Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.bmc.2014.07.040

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by

Full text of DOI:10.1016/j.bmc.2014.07.040

Bioorganic & Medicinal Chemistry 22 (2014) 5392–5409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Optimization of physicochemical properties and safety profile
of novel bacterial topoisomerase type II inhibitors (NBTIs)
with activity against Pseudomonas aeruginosa
a
a
a
a
Folkert Reck a, , David E. Ehmann , Thomas J. Dougherty , Joseph V. Newman , Sussie Hopkins ,
Gregory Stone a, Nikunj Agrawal a, Paul Ciaccio b, John McNulty b, Herbert Barthlow b, Jennifer O’Donnell b,
Kosalaram Goteti a, John Breen a, Janelle Comita-Prevoir a, Mark Cornebise a, Mark Cronin a,
Charles J. Eyermann a, Bolin Geng a, Greg R. Carr c, Lakshmipathi Pandarinathan a, Xuejun Tang d,
Andrew Cottone d, Liang Zhao a, Natascha Bezdenejnih-Snyder a
⇑
a Infection Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
b Drug Safety and Metabolism Innovative Medicines Unit, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
c AstraZeneca, Pharmaceutical Development, Chemical Sciences, Silk Road Business Park, Macclesfield, Cheshire, England SK10 4TG, United Kingdom
d Adesis Inc., 27 McCullough Drive, New Castle, DE 19720, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel
bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development
of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquino-
lones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially
against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were
optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized
analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly
improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile
in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for
the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 6 June 2014
Revised 18 July 2014
Accepted 26 July 2014
Available online 4 August 2014
Keywords:
Bacterial toposisomerases
Gyrase
Pseudomonas aeruginosa
hERG
1. Introduction
We were interested in bacterial type II topoisomerases (DNA
gyrase and topoisomerase IV), which are clinically proven antibac-
The emergence of multi-drug-resistant Gram-negative patho-
gens is an area of increasing concern in the medical community1
and provides the impetus to discover new classes of antibacterial
agents that are not cross resistant with existing drugs. Efforts
aimed at addressing resistance have focused on both novel targets,
as well as novel molecules that inhibit established targets through
a different mode of action.
terial targets for the fluoroquinolone class of drugs that target the
GyrA and ParC subunits of DNA gyrase and topoisomerase IV,
respectively. The clinical utility of fluoroquinolones is severely lim-
ited by mutations in the quinolone-resistance determining region
(QRDR) of the targets.2,3 The new class of NBTIs (novel bacterial
type II topoisomerase inhibitors) engage with the GyrA/ParC sub-
units of bacterial type II topoisomerases through a different mode
of inhibition that is not impacted by QRDR mutations.4–18 Other
classes of inhibitors of the GyrA/ParC subunits of bacterial type II
topoisomerases that do not show cross resistance with fluoroquin-
olones have been described.19–23
The pharmacophore of the NBTIs suffers from a hERG liability
and the mitigation of this risk has been the focus in the lead optimi-
zation phase of our program.8,10 We recently reported on efforts to
optimize physicochemical properties of NBTIs for selective Gram-
positive activity, during which the pKa of the compounds was
Abbreviations: NBTI, novel bacterial type II topoisomerase inhibitor; QRDR,
quinolone-resistance determining region; hERG, the potassium ion channel
encoded by the ‘human Ether-a-go-go-Related Gene’; LHS, bicyclic aromatic left-
hand side; RHS, aromatic right-hand side; MIC, minimal inhibitory concentration;
MIC90, minimum inhibitory concentration required to inhibit the growth of 90% of
organisms; CFU, colony forming units; ND, not determined; fCmax, protein binding
adjusted (free) peak plasma concentration.
⇑
Corresponding author. Tel.: +1 510 923 5314; fax: +1 510 923 3360.
E-mail address: Folkert.Reck@Novartis.com (F. Reck).
http://dx.doi.org/10.1016/j.bmc.2014.07.040
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5393
reduced and compounds displayed an improved cardiac safety pro-
file.10 The physicochemical property space for drugs targeting
Gram-negative bacteria differs for compounds targeting Gram-
positive bacteria, which provides guidelines for different strategies
of optimization.24 In particular, optimization of molecules targeting
intracellular targets in Gram-negative pathogens requires consider-
ation of drug permeability through porins of the outer membrane as
well as permeability by passive diffusion through the inner mem-
brane. In addition, many Gram-negative pathogens possess sophis-
ticated efflux pump systems.25 We report herein the optimization
of NBTIs against serious Gram-negative pathogens, with focus on
the problematic hospital pathogen Pseudomonas aeruginosa. In
depth biochemical and microbiological characterization of an
advanced lead from his series has been described elsewhere.18
agreement with the literature, which suggests that small hydride
reagents prefer to attack from the axial position.38 The cis and trans
isomers could be separated chromatographically after Boc-protec-
tion of the amino group and removal of the silyl protection of the
alcohol. Mesylation of the cis isomer 18 gave 19. Alkylation of 2c
with 19 gave predominantly the O-alkylated product, together
with the desired N-alkylated product 20 in low yield (14%). After
deprotection of the Boc-group, reductive amination was performed
with aldehyde 6 to give 22. Staudinger reduction of the hindered
azide 22 produced the target compound 23 in 12% yield. Under
the conditions of the Staudinger reaction, elimination to the
cyclohexene derivative was observed as a side reaction.
For the fluorocyclohexyl derivative 45 (Scheme 8), ketone 36
was reacted with trimethylsulfoxonium iodide to give the epoxide
37, which was opened to the alcohol with HF/pyridine. The
cis-configured alcohol 38 was obtained in 51% yield, together with
the trans-configured analog as a minor product. Oxidation of 38 to
the aldehyde 39, followed by Wittig olefination provided the olefin
40, which was converted to the final racemic compound 45 using
the same sequence of conversions as described above.
2. Chemistry
Most final compounds were assembled by reductive aminations
of chiral amines 5 with aldehydes 626 or 727 (Scheme 1).28 The
nosyl groups were removed using thiophenol, in most cases in
good yields. With fluoro-substituted heterocyclic analogs,
especially with naphthyridone 8j, care had to be taken to minimize
substitution of the fluoro group by thiophenol.
3. Results and discussion
The amine building blocks 5a–g were obtained by alkylation of
heterocycles 2a–g8,28–34 with the aziridine 328,35 (either racemic or
as the R-enantiomer), followed by deprotection of the Boc group
(Scheme 2). Quinoxalinones 2d and 2e were prepared from nitro
anilines as outlined in Schemes 3 and 4. The aziridine 3 was
obtained by aziridination of olefin 10 with N-(o-nitrophenylsulfo-
nyl)imino]phenyliodinane and Cu(OTf)2,36 followed by chiral
separation of the enantiomers (Scheme 5). A chiral route to the
R-enantiomer of 3 was developed as well and has been reported
elsewhere.35
For the N-methylated analog 31, methylation was performed on
nosylated 8h, via the protocol by Mitsunobu (Scheme 6).
Compound 23, containing a primary amine on the cyclohexyl
bridgehead, was prepared as outlined in Scheme 7. After reduction
of the commercially available acid 11 to the alcohol 12, the amino
group was converted to the azide 13 using triflic azide in toluene.37
The alcohol was protected to 14 and cleavage of the ketal produced
the ketone 15 in good yield. Reductive amination with ammonium
acetate gave the cis isomer as the major product. This is in
Our lead for this effort was compound 1 (Fig. 1), which displays
potent broad spectrum antibacterial activity.10 However, 1 is inhib-
iting the hERG cardiac channel (IC50 = 35 lM, Table 1), and caused
prolongation of the corrected QT (QTc) interval in a dog toxicology
study (Fig. 4). We aimed to reduce the changes in QTc by reducing
hERG inhibition, while retaining Gram-negative activity, especially
against multi-drug resistant P. aeruginosa. We have previously
reported that addressing the hERG issue through reduction of pKa
in analogs of 1 with substitution in the piperidine moiety led to
reduced Gram-negative activity.10 This effect may be due to
reduced permeability for less basic compounds through porins in
the outer membrane of Gram-negative bacteria, which favor
positively charged molecules.25,39,40 Agents active against
Gram-negative organisms generally fall within a lower logD range
than for drugs against Gram-positive organisms,24 and this, in our
experience, is especially true for P. aeruginosa. We chose therefore
to explore further reduction in logD in our NBTI compounds,
specifically through dibasic compounds that may transit better
through the porins of Gram-negative bacteria.
O
O
O
NH
N
O
NH
N
H
N
O
H
N
O
Nos
N
N
HN
or
H2N
N
N
H
O
N
H
z
O
z
6
O
7
O
O
R1
R2
N
A
N
X
O
R1
R2
B
5a-g
Y
X
Y
8a-j
9a-j
a: X= C; Y= C; Z= N; R1= OMe; R2= H
b:
X= CMe; Y= C; Z= N; R1= F; R2= H
c: X= CMe; Y= C; Z= C; R1= F; R2= H
d:
X= N; Y= C; Z= N; R1= OMe; R2= H
e: X= N; Y= C; Z= C; R1= OMe; R2= H
f
: X= N; Y= C; Z= C; R1= F; R2= H
g: X= N; Y= C; Z= N; R1= F; R2= H
h
: X= N; Y= C; Z= N; R1= OMe; R2= F
i: X= C; Y= N; Z= C; R1= OMe; R2= H
j
: X= C; Y= N; Z= C; R1= F; R2= H
Scheme 1. Reagents and conditions: (A) Molecular sieves 3 Å, CHCl3/MeOH, 70 °C, then NaBH(OAc)3, 0 °C to room temperature; (B) K2CO3, thiophenol, DMF, room
temperature.

5394
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
Nos
O
HN
NHBoc
NO2
H
N
O
O
O
R1
R2
S
A
H
N
R1
R2
N
N
+
O
Y
X
Y
X
O
2a-g
3
4a-g
B
a: X= C; Y= C; R1= OMe; R2= H
b:
X= CMe; Y= C; R1= F; R2= H
c: X= N; Y= C; R1= OMe; R2= H
Nos
HN
d
: X= N; Y= C; R1= OMe; R2= F
e: X= N; Y= C; R1= F; R2= H
NH2
f
: X= C; Y= N; R1= OMe; R2= H
g: X= C; Y= N; R1= F; R2= H
O
R1
R2
N
Y
X
5a-g
Scheme 2. Reagents and conditions: (A) NaH, DMF, room temperature; (B) TFA/DCM, room temperature.
for introduction of a second basic group in the 2-position of the
NH2
NH2
O
F
O
F
NH2
NO2
B
NH2
NO2
F
F
A
ethyl bridge such as in compound 9e (Table 1),28,41 which also leads
to a reduction in logD (from 0.6 for 1 to ꢀ0.36 for 9e). The pKa of the
primary amine in compounds 9e was ꢁ7.5, indicating partial pro-
tonation at physiological pH (Table 1). The change from a piperidine
to a cyclohexyl linker increased the pKa of the secondary amine,
from 8.42 in 1 to 9.09 in 9e (the third pKa value for 9e at 10.66 is
associated with deprotonation of the NH functionality of the oxaz-
inone RHS, denoting a weak acid). The pKa of the secondary amine in
9e could be lowered by ꢁ0.8 log units to match the pKa of the lead 1
by incorporation of pyrimidine right-hand-side (RHS) heterocycle,
without large changes in logD (compare analogs 9b and 9d to 9c
and 9e). Fine tuning of the pKa of the more basic secondary amine
was aimed at avoiding strongly basic groups that could engender
a safety risk42 and to retain good permeability through the inner
membrane of Gram-negative organisms. Introduction of a fluorine
in the bridgehead of the cyclohexyl moiety (compound 45) led to
a decrease in pKa for both amine moieties of 45 relative to the des-
fluoro analog 9e (pKas 8.57 and 5.87, vs 9.09 and 7.55, respectively).
Inhibition of hERG was measured by the IonWorks assay43 and
was found to be significantly reduced with all dibasic amin-
ocyclohexyl analogs relative to 1, tracking roughly with the
reduced logD (Table 1). Compounds were difficult to rank-order
by inhibition of hERG, due to solubility limitations in the IonWorks
25
26
24
H
H
N
F
N
O
O
F
O
C
+
N
O
N
27
2d
E
F
N
Cl
O
F
N
Cl
D
+
N
O
N
29
28
Scheme 3. Reagents and conditions: (A) KOH, MeOH, room temperature, quant; (B)
Pd/C, H2, EtOH/HOAc, room temperature; (C) 2-oxoacetate, toluene, room temper-
ature; (D) POCl3, 100 °C; (E) 5 M HCl, 125 °C, 64%.
assay at concentrations above 333 lM. However, the most polar
analogs such as 9i and 9j displayed no detectable inhibition of
hERG at the top concentration tested, reinforcing the link between
polarity and hERG activity.
In this context, we were interested in the trans amino
ethyl cyclohexyl linker in combination with the N-linked left-
hand-side (LHS) NBTI scaffold,9,10,28,41 because this linker allows
H
N
H
N
F
NO2
NH2
F
NO2
N
F
O
F
O
B
A
C
OEt
N
H
N
O
32
33
2e
34
Scheme 4. Reagents and conditions: (A) Ethylglyoxylate, toluene, reflux; (B) Pd/C, H2, EtOH; (C) NaOH, H2O2, 80 °C.
NO2
NO2
O
S
O
S
O
N
O
N
A
H
N
H
N
H
N
+
O
O
O
3 (S)
O
O
10
3 (R)
O
Scheme 5. Reagents and conditions: (A) [N-(o-nitrophenylsulfonyl)imino]phenyliodinane,36 Cu(OTf)2, CH3CN, room temperature.

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5395
H
N
O
H
N
O
Nos
O
N
R
N
HN
N
N
H
N
H
O
N
O
N
O
F
N
A
O
O
F
N
N
N
30
: R= Nos
8h
B
31: R=H
Scheme 6. Reagents and conditions: (A) P(Ph)3, DIAD, THF/MeOH, room temperature; (B) K2CO3, thiophenol, DMF, room temperature.
O
TBDPSiO
O
O
HO
O
O
C
HO
HO
O
O
B
O
O
A
N3
N3
H2N
H2N
13
14
12
11
TBDPSiO
TBDPSiO
TBDPSiO
F
E
D
NHBoc
NH2
O
N3
N3
N3
17
NHR
16
15
18
N3
O
I
H
NHBoc
G
NHBoc
O
N
N
MsO
HO
N3
N3
19
20: R= Boc
21: R= H
J
H
N
H
N
O
O
N
N
N
N
H
O
H
NH2
O
N3
L
O
O
N
N
K
N
N
O
O
23
22
Scheme 7. Reagents and conditions: (A) LAH, THF, reflux, 91%; (B) TflN3, NaHCO3, CuSO4, toluene/DMF/water, room temperature, 79%; (C) TBDPSCl, imidazole, DMF, room
temperature, quant; (D) p-TsOH, acetone, 80 °C, 75%; (E) NH4OAc, NaBH3CN, MeOH, room temperature, quant; (F) NaHCO3, Boc2O, EtOAc/water, room temperature, 78%; (G)
TBAF, THF, room temperature, 52%; (H) MsCl, NEt3, CH2Cl2, 0 °C; (I) 2c,8,31,32 NaH, DMF, room temperature ꢀ40 °C, 14%; (J) TFA, CH2Cl2, 0 °C, 98%; (K) 6,26 molecular sieves 3 Å,
DMF, 60 °C, then NaBH(OAc)3, 0 °C to room temperature, 69%; (L) P(Ph)3, CH3CN/water, room temperature, 12%.
C
B
A
NHBoc
NHR
NHBoc
O
NHBoc
O
NHBoc
O
HO
F
F
39
38
37
36
NO2
NH
NO2
O
S
O
S
O
O
D
NHBoc
E
N
F
NHBoc
F
O
N
O
F
F
40
41
N
42: R= Boc
43: R= H
G
H
H
N
NO2
NH
N
O
O
S
O
H2N
N
N
H
I
N
N
H
O
H
O
O
F
F
O
O
O
N
O
N
N
44
45
N
Scheme 8. Reagents and conditions: (A) tBuOK, (CH3)3SOI, THF, 98%; (B) 70% HF/pyridine, DCM, ꢀ78 °C, 51%; (C) Dess–Martin, DCM, 0 °C to rt, 56%; (D) Ph3P(CH3)Br,
K((CH3)3Si)2N, 0 °C, 57%; (E) o-NO2 PhSO2 NIPh, Cu(OTf)2, molecular sieves, CH3CN, 15%; (F) 2c, NaH, DMF, 54%; (G) TFA, DCM, quant; (H) 6, NaBH(OAc)3, DIEA, DMF, 80%; (I)
PhSH, K2CO3, DMF, 29%.

