Novel DNA gyrase inhibiting spiropyrimidinetriones with a benzisoxazole scaffold: Sar and in vivo characterization

Article abstract of DOI:10.1021/jm501174m

The compounds described herein with a spirocyclic architecture fused to a benzisoxazole ring represent a new class of antibacterial agents that operate by inhibition of DNA gyrase as corroborated in an enzyme assay and by the inhibition of precursor thymi

Full text of DOI:10.1021/jm501174m

Article
pubs.acs.org/jmc
Novel DNA Gyrase Inhibiting Spiropyrimidinetriones with a
Benzisoxazole Scaffold: SAR and in Vivo Characterization
Gregory S. Basarab,* Patrick Brassil,† Peter Doig, Vincent Galullo, Howard B. Haimes,‡ Gunther Kern,
Amy Kutschke, John McNulty,§ Virna J. A. Schuck,∥ Gregory Stone, and Madhusudhan Gowravaram⊥
Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: The compounds described herein with a spirocyclic
architecture fused to a benzisoxazole ring represent a new class of
antibacterial agents that operate by inhibition of DNA gyrase as
corroborated in an enzyme assay and by the inhibition of precursor
thymidine into DNA during cell growth. Activity resided in the
configurationally lowest energy (2S,4R,4aR) diastereomer. Highly active
compounds against Staphylococcus aureus had sufficiently high solubility,
high plasma protein free fraction, and favorable pharmacokinetics to
suggest that in vivo efficacy could be demonstrated, which was realized with
compound (−)-1 in S. aureus mouse infection models. A high drug
exposure NOEL on oral dosing in the rat suggested that a high therapeutic
margin could be achieved. Importantly, (−)-1 was not cross-resistant with other DNA gyrase inhibitors such as fluoroquinolone
and aminocoumarin antibacterials. Hence, this class shows considerable promise for the treatment of infections caused by
multidrug resistant bacteria, including S. aureus.
class of compounds, hereafter referred to as spiropyrimidine-
trione (SPT) antibacterials, are structurally and biochemically
differentiated from any class of DNA gyrase inhibitors.29 We
therefore set out to assess the potential for developing an
efficacious drug by identifying analogues with potent
antibacterial activity and sufficiently good DMPK attributes to
evaluate their efficacy in mouse models of infection and to
assess in vivo tolerability.
INTRODUCTION
■
The persistence and escalation of bacterial resistant to current
drug regimens continues to prompt efforts toward identifying
novel mode-of-action antibacterial agents.1−4 Accordingly,
targets essential for bacterial viability have been screened
against compound libraries,5,6 probed with substrate, product,
or transition state mimetics,7 or structurally enabled for
computational based inhibitor design.8−10 An alternative
approach to addressing resistance would be to exploit an
established target by introducing a novel binding mode and
thereby a novel mode-of-inhibition. Here, we disclose initial
work around compounds with a benzisoxazole scaffold fused
with a piperidine ring that displays a spiropyrimidinetrione
pharmacophore.11 A key reaction in the synthesis of this class of
compounds is the tertiary amino effect reaction (T-reaction)
RESULTS AND DISCUSSION
■
Synthesis of Target Compounds. Three patent applica-
tions from Pfizer have disclosed PNU-286607 analogues, with
most of them having two fluorine atoms and a heteroaryl
substituent on the benzene scaffold as shown in Figure 2.16,30,31
The benzisoxazole scaffold was designed to place an oxygen
atom in the same position as the one fluorine atom, thereby
either serving as an isosteric replacement or maintaining the
capability to accept an admittedly weak hydrogen bond. The
positioning of an oxygen atom in place of a fluorine atom
through an isoxazole fusion is, to our knowledge, unprece-
dented in the literature. The distal substituent on the
benzisoxazole scaffold enters into space nearby to, but offset
from, the heteroaryl substituent of the compounds of Figure 2,
thereby probing a differentiated binding environment. The
synthesis of target spirocycles with a methyl substituent on the
benzisoxazole (Table 1) utilized either meso-dimethylmorpho-
line to afford racemic product (Scheme 1) or chiral (R,R)-
wherein the Knovenagel adduct of a precursor aromatic
̈
aldehyde undergoes a [1,5]-hydride shift with an ortho-
dialkylamine substituent.12−14 In this case, barbituric acid
serves to form the Knovenagel adduct, and a Mannich
̈
cyclization following the hydride shift leads to a spiropyr-
imidinetrione (Figure 1). Previously, PNU-286607 (Figure 2),
which shares the spiropyrimidinetrione pharmacophore, was
shown to be effective against fluoroquinolone-resistant bacteria
despite inhibition of the same bacterial targets, that is, the
bacterial type II topoisomerases, DNA gyrase, and topoisomer-
ase IV (Topo IV).15,16 The course of substrate binding and
conformational transformations during the catalytic process for
type II topoisomerases leads to several possible modes-of-
inhibition,17−21 and indeed different classes of antibacterial
agents are known to inhibit the two enzymes.22−28 This new
Received: July 31, 2014
© XXXX American Chemical Society
A
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Figure 1. T-reaction for the synthesis of spiropyrimidinetriones (SPTs).
