-hydrochloride Salt (R,S-8d) and 6-
[({(3S,4R)-3-Fluoro-1-[2-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)-
ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one, Bis-hydrochloride Salt (S,R-8d). A solution of
4c (0.53 mmol) in dichloroethane/methanol (1:1, 20 mL) was
neutralized with N,N-diisopropylethylamine. 611 (113 mg, 0.63
mmol) was added, and the reaction was stirred at reflux over 3 Å
molecular sieves overnight. The reaction mixture was cooled to 0 °C,
and sodium cyanoborohydride (40 mg, 0.63 mmol) was added. The
mixture was stirred at room temperature for 2 h, then filtered through a
fritted funnel and concentrated under reduced pressure. The residue was
taken up in ethyl acetate and washed with saturated sodium bicarbonate
followed by saturated sodium chloride. The organic extracts were dried
over magnesium sulfate and concentrated under reduced pressure.
Reverse phase chromatography with water/methanol/trifluoroacetic
acid yielded the product as trifluoroacetic acid salt. The salt was
dissolved in water and chloroform and basified with saturated sodium
carbonate. The layers were separated, and the aqueous phase was
extracted with chloroform. The combined organic phases were dried
over magnesium sulfate and concentrated under reduced pressue to give
a racemic mixture of the free bases of the title compositions as a solid: 26
mg (10%); MS (ESP) m/z 483 (MH+); 1H NMR (CDCl3-d) δ 1.70 (m,
4H); 2.35 (m, 2H); 2.64 (m, 1H); 2.73 (m, 2H); 3.04 (m, 1H); 3.31 (m,
1H); 3.86 (s, 2H); 3.92 (s, 3H); 4.35 (m, 2H); 4.63 (s, 2H); 4.83 (m,
1H); 6.86 (m, 1H); 6.92 (m, 1H); 6.98 (m, 1H); 7.21 (m, 1H); 7.77 (m,
1H); 8.11 (s, 1H). The racemic mixture was separated by chiral
chromatography (HPLC, Chiralcel OJ, 250 × 20 mm, 10 μ mobile
phase: 50% hexane, 25% ethanol, 25% methanol, 0.1% diethylamine).
The free base of the 3S,4R enantiomer eluted first. The hydrochloride
salts of both enantiomers were prepared by dissolving the free bases in
dichloromethane (2 mL) and adding 2.2 equiv of 4 N HCl in dioxane.
The resulting colorless precipitates were collected by filtration and dried
to give the HCl salts of the enantiomers as colorless solids.
2H); 3.17 (ddd, 1H); 3.27 (m, 1H); 3.51−3.75 (m, 4H); 3.91 (s, 3H);
3.99 (m, 1H); 4.28 (m, 2H); 4.50−4.62 (m, 2H); 4.71 (s, 2H); 4.80 (m,
1H); 6.90 (d, 1H); 7.08 (d, 1H); 7.11 (dd, 1H); 7.34 (d, 1H); 7.79 (d,
1H); 8.05 (s, 1H).
1-[2-((3S,4R)-3-Methoxy-4-{[(3-oxo-3,4-dihydro-2H-pyrido-
[3,2-b][1,4]oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-2-
oxo-1,2-dihydroquinoline-7-carbonitrile (S,R-8j). 4i (93 mg, 0.29
mmol), 611 (51 mg, 0.29 mmol), and sodium triacetoxyborohydride
(180 mg, 0.86 mmol) were reacted using the general procedure for 8 to
give 80 mg (57%) of the title compound as an off-white solid: MS (ESP)
1
m/z 489 (MH+); H NMR (DMSO-d6) δ 1.36−1.51 (m, 1H); 1.59−
1.75 (m, 1H); 2.23−2.37 (m, 1H); 2.56 (t, 2H); 2.60−2.80 (m, 3H);
3.17 (s, 3H); 3.26−3.32 (m, 2H); 3.68 (q, 2H); 4.25−4.45 (m, 2H);
4.61 (s, 2H); 6.78 (d, 1H); 6.99 (d, 1H); 7.29 (d, 1H); 7.65 (dd, 1H);
7.91 (d, 1H); 8.00 (d, 1H); 8.10 (s, 1H); 11.20 (s, 1H).
6-[({(3S,4R)-3-Methoxy-1-[2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b]-
[1,4]oxazin-3(4H)-one (S,R-8k). 4j (160 mg, 0.48 mmol), 611 (85 mg,
0.48 mmol), and sodium triacetoxyborohydride (310 mg, 1.44 mmol)
were reacted as described in the general procedure for 8 to give 139 mg
(58%) of the product: MS (ESP) m/z 495 (MH+); 1H NMR (DMSO-
d6) δ 1.36−1.53 (m, 1H); 1.59−1.77 (m, 1H); 2.24−2.37 (m, 1H);
2.40−2.46 (m, 1H); 2.60 (t, 2H); 2.65−2.84 (m, 2H); 3.14−3.21 (m,
3H); 3.30−3.34 (m, 2H); 3.67 (q, 2H); 3.92 (s, 3H); 4.22−4.45 (m,
2H); 4.61 (s, 2H); 6.92−7.10 (m, 3H); 7.30 (d, 1H); 7.75 (d, 1H); 8.04
(s, 1H); 11.15−11.27 (m, 1H).
1-[2-(3-Methoxy-4-{[(3-oxo-3,4-dihydro-2H-pyrido[3,2-b]-
[1,4]oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-2-oxo-1,2-
dihydroquinoline-7-carbonitrile, trans-Enantiomer 1 (8l). 4k
(105 mg, 0.32 mmol), 611 (57 mg, 0.32 mmol), and sodium
triacetoxyborohydride (205 mg, 0.97 mmol) were reacted as described
in the general procedure for 8. Chromatography on silica gel with a
gradient of 2−10% methanol in dichloromethane containing 0.25%
ammonium hydroxide gave 88 mg (56%) of the title compound as an
1
off-white solid: MS (ESP) m/z 489 (MH+); H NMR (DMSO-d6) δ
6-[({3-Fluoro-1-[2-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)-
ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one, trans-Enantiomer B (8f). 4e and 611 were reacted
1.00−1.15 (m, 1H); 1.76 (m, 1H); 1.87 (d, 1H); 1.99 (m, 1H); 2.22−
2.34 (m, 1H); 2.59 (m, 2H); 2.78−2.96 (m, 2H); 3.25−3.33 (m, 2H);
3.30 (s, 3H); 3.55−3.77 (m, 2H); 4.30−4.50 (m, 2H); 4.61 (s, 2H); 6.79
(d, 1H); 6.97 (d, 1H); 7.28 (d, 1H); 7.66 (dd, 1H); 7.91 (d, 1H); 8.01
(d, 1H); 8.12 (s, 1H); 11.21 (s, 1H).