5396
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
H
The antibacterial activity of the compounds was determined
O
N
N
against the Gram-negative pathogens P. aeruginosa and E. coli and
the Gram-positive organism S. aureus (Table 1). In general, the
amino cyclohexyl analogs showed potent activity against the
Gram-negative organisms. Activity against P. aeruginosa was simi-
lar or better compared to the lead 1 for the best analogs (9c, 9d, 9e,
9f, 9g, 9i). Plasma protein binding was generally lower than for 1,
in the range of 60–70% free fraction for most compounds (Table 1).
Activity against S. aureus was generally weaker for this set than for
lead 1, and tracked with lower logD. Compound 45, which is less
basic and has a relatively high logD, was more potent against S.
aureus than against Gram-negatives. Analog 23, with an amino
group on the bridgehead of the cyclohexyl moiety showed rela-
tively weak antibacterial activity against both Gram-negative
organisms and S. aureus, despite similar potency against topoiso-
merase IV. We were unable to measure pKa and logD for 23, but
would expect 23 to be strongly dibasic. This may impair its ability
to permeate through the inner membrane of Gram-negative organ-
isms and the lipid bilayer of S. aureus. It is interesting to note in this
respect that a small molecule X-ray structure of 9g indicated an
intramolecular H-bond between the amino group and the carbonyl
N
H
O
N
O
O
N
N
Figure 1. N-linked aminopiperidine lead 1.
We determined inhibition of the topoisomerase IV target in
Escherichia coli. Most of the S-amino ethyl cyclohexyl analogs
showed IC50s in the low nanomolar range, similar to the amino
piperidine lead 1 (Table 1). The corresponding R-isomers were less
active (35 vs 9i), as were compounds with substitutions on the pri-
mary amine (compare 31 and 9h). Docking of N-methyl analog 31
into the published crystal structure of the Staphylococcus aureus
gyrase/DNA complex4 indicated a steric clash of methyl group with
the DNA (Fig. 2).
Analogs with either a fluoro- or an amino-substituent on the
bridgehead of the cyclohexyl moiety (45 and 23) showed good
potency against topoisomerase IV.
Table 1
SAR of amino ethyl cyclohexyl NBTIs versus amino piperidine 1
H
N
O
N
NH
O
N
N
N
O
O
1
H
H
N
O
O
H
N
N
O
R2
NH
N
N
N
NH2
O
R3
NH
NH
O
NH
O
N
z
O
R4
O
O
F
N
N
R1
N
O
O
N
Y
X
Y
X
9h: (R), R2= H
31
9a-g, i, j
R1, X, Y, Z see table
: racemic, R= Me
35: R3= (S) NH2; R4= H; X= C; Y= N
: R3= NH2; R4= F; X= N; Y= C
23: R3= H; R4= NH2; X= N; Y= C
45
Compd
R1
X
C
CMe
CMe
N
Y
Z
P. aeruginosaa
MIC
E. colib
MIC
S. aureusc
MIC
Fud (%)
E. coli TopoIVe
IC50 (nM)
logDf
pKa
hERGg
IC50
(lM)
1
1
2
2
0.5
0.5
0.5
0.25
0.5
2
1
2
>64
16
8
0.13
0.5
0.5
0.5
0.2
0.5
<0.06
<0.06
2
0.5
1
16
8
2
0.03
8
4
1
1
1
1
1
4
26
71
66
64
60
70
70
73
56
78
ND
ND
>70
68
ND
2.2
5
2
3
2
1
28
3
4
2
0.60
8.42/5.56/10.46
ND
35
9a
9b
9c
9d
9e
9f
9g
9h
9i
OMe
F
F
OMe
OMe
F
C
C
C
C
C
C
C
N
N
C
N
C
ꢀ0.54
>100
>333
>333
>333
>333
174
>333
>333
>333
>100
ND
0.06
8.42/7.59/9.39
9.21/7.81/10.58
8.33/7.45/9.34
9.09/7.55/10.66
ND
8.25/7.28/9.28
ND
9.09/7.66/10.66
ND
ꢀ0.05
ꢀ0.48
ꢀ0.36
ꢀ0.41
ꢀ0.32
ꢀ0.42
ꢀ0.86
ꢀ1.02
ND
N
N
N
C
N
F
OMe
F
C
C
N
N
C
C
8
9j
16
64
128
0.5
2
34
39
33
5
31
35
45
23
ND
ND
OMe
C
N
C
ꢀ0.84
>100
>33
ND
0.25
8.57/5.87/10.51
ND
8
1
2
ND
Minimum inhibitory concentration (MIC, lg/mL): lowest drug concentration that reduced growth by 90% or more.
ND: not determined. Aqueous solubility was >333
lM for all compounds (results not shown).
a
Pseudomonas aeruginosa strain PAO1.
Escherichia coli strain W3110.
Methicillin-susceptible Staphylococcus aureus.
Fraction unbound, human,% free.
b
c
d
e
Escherichia coli topoisomerase IV IC50
.
f
Partition coefficient at pH 7.4.
g
43
hERG IonWorks IC50
.

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5397
Figure 2. Docking of the R-enantiomer of compound 31 into the crystal structure of S. aureus gyrase. Intermolecular and intramolecular hydrogen-bonds distances are shown
in yellow. The N-methyl group in compound 31 has an unfavorable polar-nonpolar interaction with the C11 DNA base. The docking model was generated using Schrodinger’s
GLIDE software.
population of P. aeruginosa and E. coli strains, including fluoroquin-
olone-resistant isolates (Table 2), but were less active versus
K. pneumoniae. In our experience, optimal cellular potency against
K. pneumoniae for this chemical series requires higher logD values
relative to P. aeruginosa.
The pharmacokinetic properties of 9g and 9i following iv dosing
were determined in rat and dog (Table 3). Both compounds showed
moderate in vivo clearance and high volumes of distribution.
Clearance in rat was significantly reduced for 9g and 9i relative
to 1. Bioavailability in rat was lower for 9g than for 1 (2 vs 18%
respectively), despite the higher clearance of 1, likely due to lower
Figure 3. Small molecule X-ray structure of compound 9g. Intramolecular H-bonds
indicated between the carbonyl of the LHS and the primary amine, and between the
secondary amine and the pyrimidine nitrogen (indicated by yellow lines) may have
relevance for the conformation in solution and may be important for permeability
permeability through lipid bilayers with 9g (another indication of
this effect is the reduced Gram-positive activity of 9g relative to 1).
Compound 9g was selected for efficacy studies in an immuno-
compromised mouse thigh model of P. aeruginosa infection
(Table 4). Dosing with 9g at 200 mg/kg/day resulted in a 2 log10
reduction in bacterial tissue burden. From a pharmacokinetic–
pharmacodynamic (PK–PD) analysis,46 the AUC/MIC ratio associ-
ated with this dose was 21.
Secondary pharmacology against a range of cardiac channels
and selected targets from the AstraZeneca secondary pharmacol-
ogy screen that were hit by 1 is shown in Table 5. Whereas 1 exhib-
ited measurable inhibition of both the hERG and sodium ion-gated
channels, as well as against NET, 5-HT4 and SERT, both 9g and 9i
did not exhibit detectable inhibition of any channel in this panel
at the top concentrations tested and showed a cleaner profile
against non-ion channel hits. Interestingly, fluorinated analogs
such as 9g displayed consistently a cleaner non-ion channel
secondary pharmacology profile than the corresponding methoxy
analogs such as 9i, despite the higher logD. Compound 9g was also
better tolerated in rat than 1 or 9i (results not shown).
through the inner membrane of Gram-negative bacteria.
Table 2
a
MIC90
(l
g/mL) values versus key Gram-negative pathogens
Organism (number of strains)
1
9g
9i
Levofloxacin
Pseudomonas aeruginosa (20)
Escherichia coli (20)
Klebsiella pneumoniae (20)
4
0.25
4
4
1
16
4
0.5
8
16
16
64
a
MIC90 values of clinical isolates of the indicated organisms. MIC90 is the con-
centration of compound that will inhibit at least 90% of the organisms.
functionality (Fig. 3), which would not be possible with 23. Intra-
molecular H-bonds can reduce hydration and are known to
increase permeability through lipid bilayers.44,45
Compounds 9g and 9i were selected for further profiling based
on an improved hERG IC50 and potency against Gram-negative
organisms. Both compounds showed excellent activity against a
Table 3
Pharmacokinetics in rat and doga
Compound
Clearance (mL/min/kg)
Volume (L/kg)
Half-life (h)
AUC (iv)
lg h/mL
Bioavailability (%)
Rat
1
9g
9i
252
46
47
23
9
13
1.9
10
9
0.19
3.3
3.9
18
2
ND
Dog
1
9g
9i
8.5
14
14
2
4
2.7
13
9
8
6.8
12
3.8
ND
ND
ND
a
Pharmacokinetic parameters following administration of 10 mg/kg iv infusion.

5398
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
Table 4
Exposure and efficacy of compound 9g in an immunocompromised mouse thigh modela
Compound 9g
Log reduction in CFU/g thigh versus start of treatmentb
3.5
0.2
1.3
2.5
AUC1 (
130
14.8
20.1
29.9
lg/h/mL)
Free AUC/MICc
Levofloxacin 160 mg/kg/day
100 mg/kg/day
150 mg/kg/day
65
12
16
23
200 mg/kg/day
a
b
c
In vivo activity against P. aeruginosa PAO1 (ARC545). Vehicle control grew 1.5 log in the first 10 h post-infection and the mice were sacrificed for humane reasons.
Relative to starting inoculum.
Free AUC/MIC ratios based upon an MIC = 1 lg/mL and a mouse protein binding value of 21.5%.
were studied in the anesthetized guinea pig and in dog (Fig. 4).
In the guinea pig, 9g caused a statistically significant change in
monophasic action potential duration at 90% repolarization
Table 5
Secondary pharmacology panel results for compound 9g and 9i compared to 1
Secondary pharmacology target
1 IC50
(lM) 9g IC50
(l
M) 9i IC50 (lM)
(MAPD90) at a free drug concentration of 86
represents a leftward-shift relative to the hERG IC50 value of
>333 M. However, inspection of the dose–response curve for 9g
vs the hERG channel revealed ꢁ40% inhibition at 333 M. With this
lM (EC10). This effect
Norepinephrine transporter
(NET)
61a
9a
52a
Serotonin 5HT 4 receptor (5-HT4) 99a
7a
32a
l
Serotonin transporter (SERT)
Potassium channel, IKr (hERG)
Potassium channel, IKs (KvLQTS)
Potassium channel, IKur (Kv1.5)
Potassium channel, Ito (Kv4.3)
Sodium channel (Nav1.5)
Calcium channel, (Cav1.2)
Calcium channel, (Cav3.2)
Cyclic nucleotide-gated 4 (HCN4) >100
77a
35
32a
32a
l
>333
>100
>100
>333
>333
>100
>100
>100
>333
>333
ND
>333
>333
>333
ND
in mind, the effect seen in the guinea pig assay is consistent with
the hypothesis that 10–20% inhibition of hERG may be sufficient
to cause a detectable signal for QT prolongation in vivo.47 Prolon-
gation of the QTc interval was next investigated in a telemetered
dog study. A statistically significant 9% prolongation effect was
>100
>100
>100
92
>333
>100
seen in the dog with compound 9g at 56 lM, which is similar to
ND
the exposure that produced an effect in the guinea pig model. In
the dog model, the concentration where 9g gave a QTc signal rep-
resents roughly a 3-fold improvement over 1. For 9g, the resulting
margin from MAPD90 EC10 and QTc changes to predicted human
fCmax was estimated to be in the range of ꢁ2–4, depending on
the efficacy endpoint (one to two log reduction of bacterial load
in the P. aeruginosa thigh model).
a
% Inhibition of receptor binding at 30
lM test compound; aqueous solubility
was >333 M for all compounds (results not shown).
l
Predicted free Cmax, 9g
IonWorks hERG, 1
IonWorks hERG, 9g
GP MAP, 9g
100
75
50
25
0
20
15
10
5
Dog telemetry, 1
4. Conclusions
Dog telemetry, 9g
The NBTI lead 1 was optimized for reduced hERG channel activ-
ity and for activity against the Gram-negative organism P. aerugin-
osa by introduction of a second basic group in the linker that both
reduces the logD of the compounds and allows formation of an
intramolecular H-bond. The resulting optimized amino ethyl cyclo-
hexyl NBTI 9g shows a significantly reduced inhibition of hERG rel-
ative to 1. The effects of compound 9g on the monophasic action
potential (MAP) were studied in the guinea pig model and QT pro-
longation was studied in dog and the results indicated reduced
potential for QT prolongation for 9g relative to earlier analogs such
as 1.10
Compounds 9g and 9i have excellent potency against P. aerugin-
osa, including fluoroquinolone-resistant isolates, with MIC90s of
4 lg/mL. Compound 9g was evaluated in a neutropenic mouse
0
1
10
100
1000
thigh infection model against P. aeruginosa and was found to be
efficacious with a 2.5 log reduction in CFU associated with a free
plasma AUC/MIC ratio of 21, a ratio similar to that seen for fluoro-
quinolones.48 The pharmacokinetic properties of 9g and 9i were
studied in rat and dog and showed moderate clearance and high
volumes of distribution. Clearance in rat was significantly reduced
with 9g and 9i relative to 1. This compound class thus shows
promise for the development of novel antimicrobial agents against
drug-resistant P. aeruginosa.
[9g] or [1], (µM)
Figure 4. Effects on cardiac repolarization potential (MAPD90) of the guinea pig and
QT prolongation of the dog for compounds 9g and 1: In the guinea pig model, 9g
and vehicle control were infused intravenously over two 17-min periods and MAP
duration at 90% repolarization (MAPD90
potential duration were observed for 9g (closed circles), with an EC10 of 71
)
was determined. Increases in action
M free
l
(for in vivo studies x-axis concentrations are corrected to account for protein
binding). In the dog studies 1 and 9g were dosed intravenously and data are
presented as a percent change from control recordings. Details on the calculations
of cardiac parameters, including vehicle control data, can be found in the Section 5.
Ion Works hERG dose response data for
comparison. The predicted free Cmax value for the human therapeutic dose of 9g,
22 M, is indicated by the vertical dashed black line.
1 and 9g are plotted (triangles) for
5. Experimental section
l
5.1. Minimum inhibitory concentration testing
Cardiovascular effects were of particular concern, as they were
observed for 1 and related compounds of this class.10 Therefore,
effects of compound 9g on the cardiac repolarization potential
Minimum inhibitory concentrations (MICs) were determined
by broth microdilution according to the Clinical and Laboratory