the anion of acetone oxime, and treatment with aqueous acid
hydrolyzed both the oxime and silyl ether, enabling subsequent
cyclization to the corresponding benzisoxazoles.33 Oxidation
with MnO2 afforded aldehydes 16a and 16b. The final T-
reaction was achieved by heating barbituric acid with the
corresponding aldehyde at the appropriate temperature. In the
case of 16a, heating in 2-propanol at 80 °C effected clean
conversion to racemic ( )-1, and the individual enantiomers
(−)-1 and (+)-1 were separated by chiral supercritical-fluid
chromatography (SFC). Temperatures above 80 °C resulted in
conversion to other diastereomers and were therefore avoided.
By contrast, the T-reaction with 16b and barbituric acid was
carried out at 120 °C to equilibrate diastereomers to give a 9:1
thermodynamic mixture. The major diastereomer was obtained
by crystallization as the hydrate from aqueous methanol,
matching the properties of (−)-1 described in Scheme 1. The
configuration was confirmed from a single-crystal X-ray
structure of (−)-1, showing the morpholine ring in a lower
energy chair conformation with the two pendant methyl
substituents in equatorial orientations (Figure 3) and matching
that of the (−)-enantiomer of PNU-286607.15 The config-
uration and conformation were corroborated by NOESY NMR
examination of (−)-1 (see Supporting Information) wherein
Figure 2. PNU-286607 and analogues.
dimethylmorpholine to afford chiral product (Scheme 2).15
Along both routes, compounds 14a and 14b were made by
regioselective displacement of the 2-position fluorine atom of
13, followed by reduction to the alcohol and protection as
TBDPS ethers. This set up a fluorine directed ortho-lithiation32
followed by quenching with the Weinreb amide of acetic acid to
afford methyl ketones 15a and 15b. The use of MTBE over
other ethereal solvents proved important toward maximizing
the yield, which was otherwise compromised by the return of
starting material, presumably due to quenching by non-
productive deprotonation of the Weinreb amide. Each fluorine
atom ortho to the acetyl group of 15a and 15b was displaced by
Table 1. Influence of Stereochemistry on DNA Gyrase Inhibitory Potency, PPB, Solubility, and MIC
a
b
c
d
e
f
Spn: S. pneumoniae. Spy: S. pyogenes. Methicillin sensitive S. aureus. Methicillin resistant, quinolone resistant S. aureus. H. influenzae. E. coli.
g
Not determined.
B
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a
Scheme 1
a
Reagents and conditions: (a) LHMDS, THF, −78 °C → rt; (b) NaBH4, I2, EtOH; (c) t-Bu(CH3)2Si-Cl, imidazole; (d) s-BuLi,
CH3C(O)N(OMe)Me, THF; (e) acetone−oxime, KO-t-Bu, THF, then 2.5% HCl, EtOH; (f) MnO2, CH2Cl2, 3d; (g) i-PrOH, 80 °C, 16 h;
(h) separate isomers, 120 °C; (i) 3:1 AcOH−water.
a
Scheme 2
a
Reagents and conditions: (a) LHMDS, THF, −78 °C → rt; (b) NaBH4, I2, EtOH; (c) t-Bu(CH3)2Si-Cl, imidazole; (d) s-BuLi,
CH3C(O)N(OMe)Me, MTBE; (e) acetone−oxime, KO-t-Bu, THF, then 2.5% HCl, EtOH; (f) MnO2, CH2Cl2, 3 d; (g) 3:1 AcOH−water,
120 °C; (h) separate isomers.