6-[({3-Methoxy-1-[2-(7-methoxy-2-oxoquinoxalin-1(2H)-yl)-
ethyl]piperidin-4-yl}amino)methyl]-2H-pyrido[3,2-b][1,4]-
oxazin-3(4H)-one, trans-Enantiomer 1 (8m). 4l (75 mg, 0.23
mmol), 611 (40 mg, 0.23 mmol), and sodium triacetoxyborohydride
(150 mg, 0.69 mmol) were reacted as described in the general procedure
for 8 to give 73 mg (66%) of the product: [α]D = +17.5 (methanol, c =
0.56); MS (ESP) m/z 495 (MH+); 1H NMR (DMSO-d6) δ 1.04−1.22
1
as described in the procedure for 8b: MS (ESP) m/z 483 (MH+); H
NMR (DMSO-d6) δ 1.14−1.29 (m, 1H); 1.82−1.95 (m, 1H); 2.04−
2.20 (m, 2H); 2.52−2.61 (m, 1H); 2.60−2.70 (m, 2H); 2.78−2.89 (m,
1H); 3.16−3.27 (m, 1H); 3.67−3.80 (m, 2H); 3.88−3.96 (m, 3H);
4.29−4.43 (m, 2H); 4.30 (m, 1H); 4.61 (s, 2H); 6.96−7.06 (m, 3H);
7.30 (d, 1H); 7.75 (d, 1H); 8.00−8.09 (m, 1H); 11.19 (s, 1H).
1-[2-((3R,4S)-3-Hydroxy-4-{[(3-oxo-3,4-dihydro-2H-pyrido-
[3,2-b][1,4]oxazin-6-yl)methyl]amino}piperidin-1-yl)ethyl]-2-
oxo-1,2-dihydroquinoline-7-carbonitrile, Bis-hydrochloride
Salt (R,S-8g). 611 and 4f (3R,4S isomer) were reacted as described
M
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(m, 1H); 1.78 (t, 1H); 1.83−1.93 (m, 1H); 2.00 (m, 1H); 2.22−2.35
(m, 1H); 2.62 (t, 2H); 2.81−3.00 (m, 2H); 3.20−3.30 (m, 4H); 3.56−
3.78 (m, 2H); 3.92 (s, 3H); 4.26−4.44 (m, 2H); 4.61 (s, 2H); 6.92−7.08
(m, 3H); 7.29 (d, 1H); 7.75 (d, 1H); 8.05 (s, 1H); 11.21 (s, 1H).
3-Hydroxy-2-oxo-1,2,3,4-tetrahydroquinoline-7-carbonitrile
(10). Ethyl 3-(4-cyano-2-nitrophenyl)-2-oxopropanoate 924 (6.5 kg,
24.8 mol) and acetonitrile (21 L) were stirred at 22 °C, sodium
borohydride (0.30 kg, 7.9 mol) was added in portions, and the mixture
was then stirred for 1 h at 24 °C. Acetic acid (65 L) was charged to the
solution, and the internal temperature was raised to 65 °C. Iron (3.3 kg)
was added to the solution in portions (6 × 0.5 kg) over 1 h. After a
further 1 h, the product was isolated by filtration, washed sequentially
with water (3 × 25 L) and ethanol (29 L), and dried under reduced
pressure to give the product as a beige solid: 3.07 kg (66%); mp >250
°C; MS (ESP) m/z 189 (MH+); 1H NMR (DMSO-d6) δ 2.90−3.20 (m,
2H); 4.10−4.20 (m, 1H); 5.65 (d, 1H); 7.15 (s, 1H); 7.35−7.45 (m,
2H); 10.38 (s, 1H).
cis( )-tert-Butyl-4-{benzyl[(benzyloxy)carbonyl]amino}-3-
fluoropiperidine-1-carboxylate (14). To a mixture of 1113 (1.1 g,
3.6 mmol) in dioxane (20 mL) and saturated sodium carbonate (10 mL)
at 0 °C was added dropwise benzyl chloroformate (0.76 mL, 5.4 mmol),
and the reaction mixture was stirred at 0 °C for 1 h. Ethyl acetate (20
mL) and brine (20 mL) were added, and the layers were separated. The
aqueous phase was extracted once with ethyl acetate, and the combined
organic phases were dried over magnesium sulfate and concentrated
under reduced pressure to give the product as a colorless solid: 1.4 g
(89%); MS (ESP) m/z 343 (MH+-Boc). 1H NMR (CDCl3-d) δ 1.46 (s,
9H); 1.46 (m, 1H); 2.00 (m, 1H); 2.91 (m, 2H); 4.33 (m, 4H); 4.86 (m,
2H); 5.16 (m, 2H); 7.28 (m, 10H).
(3S,4R)-tert-Butyl-4-(benzyloxycarbonylamino)-3-fluoropi-
peridine-1-carboxylate (15). To a mixture of (3S,4R)-tert-butyl-4-
amino-3-fluoropiperidine-1-carboxylate 1214,15 (5.1 g, 23.37 mmol) in
dioxane (150 mL) and saturated sodium carbonate (50 mL) at 0 °C was
added benzyl chloroformate (5.00 mL, 35.05 mmol). After 15 min, the
reaction mixture was diluted with ethyl acetate and saturated sodium
chloride. The layers were separated, and the organic extracts were dried
over magnesium sulfate, filtered, and concentrated under reduced
pressure. Chromatography on silica gel with 0−50% ethyl acetate in
hexanes gave the product as an off-white solid: 8 g (97%); MS (ESP) m/
z 353 (MH+); 1H NMR (CDCl3-d) δ 1.44 (m, 9H); 1.73 (m, 2H); 2.80
(m, 2H); 3.60 (m, 1H); 4.30 (m, 2H); 4.65 (m, 1H); 5.06 (m, 1H); 5.09
(s, 2H); 7.34 (m, 5H).
trans( )-tert-Butyl-4-{benzyl[(benzyloxy)carbonyl]amino}-3-
fluoropiperidine-1-carboxylate (16). To a solution of trans( )-tert-
butyl-(4-benzylamino)-3-fluoropiperidine-1-carboxylate 1313 (10.3 g,
33.4 mmol) in 1,4-dioxane (100 mL) and sodium carbonate (5.31 g,
50.1 mmol) in water (20 mL) was added benzyl chloroformate (5.89
mL, 41.8 mmol) dropwise at 0 °C. The mixture was allowed to warm to
room temperature and stirred for 2 h. The reaction mixture was then
concentrated to near dryness and diluted with ethyl acetate. The organic
phase was washed with water and brine, then dried over sodium sulfate.