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5399
Standards Institute guidelines.49 Bacterial cultures were incubated
at 35 °C on blood agar plates (Remel #01202) for 18–20 h prior to
MIC determinations. Culture media for bacterial MIC determina-
tions was Mueller Hinton Broth, Difco #275730 in 96 well microti-
ter plates. Compounds were dissolved in 100% DMSO and diluted
to 2% DMSO (v/v) in culture medium in doubling dilutions from
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.
64 to 0.06 lg/mL. After inoculation, plates were incubated for
18–20 h, and optical density of the wells was read by spectropho-
tometry at 620 nm. The MIC is the concentration at which >90%
inhibition of growth (OD) has occurred.
5.8. Pseudomonas aeruginosa neutropenic thigh infection
model
Compound 9g was studied in a neutropenic mouse thigh infec-
tion model as described in Mills et al.50 Briefly, mice were rendered
neutropenic by injecting cyclophosphamide (Sigma–Aldrich, St.
Louis MO) intraperitoneally 4 days (150 mg/kg of body weight)
and 1 day (100 mg/kg) before experimental infection. Mice were
infected with P. aeruginosa PAO1 to achieve a target inoculum of
5 ꢂ 105 CFU. Groups of five animals each received an intraperito-
neal injection of 9g at the doses specified in Table 4, prepared in
5% dextrose with lactic acid pH 5.0, on a bid, q12 regime starting
2 h after infection. An additional group of five mice received vehi-
cle alone. Efficacy was determined 24 h after the start of treatment.
Thighs were removed, weighed, homogenized and aliquots plated
onto tryptic soy agar plates and incubated at 37 °C overnight for
CFU determination.
5.2. Topoisomerase IV assay
IC50 assays measuring the ATP-ase activity of E. coli TopoIV were
performed as described previously.8
5.3. Plasma protein binding
Plasma protein binding was determined using the Dianorm
equilibrium dialysis chamber. Compound (10 lM concentration)
was spiked in the plasma chamber (donor side), 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 rep-
resents the free fraction. LC/MS–MS quantitative sample analysis
was achieved using an Ace C18 50 ꢂ 4.6 mm column (MacMod,
PA) and electrospray ionization MRM detection (PE Sciex API
4000 mass spectrometer, Applied Biosystems CA). Plasma samples
5.9. IonWorks
Electrophysiologic measurements were conducted in ion chan-
nel expressing CHO or HEK cells using an automated, 384-well
PatchPlate™ device (IonWorks™ (Schroeder et al., 2003)) and
based around the method described by Bridgland-Taylor et al.,
2006. Initially, a pre-compound current was evoked in each cell
by a voltage pulse specific to each channel type. Test compounds,
vehicle or positive control were then added to each well and after
ꢁ3 min the voltage pulse was re-applied to generate a post-com-
pound current. For each IonWorks™ run, the current amplitude
in each cell was compared in the presence of test compound for
the same cell. All data were then scaled by defining the effect of
0.33% DMSO as 0% inhibition and the effect of the supra-maximal
blocking concentration of the positive control as 100% inhibition.
Percentage inhibition data from up to 4 cells for each test concen-
tration was determined from at least two runs.
(50
lL) were treated with methanol (150 lL) containing an inter-
nal standard to precipitate the protein. Concentration determina-
tion was based on a standard curve (10 nM to 10
processed by the Analyst Version 1.4.1. software.
lM), data
5.4. LogD determination
The partition coefficient (logD) 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.
5.5. pKa determination
pKas were determined at Sirius Analytical Instruments Ltd (For-
est Row Business Park, Station Road, Forest Row, East Sussex, TH18
5DW) by a Gold Standard pH metric assay on a Sirius T3 automated
system in triplicate. The accuracy of the measurement was approx-
imately 0.02 log units.
5.10. Guniea pig
Cardiac electrophysiological characterization of compounds
was performed by the method described by.51 Male guinea pigs
were anaesthetized with NembutalÒ 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 record-
ing of lead II electrocardiogram. The chest was opened and a bipo-
lar electrode was clipped to the left atrial appendage for cardiac
pacing. A suction electrode for recording of the monophasic action
potential (MAP) was positioned on the left ventricular epicardial
wall. For the cardiac electrophysiological characterization of the
drug, the MAP duration at 90% repolarization (MAPD90) were
recorded during cardiac pacing. The compound was infused intra-
venously with two 17-min infusion periods and cardiovascular sig-
nals were continuously recorded. The first infusion period was
administered at 17 mg/kg at a rate of 0.1 mL/kg min; the second
5.6. Animals
Wistar Han rats for pharmacokinetic studies were obtained
from Charles River Laboratories (Raleigh, NC). CD-1 mice were
obtained from Charles River Laboratories (Kingston, NY). All ani-
mals were housed and acclimated in the animal facility on site
before each study. All experimental procedures were conducted
in accordance with protocols approved by the Institutional Animal
Care and Use Committee.
5.7. Pharmacokinetic studies
Pharmacokinetic properties of selected compounds were stud-
ied in male rat and Beagle dog. Plasma pharmacokinetics were
determined from 0 to 24 h following 15 min iv infusions at
infusion period was administered at 51 mg/kg, at
a rate of
0.2 mL/kg min. Blood samples were collected at each dose level
for determination of plasma concentration. Calculation of% change
in MAPD90 was relative to the time zero baseline for each animal.
10 mg/kg. Serial 200
lL samples of whole blood were taken at time
intervals. Concentration of compound in plasma was determined

5400
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
The vehicle employed was 5% dextrose in water and the change in
MAPD90 was less than 2% during vehicle alone infusion.
temperature and diluted with water (50 mL). The brown precipi-
tate was collected by filtration and washed with water to give
the desired product as a light brown solid, 1.98 g (64%). MS (ESP)
m/z 195 (MH+); 1H NMR (DMSO-d6) d: 12.38 (br s, 1H); 8.05 (s,
1H); 7.65 (d, 1H); 6.92 (m, 1H); 3.92 (s, 3H).
5.11. Dog telemetry
For compound 1, non-invasive ECG telemetry data was recorded
continuously from a small, lightweight transmitter that was
housed in a standard animal jacket. The transmitter was connected
to four adhesive skin electrodes producing an approximate Lead II
configuration. Each dog was single housed in pens during record-
ings fitted with appropriate radio antennae to minimize distur-
bances. For compound 9g, dogs were surgically implanted with
telemetric transmitter in the peritoneal cavity. The electrodes of
transmitters were placed in Lead II configuration and a sensor
catheter was introduced into the femoral artery to measure blood
pressure. For both compounds, recordings were made once during
the pre-study period, on Day 1 and at end of the repeat dose period.
Heart rate (RR interval) and ECG intervals (PR, QRS, QT, QTcV and
QTcR) were measured or calculated (QTcV is QT adjusted for heart
rate using Van de Water’s correction, QTcR is QT adjusted for heart
rate using an individual animals correction. The% change in QTcR
was calculated relative to the matched time point from the same
animal dosed with the vehicle control. Vehicle for compound 1
was 5% w/v dextrose in water, adjusted to pH 4 and route of
administration was by intravenous infusion. Dosing duration was
1 h at a rate of 5 mL/kg/h. Vehicle for compound 9g was 5% manni-
tol/0.1 M tartaric acid in water, adjusted to pH 4 and route of
administration was by intravenous infusion. Dosing duration was
4 h at a rate of 2 mL/kg/h. Data plotted on Figure 4 are from mea-
surements made at completion of infusion, 1 h for compound 1 and
6.2. 7-Fluoro-1H-quinoxalin-2-one (2e)
To a solution of sodium hydroxide (597.1 g) in water (6.7 L) was
added 34 followed by a solution of 3 wt % hydrogen peroxide in
water (6.7 L). The reaction mixture was slowly heated to 80 °C
and maintained at this temperature for 4 h. Then the heating
source was removed and acetic acid (860 mL) was added dropwise.
The suspension was stirred overnight at room temperature and the
precipitate was collected by suction filtration and dried under vac-
uum at 60 °C to afford the product as an off-white solid (522 g). MS
(ESP) m/z 165 (MH+); 1H NMR (DMSO-d6) d: 12.44 (br s, 1H); 8.13
(s, 1H); 7.83 (m, 1H); 7.79 (m, 1H); 7.16 (m, 1H); 7.04 (m, 1H).
6.3. trans-tert-Butyl 4-(1-(2-nitrophenylsulfonyl)aziridin-2-yl)-
cyclohexylcarbamate (3)
To a solution of copper(II) triflate (643, 1.78 mmol) in dry ace-
tonitrile (2 mL) was added a solution of commercially available
tert-butyl 4-vinylcyclohexylcarbamate 10 (4.0 g, 17.75 mmol) in
dry acetonitrile (4 mL, heated till dissolved). The mixture was
warmed gently to enhance solubility, then cooled to room temper-
ature
and
[N-(o-nitrophenylsulfonyl)imino]phenyliodinane36
(7.2 g, 17.75 mmol) was added. The reaction mixture became exo-
thermic after several minutes. The mixture was stirred at room
temperature under nitrogen for 3 h, then concentrated under
reduced pressure. The crude product was taken up in dichloro-
methane and the solids were removed by filtration. The filtrate
was chromatographed on silica gel eluting with 10–50% acetone
in hexanes to give 2.0 g (27%) of the racemic product as an off-
white solid. MS (ESP) m/z 426 (MH+); 1H NMR (DMSO-d6) d:
0.98–1.12 (m, 5H); 1.36 (m, 10H); 1.45–1.53 (m, 1H); 1.60–1.65
(m, 1H); 1.66–1.79 (m, 2H); 2.63–2.69 (m, 1H); 2.68–2.75 (m,
1H); 3.09 (br s, 1H); 6.64 (d, 1H); 7.88–7.95 (m, 1H); 7.96–8.02
(m, 1H); 8.03–8.07 (m, 1H); 8.15 (dd, 1H).
4 h for compound 9g, when plasma concentrations were at Cmax
.
6. 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. Aluminium-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 Labora-
tory Devices, Inc and are uncorrected. 1H NMR spectra were
recorded at 300 MHz or 400 MHz. Chemical shifts are reported in
ppm (d) relative to solvent. The purity of tested compounds was
assessed by LC-MS. Reverse Phase HPLC was carried out using
The R and S enantiomers of 3 were separated by chiral HPLC
(Chiralcel OJ column, 20 ꢂ 250 mm, 10
l, 1:1 isopropanol:
hexanes, 10 mL/min) to give the (S) enantiomer of 3 as first eluting
enantiomer and the (R) enantiomer of
enantiomer.
3 as second eluting
YMC Pack ODS-AQ (100 ꢂ 20 mm ID, S-5
l particle size, 12 nm pore
6.4. (S)-trans-tert-Butyl 4-(1-(2-nitrophenylsulfonyl)aziridin-2-
yl)-cyclohexylcarbamate (3,(S))
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 at least 95%.
[
a]
D,S
+38 (c 0.1, in methanol).
6.5. (R)-trans-tert-Butyl 4-(1-(2-nitrophenylsulfonyl)aziridin-2-
yl)-cyclohexylcarbamate (3,(R))
6.1. 6-Fluoro-7-methoxyquinoxalin-2(1H)-one (2d)
The reaction mixture of 26 was filtered through a 0.45 lm
membrane into a flask containing ethyl 2-oxoacetate, 50% in tolu-
ene (31.9 mL, 161.16 mmol). The filter cake was into the reaction
mixture with ethanol (30 mL) and the mixture was stirred for
3 days at room temperature. The mixture was filtered through a
[
a
]
ꢀ38 (c 0.1, in methanol).
D,R
7. General procedure for 4, by alkylation of 2
0.45
l
m membrane and the solid was washed with ethanol
To a solution of 2 (2 mmol) in DMF (10 mL) was added sodium
hydride (120 mg, 3.0 mmol), and the mixture was stirred at room
temperature for 15 min under nitrogen. A solution of 3 (850 mg,
2.00 mmol) in DMF (6 mL) was added and the mixture was stirred
at room temperature over night. The mixture was quenched with
ice water and extracted with ethyl acetate (100 mL). The organic
phase was washed with brine twice (100 mL) and dried over
magnesium sulfate, then concentrated under reduced pressure.
(2 ꢂ 50 mL) and hexanes (50 mL) to give the product together with
the regioisomer 27 as a yellow solid, 11.80 g, ꢁ1:1 mixture. This
mixture was converted to the chlorides 28 and 29 as described
under the preparation for 28, at which stage the regioisomers
could be separated by chromatography on silica gel. A suspension
of 28 (3.37 g, 15.85 mmol) in HCl (5 M, 100 mL) was heated to
125 °C bath temperature for 2 h. The mixture was cooled to room