Figure 3. (left) X-ray structure of (−)-1 (as water hydrate, Ortep representation). (right) Side view showing morpholine chair conformation of
(−)-1 with equatorial methyl groups (water and hydrogen atoms removed for clarity).
large coupling constants (12 and 9 Hz) were seen for the C2
and C3 morpholine hydrogen atoms (see Figure 3 for
numbering) to the adjacent C1 and C4 hydrogen atoms
indicative of trans-diaxial relationships, placing the methyl
substituents in equatorial orientations. A through-space NOE
was seen between the same two morpholine C2 and C3
hydrogen atoms, achieved by their axial orientations. A notable
through-space NOE was seen between the C4 hydrogen atom
and one of the C6 benzylic hydrogen atoms. Hence, solution
and solid-state structures match closely. The configuration of
(−)-1 proved stable through prolonged (multiday) heating at
80 °C in solution and very much longer (greater than one year)
at room temperature in the solid state. The minor diastereomer
(+)-2 was isolated by chromatography of the mother liquor
C
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Table 2. Structure−Activity Relationships
MIC (μM)
c
human
PPB
(% fu)
Eco FP
IC50
(μM)
MSSA
+
d
f
solubility
(μM)
MRQR
Sau
Eco
a
b
c
e
f
compd
(−)-1
)-3
R
X
Y
Spn
8.8
50
12
3.1
Spy
6.2
25
MSSA
serum
Hin
Eco
tolC−
−CH3
−CH3
−CF3
−CF2H
−CH2OCH3
cyclopropyl
t-butyl
F
O
9.4
3
>1000
330
0.29
0.37
0.61
0.48
1.6
1.0
6.2
3.1
50
2.0
12
1.0
45
200
25
≤0.2
≤0.2
≤0.2
≤0.2
≤0.2
≤0.2
3.1
(
Cl
F
F
F
F
F
F
F
F
F
O
3.1
(−)-4
(−)-5
O
<1
2.8
9.6
5.2
4.2
<1
<1
9.0
13
53
3.1
1.2
6.2
6.2
0.78
0.39
3.1
100
3.1
1.6
0.39
6.2
12
1.6
O
>1000
180
0.62
25
g
(
(
(
(
)-6
)-7
)-8
)-9
O
12
25
ND
12
3.1
200
200
100
>200
50
O
29
0.34
4.0
1.6
ND
ND
50
O
770
200
100
12
50
200
6.2
18
18
−CH3
CH2
O
<1.0
>1000
510
0.34
0.21
0.97
0.26
>32
0.27
25
25
>200
25
6.2
(−)-10
(−)-11
(−)-12
linezolid
−Cl
0.39
3.1
12
0.78
3.1
50
25
6.2
−OCH3
−NH2
O
25
6.2
3.1
3.1
1.5
ND
ND
12
3.1
1.6
23
<0.1
200
100
>200
≤0.2
≤0.2
20
O
330
6.2
25
71
>1000
240
3.1
1.5
6.2
6.2
96
ciprofloxacin
74
0.91
ND
0.06
<0.1
a
b
c
d
e
f
Spn: S. pneumoniae. Spy: S. pyogenes. Methicillin-sensitive S. aureus. Methicillin-resistant, quinolone-resistant S. aureus. H. influenzae. E. coli.
g
Not determined.
a
Scheme 3
a
Reagents and conditions: (a) s-BuLi, THF, −78 °C; (b) PCC, DCM, 0 °C; (c) 2.5% HCl, EtOH; (d) MnO2, CH2Cl2, 3d; (e) i-PrOH, 80 °C, 16h;
(f) NH2OH.HCl, pyridine, 90 °C; (g) Cs2CO3, DMF, 100 °C.