Chromatography on silica with 20% ethyl acetate in hexanes gave the
product as a solid (12.5 g, 94%): MS (ESP) m/z 343 (MH+-Boc); 1H
NMR (CDCl3-d) δ 1.45 (s, 9H); 1.67 (d, 2H); 1.84 (m, 1H); 2.59−2.75
(m, 2H); 3.91−4.07 (m, 2H); 4.48 (d, 2H); 4.63 (d, 1H); 5.18 (s, 2H);
7.20−7.34 (m, 10H).
by filtration and dried under reduced pressure to give 5.9 g of the
product (90%): MS (ESP) m/z 253 (MH+).
trans-( )-Benzylbenzyl(3-fluoropiperidin-4-yl)carbamate
hydrochloride (19). To a solution of 16 (12.05 g, 28.2 mmol) in
dichloromethane (50 mL) at 0 °C was added hydrogen chloride (1 M in
diethyl ether, 56.5 mL, 56.5 mmol). The mixture was stirred at room
temperature for 1 h. The solid was isolated by filteration and washed
with diethyl ether to give the mono-hydrochloride salt of the product
(10.1 g, 95%): MS (ESP) m/z 343 (MH+); 1H NMR (DMSO-d6) δ 1.68
(m, 1H); 2.00−2.15 (m, 1H); 3.08 (m, 1H); 3.18 (m, 1H); 3.34 (m,
2H); 3.50 (m, 1H); 4.34−4.49 (m, 2H); 4.65 (m, 1H); 5.02 (s, 1H);
5.14 (d, J = 19.40 Hz, 2H); 7.15−7.30 (m, 8H); 7.32 (m, 2H).
cis( )-Benzylbenzyl[1-(2-{[tert-butyl(dimethyl)silyl]oxy}-
ethyl)-3-fluoropiperidin-4-yl]carbamate (20). A mixture of 17 (4.3
g, 6.1 mmol), (2-bromoethoxy)-tert-butyldimethylsilane (9.8 mL, 45.7
mmol), and cesium carbonate (9.9 g, 30.4 mmol) in acetonitrile (150
mL) was heated at 60 °C overnight. The reaction mixture was filtered
and concentrated under reduced pressure. Chromatography on silica gel
with hexanes/ethyl acetate (3:2) afforded the product as a colorless oil:
5.2 g (91%); MS (ESP) m/z 501 (MH+).
cis( )-1-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)-3-fluoropi-
peridin-4-amine (21). 20 (5.2 g, 10.4 mmol) was hydrogenated in
anhydrous methanol (15 mL) on palladium hydroxide 20 wt % on
carbon (31 mg) for 24 h, then filtered through Celite and concentrated
under reduced pressure to give the product as a colorless oil: 2.8 g
(98%); MS (ESP) m/z 277 (MH+).
cis( )-tert-Butyl-[1-(2-{[tert-butyl(dimethyl)silyl]oxy}ethyl)-
3-fluoropiperidin-4-yl]carbamate (22). 21 (2.8 g, 10.4 mmol) and
di-tert-butyldicarbonate (3.4 g, 15.6 mmol) were combined in
tetrahydrofuran (50 mL) at room temperature. After 90 min, the
reaction mixture was concentrated under reduced pressure. Chroma-
tography on silica gel with hexanes/ethyl acetate (3:2) afforded the
product as a colorless oil: 3.2 g (82%); 1H NMR (CDCl3-d) δ 0.03 (s,
6H); 0.86 (s, 9H); 1.43 (s, 9H); 1.77 (m, 2H); 2.25 (m, 1H); 2.37 (m,
1H); 2.58 (m, 2H); 2.95 (m, 1H); 3.26 (m, 1H); 3.62 (m, 1H); 3.74 (m,
2H); 4.65 (m, 1H); 4.83 (m, 1H).
Benzyl (3S,4R)-1-(2-tert-Butyldimethylsilyloxy)ethyl)-3-fluo-
ropiperidin-4-ylcarbamate (23). To a stirred mixture of 18 (5.9 g,
20.43 mmol) and cesium carbonate (33.3 g, 102.17 mmol) in
acetonitrile (300 mL) at room temp was added (2-bromoethoxy)(tert-
butyl)dimethylsilane (21.92 mL, 102.17 mmol). The reaction was
stirred at 60 °C overnight, then filtered through a fritted funnel and
concentrated. Chromatography on silica gel with 0−50% ethyl acetate in
hexanes afforded the title compound as a yellow oil: 8 g (95%); MS
(ESP) m/z 411 (MH+).
(3S,4R)-1-(2-tert-Butyldimethylsilyloxy)ethyl)-3-fluoropiperi-
din-4-amine (24). A solution of 23 (8 g, 19.48 mmol) in ethanol (100
mL) was hydrogenated on palladium on carbon (10%, activated, 1.037
g) under normal pressure at room temp overnight. The reaction mixture
was filtered through a 0.45 μm membrane, and solvent was evaporated
under reduced pressure to give the product as an oil (5 g, 93%): MS
1
(ESP) m/z 277 (MH+); H NMR (CDCl3-d) δ 0.04 (s, 6H); 0.87 (s,
9H); 1.75 (m, 4H); 2.35 (m, 2H); 2.56 (m, 2H); 2.81 (m, 2H); 3.15 (m,
1H); 3.74 (m, 2H); 4.57 (m, 1H).
trans( )-Benzylbenzyl-1-(2-{[tert-butyl(dimethyl)silyl]oxy}-
ethyl)-3-fluoropiperidin-4-yl]carbamate (25). A mixture of 19
(7.98 g, 21.1 mmol), (2-bromoethoxy)-tert-butyldimethylsilane (6.85 g,
27.5 mmol) and cesium carbonate (17.9 g, 55.0 mmol) in acetonitrile
(60 mL) was heated to 60 °C for 12 h. The reaction mixture cooled to
room temperature and concentrated under reduced pressure to dryness.