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5401
7.1. (R)-trans-tert-Butyl 4-(2-(7-methoxy-2-oxoquinolin-1(2H)-
yl)-1-(2-nitrophenylsulfonamido)ethyl)cyclohexylcarbamate
(4a)
(49%) of the product as a colorless solid. MS (ESP) m/z 612
(MNa+); 1H NMR (DMSO-d6) d: 8.05 (s, 1H); 7.92 (d, 1H); 7.68–
7.52 (m, 6H); 7.11 (ddd, 1H); 6.66 (d, 1H); 4.23 (dd, 1H); 4.09
(dd, 1H); 3.69 (m, 1H); 3.12 (m, 1H); 1.86–1.71 (m, 4H); 1.51 (m,
1H); 1.38 (s, 9H); 1.22–0.94 (m, 4H).
Compound 2a29 was reacted with sodium hydride and 3, (R)-
enantiomer, following the general procedure for 4. Chromatogra-
phy was done on silica gel with 10–80% ethyl acetate in hexanes
to give the product in 83% yield as a pale yellow solid. MS (ESP)
m/z 601 (MH+); 1H NMR (CDCl3) d: 1.04–1.22 (m, 2H); 1.22–1.42
(m, 4H); 1.46 (s, 9H); 1.64–1.78 (m, 1H); 1.82–1.95 (m, 1H);
2.06–2.22 (m, 3H); 3.41 (br s, 1H); 3.95 (s, 3H); 4.05 (d,
J = 14.32 Hz, 1H); 4.38 (br s, 1H); 4.76 (t, J = 12.15 Hz, 1H); 6.21
(d, J = 7.16 Hz, 1H); 6.26 (d, J = 9.42 Hz, 1H); 6.71 (s, 1H); 6.79
(dd, J = 8.67, 2.26 Hz, 1H); 7.19–7.26 (m, 1H); 7.28–7.32 (m, 1H);
7.41 (td, J = 7.72, 1.32 Hz, 1H); 7.50–7.61 (m, 2H).
7.6. trans-tert-Butyl 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-
1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)-
cyclohexylcarbamate (4f)
7-Methoxy-1,5-naphthyridin-2(1H)-one 2f52 (0.444 g, 2.52
mmol) was reacted with sodium hydride (101 mg, 2.52 mmol)
and 3, racemic, (715 mg, 1.68 mmol) following the general proce-
dure for 4. Chromatography was done on silica gel with 10–50% ace-
tone in hexanes to give 740 mg (73%) of the product as an off-white
solid. MS (ESP) m/z 602 (MH+); 1H NMR (DMSO-d6) d: 1.01–1.20 (m,
4H); 1.38 (s, 9H); 1.47–1.59 (m, 1H); 1.73–1.94 (m, 4H); 3.14 (br s,
1H); 3.66–3.78 (m, 1H); 3.98 (s, 3H); 4.12 (br s, 1H); 4.27–4.39 (m,
1H); 6.48–6.58 (m, 1H); 6.65–6.75 (m, 1H); 7.39–7.45 (m, 1H);
7.47–7.69 (m, 5H); 7.76–7.89 (m, 1H); 8.11 (s, 1H).
7.2. (R)-trans-tert-Butyl 4-(2-(7-fluoro-4-methyl-2-oxoquinoli-
n-1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)cyclohexylcar-
bamate (4b)
7-Fluoro-4-methylquinolin-2(1H)-one 2b30 (354 mg, 2.00 mmol)
was reacted with sodium hydride (120 mg, 3.0 mmol) and 3, (R) iso-
mer, following the general procedure for 4. Chromatography was
done on silica gel with hexanes/acetone 2:1 to give 640 mg (53%)
of the product as a colorless solid. MS (ESP) m/z 601 (MH+); 1H
NMR (DMSO-d6) d: 7.40–7.75 (m, 7H); 6.97 (t, 1H); 6.68 (d, 1H);
6.29 (s, 1H); 4.00–4.30 (m, 2H); 3.75 (s, br s, 1H); 3.16 (m, 1H);
2.27 (s, 3H); 1.82 (m, 4H); 1.50 (m, 1H); 1.38 (s, 9H); 1.00–1.25
(m, 4H).
7.7. (R)-trans-tert-Butyl 4-(2-(7-fluoro-2-oxo-1,5-naphthyridin-
1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)cyclo-
hexylcarbamate (4g)
7-Fluoro-1,5-naphthyridin-2(1H)-one 2 g52 (680 mg, 4.14
mmol) was reacted with sodium hydride (199 mg, 4.97 mmol)
and 3, (R) isomer, (1.76 g, 4.14 mmol) following the general proce-
dure for 4. Chromatography was done on silica gel with 20–100%
ethyl acetate in hexanes to give 720 mg of the product as a yellow
foam. MS (ESP) m/z 612 (MNa+); 1H NMR (CDCl3) d: 8.25 (m, 1H);
7.96 (m, 1H); 7.56–7.40 (m, 5H); 7.33 (m, 1H); 6.59 (d, 1H); 4.56
(m, 1H); 4.39 (m, 1H); 4.14 (m, 1H); 3.88 (m, 1H); 1.96 (m, 2H);
1.75 (m, 2H); 1.58 (m, 2H); 1.36 (s, 9H); 1.20–1.00 (m, 3H).
7.3. (R)-trans-tert-Butyl 4-(2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)-
cyclohexylcarbamate (4c)
Compound 2c8,31,32 was reacted with sodium hydride and 3, (R)-
enantiomer, following the general procedure for 4. Chromatogra-
phy was done on silica gel with 10–50% acetone in hexanes to give
8. General procedure for 5, by deprotection of 4
the product in 48% yield as an off-white hard foam. [a]D +6.0 (c 0.1,
A solution of 4 (1 mmol) in dichloromethane (3 mL) was treated
with trifluoroacetic acid (1 mL) at room temperature for 2 h. The
solvent was removed under reduced pressure and the residue
codistilled with methanol twice, to give the products as bis-trifluo-
roacetate salts.
in methanol). MS (ESP) m/z 602 (MH+); 1H NMR (DMSO-d6) d:
0.98–1.18 (m, 4H); 1.37 (s, 9H); 1.45–1.58 (m, 1H); 1.72–1.92 (m,
4H); 3.10 (br s, 1H); 3.73 (br s, 1H); 3.92 (s, 3H); 4.13–4.31 (m,
2H); 6.61–6.71 (m, 1H); 6.86 (dd, 1H); 7.04 (s, 1H); 7.46–7.68
(m, 5H); 7.86–7.97 (m, 2H).
8.1. (R)-N-(1-(4-Aminocyclohexyl)-2-(7-methoxy-2-
oxoquinolin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide
trifluoroacetate salt (5a)
7.4. trans-tert-Butyl-4-(2-(6-fluoro-7-methoxy-2-
oxoquinoxalin-1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)-
cyclohexylcarbamate (4d)
Compound 4a (1.14 g, 1.9 mmol) was reacted with trifluoroace-
tic acid according to the general procedure for 5 to give the product
as a pale yellow solid 1.05 g (90%). MS (ESP) m/z 501 (MH+); 1H NMR
(DMSO-d6) d: 1.10–1.36 (m, 4H); 1.49–1.63 (m, 1H); 1.87–2.05 (m,
4H); 2.96 (br s, 1H); 3.76–3.86 (m, 1H); 3.89 (s, 3H); 4.10–4.37 (m,
2H); 6.25 (d, J = 9.42 Hz, 1H); 6.74 (dd, J = 8.57, 1.98 Hz, 1H); 6.97
(s, 1H); 7.35 (d, J = 8.67 Hz, 1H); 7.45 (d, J = 3.96 Hz, 2H); 7.50–
7.58 (m, 2H); 7.59–7.64 (m, 1H); 7.65–7.87 (m, 4H).
Compound 2d (0.412 g, 2.12 mmol) was reacted with sodium
hydride (84 mg, 2.1 mmol) and 3, racemic, following the general
procedure for 4. Chromatography was done on silica gel with
20–100% ethyl acetate in hexanes to give 520 mg (48%) of the
product as a yellow foam. MS (ESP) m/z 620 (MH+); 1H NMR
(DMSO-d6) d: 7.99 (s, 1H); 7.96 (s, 1H); 7.66 (m, 2H); 7.56 (m,
2H); 7.43 (d, 1H); 7.14 (d, 1H); 6.69 (d, 1H); 4.30 (d, 1H); 4.11 (t,
1H); 4.04 (s, 3H); 3.73 (m, 1H); 1.84 (m, 4H); 1.50 (m, 1H); 1.37
(s, 9H); 1.15 (m, 4H).
8.2. (R)-trans-N-(1-(4-Aminocyclohexyl)-2-(7-fluoro-4-methyl-
2-oxoquinolin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide,
trifluoroacetic acid salt (5b)
7.5. (R)-trans-tert-Butyl 4-(2-(7-fluoro-2-oxoquinoxalin-1(2H)-
yl)-1-(2-nitrophenylsulfonamido)ethyl)cyclohexylcarbamate
(4e)
Compound 4b (640 mg, 1.06 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as a colorless solid (740 mg). MS (ESP) m/z 503 (MH+);
1H NMR (DMSO-d6) d: 1.26 (m, 4H); 1.50–1.66 (m, 3H); 1.79–
2.04 (m, 4H); 2.26 (s, 3H); 3.00 (m 1H); 3.79 (m, 1H); 4.10 (m,
2H); 6.28 (s, 1H); 6.80–7.04 (m, 1H); 7.30–7.76 (m, 10H).
7-Fluoro-4-methylquinolin-2(1H)-one 2e (171 mg, 1.04 mmol)
was reacted with sodium hydride (50 mg, 1.25 mmol) and 3, (R)
isomer, following the general procedure for 4. Chromatography
was done on silica gel with hexanes/acetone 2:1 to give 300 mg

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F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
8.3. (R)-trans-N-(1-(4-Aminocyclohexyl)-2-(7-methoxy-2-
oxoquinoxalin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide,
trifluoroacetic acid salt (5c)
general procedure for 8 to give 0.36 g (67%) of the product as a
pale yellow solid. MS (ESP) m/z 664 (MH+); 1H NMR (DMSO-d6)
d: 0.90–1.20 (m, 4H); 1.47–1.62 (m, 1H); 1.79–2.01 (m, 4H);
2.35 (t, J = 10.27 Hz, 1H); 3.70–3.84 (m, 3H); 3.89 (s, 3H); 4.24
(br s, 2H); 4.72 (s, 2H); 6.25 (d, J = 9.42 Hz, 1H); 6.73 (dd,
J = 8.67, 1.88 Hz, 1H); 6.95 (br s, 1H); 7.35 (d, J = 8.67 Hz, 1H);
7.42–7.49 (m, 2H); 7.49–7.58 (m, 2H); 7.58–7.65 (m, 1H); 8.23
(s, 1H).
Compound 4c (570 mg, 0.95 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as colorless hard foam, 580 mg (quant). MS (ESP) m/z
502 (MH+).
8.4. N-trans-(1-(4-Aminocyclohexyl)-2-(6-fluoro-7-methoxy-2-
oxoquinoxalin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide (5d)
9.2. (R)-N-(2-(7-Fluoro-4-methyl-2-oxoquinolin-1(2H)-yl)-1-
trans-(4-((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-
yl)methylamino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8b)
Compound 4d (520 mg, 0.84 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as an orange foam (quant). MS (ESP) m/z 520 (MH+).
Compound 5b (725 mg, 1.44 mmol), N,N-diisopropylethylamine
(0.25 mL, 1.6 mmol), 753 (0.257 g, 1.44 mmol) and sodium triacet-
oxyborohydride (1.523 g, 7.19 mmol) were reacted following the
general procedure for 8 to give 550 mg of the product as a colorless
solid (58%). MS (ESP) m/z 666 (MH+); 1H NMR (methanol-d4) d:
1.16 (m, 5H); 1.58 (m, 1H); 1.79 (m, 1H); 1.99 (m, 3H); 2.17 (s,
3H); 2.44 (m, 1H); 3.78–3.93 (m, 6H); 4.61 (s, 2H); 6.10 (s, 1H);
6.83 (t, J = 9.8 Hz,1H); 6.94 (d, J = 9.8 Hz, 1H), 7.14 (t, J = 7.5 Hz,
1H); 7.30–7.50 (m, 4H); 8.08 (s, 1H).
8.5. (R)-trans-N-(1-(4-Aminocyclohexyl)-2-(7-fluoro-2-
oxoquinoxalin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide,
trifluoroacetic acid salt (5e)
Compound 4e (295 mg, 0.50 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as a colorless oil, 233 mg (quant). MS (ESP) m/z 490 (MH+).
8.6. trans-N-(1-(4-Aminocyclohexyl)-2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide,
trifluoroacetic acid salt (5f)
9.3. (R)-N-(2-(7-Fluoro-4-methyl-2-oxoquinolin-1(2H)-yl)-1-
trans-(4-((3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-
yl)methylamino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8c)
Compound 4f (655 mg, 1.09 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as a colorless oil, 670 mg (quant). MS (ESP) m/z 502 (MH+).
Compound 5b (620 mg, 0.99 mmol), triethylamine (1 mL,
0.71 mmol), 626 and sodium triacetoxyborohydride were reacted
following the general procedure for 8 to give 237 mg of the product
as a colorless hard foam (29%). MS (ESP) m/z 665 (MH+); 1H NMR
(DMSO-d6) d: 11.15 (br s, 1H); 7.25–7.60 (m, 7H); 6.90–7.05 (m,
2H); 6.28 (s, 1H); 4.61 (s, 2H); 4.24 (m, 1H); 4.07 (m, 1H); 3.75
(m, 3H); 3.17 (d, 1H); 2.33 (m, 1H); 2.26 (m, 3H); 1.93 (m, 4H);
1.51 (m, 1H); 1.05 (m, 4H).
8.7. (R)-trans-N-(1-(4-Aminocyclohexyl)-2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide (5g)
Compound 4g (582 mg, 0.99 mmol) was reacted with trifluoro-
acetic acid according to the general procedure for 5 to give the
product as a colorless oil, 483 mg (quant). MS (ESP) m/z 490 (MH+).
9.4. (R)-trans-N-(2-(7-Methoxy-2-oxoquinoxalin-1(2H)-yl)-
1-(4-((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-yl)
methylamino)-cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8d)
9. General procedure for 8, by reductive amination of 5
A solution of 5 (0.20 mmol) and 1 equiv aldehyde, either 3-oxo-
3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde 626 or
7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazine-2-carbaldehyde
753 in dry DMF (2 mL) were heated over freshly activated 3 Å
molecular sieves (pearled) at 60 °C for 3 h. The reaction mixture
was cooled to 0 °C, and sodium triacetoxy borohydride (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 dichloro-
methane/methanol (9:1, 50 mL) and saturated aqueous sodium
hydrogencarbonate solution (5 mL). The aqueous phase was back
extracted twice with dichloromethane/methanol (9:1, 2 ꢂ 20 mL)
and the combined organic phases were dried over sodium sulfate
and concentrated under reduced pressure. Chromatography was
done on silica gel with a gradient of 0–20% methanol in CH2Cl2,
containing 0.25% NH4OH.
Compound 5c (725 mg, 1.44 mmol), N,N-diisopropylethyl-
amine, 753 and sodium triacetoxyborohydride were reacted follow-
ing the procedure for 8b to give the product in 80% yield as a
colorless solid. MS (ESP) m/z 665 (MH+); 1H NMR (DMSO-d6) d:
0.93–1.18 (m, 4H); 1.55 (br s, 1H); 1.80–1.98 (m, 4H); 2.35 (br s,
1H); 3.69–3.80 (m, 2H); 3.92 (s, 3H); 4.10–4.31 (m, 2H); 4.72 (s,
2H); 6.84 (dd, 1H); 7.04 (s, 1H); 7.43–7.66 (m, 5H); 7.89 (s, 1H);
7.96 (s, 1H); 8.23 (s, 1H).
9.5. (R)-trans-N-(2-(7-Methoxy-2-oxoquinoxalin-1(2H)-yl)-1-(4-
((3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl-
amino)-cyclohexyl)ethyl)-2-nitrobenzenesulfonamide (8e)
Compound 5b ethyldiisopropylamine, 626 and sodium triacet-
oxyborohydride were reacted following the procedure for 8b to
give the product in 71% yield as a colorless solid. [a] +20 (c 0.1,
D
9.1. (R)-N-(2-(7-Methoxy-2-oxoquinolin-1(2H)-yl)-1-(4-((7-oxo-
7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-yl)methylamino)-
cyclohexyl)ethyl)-2-nitrobenzenesulfonamide (8a)
in methanol); MS (ESP) m/z 664 (MH+); 1H NMR (DMSO-d6) d:
0.94–1.12 (m, 4H); 1.50–1.61 (m, 1H); 1.83–1.98 (m, 4H); 2.25–
2.37 (m, 1H); 3.67–3.76 (m, 2H); 3.92 (s, 3H); 4.12–4.30 (m, 2H);
4.61 (s, 2H); 6.81–6.88 (m, 1H); 6.98–7.06 (m, 2H); 7.27–7.32 (m,
1H); 7.45–7.64 (m, 6H); 7.89 (s, 1H); 11.19 (br s, 1H).
Compound 5a (500 mg, 0.8 mmol), N,N-diisopropylethylamine,
753 and sodium triacetoxyborohydride were reacted following the