from the crystallization, and the configuration was assigned by
NMR. There was slow conversion of the higher energy (+)-2 to
(−)-1 at room temperature in DMSO solution, so its
characterization was best carried out soon after isolation. In
the solid state, the configuration of (+)-2 was maintained at
room temperature for over a year. Finally, heating the inactive
diastereomer (+)-1 at 120 °C effected equilibration to a
thermodynamic 9:1 mixture of (+)-1 and (−)-2, which were
separated by chromatography. With three stereocenters, only
two of the four possible diastereomers for the chiral scaffold
could be isolated although all would be sampled under
thermodynamic equilibrating conditions (120 °C). Enan-
tiomers (−)-1 and (+)-1 represent the lowest energy
configuration, while enantiomers (−)-2 and (+)-2 are next
lowest in energy. The morpholine of (+)-2 primarily exists in a
chair conformation, and the C14 methyl group (using the
numbering in Figure 3) has an axial orientation as delineated by
a diagnostic 1,3-axial NOE to the C3 ether C−H (see
Supporting Information for NOESY and modeled conforma-
tion). A through-space NOE is seen between the axial C4 and
C1 hydrogen atoms, and the two C1 hydrogen atoms show
small 3.0 and 3.3 coupling constants to the C2 hydrogen atom
in line with the latter being equatorial. Finally, the C3 and C2
hydrogen atoms showed a coupling constant (6.8 Hz)
consistent with a trans-diaxial orientation. There is also minor
(25%) conformation for (+)-2 detected through an NOE
between the C15 methyl group and the C2 hydrogen atom
consistent with a ring flip to a boat conformation and
accounting for an averaging of coupling constants overall.
There are four diastereomers otherwise (two enantiomeric
pairs) that are likely sampled in the course of the T-reactions
depending on substrates and conditions. However, their
propensity to convert to the more stable diastereomers via
the retro-Mannich iminium intermediate (Figure 1) precluded
their isolation. Neither of these two diastereomers would be
able to orient the two morpholine methyl groups in lower
energy equatorial positions without significant distortions to
the overall molecular shape. Note that when the temperature
was not too high (80 °C or below), the racemate ( )-1 did not
equilibrate to ( )-2. Shorter reaction times (1 h) for the
conversion of 16a to ( )-1 afforded a third diastereomer (as
seen by NMR) that could not be isolated from a mixture with
( )-1 due to its facile conversion to ( )-1 during all attempts
at chromatographic separation. Compounds 3, 4, 5, and 6 of
Table 2 were constructed similarly via Scheme 1 or Scheme 2
with some modifications (for 5 and 6) in the assembly of the
benzisoxazole ring (see Experimental Section).
A variation from the sequence of Schemes 1 and 2 involved
reaction of the lithium anion derived from 14a with aldehydes
followed by oxidation to the carbonyl as exemplified in Scheme
3 for the synthesis of ( )-7. In this particular sequence, the
benzisoxazole was built after the assembly of the spiropyr-
D
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a
Scheme 4
a
Reagents and conditions: (a) TBDPS-Cl, imidazole; (b) s-BuLi, CH3C(O)N(OMe)Me, THF, −78 °C; (c) acetone−oxime, KO-t-Bu, THF, 0
°C; (d) 2.5% HCl, EtOH; (e) MnO2, CH2Cl2, 3 d; (f) DIEA, CH3CN, H2O, reflux; (g) i-PrOH, 80 °C.
a
Scheme 5
a
Reagents and conditions: (a) HCl, MeOH, reflux; (b) NH2OH, dioxane, rt; (c) CDI, THF, reflux; (d) POCl3, 140 °C; (e) LTMP, DMF, −78 °C
(f) DIEA, butyronitrile, H2O, reflux; (g) AcOH, H2O, 120 °C; (h) HOCH2CH2OH, p-TsOH, toluene, reflux; (i) NaOMe, MeOH, 100 °C; (j)
NaN3, DMSO, 100 °C; (k) Ph3P, AcOH, H2O, rt then barbituric acid, 120 °C.
imidinetrione, reflecting the flexibility of the reaction sequence
order for the construction of target molecules, albeit the lower
yield made the route less efficient. Compound ( )-8 was also
made in an analogous route to ( )-7. Scheme 4 exemplifies
another sequence variation wherein the benzisoxazole ring was
assembled before the introduction of an amine, enabling late
stage variation of the amine portion of compounds (other
analogues not included herein). The route complemented that
of Schemes 1, 2, and 3, which were more amenable toward
variation of benzisoxazole substituents. Protection of benzyl-
alcohol 20 was followed by fluorine directed ortho-lithiation and
Weinreb amide quenching to afford methylketone 21. The
fluorine atom adjacent to the acetyl group was selectively
displaced by the anion of acetone oxime, and subsequent acid
treatment led to alcohol 22. Oxidation of 22 to the aldehyde
followed by fluorine displacement with the dimethylpiperidine
afforded 23. The final T-reaction with barbituric acid was
performed as described in Scheme 1 for ( )-1. In this case, a
9:1 ratio of diastereomers for ( )-9 was obtained even at the
lower 80 °C temperature, with the major diastereomer being
isolated by fractional crystallization.