The residue was diluted with ethyl acetate and washed with water and
brine. The organic phase was dried over sodium sulfate and
concentrated under reduced pressure. Chromatography on silica with
10% acetone in hexanes gave the product as oil (8.9 g, 84%): MS (ESP)
m/z 501 (MH+); 1H NMR (CDCl3-d) δ 0.04−0.07 (s, 6H); 0.77−0.88
(s, 9H); 1.58−1.74 (m, 2H); 2.05−2.20 (m, 2H); 2.44−2.58 (m, 2H);
2.69−2.84 (m, 1H); 3.24 (m, 1H); 3.65 (s, 2H); 4.44−4.59 (m, 3H);
5.11 (s, 2H); 7.13−7.28 (m, 9H); 7.34 (m, 2H).
cis( )-Benzylbenzyl[1-(2-{[tert-butyl(dimethyl)silyl]oxy}-
ethyl)-3-fluoropiperidin-4-yl]carbamate (17). To a solution of 14
(6 g, 13.5 mmol) in dichloromethane (50 mL) at 0 °C was added 4 N
HCl in dioxane (6.8 mL). The reaction mixture was stirred at room
temperature overnight. The precipitate was collected by filtration to
afford the product as a colorless solid: 4.4 g (86%): MS (ESP) m/z 343
(MH+).
Benzyl-(3S,4R)-3-fluoropiperidin-4-ylcarbamate, Hydro-
chloride Salt (18). To solution of 15 (8 g, 22.7 mmol) in
dichloromethane (200 mL) at 0 °C was added 4 M hydrogen chloride
in dioxane (11.35 mL, 45.4 mmol). The reaction mixture was allowed to
warm to room temperature and stirred overnight. Another equivalent of
4 M hydrogen chloride in dioxane was added, and the reaction was
stirred for another 4 h. The resulting colorless precipitate was collected
cis( )-tert-Butyl-[3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]-
carbamate (26). To a solution of 22 (530 mg, 1.4 mmol) in
tetrahydrofuran (10 mL) at 0 °C was added tetrabutylammonium
N
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fluoride (1 M in THF, 2.8 mL). After 30 min, the reaction was quenched
with saturated sodium bicarbonate and extracted twice with ethyl
acetate, dried over magnesium sulfate, and concentrated. Silica gel
chromatography with 2.5% methanol in ethyl acetate afforded the
product as a colorless solid: 314 mg (85%); 1H NMR (CDCl3-d) δ 1.43
(s, 9H); 1.81 (m, 2H); 2.30 (m, 1H); 2.36 (m, 1H); 2.59 (m, 2H); 2.75
(m, 1H); 2.95 (m, 1H); 3.24 (m, 1H); 3.61 (m, 2H); 3.71 (m, 1H); 4.68
(m, 1H); 4.85 (m, 1H).
concentrated under reduced pressure to give the product as a colorless
solid: 3.43 g (49%); MS (ESP) m/z 339 (MH+); 1H NMR (DMSO-d6)
δ 1.56 (m, 1H); 1.71 (m, 1H); 2.25−2.63 (m, 6H); 3.67 (m, 2H); 4.35
(dd, 2H); 5.06 (m, 1H); 6.78 (d, 1H); 7.64 (dd, 1H); 7.90 (d, 1H); 8.00
(d, 1H); 8.08 (br s, 1H).
1-(2,2-Diethoxyethyl)-2-oxo-1,2-dihydroquinoline-7-car-
bonitrile (33). A mixture of 1c (35.0 g, 201 mmol), 2-bromo-1,1-
diethoxyethane (44.1 mL, 281 mmol), and cesium carbonate (78.5 g,
241 mmol) in dry NMP (200 mL) was stirred at 70 °C overnight. The
reaction mixture was diluted with water (350 mL) and extracted with
butyl acetate (2 × 350 mL). The combined organic phases were filtered
through Celite and washed with water (1 × 175 mL). The butyl acetate
solution was concentrated to 140 mL and diluted with iso-hexane (525
mL). The precipitate was isolated by filtration and washed with iso-
hexane (70 mL). This gave 34 g (60%) of the product as a colorless solid
after drying, which was used without further purification: MS (ESP) m/z
309 (MH+); 1H NMR (DMSO-d6) δ 0.96 (t, 6H); 3.34−3.47 (m, 2H);
3.56−3.73 (m, 2H); 4.39 (d, 2H); 4.72 (t, 1H); 6.80 (d, 1H); 7.62 (d,
1H); 7.89 (d, 1H); 8.02 (d, 1H); 8.13−8.22 (m, 1H).
2-Oxo-1-(2-oxoethyl)-1,2-dihydroquinoline-7-carbonitrile
(34). To a solution of 33 (21.5 g, 75.1 mmol) in acetonitrile (230 mL)
was added concentrated hydrochloric acid (2 equiv, 12.5 mL) at room
temp. After 1 h, the resulting precipitate was collected by filtration. This
gave 16 g (100%) of the product as a colorless solid after drying, which
was used without further purification: MS (ESP) m/z 213 (MH+); 1H
NMR (DMSO-d6) δ 5.25−5.38 (m, 2H); 6.82 (d, 1H); 7.67 (d, 1H);
7.95 (d, 1H); 8.02−8.14 (m, 2H); 9.64−9.74 (m, 1H).