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5403
9.6. (R)-trans-N-(2-(7-Fluoro-2-oxoquinoxalin-1(2H)-yl)-1-(4-
((3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl-
amino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide (8f)
solid (41%). MS (ESP) m/z 652 (MH+); 1H NMR (MeOD) d: 8.41 (d,
1H); 7.98 (m, 1H); 7.76 (d, 1H); 7.63 (m, 3H); 7.55 (m, 1H); 7.31
(d, 1H); 7.01 (d, 1H); 6.76 (d, 1H); 4.69 (s, 2H); 4.40 (m, 2H);
3.95 (m, 1H); 3.83 (s, 2H); 2.49 (m, 1H); 2.06 (m, 4H); 1.68 (m,
1H); 1.33–1.12 (m, 4H).
Compound 5e ethyldiisopropylamine, 626 and sodium triacet-
oxyborohydride were reacted following the procedure for 8b to
give the product in 71% yield as a colorless solid. [a] +20 (c 0.1,
D
10. General procedure for 9 by deprotection of 8
in methanol); MS (ESP) m/z 652 (MH+); 1H NMR (DMSO-d6) d:
11.15 (br s, 1H); 8.04 (s, 1H); 7.89 (br s, 1H); 7.66–7.49 (m, 6H);
7.30 (d, 1H); 7.09 (m, 1H); 7.02 (d, 1H); 4.61 (s, 2H); 4.24 (m,
1H); 4.08 (m, 1H); 3.71 (m, 3H); 2.31 (m, 1H); 1.99–1.75 (m,
4H); 1.57 (m, 1H); 1.16–0.88 (m, 4H).
To a solution of 8 (0.68 mmol) in anhydrous DMF (5 mL) was
added anhydrous K2CO3 (467 mg, 3.38 mmol) and thiophenol
(0.348 mL, 3.38 mmol). The mixture was stirred at room tempera-
ture for 2 h under a blanket of nitrogen. The volatile portion of the
mixture was removed by rotary evaporation and saturated
aqueous NaHCO3 (20 mL) was added to the resulting residue. The
residue was extracted with MeOH/CH2Cl2 (10%, twice with
100 mL). The organic layer was dried over Na2SO4 and concen-
trated under reduced pressure.
9.7. (R)-trans-N-(2-(7-Fluoro-2-oxoquinoxalin-1(2H)-yl)-1-(4-
((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-
yl)methylamino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8g)
Compound 5e (143 mg, 0.24 mmol), N,N-diisopropylethyl-
amine, 753 and sodium triacetoxyborohydride were reacted follow-
ing the procedure for 8b to give 106 mg (68%) of the product as a
colorless hard foam. MS (ESP) m/z 653 (MH+); 1H NMR (DMSO-
d6) d: 8.25 (s, 1H); 8.04 (s, 1H); 7.87 (br s, 1H); 7.66–7.49 (m,
6H); 7.08 (ddd, 1H); 4.73 (s, 2H); 4.23 (dd, 1H); 4.08 (m, 1H);
3.76 (s, 2H); 3.69 (m, 1H); 2.35 (m, 1H); 1.99–1.77 (m, 4H); 1.58
(m, 1H); 1.18–0.91 (m, 4H).
10.1. (R)-2-(trans-(4-(1-Amino-2-(7-methoxy-2-oxoquinolin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-6H-pyrimido[5,4-
b][1,4]oxazin-7(8H)-one (9a)
Compound 8a (420 mg, 0.60 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with a gradient of 0–25% MeOH in CH2Cl2, containing 0.25% NH4-
OH, to give the product as a colorless solid (214 mg, 71%). MS
(ESP) m/z 479 (MH+); 1H NMR (DMSO-d6) d: 1.29–1.53 (m, 4H);
1.53–1.69 (m, 1H); 1.84 (br s, 1H); 2.07 (d, J = 14.51 Hz, 1H);
2.13–2.30 (m, 2H); 3.06 (t, J = 11.21 Hz, 1H); 3.48 (dd, J = 9.32,
5.56 Hz, 2H); 3.94 (s, 3H); 4.22 (s, 2H); 4.35–4.52 (m, 1H); 4.52–
4.68 (m, 1H); 4.79 (s, 2H); 6.46 (d, J = 9.42 Hz, 1H); 6.93 (dd,
J = 8.67, 1.88 Hz, 1H); 7.02 (s, 1H); 7.68 (d, J = 8.67 Hz, 1H); 7.87
(d, J = 9.61 Hz, 1H); 8.36 (s, 1H).
9.8. N-(2-(6-Fluoro-7-methoxy-2-oxoquinoxalin-1(2H)-yl)-1-
trans-(4-((7-oxo-7,8-dihydro-6H-pyrimido[5,4-b][1,4]oxazin-2-
yl)methylamino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8h)
Compound 5h (520 mg, 1.00 mmol), 753 (0.179 g, 1.00 mmol)
and sodium triacetoxyborohydride (528 mg, 2.5 mmol) were
reacted following the general procedure for 8 to give 295 mg of
the product as an off white solid (44%). MS (ESP) m/z 683 (MH+);
1H NMR (DMSO-d6) d: 0.8–1.2 (m, 4H); 1.52 (m, 1H); 1.90 (m,
4H); 2.36 (m, 1H); 3.17 (s, 1H); 3.71 (m, 1H); 3.76 (s, 2H); 4.04
(d, 2H); 4.05–4.29 (m, 3H); 4.73 (s, 2H); 7.14 (d, 1H); 7.37–7.65
(m, 5H); 7.97 (s, 1H); 8.25 (s, 1H).
10.2. (R)-2-(trans-[4-(1-Amino-2-(7-fluoro-4-methyl-2-
oxoquinolin-1(2H)-yl)ethyl)cyclohexylamino]-methyl)-6H-
pyrimido[5,4-b][1,4]oxazin-7(8H)-one (9b)
Compound 8b (450 mg, 0.68 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with a gradient of 0–25% MeOH in CH2Cl2, containing 0.25% NH4-
9.9. trans-N-(2-(7-Methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)-1-
(4-((3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-
yl)methylamino)-cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8i)
OH, to give the product as a colorless solid (130 mg, 40.0%). [a]
+43 (c 0.1, MeOH). MS (ESP) m/z 481 (MH+); 1H NMR (DMSO-d6)
d: 0.87–1.18 (m, 5H); 1.48 (m, 1H); 1.70–1.98 (m, 4H); 2.18–2.38
(m, 4H); 2.77 (m, 1H); 3.10 (s, 1H); 3.69 (s, 2H); 3.90–4.08 (m,
1H); 4.09–4.23 (m, 1H); 4.65 (s, 2H); 6.40 (s, 1H); 7.05 (m, 1H);
7.37 (dd, 1H); 7.74 (dd, 1H), 8.16 (s, 1H).
Compound 5f (795 mg, 1.09 mmol), ethyldiisopropylamine
(0.758 mL, 4.36 mmol), 626 (194 mg, 1.09 mmol) and sodium tri-
acetoxyborohydride (693 mg, 3.27 mmol) were reacted following
the procedure for 8b to give 555 mg of the product as an off-white
solid (77%). MS (ESP) m/z 664 (MH+); 1H NMR (DMSO-d6) d: 0.98–
1.13 (m, 4H); 1.57 (br s, 1H); 1.86–2.01 (m, 4H); 2.30 (br s, 1H);
3.68–3.77 (m, 2H); 3.99 (s, 3H); 4.12 (br s, 1H); 4.26–4.37 (m,
1H); 4.61 (s, 2H); 6.52 (d, 1H); 7.01 (d, 1H); 7.30 (d, 1H); 7.40–
7.64 (m, 6H); 7.77 (br s, 1H); 8.09 (d, 1H); 11.15 (br s, 1H).
10.3. (R)-trans-6-((4-(1-Amino-2-(7-fluoro-4-methyl-2-
oxoquinolin-1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one (9c)
Compound 8c (235 mg, 0.35 mmol) was reacted following the
general procedure for 9. The crude product was then purified by
reverse phase HPLC using water/trifluoroacetic acid (0.1%) with
acetonitrile gradient of 5–95%. The trifluoroacetic acid salt of the
product was redissolved in dichloromethane (100 mL) and basified
by a potassium carbonate solution (20 mL) and extracted with
dichloromethane (2 ꢂ 100 mL), the organic was dried over magne-
sium sulfate and concentrated under reduced pressure to give the
product as a colorless solid, 42 mg (24.8%). MS (ESP) m/z 480
(MH+); 1H NMR (DMSO-d6) d: 0.90–1.42 (m, 6H); 1.55 (m, 1H);
1.92 (m, 3H); 2.30 (t, 1H); 2.42 (s, 3H); 2.84 (s, 1H); 3.69 (s, 2H);
4.00–4.40 (m, 2H); 4.60 (s, 2H); 6.48 (s, 1H); 7.02 (d, 1H); 7.13 (t,
1H); 7.28 (d, 1H); 7.44 (d, 1H); 7.81 (t, 1H).
9.10. (R)-trans-N-(2-(7-Fluoro-2-oxo-1,5-naphthyridin-1(2H)-
yl)-1-(4-((3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-
yl)methylamino)cyclohexyl)ethyl)-2-nitrobenzenesulfonamide
(8j)
Compound 5g (483 mg, 0.99 mmol), ethyldiisopropylamine
(0.652 mL, 3.95 mmol), 626 (211 mg, 1.18 mmol) and sodium tri-
acetoxyborohydride (627 mg, 2.96 mmol) were reacted following
the procedure for 8b to give 215 mg of the product as an off-white

5404
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
10.4. (R)-trans-2-((4-(1-Amino-2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-6H-pyrimido[5,4-
b][1,4]oxazin-7(8H)-one (9d)
The racemic material was separated on a Chiralpak IB column with
50% hexanes in EtOH/MeOH (1:1), containing 0.1% diethylamine.
The desired R-enantiomer eluted second. MS (ESP) m/z 498
(MH+); 1H NMR (DMSO-d6) d: 0.8–1.4 (m, 7H); 1.73 (d, 1H); 1.92
(m, 3H); 2.35 (m, 1H); 2.89 (m, 1H); 3.75 (s, 2H); 4.00 (s, 3H);
4.21 (d, 2H); 4.72 (s, 2H); 7.17 (d, 1H); 7.68 (d, 1H); 8.10 (s, 1H);
8.24 (s, 1H).
Compound 8d was reacted following the general procedure for
9. Chromatography was done on silica gel with a gradient of 5–15%
methanol in dichloromethane containing 0.25% ammonium
hydroxide to give the product as a colorless solid in 45% yield.
MS (ESP) m/z 480 (MH+); 1H NMR (DMSO-d6) d: 1H NMR (DMSO-
d6) d: ppm 0.95–1.36 (m, 6H); 1.62–1.75 (m, 1H); 1.83–2.03 (m,
4H); 2.29–2.46 (m, 2H); 2.91 (br s, 1H); 3.78 (s, 2H); 3.90 (s, 3H);
4.12–4.23 (m, 2H); 4.73 (s, 2H); 6.93–7.04 (m, 2H); 7.74 (d, 1H);
8.04 (s, 1H); 8.24 (s, 1H).
10.9. (R)-trans-6-((4-(1-Amino-2-(7-methoxy-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one (9i) and (S)-trans-6-((4-(1-
amino-2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-
yl)ethyl)cyclohexylamino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-
3(4H)-one (35)
10.5. (R)-trans-6-((4-(1-Amino-2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one (9e)
Compound 8i (550 mg, 0.83 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with 20% methanol in dichloromethane, containing 1% ammonium
hydroxide to give 255 mg of the racemic product as a light yellow
solid, (64%). MS (ESP) m/z 479 (MH+); 1H NMR (DMSO-d6) d: 0.96–
1.32 (m, 6H); 1.61–1.71 (m, 1H); 1.84–2.02 (m, 4H); 2.24–2.40 (m,
2H); 2.79–2.89 (m, 1H); 3.70 (s, 2H); 3.96 (s, 3H); 4.12–4.31 (m,
2H); 4.61 (s, 2H); 6.65 (d, 1H); 7.02 (d, 1H); 7.29 (d, 1H); 7.43 (s,
1H); 7.85 (d, 1H); 8.27 (d, 1H); 11.12 (br s, 1H).
Compound 8e was reacted following the general procedure for
9. Chromatography was done on silica gel with a gradient of 5–
15% methanol in dichloromethane containing 0.25% ammonium
hydroxide to give the product as a colorless solid in 58% yield.
MS (ESP) m/z 479 (MH+); 1H NMR (DMSO-d6) d: 0.94–1.35 (m,
6H); 1.62–1.74 (m, 1H); 1.84–2.03 (m, 4H); 2.23–2.40 (m, 2H);
2.90 (br s, 1H); 3.71 (s, 2H); 3.90 (s, 3H); 4.17 (d, 2H); 4.61 (s,
2H); 6.95–7.06 (m, 3H); 7.30 (d, 1H); 7.74 (d, 1H); 8.03 (s, 1H);
11.15 (br s, 1H).
The racemic material was separated on a Chiralcel OJ column,
20 ꢂ 250 mm, 10
l, with 40% 1:1 methanol/ethanol, 60% hexanes,
10 mL/min) to give 9i as the first eluting enantiomer, followed by
35.
10.6. (R)-trans-6-((4-(1-Amino-2-(7-fluoro-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one (9f)
10.10. (R)-trans-6-((4-(1-Amino-2-(7-fluoro-2-oxo-1,5-
naphthyridin-1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one (9j)
Compound 8f (2.65 g, 4.07 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with a gradient of 5–15% methanol in dichloromethane containing
0.25% ammonium hydroxide. The crude product was triturated in a
mixture of EtOH/hexanes overnight, while stirring vigorously. The
solid was collected by filtration to give the product as an off-white
Compound 8j (72 mg, 0.11 mmol) was reacted following the
general procedure for 9, except the temperature was 0 °C, and
the reaction time 3 h. Chromatography was done on silica gel with
0–10% methanol in dichloromethane, containing 0.25% ammonium
hydroxide to give 14 mg (27%) of the product as a light yellow
foam. MS (ESP) m/z 467 (MH+); 1H NMR (MeOD) d: 8.52 (d, 1H);
7.98 (m, 2H); 7.29 (d, 1H); 6.96 (m, 2H); 4.67 (s, 2H); 4.36 (m,
2H); 3.82 (m, 2H); 3.07 (m, 1H); 2.51 (m, 1H); 2.11 (m, 3H); 1.81
(m, 1H); 1.35 (m, 6H).
solid, 958 mg (51%). [a] +31.9 (c 1, methanol). MS (ESP) m/z 467
D
(MH+); 1H NMR (DMSO-d6) d: 11.16 (br s, 1H); 8.18 (s, 1H); 7.86
(dd, 1H); 7.56 (dd, 1H); 7.30 (d, 1H); 7.22 (dd, 1H); 7.03 (d, 1H);
4.61 (s, 2H); 4.21–4.05 (m, 2H); 3.72 (s, 2H); 2.88 (m, 1H); 2.35
(m, 1H); 2.02–1.83 (m, 4H); 1.64 (m, 1H); 1.35–0.93 (m, 4H).
10.11. 2-(8-Amino-1,4-dioxaspiro[4.5]decan-8-yl)ethanol (12)
10.7. (R)-trans-2-((4-(1-Amino-2-(7-fluoro-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-6H-pyrimido[5,4-
b][1,4]oxazin-7(8H)-one (9g)
To a mixture of 2-(8-amino-1,4-dioxaspiro[4.5]decan-8-yl)ace-
tic acid (from Discovery Chemscience) (4.2 g, 19.51 mmol) in THF
(100 mL) was added LAH (1.851 g, 48.78 mmol). After the exother-
mic reaction had subsided, the mixture was heated to reflux for
3 h. The mixture was cooled to 0 °C and water (5 mL, in 20 mL
THF) was added dropwise under nitrogen. The mixture was stirred
Compound 8g (103 mg, 0.16 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with a gradient of 5–15% methanol in dichloromethane containing
0.25% ammonium hydroxide to give 30 mg (40%) of the product as
for 30 min, then filtered through a 0.45 lm membrane and washed
a slightly yellow solid. [
a
]
D +34.8 (c 1, methanol). MS (ESP) m/z 468
with ethyl acetate (3 ꢂ 10 mL). Filtrate and wash were combined
and concentrated under reduced pressure. Chromatography was
done on silica gel with CH2Cl2/MeOH 4:1, containing 1% ammo-
nium hydroxide to give 3.56 g of the product as a colorless solid
(91%). MS (ESP) m/z 202 (MH+); 1H NMR (DMSO-d6) d: 3.83 (s,
4H); 3.56 (m, 2H); 1.72 (m, 2H); 1.50–1.35 (m, 8H). (The OH and
NH2 protons were exchanged).
(MH+); 1H NMR (DMSO-d6) d: 8.25 (s, 1H); 8.18 (s, 1H); 7.86 (dd,
1H); 7.56 (m, 1H); 7.23 (ddd, 1H); 4.73 (s, 2H); 4.13 (m, 2H);
3.78 (s, 2H); 2.86 (m, 1H); 2.37 (m, 1H); 2.02–1.82 (m, 4H); 1.64
(m, 1H); 1.38–0.95 (4H).
10.8. (R)-2-(trans-(4-(1-Amino-2-(6-fluoro-7-methoxy-2-
oxoquinoxalin-1(2H)-yl)ethyl)cyclohexylamino)methyl)-6H-
pyrimido[5,4-b][1,4]oxazin-7(8H)-one (9h)
10.12. 2-(8-Azido-1,4-dioxaspiro[4.5]decan-8-yl)ethanol (13)
Compound 8h (300 mg, 0.44 mmol) was reacted following the
general procedure for 9. Chromatography was done on silica gel
with 20% methanol in dichloromethane, containing 1% ammonium
hydroxide to give the product as a light yellow solid, 85 mg (39%).
A triflic azide stock solution was prepared from sodium azide
(4.60 g, 70.75 mmol) and triflic anhydride (5.98 mL, 35.38 mmol)
in toluene according to the protocol by Titz et al.37 (caution! Triflic
azide is potentially explosive.) To
a mixture of 12 (3.56 g,