The synthesis of (−)-10 with a chlorobenzisoxazole
substituent and analogues derived from SNAr displacement of
the chlorine are outlined in Scheme 5. To this end, salicylic acid
24 was converted to 26 by procedures set out in the literature
for the synthesis of chlorobenzisoxazoles.33,34 An aldehyde was
introduced by sequential fluorine directed lithiation and
quenching with DMF, affording 27. Displacement of the
fluorine atom adjacent to the carboxaldehyde followed by the
T-reaction with barbituric acid afforded (−)-10 after separation
from the analogous minor diastereomer described in Scheme 2.
The aldehyde of 28 was protected as the ketal (29) for an SNAr
displacement of chlorine with methoxide and azide. The methyl
ether 30 was transformed to (−)-11 by in situ ketal hydrolysis
and T-reaction. Azide 31 was converted to (−)-12 by treatment
with Ph3P and barbituric acid, reducing the azide to the amine,
hydrolyzing the ketal, and carrying out the T-reaction in a
single reaction vessel.
Structure−Activity Relationships. There are eight
possible diastereomers with the three chiral centers around
the morpholine ring of ( )-1, and ultimately four of the
diastereomers (two enantiomer pairs) were isolated and
characterized biologically. As mentioned, the other diaster-
E
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eomers could not be isolated due to instability relative to
configurational isomerization. Compound inhibitory potency
against Escherichia coli DNA gyrase was determined in an assay
where differential binding of a fluorescence tagged DNA chain
with relaxed and supercoiled DNA was monitored.35 The
highest inhibitory potency resided in (−)-1, correlating with
higher antibacterial activity against the Gram-positive Staph-
ylococcus aureus, Streptococcus pneumoniae, and Streptococcus
pyogenes and the Gram-negative Haemophilus influenzae and E.
coli (Table 1). The wild-type E. coli MIC was considerably
higher compared to other bacteria, probably due to low
membrane permeability, as indicated by the much lower MIC
against the tolC− E. coli mutant in which the compound efflux
machinery is disrupted. The lower activity against E. coli and
other serious Gram-negative pathogens is therefore likely due
to the limited capability for compounds to enter and remain in
the bacteria. Because the net epimerization of the C14 methyl
group (Figure 3) in going from (−)-1 to (−)-2 led to a
reorientation from equatorial to axial positions and about a 25%
population of the boat conformation, it is presumed the
predominate conformations of the two compounds are quite
similar and accessible at room temperature. The diminished
activity for (−)-2 would presumably result from a disfavored
steric interaction with the target due to the axial methyl group.
The 25% boat conformer was also presumably not well
tolerated for target binding, perhaps in part due the
reorientation of the C15 methyl group to the axial position.
Also seen in Table 1, there was a measure of differential
recognition for binding to human plasma proteins in that free
fraction (fraction unbound or fu) for (−)-1 was 2−3-fold higher
than that for opposite enantiomer (+)-1. This trend for the
(−)-enantiomer, the active diastereomer, showing a higher fu
than the racemate, which in turn showed a higher fu than the
(+)-enantiomer, was seen consistently across many other SPT
analogues (data not shown). A higher fu would presumably
correlate with improved in vivo activity as free drug is presumed
necessary for expression of antibacterial activity. Conversely, a
lower fu can be beneficial for protection from clearance
mechanisms and lead to more prolonged drug exposure. Hence,
a balance must be achieved for antibacterial activity, PPB, and
in vivo clearance for advancing a drug candidate.36−38 Related
to this, the extent of PPB can influence the level of free drug
that would interact with secondary host targets including those
involved in drug−drug interactions, which is a concern for drug
safety in vivo.37,39 Finally, consideration of solubility is
important toward the viability of a drug candidate, especially
for antibacterial agents where relatively high doses are
administered.40
increased lipophilicity contributed to notably low solubility and
low fu, perhaps leading to the higher MIC values. Similarly,
replacement of the fluorine atom of (−)-1 with a chlorine
(compound ( )-3) maintained inhibitor potency, but the
antibacterial activity was lower. The bulky t-butyl substituent of
( )-8 was less well tolerated for binding potency, and therefore
MIC values were higher. Having a CF2H, Cl, or OCH3 in place
of CH3 on the benzisoxazole (compounds (−)-5, (−)-10, and
(−)-11) led to similar antibacterial activity across S. aureus, S.
pneumoniae, S. pyogenes, and H. influenzae. The more polar NH2
substituent of (−)-12 led to decreased activity, in particular
against S. aureus, despite high enzyme inhibitory potency, in
line with general observations that higher polarity decreases
permeability through lipophilic bacterial membranes.