tert-Butyl-(cis( ))-4-azido-3-hydroxypiperidine-1-carboxy-
late (35). To a mixture of 31 (2.1 g, 14.8 mmol) and potassium
hydroxide (2.5 g, 44 mmol) in isopropyl alcohol (20 mL) and
dichloromethane (25 mL) was added at 0 °C a solution of di-tert-
butyldicarbonate (3.9 g, 17.7 mmol) in dichloromethane (10 mL). The
cooling was removed, and it was stirred 2 h at room temperature. It was
quenched with water (50 mL), and isopropyl alcohol and dichloro-
methane were removed under reduced pressure. It was neutralized with
potassium phosphate buffer (1 M, pH 7, 100 mL), extracted with ethyl
acetate twice (2 × 300 mL), and the combined organic phases were
dried over sodium sulfate. Solvent was removed under reduced pressure,
and the residue was titurated from hexanes (∼20 mL) to give 0.966 g of
product as a colorless solid. Chromatography of the mother liquors with
hexanes/ethyl acetate (5:1) afforded an additional 0.353 g of product
(35%): MS (ESP) m/z 265 (MNa+); 1H NMR (DMSO-d6) δ 1.39 (s,
9H); 1.58 (m, 1H); 1.74 (m, 1H); 3.20−3.40 (m, 4H); 3.69 (m, 2H);
5.40 (d, 1H).
tert-Butyl-(cis( ))-4-azido-3-{[tert-butyl(dimethyl)silyl]oxy}-
piperidine-1-carboxylate (36). A mixture of 35 (1.76 g, 7.25 mmol)
and imidazole (0.74 g, 10.9 mmol) in DMF (7 mL) at 0 °C was treated
with tert-butyldimethylsilyl chloride (1.3 g, 8.7 mmol). Cooling was
removed, and the mixture was stirred overnight at room temperature. It
was cooled to 0 °C and quenched with phosphate buffer (1M, pH 7, 20
mL). After 15 min, the mixture was diluted with ethyl acetate (100 mL),
the organic phase was washed with water (2 × 50 mL) and dried over
sodium sulfate. Chromatography on silica gel with hexanes/ethyl acetate
(9:1) gave the product as a colorless oil: 2.3 g (89%); 1H NMR (DMSO-
d6) δ 0.10 (s, 6H); 0.87 (s, 9H); 1.37 (s, 9H); 1.56−1.80 (m, 2H); 3.09−
3.30 (m, 2H); 3.46 (m, 2H); 3.62 (m, 1H); 3.88 (m, 1H).
(3S,4R)-1-(2-(tert-Butyldimethylsilyloxy)ethyl)-3-fluoropiper-
idin-4-amine (27). The compound was prepared from 24 using the
procedure for the synthesis of racemic 26 from 21.
Benzylbenzyl[3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]-
carbamate, trans-Enantiomer B (28). A solution of tetrabutylam-
monium fluoride in tetrahydrofuran (1 M, 21.3 mL, 21.3 mmol) was
added to 25 (8.9 g, 17.8 mmol) in tetrahydrofuran (20 mL) at 0 °C. The
solution was allowed to warm to room temperature and stirred for 1 h.
The mixture was then cooled to 0 °C and quenched with water. The
mixture was extracted with ethyl acetate and washed with brine. The
combined organic phase was dried over sodium sulfate and concentrated
under reduced pressure. Chromatography on silica with 40% acetone in
hexanes gave the racemic product as an oil (5.1 g, 74%): MS (ESP) m/z
387 (MH+); 1H NMR (DMSO-d6) δ 1.55 (m, 1H); 1.67 (m, 1H); 2.02
(m, 2H); 2.40 (m, 2H); 2.74 (m, 1H); 3.14−3.28 (m, 2H); 3.43 (m,
2H); 3.93 (m, 1H); 4.40 (t, 2H); 4.50 (m, 1H); 5.06 (m, 2H); 7.15 (m,
1H); 7.20−7.31 (m, 8H); 7.36 (m, 1H). The racemic mixture was
separated on a Chiralpak AD column (500 × 20 mm, 20 μm) with
ethanol/methanol (1:1), containing 0.1% diethyl amine. trans-
Enantiomer B (27) was the second eluting enatiomer. The chiral purity
(using an analytical method equivalent to the preparative method
described above) was determined to be >98% ee.
tert-Butyl-[3-fluoro-1-(2-hydroxyethyl)piperidin-4-yl]-
carbamate, trans-Enantiomer B (29). A mixture of 28 (5.6 g, 14.4
mmol) and palladium hydroxide on carbon (20%, 0.5 g) in methanol
was stirred under an atmosphere of hydrogen overnight at normal
pressure. Hydrogen was removed by purging with nitrogen, di-tert-
butyldicarbonate (3.5 g, 15.8 mmol) was added, and the mixture was
stirred under nitrogen for 1 h. The reaction mixture was filtered through
Celite and the filtrate concentrated to dryness under reduced pressure.
Chromatography on silica gel with 10% methanol (containing 0.1%
ammonium hydroxide) in ethyl acetate gave 2.9 g (76%) of product as
an oil: 1H NMR (CDCl3-d) δ 1.36−1.55 (m, 10H); 2.02−2.31 (m, 3H);
2.52−2.64 (m, 2H); 2.72−2.82 (m, 2H); 3.09−3.20 (m, 1H); 3.60 (t,
3H); 4.31 (m, 1H); 4.80 (d, 1H).
4-Azidopiperidin-3-ol (31).25 To a solution of ethyl 4-azido-3-
(tert-butyldimethylsilyloxy)piperidine-1-carboxylate 3025 (22.3 g, 67.89
mmol) in ethanol (300 mL) was added potassium hydroxide (38.1 g,
678.9 mmol) in water (75 mL). The mixture was heated at 90 °C for 6 h.
Most of the solvent was removed under reduced pressure, and the
residue was diluted with brine (30 mL). The mixture was extracted with
dichloromethane (3 × 200 mL), dried over sodium sulfate, and
concentrated under reduced pressure. The residue was dried under high
vacuum to remove most of the silanol. The residue was taken up in ether
(∼100 mL), hexanes were added (∼150 mL), and most of the ether was
removed under reduced pressure. The precipitate was collected by
filtration and washed with hexanes to give the product as a slightly pink
solid: 4.18 g (43%); 1H NMR (DMSO-d6) δ 5.00 (d, 1H); 3.63 (m, 1H);
3.50 (m, 1H); 2.75−2.60 (m, 2H); 2.60−2.50 (m, 1H); 2.50−2.35 (m,
1H); 1.85 (m, 1H); 1.67 (m, 1H); 1.51 (m, 1H).