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5405
17.69 mmol), sodium bicarbonate (5.94 g, 70.75 mmol) and
10.16. tert-Butyl 4-azido-4-(2-(tert-butyldiphenylsilyloxy)-
copper(II) sulfate pentahydrate (0.177 g, 0.71 mmol) in water
(23 mL) was added the triflic azide solution, followed by addition
of methanol (170 mL). The mixture was stirred over night at room
temperature, then filtered and washed with methanol (2 ꢂ 50 mL).
Filtrate and wash were combined and concentrated under reduced
pressure to ꢁ30 mL volume. This aqueous mixture was extracted
with dichloromethane three times (3 ꢂ 70 mL) and the combined
organic phases were dried over sodium sulfate and concentrated
under reduced pressure, keeping the temperature of the water bath
below 25 °C. The residue was chromatographed on silica gel with
hexanes/acetone 4:1 to give the product as a colorless oil, 3.16 g
(79%). 1H NMR (DMSO-d6) d: 4.51 (t, 1H); 3.86 (s, 4H); 3.54 (m,
2H); 1.75 (t, 2H); 1.60–1.50 (m, 8H).
ethyl)cyclohexylcarbamate (17)
To a biphasic mixture of 16 (4.42 g, 10.46 mmol) in ethyl ace-
tate (80 mL) and sodium bicarbonate (2.64 g, 31.38 mmol) in water
(80 mL) was added BOC2O (2.91 mL, 12.55 mmol) (in ethyl acetate,
30 mL) and the mixture was rapidly stirred at room temperature
for 3 d. The organic phase was separated, the aqueous phase
extracted once with ethyl acetate (30 mL) and the combined
organic phases were tried over sodium sulfate and concentrated
under reduced pressure. Chromatography was done on silica gel
with hexanes/ethyl acetate 10:1 to give the product as a colorless
oil, 4.28 g (78%). The product was obtained as a mixture of ꢁ1:2 of
the cis- and trans-isomers. MS (ESP) m/z 523 (MH+); 1H NMR
(DMSO-d6) d: 7.66 (m, 4H); 7.42 (m, 7H); 3.76 (m, 2H); 1.85 (m,
2H); 1.75–1.50 (m, 4H); 1.50–1.20 (m, 5H); 1.37 (s, 9H); 1.00 (s,
9H).
10.13. (2-(8-Azido-1,4-dioxaspiro[4.5]decan-8-yl)ethoxy)(tert-
butyl)diphenylsilane (14)
10.17. cis-tert-Butyl 4-azido-4-(2-hydroxyethyl)-
cyclohexylcarbamate (18)
To a solution of 13 (250 mg, 1.10 mmol) in DMF (2 mL) was
added a solution of imidazole (195 mg, 2.86 mmol) and tert-butyl-
diphenylsilyl chloride (0.367 mL, 1.43 mmol) in DMF (1 mL) at
room temperature. After 30 min the reaction mixture was diluted
with ethyl acetate (30 mL) and washed with potassium phosphate
buffer pH 7 (1 M, 20 mL), then washed with water (30 mL). The
organic phase was dried over sodium sulfate and concentrated
under reduced pressure to give 610 mg of the product as a color-
less oil (quant). The crude product was taken to the next step
without further purification. NMR (DMSO-d6) d: 7.75–7.45 (m,
10H); 3.92 (s, 4H); 3.82 (t, 2H); 1.94 (t, 2H); 1.75–1.55 (m, 8H);
1.05 (s, 9H).
To a solution of 17 (4.25 g, 8.13 mmol) in THF (60 mL) was added
a solution of tetrabutylammonium fluoride in THF (1 M, 10.57 mL,
10.57 mmol) at room temperature. After 1d, the mixture was con-
centrated under reduced pressure, the residue was taken up in ethyl
acetate (200 mL) and washed with potassium phosphate buffer pH7
(1 M, 100 mL), then dried over sodium sulfate and concentrated
under reduced pressure. Chromatographed was done on silica gel
with hexanes/acetone 4:1. The first eluting peak was the product
with the trans-configuration (0.57 g, 25%). The second eluting peak
was the desired cis-isomer (azido- and –NHBoc groups on the same
side of the ring) and was collected to give the product as a colorless
oil, 1.212 g (52%). MS (ESP) m/z 523 (MH+); 1H NMR (DMSO-d6) d:
6.75 (d, 1H); 4.50 (t, 1H); 3.53 (m, 2H); 3.20 (m, 1H); 1.74–1.25
(m, 10H); 1.37 (s, 9H). The cis configuration for the major product
was confirmed by NOE NMR studies.
10.14. 4-Azido-4-(2-(tert-butyldiphenylsilyloxy)-
ethyl)cyclohexanone (15)
A solution of 14 (6.5 g, 13.96 mmol) and p-TsOH (0.425 g,
2.23 mmol) in acetone (300 mL) was heated to 80 °C bath temper-
ature over night. The mixture was cooled with an ice bath and pyr-
idine (0.271 mL, 3.35 mmol) was added once the temperature
reached room temperature. The mixture was concentrated under
reduced pressure, the residue taken up in ethyl acetate (200 mL)
and washed with phosphate buffer pH 7 (1 M, 50 mL), then with
water (50 mL), dried over sodium sulfate and concentrated under
reduced pressure. Chromatography was done on silica gel with
hexanes/acetone 9:1 to give the product as a colorless oil, 4.41 g
(75%). MS (ESP) m/z 422 (MH+); 1H NMR (DMSO-d6) d: 7.64 (m,
4H); 7.46 (m, 6H); 3.82 (t, 2H); 2.41 (m, 2H); 2.18 (m, 2H); 1.99
(m, 6H); 1.00 (s, 9H).
10.18. cis-2-(1-Azido-4-(tert-
butoxycarbonylamino)cyclohexyl)ethyl methanesulfonate (19)
To a solution of 18 (600 mg, 2.11 mmol) in dichloromethane
(10 mL) was added TEA (0.412 mL, 2.95 mmol) and the mixture
was cooled to 0 °C. A solution of MsCl (0.197 mL, 2.53 mmol) in
dichloromethane (3 mL) was added dropwise. After one h the mix-
ture was quenched with phosphate buffer pH 7 (1 M, ꢁ50 mL) and
dichloromethane was removed under reduced pressure. The aque-
ous mixture was extracted with ethyl acetate (50 mL) and the
extract was washed once more with phosphate buffer as above,
then dried over sodium sulfate and concentrated under reduced
pressure. The colorless oil was taken up in DMF (5 mL) and used
without further purification directly in the next step. MS (ESP)
m/z 385 (MNa+).
10.15. 4-Azido-4-(2-(tert-butyldiphenylsilyloxy)-
ethyl)cyclohexanamine (16)
A mixture of 15 (4.41 g, 10.46 mmol) and ammonium acetate
(8.06 g, 104.60 mmol) in methanol (100 mL) was treated with
sodium cyanoborohydride (0.657 g, 10.46 mmol) and the mixture
was stirred at room temperature for 18 h. The mixture was concen-
trated under reduced pressure, the residue was taken up in dichlo-
romethane (300 mL) and washed with saturated sodium
bicarbonate solution (50 mL, pH adjusted to 10 with 15% NaOH).
The aqueous phase was back-extracted twice with dichlorometh-
ane (2 ꢂ 100 mL) and the combined organic phases were dried
over sodium sulfate and concentrated under reduced pressure to
give the product as a colorless oil, 4.5 g (quant). MS (ESP) m/z
423 (MH+); 1H NMR (DMSO-d6) d: 7.65 (m, 4H); 7.43 (m, 6H);
3.77 (m, 2H); 1.86 (m, 2H); 1.80–1.50 (m, 4H); 1.44 (m, 3H); 1.15
(m, 2H); 1.00 (s, 9H).
10.19. cis-tert-Butyl 4-azido-4-(2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylcarbamate (20)
To a suspension of 7-methoxyquinoxalin-2(1H)-one 2c8,31,32
(558 mg, 3.17 mmol) in DMF (10 mL) at 0 °C was added NaH, 60%
in oil (135 mg, 3.38 mmol) and the mixture was stirred for
30 min. A solution of 18 (765 mg, 2.11 mmol) in DMF (5 mL) was
added and the mixture was stirred at room temperature. The mix-
ture was stirred for 2 days at room temperature and then for 5 d at
40 °C. The mixture was cooled to room temperature, diluted with
ethyl acetate (100 mL) and quenched with potassium phosphate
buffer pH7 (1 M, 50 mL). The organic phase was washed with
water (2 ꢂ 50 mL), dried over sodium sulfate and concentrated

5406
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
under reduced pressure. Chromatography was done on silica gel
with hexanes/acetone 3:1. The first eluting spot was O-alkylated
material which was the major product, the desired N-alkylated
product eluted second, 134 mg colorless hard foam (14%). MS
(ESP) m/z 465 (MNa+); 1H NMR (DMSO-d6) d: 8.05 (s, 1H); 7.78
(d, 1H); 7.02 (dd, 1H); 6.92 (m, 1H); 6.80 (d, 1H); 4.31 (m, 2H);
3.94 (s, 3H); 1.95–1.30 (m, 11H); 1.39 (s, 9H).
yellow solid, 10.77 g (quant). MS (ESP) m/z 187 (MH+); 1H NMR
(DMSO-d6) d: 7.75 (d, 1H); 7.52 (br s, 2H); 6.63 (d, 1H); 3.87 (s, 3H).
10.24. 4-Fluoro-5-methoxybenzene-1,2-diamine (26)
A mixture of 25 (4 g, 21.49 mmol) in ethanol (100 mL) and ace-
tic acid (10.0 mL) was hydrogenated on Pd/C, wet, 10% (1 g,
9.40 mmol) at normal pressure and room temperature for 3 h.
10.20. cis-1-(2-(4-Amino-1-azidocyclohexyl)ethyl)-7-
methoxyquinoxalin-2(1H)-one (21)
The mixture was filtered through a 0.45 lm membrane directly
into the reaction mixture for 2d MS (ESP) m/z 157 (MH+).
To a solution of 20 (133 mg, 0.30 mmol) in dichloromethane
(2 mL) was added trifluoroacetic acid (1.0 mL) at 0 °C. The cooling
bath was removed and the mixture was stirred at room temperature
for 2 h. The mixture was concentrated under reduced pressure and
the residue was codistilled with dichloromethane twice. The resi-
due was taken up in dichloromethane (50 mL) and washed with
saturated sodium bicarbonate solution (7 mL, 1 M, pH adjusted to
ꢁ10 with 15% NaOH). The aqueous phase was back extracted with
dichloromethane twice (2 ꢂ 50 mL) and the combined organic
phases were dried over sodium sulfate and concentrated under
reduced pressure to give the product as a slightly yellow oil,
101 mg (98%). MS (ESP) m/z 343 (MH+).
10.25. 2-Chloro-6-fluoro-7-methoxyquinoxaline (28)
A suspension of a mixture of 2d (5.9 g, 30.39 mmol) and 7-flu-
oro-6-methoxyquinoxalin-2(1H)-one 27 (5.9 g, 30.39 mmol) in
POCl3 (70 mL, 750.98 mmol) was heated at 100 °C bath tempera-
ture for 1 h. The mixture was cooled to room temperature and
poured onto ice and dichloromethane (ꢁ200 mL) was added. The
pH of the mixture was brought to ꢁ8 by addition of solid sodium
carbonate in portions under stirring, while maintaining the tem-
perature of the mixture between room temperature and 10 °C with
ice. The mixture brought to room temperature and was diluted
with dichloromethane (400 mL) and with water (400 mL). The
organic phase was separated and washed once with saturated
aqueous bicarbonate solution (300 mL), then dried over sodium
sulfate. Chromatography was done on silica gel with hexanes/ethyl
acetate 10:1. The higher migrating product was isolated to give a
colorless solid, 3.37 g. MS (ESP) m/z 213/215 (MH+); 1H NMR
(DMSO-d6) d: 8.87 (s, 1H); 7.99 (d, 1H); 7.66 (d, 1H); 4.05 (s, 3H).
10.21. cis-6-((4-Azido-4-(2-(7-methoxy-2-oxoquinoxalin-1(2H)-
yl)ethyl)cyclohexylamino)methyl)-2H-pyrido[3,2-b][1,4]oxazin-
3(4H)-one (22)
Compound 21 (101 mg, 0.29 mmol), 3-oxo-3,4-dihydro-2H-pyr-
ido[3,2-b][1,4]oxazine-6-carbaldehyde 626 (52.5 mg, 0.29 mmol)
and sodium triacetoxyborohydride (156 mg, 0.74 mmol) were
reacted following the general procedure for 8. Chromatography
was done on silica gel with dichloromethane/methanol 10:1 to
give the product as a colorless hard foam, 130 mg (69%). MS
(ESP) m/z 505 (MH+); 1H NMR (DMSO-d6) d: 11.18 (s, 1H); 8.05
(s, 1H); 7.78 (d, 1H); 7.31 (m, 1H); 7.04 (m, 2H); 6.91 (m, 1H);
4.63 (s, 2H); 4.32 (m, 2H); 3.93 (s, 3H); 3.80 (br s, 2H); 3.30 (m,
2H); 1.90 (m, 5H); 1.56 (m, 2H); 1.35 (m, 2H).
10.26. N-(trans-2-(6-Fluoro-7-methoxy-2-oxoquinoxalin-1(2H)-
yl)-1-((1s,4R)-4-((7-oxo-7,8-dihydro-6H-pyrimido[5,4-
b][1,4]oxazin-2-yl)methylamino)cyclohexyl)ethyl)-N-methyl-2-
nitrobenzenesulfonamide (30)
To a mixture of 8h (100 mg, 0.15 mmol) in THF (5 mL) and
triphenylphosphine (45 mg, 0.17 mmol) and methanol (18
0.44 mmol) was added diisopropylazodicarboxylate (28.8
l
l
L,
L,
0.15 mmol) at room temperature. After 2 h, additional 20
lL of
10.22. cis-6-((4-Amino-4-(2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl)cyclohexylamino)methyl)-2H-pyrido[3,2-
b][1,4]oxazin-3(4H)-one (23)
diisopropylazodicarboxylate (20 L) and triphenylphosphine
l
(20 mg) were added. The reaction mixture was concentrated under
reduced pressure. The residue was purified by reverse phase HPLC,
eluting with methanol/TFA to give the TFA salt of the product as a
red foam, 101 mg. MS (ESP) m/z 697 (MH+).
A solution of 22 (80 mg, 0.16 mmol) and triphenylphosphine
(49.9 mg, 0.19 mmol) in acetonitrile (5 mL) and water (0.55 mL)
was stirred at room temperature for 5 days. The temperature was
increased to 45 °C and the mixture was stirred for another 3 days.
The reaction mixture was cooled to room temperature and concen-
trated under reduced pressure. The residue was codistilled with
methanol twice to remove water. Chromatography was done on
silica gel with CH2Cl2/MeOH 8:1, containing 1% ammonium
hydroxide. This gave 9 mg (12%) of the desired product as a color-
less hard film. MS (ESP) m/z 505 (MH+); 1H NMR (MeOD) d: 8.05 (s,
1H); 7.80 (m, 1H); 7.33 (m, 1H); 7.09–7.01 (m, 3H); 4.67 (m, 2H);
4.40 (m, 2H); 4.05 (m, 2H); 3.97 (m, 3H); 2.92 (m, 1H); 2.02 (m,
2H); 1.91 (m, 4H); 1.69 (m, 4H).
10.27. trans-2-((4-(2-(6-Fluoro-7-methoxy-2-oxoquinoxalin-
1(2H)-yl)-1-(methylamino)ethyl)cyclohexylamino)methyl)-6H-
pyrimido[5,4-b][1,4]oxazin-7(8H)-one (31)
Compound 30 (100 mg, 0.14 mmol), potassium carbonate
(40 mg, 0.29 mmol) and benzenethiol (30 mg, 0.27 mmol) were
reacted following the general procedure for 9. The crude material
was purified by reverse phase HPLC with methanol/ammonia to
give the product as a yellow solid, 11 mg (15%). MS (ESP) m/z
512 (MH+); 1H NMR (DMSO-d6) d: 0.9–1.4 (m, 6H); 1.74 (d, 1H);
1.98 (m, 3H); 2.39 (m, 1H); 2.90 (m, 2H); 3.84 (s, 2H); 4.00 (s,
3H); 4.22 (d, 2H); 4.85 (s, 2H); 7.17 (d, 1H); 7.68 (d, 1H); 8.10 (s,
1H); 8.28 (s, 1H).
10.23. 4-Fluoro-5-methoxy-2-nitroaniline (25)
Toa suspensionof commercially available4,5-difluoro-2-nitroan-
iline 24 (10 g, 57.44 mmol) in methanol (200 mL) was added KOH,
2 M (60 mL, 120 mmol) and the mixture was stirred vigorously at
room temperature for 20 h. The mixture was concentrated under
reduced pressure, the residue was taken up in dichloromethane
(500 mL) and washed with water (100 mL), dried over sodiumsulfate
and concentrated under reduced pressure to give the product as a
10.28. 4-Fluoro-2-nitro-phenylimino)-acetic acid ethyl ester
(33)
A solution of 4-fluoro-2-nitroaniline (1.50 kg, 9.6 mol) and ethyl
glyoxylate (1.96 kg, 50 wt % in toluene, 9.6 mol) in toluene (12.6 L)
was refluxed in a 22-L flask equipped with a Dean–Stark apparatus
for 48 h and evaporated in vacuo to give the product as a dark