In Vitro Microbiology. Analysis of incorporation of
radioactive precursors in a S. aureus supported the topoisomer-
ase mode-of-action for (−)-1 (Figure 4).38,42 The lowest IC50
Figure 4. Inhibition of radioactive precursor incorporation by (−)-1 in
S. aureus.
(0.06 μg/mL) was seen for thymidine incorporation indicating
DNA biosynthesis inhibition. The IC50 for incorporation of
uridine and N-acetylglucosamine, indicative of the disruption of
RNA and cell wall biosynthesis, was much higher at
approximately 5 and 8 μg/mL, respectively, and would be
expected to follow the disruption of DNA biosynthesis. This
data correlated well with similar studies carried out for PNU-
286607, where DNA biosynthesis inhibition was also
inhibited.11 This profile was similar to other antibacterial
drugs that inhibit type II topoisomerases.24,27,43,44 Despite a
similar inhibition profile, low MIC values were seen against a
methicillin resistant, quinolone resistant S. aureus isolate
(MRQR in Table 2), showing that the compounds were not
cross-resistant to the fluoroquinolone class. To expand on this,
the activity of (−)-1 was surveyed against other bacterial strains
resistant to DNA gyrase inhibitors including levofloxacin and
novobiocin (Table 3). The MIC of (−)-1 was not changed for
the isolates, indicating that the mode of DNA gyrase inhibition
of this class differs from the other two classes. Therefore, an
SPT compound should not be compromised by pre-existing
clinical resistance to the two drugs. Although the documenta-
tion of clinical strains of bacteria resistant to novobiocin is
sparse, numerous laboratory spontaneous resistant mutants
have been reported,27,45 and all those tested including those of
Table 3 were not cross-resistant to (−)-1. Hence, (−)-1 does
not share the mode-of-inhibition with novobiocin, namely that
of binding to the ATP binding site of the topoisomerases. More
broadly, there is no reduced susceptibility with (−)-1 to other
classes of antibacterials including linezolid, vancomycin, and
doripenem (data not included). Finally, resistance frequencies
for (−)-1 in S. aureus (MSSA) cultures at concentrations of
Table 2 shows the data for a series of modifications on the
structure of 1 wherein enzyme inhibition potencies are often
maintained but physical properties vary, influencing the
microbiological profile. The table also includes MIC data for
a methicillin sensitive S. aureus strain (MSSA) in the presence
of 50% human serum as a measure of the influence of plasma
protein on MIC; generally, more lipophilic compounds with a
lower fu show a larger shift to a higher MIC with serum.41 As a
dramatic example, replacement of the benzisoxazole CH3
substituent of (−)-1 with CF3 made for highly protein bound
compound (−)-4 that showed a low MIC (0.78 μM) against
MSSA under standard culture conditions but a notably higher
MIC (100 μM) when 50% human serum was added.
Replacement of the morpholine oxygen of (−)-1 with carbon
(compound ( )-9) maintained inhibitory potency but the
F
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Table 3. Cross Resistance
genotype
MIC (μg/mL)
strain
GyrA−GyrB mutations
ParC−ParE mutations
WT−WT
WT−wild type
S80Y, E84G, V693M−N141S
WT−WT
levofloxacin
novobiocin
(−)-1
a
Sau516
WT−WT
WT−R144A
S84L, A457T−K65Q
WT−WT
0.13
0.25
>16
1
0.13
32
0.5
0.5
0.50
4
Sau2792
Sau2375
4.3
b
Spn548
1
Spn2800
Spn2485
WT−K143I, T172A
S81F−D160E, T329A
WT−WT
S79F−WT
1
>16
0.13
4
16
2
a
b
Sau: S. aureus. Spn: S. pneumoniae.