1-[2-cis( )-(4-Azido-3-hydroxypiperidin-1-yl)ethyl]-2-oxo-
1,2-dihydroquinoline-7-carbonitrile (32). A mixture of 34 (4.44 g,
20.91 mmol) and 31 (3.27 g, 23 mmol) in THF (200 mL) was heated
under stirring at 75 °C for 3 h. The mixture was cooled to room
temperature and sodium triacetoxyborohydride (13.30 g, 62.73 mmol)
was added. It was stirred for 2 h at room temperature, then quenched by
addition of methanol (100 mL). The reaction mixture was stirred
overnight, filtered through a 0.45 μm membrane, and the solid residue
was washed with MeOH/dichloromethane (1:3, 2 × 20 mL). The
filtrate and wash were combined and concentrated to dryness under
reduced pressure. Chromatography was done on silica gel with hexanes/
acetone (1:1 to 1:2). Fractions containing product were pooled and
(cis( ))-4-Azido-3-{[tert-butyl(dimethyl)silyl]oxy}piperidine
(37). A solution of 36 (2.3 g, 6.45 mmol) in dichloromethane (50 mL)
was treated at 0 °C with trifluoroacetic acid (5 mL). After 3 h, the
mixture was concentrated under reduced pressure and the residue was
co-distilled twice with dichloromethane. The residue was taken up in
dichloromethane (100 mL) and washed with saturated aqueous sodium
hydrogencarbonate solution (30 mL). The aqueous phase was back-
extracted once with dichloromethane (100 mL), and the combined
organic phases were dried over sodium sulfate to give the product as a
slightly yellow oil: 1.625 g (98%); 1H NMR (DMSO-d6) δ 0.07 and 0.09
(2 × s, 6H); 0.88 (s, 9H); 1.49−1.73 (m, 2H); 2.45 (m, 1H); 2.56−2.69
(m, 3H); 3.65 (m, 1H); 3.79 (m, 1H).
2-(cis( ))-(4-Azido-3-{[tert-butyl(dimethyl)silyl]oxy}-
piperidin-1-yl)ethanol (38). A mixture of 37 (1.625 g, 6.34 mmol),
O
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N,N-diisopropylethylamine (1.65 mL, 9.5 mmol), and 2-bromoethanol
(0.584 mL, 8.25 mmol) in dry acetonitrile (17 mL) was heated in the
microwave oven at 70 °C for 2 h. The solvent was removed under
reduced pressure and the residue taken up in ethyl acetate (∼150 mL)
and washed with saturated aqueous sodium hydrogencarbonate solution
(∼25 mL). The aqueous phase was back-extracted once with ethyl
acetate (100 mL), and the combined organic phases were dried over
sodium sulfate. Chromatography on silica gel with dichloromethane/
methanol (20:1) gave 1.80 g (95%) of product as a colorless oil: MS
(ESP) m/z 301 (MH+); 1H NMR (DMSO-d6) δ 0.08 (s, 6H); 0.87 (s,
9H); 1.65 (m, 2H); 2.18 (m, 1H); 2.25−2.60 (m, 5H); 3.44 (m, 2H);
3.73 (m, 1H); 3.91 (m, 1H); 4.35 (m, 1H).
2-(cis( ))-(4-Azido-3-{[tert-butyl(dimethyl)silyl]oxy}-
piperidin-1-yl)ethyl methanesulfonate (39). 38 (1.8 g, 6 mmol)
was reacted with triethylamine (1.18 mL, 8.4 mmol) and methane-
sulfonyl chloride (0.556 mL, 7.2 mmol) as described for 2b. The crude
preparation of the mesylate was used without delay for the next step: MS
(ESP) m/z 379 (MH+).
1-[2-(cis( ))-4-Azido-3-{[tert-butyl(dimethyl)silyl]oxy}-
piperidin-1-yl)ethyl]-7-methoxyquinoxalin-2(1H)-one (40). 1b
(0.528 g, 3.0 mmol), 39 (3 mmol), and sodium hydride (in oil, 60%, 132
mg) were reacted following the general procedure for 3 to give the
product as a hard foam: 0.721 g (52%); MS (ESP) m/z 459 (MH+); 1H
NMR (DMSO-d6) δ 0.03 and 0.05 (2 × s, 6H); 0.82 (s, 9H); 1.65 (m,
2H); 2.25−2.70 (m, 6H); 3.70 (m, 1H); 3.90 (s, 3H); 3.83 (m, 1H);
4.24 (m, 1H); 4.39 (m, 1H); 6.96−7.00 (m, 2H); 7.73 (m, 1H); 8.02 (s,
1H).
1-[2-{(cis( ))-(4-Azido-3-hydroxypiperidin-1-yl)}ethyl]-7-me-
thoxyquinoxalin-2(1H)-one (41). A solution of 40 (0.721 g, 1.57
mmol) in THF (5 mL) was treated dropwise at room temperature with a
solution of tetrabutylammonium fluoride in THF (1M, 2.2 mL). After 1
h, saturated aqueous sodium hydrogencarbonate solution (10 mL) was
added and THF was removed under reduced pressure. It was extracted
with dichloromethane/ether (1:1, ∼200 mL). The aqueous phase was
back-extracted with dichloromethane (100 mL), and the combined
organic phases were dried over sodium sulfate. Chromatography on
silica gel with hexanes/acetone (1:1) gave the product as a colorless hard
foam: 0.507 g (94%); MS (ESP) m/z 345 (MH+); 1H NMR (DMSO-
d6) δ 1.58 (m, 1H); 1.70 (m, 1H); 2.25−2.65 (m, 6H); 3.67 (m, 2H);
3.90 (s, 3H); 4.31 (dd, 2H); 5.11 (m, 1H); 6.97−7.00 (m, 2H); 7.73 (d,
1H); 8.03 (s, 1H).
genated in methanol (50 mL) over 10% palladium on carbon (∼400
mg) at normal pressure for 1 h. The reaction mixture was filtered
through Celite. The filtrate was concentrated under reduced pressure to
give 1.3 g (quant) of the product as a colorless solid: 1H NMR (DMSO-
d6) δ 0.00 (s, 6H); 0.80 (s, 9H); 1.20−1.30 (m, 1H); 1.33 (s, 9H); 1.53
(d, 1H); 2.15 (dd, 1H); 2.23−2.39 (m, 1H); 2.74 (d, 1H); 2.88 (dd,
1H); 3.20−3.30 (m, 2H); 4.08 (s, 1H); 6.58 (d, 1H).