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5407
brown oil that was used directly for the next step without further
purification.
was added in portions over 30 min. The mixture was stirred at 0 °C
for 90 min, then the cooling bath was removed and the reaction
stirred at room temperature for 2 h. The mixture was diluted with
dichloromethane, washed once with an aqueous 10% sodium thio-
sulfate solution, once with a saturated aqueous sodium bicarbon-
ate solution, dried over sodium sulfate and concentrated under
reduced pressure. Chromatography was done on silica gel with a
gradient of 10–15% ethyl acetate in hexanes to give 0.44 g
(1.8 mmol, 56%) of the desired product as a colorless solid. 1H
NMR (CDCl3) d: 1.46 (s, 11H); 1.57–1.70 (m, 1H); 1.77 (td,
J = 14.03, 4.52 Hz, 1H); 1.89–2.05 (m, 4H); 3.52 (br s, 1H); 4.46
(br s, 1H); 9.75 (d, J = 5.27 Hz, 1H); 19F NMR (CDCl3) d: ꢀ172.48
(s, 1F).
10.29. 7-Fluoro-3,4-dihydro-1H-quinoxalin-2-one (34)
In a 18-L Parr apparatus a suspension of 33 generated above in
ethanol was hydrogenated at 50 psi in the presence of 20 wt % Pd/C
(240 g, containing ca. 50 wt % water) until no hydrogen was con-
sumed. (Note: The reaction was strongly exothermic and the tem-
perature should be controlled at around 60 °C by adjusting the rate
of recharging hydrogen and by a cooling system). The reaction mix-
ture was discharged, filtered over a Celite cake, and evaporated in
vacuo to give a crude solid that was triturated with MTBE (8 L) to
afford the product (600 g) as a tan solid.
10.33. (Z)-tert-Butyl-4-fluoro-4-vinylcyclohexylcarbamate (40)
10.30. tert-Butyl 1-oxaspiro[2.5]octan-6-ylcarbamate (37)54
A suspension of methyltriphenylphosphonium bromide (0.44 g,
1.2 mmol) in dry THF (5 mL) was cooled to 0 °C and a potassium
hexamethyldisilazide solution in THF (1 M, 1.2 mL, 1.2 mmol)
added dropwise to give a bright yellow mixture. The mixture
was stirred at 0 °C for 60 min, then a solution of (Z)-tert-butyl
(1S,4S)-4-fluoro-4-formylcyclohexylcarbamate (0.15 g, 0.6 mmol)
in dry THF (5 mL) added and the yellow mixture stirred at 0 °C
for 60 min. The reaction was quenched with 5 mL of a saturated
aqueous ammonium chloride solution and extracted twice with
ethyl acetate. The combined organic phases were washed with
brine, dried over sodium sulfate and concentrated under reduced
pressure. Chromatography was done on silica gel with a gradient
of 0–5% EtOAc in hexanes to give 105 mg (0.43 mmol, 71%) of the
desired product as a colorless solid. 1H NMR (CDCl3-d) d: 1.44 (s,
9H); 1.46–1.71 (m, 4H); 1.81–1.99 (m, 4H); 3.44 (br s, 1H); 4.46
(br s, 1H); 5.10 (dd, J = 11.11, 0.94 Hz, 1H); 5.27 (dt, J = 17.52,
1.41 Hz, 1H); 5.88 (td, J = 17.05, 10.93 Hz, 1H); 19F NMR
(282 MHz, CDCl3-d) d: ꢀ159.82 (s, 1F).
To
a suspension of potassium tert-butoxide (95%, 2.4 g,
20.4 mmol) in dry THF (70 mL) was added trimethylsulfoxonium
iodide (4.75 g, 21 mmol) in one portion and the pale yellow mix-
ture was heated to reflux for 2 h. The mixture was cooled to room
temperature and a solution of tert-butyl 4-oxocyclohexylcarba-
mate (3.0 g, 14.1 mmol) in dry THF (20 mL) was added dropwise
over 5 min. The resulting pale orange mixture was heated to reflux
and stirred for 2 h. The yellow mixture was allowed to cool to room
temperature, partitioned between water (100 mL) and ethyl ace-
tate (200 mL) and the phases separated. The aqueous phase was
extracted twice with ethyl acetate, and the combined organic
phases were washed with brine, then dried over sodium sulfate
and concentrated under reduced pressure to give 3.13 g
(13.8 mmol, 98%) of the product as a waxy colorless solid. The
crude product was taken to the next step without further purifica-
tion. MS (ESP) m/z 250 (M+Na+); 1H NMR (CDCl3) d: 1.29–1.41 (m,
2H); 1.41–1.50 (m, 11H); 1.86–2.07 (m, 4H); 2.67 (s, 2H); 3.58 (d,
J = 9.98 Hz, 1H); 4.46 (br s, 1H).
10.34. (Z)-tert-Butyl-4-fluoro-4-(1-(2-
nitrophenylsulfonyl)aziridin-2-yl)cyclohexylcarbamate (41)
10.31. (Z)-tert-Butyl-4-fluoro-4-
(hydroxymethyl)cyclohexylcarbamate (38)
To a solution of 40 (0.308 g, 1.3 mmol) in dry acetonitrile (10 mL)
was added activated molecular sieves, followed by copper (II) tri-
flate (46 mg, 0.13 mmol) to give a green/blue mixture. To this was
added (N-(2-nitrobenzylsulfonyl)imino)phenyliodinane (0.77 g,
1.9 mmol) and the pale green mixture was flushed with dry nitro-
gen and stirred at room temperature for 2 h. An additional 46 mg
(0.13 mmol) copper (II) triflate was added and the tan mixture stir-
red overnight. The reaction mixture was filtered through Celite, the
filter solids washed with acetonitrile, then with ethyl acetate, the
filtrate was concentrated under reduced pressure and the residue
dissolved in dichloromethane and filtered through Celite. The fil-
trate was concentrated under reduced pressure, redissolved in min-
To a 125 mL polypropylene flask was added a 70% HF solution in
pyridine (5 mL) under dry nitrogen with stirring, and the colorless
solution cooled to ꢀ78 °C. To this was added a solution of 37 (1.0 g,
4.4 mmol) in dichloromethane (5 mL) dropwise over 10 min. The
resulting solution was stirred at ꢀ78 °C for 5 h, then poured into
an ice-cold 2 N ammonium hydroxide solution (50 mL) and the
pH of the mixture was made alkaline by addition of conc. ammo-
nium hydroxide. The mixture was extracted three times with
dichloromethane, the combined organic phases were washed with
brine, dried over sodium sulfate and concentrated under reduced
pressure. Chromatography was done on silica gel with a gradient
of 10–20% ethyl acetate in hexanes to give 0.55 g of the desired
product (2.2 mmol, 51%) as a white solid. The E isomer was also
isolated and its configuration was determined by a 2D proton
imal dichloromethane and filtered through
a cotton plug.
Chromatography of the filtrate was done on silica gel with a gradi-
ent of 0–10% acetone in hexanes to give 163 mg (0.67 mmol) recov-
ered olefin and 83 mg (0.19 mmol, 15%) of the desired product as a
colorless hard foam. MS (ESP) m/z 466 (MNa+); 1H NMR (CDCl3) d:
1.41–1.52 (m, 12H); 1.60–1.70 (m, 1H); 1.84–1.99 (m, 4H); 2.62
(d, J = 4.71 Hz, 1H); 2.89 (d, J = 7.35 Hz, 1H); 3.08 (ddd, J = 15.12,
7.21, 4.80 Hz, 1H); 3.46 (br s, 1H); 4.40 (br s, 1H); 7.71–7.82 (m,
3H); 8.18–8.26 (m, 1H); 19F NMR (CDCl3) d: ꢀ171.33 (s, 1F).
NMR
NOE-experiment.
(E)-tert-Butyl-4-fluoro-4-(hydroxy-
methyl)cyclohexylcarbamate: 1H NMR (CDCl3) d: 1.37–1.56 (m,
11H); 1.69–1.85 (m, 4H); 1.86–2.01 (m, 2H); 3.60–3.78 (m, 3H);
4.55 (br s, 1H); 19F NMR (CDCl3) d: ꢀ160.68 (s, 1F).
(Z)-tert-Butyl-4-fluoro-4-(hydroxymethyl)cyclohexylcarbamate:
1H NMR (CDCl3) d: 1.30–1.43 (m, 2H), 1.43–1.49 (m, 11H), 1.85–
2.09 (m, 4H), 3.47 (d, J = 7.72 Hz, 1H), 3.57 (d, J = 19.97 Hz, 2H),
4.43 (br s, 1H); 19F NMR (CDCl3) d: ꢀ169.91 (s, 1F).
10.35. (Z)-tert-Butyl-4-fluoro-4-(2-(7-methoxy-2-
oxoquinoxalin-1(2H)-yl)-1-(2-nitrophenylsulfonamido)ethyl)-
cyclohexylcarbamate (42)
10.32. (Z)-tert-Butyl-4-fluoro-4-formylcyclohexylcarbamate
(39)
Compound 2c8,31,32 (41 mg, 0.23 mmol) was reacted with
sodium hydride (11 mg, 0.28 mmol) and 41 (103 mg, 0.23 mmol)
A solution of 38 (0.8 g, 3.2 mmol) in dichloromethane (40 mL)
was cooled to 0 °C and Dess–Martin periodinane (2.75 g, 6.5 mmol)