Table 4. PK Properties in Mouse and Rat
compd
mouse PPB (% fu)
mouse Cl (mL/min/kg)
mouse F (%)
rat PPB (% fu)
16
7.8
4.3
rat Cl (mL/min/kg)
rat F (%)
(−)-1
(−)-4
(−)-5
19
38
46
47
58
63
95
32
71
60
87
7.8
4.4
2.3
1.0
Figure 5. CFU response as to AUC for (−)-1 in S. aureus (ARC516, MSSA) mouse thigh infection models: immunocompetent (left) and
neutropenic (right).
two, four, and eight times the MIC were determined to be 1.7
× 10−8, ≤1 × 10−9, and ≤1 × 10−9, respectively. The resistance
frequency was lower than those recorded for ciprofloxacin and
other fluoroquinolones against S. aureus.46 At two times the
MIC, an isolate was characterized and showed only a 2-fold
elevation in MIC and no changes in any of the four genes
(gyrA, gyrB, parC, or parE) encoding for type II top-
oisomerases. An inoculum of the isolate was plated in the
presence of four times the MIC of (−)-1 to afford two
spontaneous mutants at a frequency of 1.1 × 10−8, each 8-fold
elevated in MIC compared to wild-type. Genomic analysis of
the topoisomerase genes of the resultant second step mutants
showed changes in only gyrB Trp592Leu in one strain and
Ala439Ser in the other. These residues are located near the
DNA binding region of GyrB that abuts the DNA cleavage
domain of GyrA and did not confer cross-resistance to
ciprofloxacin. This data also strongly supports the novel
mode-of-inhibition of (−)-1 relative to ciprofloxacin.
In Vivo Characterization. The in vivo PK properties of
(−)-1, (−)-4, and (−)-5 were determined toward selecting a
compound for evaluation for efficacy in a S. aureus infection
study in the mouse and tolerability studies in the rat (Table 4).
A correlation was seen in the rat data in that the more highly
protein bound (−)-4 and (−)-5 were protected from clearance
relative to (−)-1 and, indeed, the free drug exposure on IV
dosing of (−)-4 and (−)-5 in the rat calculate to about 8-fold
higher than that of (−)-1. However, despite the lower fu for
(−)-4 and (−)-5 relative to (−)-1, clearances of the three
compounds were comparable in the mouse. This, combined
with the smaller MIC serum shift relative to MIC for (−)-1 and
the assumption that the shift with human serum translates to
what might be seen in the mouse, made it the better candidate
for profiling for in vivo efficacy. The compound otherwise
showed promising oral bioavailability of 58 and 71% in the
mouse and rat, respectively, supporting the capability for PO
administration.
A mouse S. aureus (MSSA) thigh infection model was carried
out in both immunocompetent and immunosuppressed mouse
models via IP administration of 10−400 mg/kg single doses of
(−)-1 (Figure 5). Compound (−)-1 was highly efficacious in a
dose dependent fashion for both mouse models, offering a
measure of in vivo validation of the scaffold series. An exposure
(AUC or area under the curve) of AUC = 20 μg·h/mL resulted
in no net change in CFU at the end of treatment when
compared to the bacterial load at the start of treatment. A
maximal response of 3-log relative to the untreated control was
achieved at 70 μg·h/mL in the immunocompetent model. In a
neutropenic (immunosuppressed) mouse model, a 3-log drop
in CFU was seen at about 120 μg·h/mL, although higher doses
to record a maximum response were not investigated.
Accounting for free drug concentrations relative to PPB, the
3-log decreases in CFU correspond to 13 μmol·h/L for the
immunocompetent mouse and 23 μg·h/L for the neutropenic
mouse.
An oral tolerability study was conducted in Wistar Hannover
rats with (−)-1 as a single daily dose and multiple doses over
14 days. In the acute single dose phase, 1000 mg/kg was not
tolerated, but doses up to 750 mg/kg were well tolerated.
Dosing for 14 days was carried out at 250, 500, and 750 mg/kg.
One animal at 750 mg/kg was prematurely sacrificed due to
G
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signs of toxicity (subdued behavior, ataxia), and no significant
histopathogical findings were seen in two of three rats dosed at
the 750 mg/kg and any of the 500 or 250 mg/kg dosed rats. In
the one rat that was sacrificed, the most significant pathologic
effects seen were marked apoptosis in the rapidly dividing tissue
such as the gastrointestinal and hemopoietic cells. At high
doses, fluoroquinolones are known to induce cell death via cell
cycle arrest and mitochondrial membrane breakdown, an effect
that may be related to the topoisomerase mode-of-inhibition of
the class.47 As seen with fluoroquinolones,48 (−)-1 inhibited
human topoisomerase IIα with an IC50 = 50 μM, 170-fold
higher than that for E. coli DNA gyrase. A NOEL of 500 mg/
kg/day for (−)-1 was established. Table 5 summarizes the
EXPERIMENTAL SECTION
■
General Considerations. All of the solvents and reagents used
were obtained commercially and used as such unless noted otherwise.