tert-Butyl-[trans( )-3-{[tert-butyl(dimethyl)silyl]oxy}-1-(2-
hydroxyethyl)piperidin-4-yl]carbamate (46). 45 (1.3 g, 3.9 mmol),
2-bromoethanol (0.36 mL, 5.1 mmol), and ethyl(diisopropyl)amine
(1.0 mL, 5.9 mmol) were reacted using the procedure described for 38
to give 1.0 g (67%) of the desired product: 1H NMR (DMSO-d6) δ 0.00
(s, 6H); 0.79 (s, 9H); 1.33 (s, 9H); 1.39 (dd, 1H); 1.46−1.58 (m, 1H);
1.71−1.82 (m, 1H); 1.82−1.93 (m, 1H); 2.33 (t, 2H); 2.72 (d, 1H);
2.80−2.90 (m, 1H); 2.99−3.16 (m, 1H); 3.32−3.47 (m, 3H); 4.36 (t,
1H); 6.56 (d, 1H).
2-[(3S,4R)-4-(Dibenzylamino)-3-methoxypiperidin-1-yl]-
ethanol (48). A mixture of cis( )N,N-dibenzyl-3-methoxypiperidin-4-
amine 4727 (1.7 g, 5.5 mmol), bromoethanol (0.5 mL, 7.1 mmol), and
N,N-diisopropylethylamine (1.4 mL, 8.3 mmol) were reacted like
described for 38, heating for 1 h at 70 °C. Chromatography on silica gel
with 5% methanol in dichloromethane containing 0.25% ammonium
hydroxide gave 1.3 g (68%) of the cis-racemic product as a colorless
solid: MS (ESP) m/z 355 (MH+); 1H NMR (DMSO-d6) δ 1.44−1.58
(m, 1H); 1.64 (d, 1H); 1.79−2.08 (m, 2H); 2.32 (t, 2H); 2.36−2.45 (m,
1H); 2.88 (d, 1H); 3.13 (d, 1H); 3.30 (s, 3H); 3.40−3.49 (m, 2H); 3.56
(s, 1H); 3.59−3.87 (m, 4H); 4.34 (s, 1H); 7.11−7.24 (m, 2H); 7.24−
7.40 (m, 8 H).
The enantiomers were separated by chiral chromatography on a chiral
cell OJ column (250 × 20 mm, 10 μm) eluting with 1:1 methanol/
ethanol and 0.1% diethylamine at 10 mL/min flow rate. The (−) isomer
(3R,4S) eluted first followed by the (+) isomer (3S,4R) (48).
tert-Butyl-[(3S,4R)-1-(2-hydroxyethyl)-3-methoxypiperidin-
4-yl]carbamate (49). A solution of 48 (3S,4R isomer) (940 mg, 2.66
mmol) and di-tert-butyldicarbonate (0.67 mL, 2.92 mmol) in methanol
(100 mL) was hydrogenated over 20% palladium hydroxide on carbon
(240 mg) overnight. The reaction mixture was filtered through Celite
and concentrated to dryness under reduced pressure. Chromatography
on silica gel with 2−10% methanol in chloroform gave 540 mg (74%) of
1
the product as a colorless oil: H NMR (DMSO-d6) δ 1.38 (s, 9H);
1.43−1.50 (m, 1H); 1.58−1.73 (m, 1H); 2.15 (d, 2H); 2.37 (t, 2H);
2.54−2.64 (m, 1H); 2.75−2.88 (m, 1H); 3.22 (s, 3H); 3.27−3.32 (m,
1H); 3.41−3.59 (m, 3H); 4.37 (t, 1H); 6.35 (d, 1H).
Benzyl-trans( )-4-[(tert-butoxycarbonyl)amino]-3-hydroxy-
piperidine-1-carboxylate (43).26 A mixture of benzyl trans( )-4-
amino-3-hydroxypiperidine-1-carboxylate 4225 (3.0 g, 12.0 mmol), di-
tert-butyldicarbonate (2.9 g, 13.2 mmol), and sodium bicarbonate (3.0 g,
36.0 mmol) in ethyl acetate/water (1:1, 100 mL) was stirred vigorously
overnight. The biphasic mixture was separated. The aqueous phase was
re-extracted with ethyl acetate. The combined organic phases were dried
over sodium sulfate and concentrated under reduced pressure to give 4.2
g (quant) of the product as a colorless solid. This material was used
Benzyl-trans( )-4-[(tert-butoxycarbonyl)amino]-3-methoxy-
piperidine-1-carboxylate (50). 43 (1.0 g, 2.86 mmol) was suspended
in 10 mL of toluene and treated with a 50 wt % solution of aqueous
sodium hydroxide (6 mL) followed by dimethylsulfate (0.33 mL, 3.43
mmol) and benzyl triethylammonium chloride (catalytic amount). The
reaction was stirred vigorously for 1 h. The reaction was quenched with
ice. The phases were separated. The aqueous phase was re-extracted
with ethyl acetate. The combined organic phases were dried over
sodium sulfate and concentrated under reduced pressure. Chromatog-
raphy on silica gel with 25−50% acetone in hexanes gave 0.78 g (78%) of
1
without further purification: H NMR (DMSO-d6) δ 1.15−1.32 (m,
1H); 1.35−1.42 (m, 9H); 1.71−1.83 (m, 1H); 2.60−2.79 (m, 1H);
2.82−2.98 (m, 1H); 3.15−3.29 (m, 2H); 3.74−3.86 (m, 1H); 3.88−3.98
(m, 1H); 5.00 (d, 1H); 5.04−5.08 (m, 2H); 6.73 (d, 1H); 7.25−7.42 (m,
5H).