5408
F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
following the general procedure for 4. Chromatography was done
on silica gel with 20–80% ethyl acetate in hexanes to give 78 mg
of the desired product as an off-white solid (54%). MS (ESP) m/z
618 (MꢀHꢀ); 1H NMR (CDCl3) d: 1.46 (s, 9H); 1.51–1.66 (m, 2H);
1.70–1.93 (m, 2H); 1.98–2.08 (m, 2H); 2.09–2.20 (m, 1H); 2.20–
2.34 (m, 1H); 3.51 (br s, 1H); 3.92 (s, 3H); 4.20 (d, J = 13.56 Hz,
1H); 4.26–4.43 (m, 1H); 4.43–4.56 (m, 1H); 4.56–4.87 (m, 1H);
6.06 (d, J = 9.04 Hz, 1H); 6.70 (d, J = 2.45 Hz, 1H); 6.86 (dd,
J = 8.85, 2.26 Hz, 1H); 7.30–7.49 (m, 2H); 7.51–7.60 (m, 2H);
7.60–7.72 (m, 2H); 19F NMR (CD2Cl2) d: ꢀ170.89 (s, 1F).
(0.03 mmol, 29%) of the desired product as a pale pink hard foam.
MS (ESP) m/z 497.1 (MH+); 1H NMR (CDCl3-d) d: 1.55–1.69 (m,
2H); 1.76–1.88 (m, 2H); 1.88–2.05 (m, 4H); 2.59 (t, J = 10.74 Hz,
1H); 3.20–3.29 (m, 1H); 3.48 (s, 2H); 3.84–3.95 (m, 6H); 4.14 (dd,
J = 13.94, 1.70 Hz, 1H); 4.64 (s, 2H); 4.70 (dd, J = 13.94, 10.36 Hz,
1H); 6.90–6.98 (m, 3H); 7.21 (d, J = 7.91 Hz, 1H); 7.79 (d,
J = 9.42 Hz, 1H); 8.15 (s, 1H) (lactam proton not seen); 19F NMR
(282 MHz, CDCl3-d) d: ꢀ165.14 (s, 1F).
Acknowledgments
10.36. (Z)-N-(1-(4-Amino-1-fluorocyclohexyl)-2-(7-methoxy-2-
oxoquinoxalin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide (43)
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 Lena Grosser, Pharmacology Depart-
ment, AstraZeneca R&D Boston for efficacy studies.
Compound 42 was reacted with trifluoroacetic acid according to
the general procedure for 5 to give the desired product as a pale
orange solid in quantitative yield. MS (ESP) m/z 520 (MH+). 1H
NMR (acetonitrile-d3) d: 1.56 (br s, 1H); 1.61–1.87 (m, 4H); 1.99–
2.12 (m, 2H); 2.19–2.37 (m, 2H); 3.30 (br s, 1H); 3.93 (s, 3H);
4.51–4.70 (m, 1H); 6.30 (d, J = 9.42 Hz, 1H); 6.57 (br s, 3H); 6.80
(d, J = 2.45 Hz, 1H); 6.89 (dd, J = 8.95, 2.54 Hz, 1H); 7.29–7.45 (m,
2H); 7.45–7.66 (m, 4H); 7.66–7.77 (m, 1H); 19F NMR (acetoni-
trile-d3) d: ꢀ171.16 (s, 1F); ꢀ76.73 (s, 3F).
References and notes
1. Livermore, D. M. Int. J. Antimicrob. Agents 2012, 39, 283.
2. Cuevas, O.; Oteo, J.; Lázaro, E.; Aracil, B.; de Abajo, F.; García-Cobos, S.; Ortega,
A.; Campos, J. J. Antimicrob. Chemother. 2011, 66, 664.
3. Pasca M. Rosalia; Dalla Valle, Claudia; De Jesus Lopes Ribeiro, Ana, Luisa;
Buroni, Silvia; Papaleo, M., Cristiana; Bazzini, Silvia; Udine, Claudia; Incandela,
M., Loreto; Daffara, Silvio; Fani, Renato; Riccardi, Giovanna; Marone, Piero;
Daffara, Silvio Microb. Drug Resist. 2012, 18, 23.
4. Bax, B. D.; Chan, P. F.; Eggleston, D. S.; Fosberry, A.; Gentry, D. R.; Gorrec, F.;
Giordano, I.; Hann, M. M.; Hennessy, A.; Hibbs, M.; Huang, J.; Jones, E.; Jones, J.;
Brown, K. K.; Lewis, C. J.; May, E. W.; Saunders, M. R.; Singh, O.; Spitzfaden, C.
E.; Shen, C.; Shillings, A.; Theobald, A. J.; Wohlkonig, A.; Pearson, N. D.; Gwynn,
M. N. Nature 2010, 466, 935.
5. Gomez, L.; Hack, M. D.; Wu, J.; Wiener, J. J. M.; Venkatesan, H.; Santillan, A.;
Pippel, D. J.; Mani, N.; Morrow, B. J.; Motley, S. T.; Shaw, K. J.; Wolin, R.; Grice, C.
A.; Jones, T. K. Bioorg. Med. Chem. Lett. 2007, 17, 2723.
6. Wiener, J. J. M.; Gomez, L.; Venkatesan, H.; Santillan, A., Jr.; Allison, B. D.;
Schwarz, K. L.; Shinde, S.; Tang, L.; Hack, M. D.; Morrow, B. J.; Motley, S. T.;
Goldschmidt, R. M.; Shaw, K. J.; Jones, T. K.; Grice, C. A. Bioorg. Med. Chem. Lett.
2007, 17, 2718.
7. Black, M. T.; Stachyra, T.; Platel, D.; Girard, A.; Claudon, M.; Bruneau, J.;
Miossec, C. Antimicrob. Agents Chemother. 2008, 52, 3339.
8. Reck, F.; Alm, R.; Brassil, P.; Newman, J.; DeJonge, B.; Eyermann, C. J.; Breault,
G.; Breen, J.; Comita-Prevoir, J.; Cronin, M.; Davis, H.; Ehmann, D.; Galullo, V.;
Geng, B.; Grebe, T.; Morningstar, M.; Walker, P.; Hayter, B.; Fisher, S. J. Med.
Chem. 2011, 54, 7834.
9. Geng, B.; Comita-Prevoir, J.; Eyermann, C. J.; Reck, F.; Fisher, S. Bioorg. Med.
Chem. Lett. 2011, 21, 5432.
10. Reck, F.; Alm, R. A.; Brassil, P.; Newman, J. V.; Ciaccio, P.; McNulty, J.; Barthlow,
H.; Goteti, K.; Breen, J.; Comita-Prevoir, J.; Cronin, M.; Ehmann, D. E.; Geng, B.;
Godfrey, A. A.; Fisher, S. L. J. Med. Chem. 2012, 55, 6916.
11. Miles, T. J.; Axten, J. M.; Barfoot, C.; Brooks, G.; Brown, P.; Chen, D.; Dabbs, S.;
Davies, D. T.; Downie, D. L.; Eyrisch, S.; Gallagher, T.; Giordano, I.; Gwynn, M.
N.; Hennessy, A.; Hoover, J.; Huang, J.; Jones, G.; Markwell, R.; Miller, W. H.;
Minthorn, E. A.; Rittenhouse, S.; Seefeld, M.; Pearson, N. Bioorg. Med. Chem. Lett.
2011, 21, 7489.
12. Miles, T. J.; Barfoot, C.; Brooks, G.; Brown, P.; Chen, D.; Dabbs, S.; Davies, D. T.;
Downie, D. L.; Eyrisch, S.; Giordano, I.; Gwynn, M. N.; Hennessy, A.; Hoover, J.;
Huang, J.; Jones, G.; Markwell, R.; Rittenhouse, S.; Xiang, H.; Pearson, N. Bioorg.
Med. Chem. Lett. 2011, 21, 7483.
13. Mitton-Fry, M. J.; Brickner, S. J.; Hamel, J. C.; Brennan, L.; Casavant, J. M.; Chen,
M.; Chen, T.; Ding, X.; Driscoll, J.; Hardink, J.; Hoang, T.; Hua, E.; Huband, M. D.;
Maloney, M.; Marfat, A.; McCurdy, S. P.; McLeod, D.; Plotkin, M.; Reilly, U.;
Robinson, S.; Schafer, J.; Shepard, R. M.; Smith, J. F.; Stone, G. G.; Subramanyam,
C.; Yoon, K.; Yuan, W.; Zaniewski, R. P.; Zook, C. Bioorg. Med. Chem. Lett. 2013,
23, 2955.
14. Miles, T. J.; Hennessy, A. J.; Bax, B.; Brooks, G.; Brown, B. S.; Brown, P.; Cailleau,
N.; Chen, D.; Dabbs, S.; Davies, D. T.; Esken, J. M.; Giordano, I.; Hoover, J. L.;
Huang, J.; Jones, G. E.; Kusalakumari Sukmar, S.; Spitzfaden, C.; Markwell, R. E.;
Minthorn, E. A.; Rittenhouse, S.; Gwynn, M. N.; Pearson, N. D. Bioorg. Med.
Chem. Lett. 2013, 23, 5437.
10.37. (Z)-N-(1-(1-Fluoro-4-((3-oxo-3,4-dihydro-2H-pyrido[3,2-
b][1,4]oxazin-6-yl)methylamino)cyclohexyl)-2-(7-methoxy-2-
oxoquinoxalin-1(2H)-yl)ethyl)-2-nitrobenzenesulfonamide (44)
Compound 43 (80 mg, 0.13 mmol), ethyldiisopropylamine, 626
(22 mg, 0.13 mmol) and sodium triacetoxyborohydride were
reacted following the procedure for 8b to give 69 mg (80%) of the
desired product as a colorless solid. MS (ESP) m/z 682 (MH+); 1H
NMR (DMF-d7) d: 1.36–1.52 (m, 2H); 1.66–1.80 (m, 1H); 1.84–
1.99 (m, 3H); 2.00–2.34 (m, 3H); 2.44–2.62 (m, 1H); 3.80 (s, 2H);
3.99 (s, 3H); 4.23 (ddd, J = 17.80, 10.36, 2.17 Hz, 1H); 4.39–4.48
(m, 1H); 4.58 (d, J = 12.24 Hz, 1H); 4.70 (s, 2H); 6.92 (dd, J = 8.95,
2.35 Hz, 1H); 7.10 (d, J = 8.10 Hz, 1H); 7.16–7.22 (m, 1H); 7.33 (d,
J = 8.10 Hz, 1H); 7.51–7.59 (m, 2H); 7.59–7.64 (m, 1H); 7.66–7.72
(m, 2H); 7.86 (s, 1H), (secondary amine and lactam protons not
seen); 19F NMR (282 MHz, DMF-d7) d: ꢀ167.29 (s, 1F).
10.38. (Z)-6-((4-(1-Amino-2-(7-methoxy-2-oxoquinoxalin-
1(2H)-yl)ethyl)-4-fluorocyclohexylamino)methyl)-2H-
pyrido[3,2-b][1,4]oxazin-3(4H)-one (45)
To a suspension of 44 (69 mg, 0.1 mmol) in DMF (2 mL) was
added 168 mg (1.2 mmol) powdered potassium carbonate (168
mg, 1.2 mmol), followed by addition of thiophenol (0.126 mL,
1.2 mmol) and the mixture stirred vigorously at room temperature
for 12 h. DMF was removed by azeotroping with tetrachloroethyl-
ene. The residue was triturated with methanol, filtered through
Celite, the filter solids washed with methanol, then with dichloro-
methane, and the filtrate concentrated under reduced pressure.
The residue was suspended in a mixture of brine and a saturated
aqueous sodium bicarbonate solution (1:1, 10 mL), the mixture
was extracted five times with 10% methanol in DCM, and the com-
bined organic phases were dried over sodium sulfate and concen-
trated under reduced pressure. Chromatography was done on
silica gel with a gradient of 0–4% methanolic 7 N ammonia in dichlo-
romethane to give 30 mg of a red oil. This was dissolved in minimal
dichloromethane and diethyl ether added dropwise until a red pre-
cipitate formed. This was filtered off and the filtrate diluted with
ether to turbidity. The mixture was placed at 0 °C for 30 min, the
supernatant pipetted away from a precipitated solid and filtered.
The filter solids and flask solids were dissolved in methanol, filtered
15. Surivet, J.; Zumbrunn, C.; Rueedi, G.; Hubschwerlen, C.; Bur, D.; Bruyere, T.;
Locher, H.; Ritz, D.; Keck, W.; Seiler, P.; Kohl, C.; Gauvin, J.; Mirre, A.; Kaegi, V.;
Dos Santos, M.; Gaertner, M.; Delers, J.; Enderlin-Paput, M.; Boehme, M. J. Med.
Chem. 2013, 56, 7396.
16. Singh, S. B.; Kaelin, D. E.; Wu, J.; Miesel, L.; Tan, C. M.; Meinke, P. T.; Olsen, D.;
Lagrutta, A.; Bradley, P.; Lu, J.; Patel, S.; Rickert, K. W.; Smith, R. F.; Soisson, S.;
Wei, C.; Fukuda, H.; Kishii, R.; Takei, M.; Fukuda, Y. ACS Med. Chem. Lett. 2014, 5,
609.
17. Mayer, C.; Janin, Y. L. Chem. Rev. (Washington, DC, U.S.) 2014, 114, 2313.
through a 0.45 lm syringe filter and conc. to give 14.3 mg

F. Reck et al. / Bioorg. Med. Chem. 22 (2014) 5392–5409
5409
18. Dougherty, T. J.; Nayar, A.; Newman, J. V.; Hopkins, S.; Stone, G. G.; Johnstone,
M.; Shapiro, A. B.; Cronin, M.; Reck, F.; Ehmann, D. E. Antimicrob. Agents
Chemother. 2014.
19. Miller, A. A.; Bundy, G. L.; Mott, J. E.; Skepner, J. E.; Boyle, T. P.; Harris, D. W.;
Hromockyj, A. E.; Marotti, K. R.; Zurenko, G. E.; Munzner, J. B.; Sweeney, M. T.;
Bammert, G. F.; Hamel, J. C.; Ford, C. W.; Zhong, W.; Graber, D. R.; Martin, G. E.;
Han, F.; Dolak, L. A.; Seest, E. P.; Ruble, J. C.; Kamilar, G. M.; Palmer, J. R.; Banitt,
L. S.; Hurd, A. R.; Barbachyn, M. R. Antimicrob. Agents Chemother. 2008, 52, 2806.
20. Barvian, K.; Basarab, G. S.; Gowravaram, M. R.; Hauck, S. I.; Zhou, F.
WO2010043893, 2010.
21. Basarab, G. S.; Dumas, J.; Hill, P. WO2009010801, 2009.
22. Basarab, G. S.; Dumas, J.; Hill, P. WO2009004382, 2009.
23. Huband, M. D.; Cohen, M. A.; Zurack, M.; Hanna, D. L.; Skerlos, L. A.; Sulavik, M.
C.; Gibson, G. W.; Gage, J. W.; Ellsworth, E.; Stier, M. A.; Gracheck, S. J.
Antimicrob. Agents Chemother. 2007, 51, 1191.
24. O’Shea, R.; Moser, H. E. J. Med. Chem. 2008, 51, 2871.
25. Nikaido, H.; Thanassi, D. G. Antimicrob. Agents Chemother. 1993, 37, 1393.
26. 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. Tetrahedron Lett. 2010, 51,
5035.
36. Soedergren, M. J.; Alonso, D. A.; Bedekar, A. V.; Andersson, P. G. Tetrahedron
Lett. 1997, 38, 6897.
37. Titz, A.; Radic, Z.; Schwardt, O.; Ernst, B. Tetrahedron Lett. 2006, 47, 2383.
38. Hutchins, R. O.; Su, W.; Sivakumar, R.; Cistone, F.; Stercho, Y. P. J. Org. Chem.
1983, 48, 3412.
39. Nikaido, H. Microbiol. Mol. Biol. Rev. 2003, 67, 593.
40. Nikaido, H.; Hancock, R. E. W. Outer Membrane Permeability of Pseudomonas
aeruginosa In Sokatch, J. R., Ed.; The Bacteria; Academic Press: Orlando, Fla,
1986; Vol. 10, pp 145–193.
41. Hubschwerlen, C.; Rueedi, G.; Surivet, J.; Zumbrunn Acklin, C. WO2009147616,
2009.
42. Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. ACS Chem. Neurosci. 2010, 1,
435.
43. Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.;
Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.; Persson, F.;
Jacobson, I.; Sullivan, M.; Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin,
J. J. Pharmacol. Toxicol. Methods 2006, 54, 189.
44. Kuhn, B.; Mohr, P.; Stahl, M. J. Med. Chem. 2010, 53, 2601.
45. Andrew, C. L.; Klemm, A. R.; Lloyd, J. B. Biochim. Biophys. Acta 1997, 1330, 71.
46. Craig, W. A. Infect. Dis. Clin. North Am. 2003, 17, 479.
47. Redfern, W. S.; Carlsson, L.; Davis, A. S.; Lynch, W. G.; MacKenzie, I.; Palethorpe,
S.; Siegl, P. K. S.; Strang, I.; Sullivan, A. T.; Wallis, R.; Camm, A. J.; Hammond, T.
G. Cardiovasc. Res. 2003, 58, 32.
48. Andes, D.; Craig, W. A. Antimicrob. Agents Chemother. 2002, 46, 1665.
49. Anonymous Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically: Approved Standard, 2nd ed. In: National
Committee for Clinical Laboratory Standards: Villanova, PA, 1993.
50. Mills, S. D.; Eakin, A. E.; Buurman, E. T.; Newman, J. V.; Gao, N.; Huynh, H.;
Johnson, K. D.; Lahiri, S.; Shapiro, A. B.; Walkup, G. K.; Yang, W.; Stokes, S. S.
Antimicrob. Agents Chemother. 2011, 55, 1088.
27. Ballell, L.; Barros, D.; Brooks, G.; Castro Pichel, J.; Dabbs, S.; Daines, R. A.;
Davies, D. T.; Fiandor Roman, J. M.; Giordano, I.; Hennessy, A. J.; Hoffman, J. B.;
Jones, G. E.; Miles, T. J.; Pearson, N. D.; Pendrak, I.; Remuinan Blanco, M. J.;
Rossi, J. A.; Zhang, L. WO2008009700, 2008.
28. Cronin, M.; Geng, B.; Reck, F. WO2010055348, 2010.
29. Sidhu, G. S.; Thyagarajan, G.; Ansari, S. Liebigs Ann. Chem. 1959, 627, 218.
30. Evertsson, E.; Inghardt, T.; Lindberg, J.; Linusson, A.; Giordanetto, F.
WO2005066132, 2005.
31. Chen, P.; Barrish, J. C.; Iwanowicz, E.; Lin, J.; Bednarz, M. S.; Chen, B. Tetrahedron
Lett. 2001, 42, 4293.
32. Curd, F. H. S.; Davey, D. G.; Stacey, G. J. J. Chem. Soc. 1949, 1271.
33. Boss, C.; Brotschi, C.; Heidmann, B.; Sifferlen, T.; Williams, J. T.
WO2012085857, 2012.
34. Breault, G.; Eyermann, C.J.; Geng, B.; Morningstar, M.; Reck, F. WO2006134378,
2006.
51. Duker, G.; Almgren, O.; Axenborg, J. Pharmacol. Toxicol. (Copenhagen) 1988, 63,
85.
52. Kiyoto, T.; Ando, J.; Tanaka, T.; Tsutsui, Y.; Yokotani, M.; Noguchi, T.; Ushiyama,
F.; Urabe, H.; Horikiri, H. WO2007138974, 2007.
53. Barfoot, C.; Davies, D. T.; Miles, T.; Pearson, N. D. WO2008128961A1, 2008.
54. Ashwell, M.; Tandon, M.; Lapierre, J.; Ali, S.; Vensel, D.; Li, C. J. WO 2007139569,
2007.
35. Carr, G.; Butterworth, S.; Walker, P.; Reck, F.; Geng, B.; Pandarinathan, L.
Synthesis 2012, 44, 875.