1
the product as a colorless oil: MS (ESP) m/z 365 (MH+); H NMR
(DMSO-d6) δ 0.55−0.68 (m, 10H); 1.04−1.19 (m, 1H); 2.17−2.46 (m,
2H); 2.55−2.64 (m, 2H); 2.67−2.80 (m, 1H); 2.85−3.07 (m, 1H);
3.10−3.31 (m, 1H); 4.09 (s, 3H); 4.32 (s, 2H); 6.45−6.61 (m, 5H).
tert-Butyl-[trans( )-3-methoxypiperidin-4-yl]carbamate
(51).14 50 (0.98 g, 2.69 mmol) was hydrogenated in methanol (50 mL)
over 10% Pd/C (400 mg) at normal pressure. After 1 h, the reaction
mixture was filtered through Celite. The filtrate was concentrated under
reduced pressure to give 0.61 g (98%) of the product as a colorless oil:
1H NMR (DMSO-d6) δ 1.14−1.29 (m, 1H); 1.34−1.42 (m, 9H); 1.68
(d, 1H); 2.11 (dd, 1H); 2.26−2.38 (m, 1H); 2.71−2.82 (m, 1H); 2.86−
2.98 (m, 1H); 3.14−3.21 (m, 3H); 3.26 (s, 3H); 6.75−6.86 (m, 1H).
tert-Butyl-[trans( )-1-(2-hydroxyethyl)-3-methoxypiperidin-
4-yl]carbamate (52). 51 (1.1 g, 4.8 mmol), 2-bromoethanol (0.44 mL,
6.2 mmol), and ethyl(diisopropyl)amine (1.25 mL, 7.2 mmol) were as
described for 37 to give 0.74 g (57%) of the product as a colorless oil: 1H
NMR (DMSO-d6) δ 1.24−1.34 (m, 1H); 1.38 (s, 9H); 1.62−1.77 (m,
Benzyl-trans( )-4-[(tert-butoxycarbonyl)amino]-3-{[tert-
butyl(dimethyl)silyl]oxy} piperidine-1-carboxylate (44). A mix-
ture of 4326 (2.0 g, 5.7 mmol), imidazole (0.58 g, 8.6 mmol), and tert-
butyl(chloro)dimethylsilane (1.0 g, 6.9 mmol) in DMF (15 mL) was
stirred at room temperature under nitrogen overnight. Water (50 mL)
was added to the reaction, and the mixture was extracted 2× with ether.
The combined organic phases were dried over sodium sulfate and
concentrated to dryness. Chromatography on silica gel with 10−25%
acetone in hexanes giving 1.8 g (69%) of the product as a colorless solid:
1H NMR (DMSO-d6) δ 0.00 (s, 6H); 0.80 (s, 9H); 1.27−1.41 (m,
10H); 1.61−1.72 (m, 1H); 2.59−3.05 (m, 2H); 3.30−3.40 (m, 2H);
3.69−3.95 (m, 2H); 4.92−5.14 (m, 2H); 6.68 (d, 1H); 7.24−7.40 (m,
5H).
tert-Butyl-(trans( )-3-{[tert-butyl(dimethyl)silyl]oxy}-
piperidin-4-yl)carbamate (45). 44 (1.8 g, 3.9 mmol) was hydro-
P
dx.doi.org/10.1021/jm300690s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H); 1.82−1.97 (m, 1H); 2.38 (t, 2H); 2.73 (d, 1H); 2.98−3.18 (m,
a novel nonfluoroquinolone inhibitor of bacterial type II top-
oisomerases. Antimicrob. Agents Chemother. 2008, 52, 3339−3349.
(9) Wallis, R. M. Themed section: QT safety reviewIntegrated risk
assessment and predictive value to humans of non-clinical repolarization
assays. Br. J. Pharmacol. 2010, 159, 115−121.
(10) Barfoot, C. W.; Brown, P.; Dabbs, S.; Davies, D. T.; Hennessy, A.
J.; Miles, T. J.; Pearson, N. D. The design of efficient and selective routes
to pyridyl analogues of 2,3-dihydro-1,4-benzodioxin-6-carbaldehyde.
Tetrahedron Lett. 2010, 51, 5038−5040.
3H); 3.27 (s, 3H); 3.46 (q, 2H); 4.39 (t, 1H); 6.78 (d, 1H).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +1 (510) 923-5314. Fax: +1 (510) 655-9910. E-mail:
folkert.reck@novartis.com.
Notes
(11) 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-carbaldehydes.
Tetrahedron Lett. 2010, 51, 5035−5037.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Camil Joubran, Analytical Chemistry
Department of AstraZeneca R&D Boston for performing NOE
and HMBC NMR experiments, AstraZeneca R&D Susceptibility
Testing Group for MIC testing, and Sussie Hopkins and Lena
Grosser, Pharmacology Department, AstraZeneca R&D Boston
for efficacy studies.
(12) Comita-Prevoir, J.; Cronin, M.; Geng, B.; Godfrey, A. A.; Reck, F.
WO2008071961, 2008.
(13) Van Niel, M. B.; Collins, I.; Beer, M. S.; Broughton, H. B.; Cheng,
S. K. F.; Goodacre, S. C.; Heald, A.; Locker, K. L.; MacLeod, A. M.;
Morrison, D.; Moyes, C. R.; O’Connor, D.; Pike, A.; Rowley, M.;
Russell, M. G. N.; Sohal, B.; Stanton, J. A.; Thomas, S.; Verrier, H.; Watt,
A. P.; Castro, J. L. Fluorination of 3-(3-(piperidin-1-yl)propyl)indoles
and 3-(3-(1-piperazinyl)propyl)indoles gives selective human 5-HT1D
receptor ligands with improved pharmacokinetic profiles. J. Med. Chem.
1999, 42, 2087−2104.
(14) Basarab, G.; Dangel, B.; Fleming, P. R.; Gravestock, M. B.; Green,
O.; Hauck, S. I.; Hill, P.; Hull, K. G.; Mullen, G.; Sherer, B.; Zhou, F.
WO2006087543A1, 2006.
(15) Basarab, G.; Ni, H.; Sherer, B.; Zhou, F. WO2007071965A2,
2007.
ABBREVIATIONS USED
■
NBTI, novel (non-fluoroquinolone) bacterial type II topo-
isomerase inhibitor; LHS, bicyclic aromatic left-hand side; RHS,
aromatic right-hand side (as positioned in Figure 1); MIC,
minimal inhibitory concentration; CFU, colony forming units;
ND, not determined; hERG, human ether-a-go-go-related gene;
MAPD90, monophasic action potential duration at 90%
(16) Aronov, A. M. Ligand structural aspects of hERG channel
Blockade. Curr. Top. Med. Chem. 2008, 8, 1113−1127.
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dx.doi.org/10.1021/jm300690s | J. Med. Chem. XXXX, XXX, XXX−XXX