1H NMR spectra were recorded in CDCl3 or DMSO-d6 solutions at
300 K using a Bruker Ultrashield 300 MHz instrument or a Bruker
Ultrashield 400 MHz instrument. 13C NMR spectra were recorded in
DMSO-d6 solutions at 300 K and 126 MHz using a Bruker DRX-500
500 MHz instrument with a QNP cryoprobe or at 101 MHz using a
Bruker Ultrashield 400 MHz instrument or at 75.5 MHz using a
Bruker Ultrashield 300 MHz instrument. 19F NMR spectra were
recorded at 282 MHz in CDCl3 or DMSO-d6 solutions at 300 K using
a Bruker Ultrashield 300 MHz instrument. Chemical shifts are
reported as parts per million relative to TMS (0.00) for H and 13C
1
NMR and CFCl3 for 19F NMR. High-resolution mass spectra (HRMS)
were obtained using a hybrid quadrupole time-of-flight mass
spectrometer (microTOFq II, Bruker Daltonics) in ESI+ mode. Silica
gel chromatographies were performed on an ISCO Combiflash
Companion instruments using ISCO RediSep flash cartridges (particle
size: 35−70 μm) or Silicycle SiliaSep flash cartridges (particle size:
40−63 μm). Preparative reverse phase HPLC was carried out using
YMC Pack ODS-AQ (100 mm × 20 mm ID, S-5 μ particle size, 12 nm
pore size) on Agilent instruments. When not indicated, compound
intermediates and reagents were purchased from chemical supply
houses. All final compounds (compounds 1−12) were determined to
be greater than 95% pure via analysis by reversed phase UPLC-MS
(retention times, RT, in minutes) was used with a Waters Acquity
UPLC instrument with DAD and ELSD and a UPLC HSS T3, 2.1 mm
× 30 mm, 1.8 μm column and a gradient of 2−98% acetonitrile in
water with 0.1% formic acid over 2.0 min at 1 mL/min. Injection
volume was 1 μL and the column temperature was 30 °C. Detection
was based on electrospray ionization (ESI) in positive and negative
polarity using a Waters ZQ mass spectrometer (Milford, MA, USA),
diode-array UV detector from 210 to 400 nm, and evaporative light
scattering detector (Sedex 75, Sedere, Alfortville Cedex, France).
rel-2-((2R,6S)-2,6-Dimethyl-morpholin-4-yl)-3,4-difluoro-benzoic
Acid. LHMDS (1 M in THF, 156 mL, 156 mmol) was added to a
stirred solution of 2,3,4-trifluorobenzoic acid (25.0 g, 142 mmol) in
250 mL of THF at −78 °C under N2 and the solution stirred for 45
min. In a separate reaction flask, lithium bis(trimethylsilyl)amide (1 M
in THF, 156 mL, 156 mmol) was added to a solution of cis-2,6-
dimethylmorpholine (17.4 mL, 142 mmol) in 200 mL of THF and
stirred for 45 min at −78 °C. This latter solution was added to the first
solution, and stirring was continued for 1 h at −78 °C. Stirring was
continued for an additional 12 h with warming to room temperature.
Solvents were evaporated, and the residue was dissolved in EtOAc,
which was washed with 1 N HCl, water, and brine. The organic layer
was dried (Na2SO4) and concentrated to give the title compound as a
semisolid. Yield: 27.5 g, (72%). UPLC (ESI) m/z (M + H)+: 271.2 for
Table 5. Toxicokinetic Results (Day 14 of 14 Day Repeat
Dosing) for (−)-1
dose (mg/kg)
no. of animals
Cmax (μmol/L)
AUC (μmol·h/L)
250
500
750
4 (2 M, 2 F)
4 (2 M, 2 F)
4 (2 M, 2 F)
290 150
440 82
487 180
2300 970
4400 890
4600 1500
toxicokinetic exposures of the drug in blood sample at day 14
from four treated rats at the three doses. Increasing the dose
from 500 to 750 mg/kg did not proportionately increase drug
exposure, likely due to solubility and/or absorption limit. On
the basis of the NOEL, the Cmax and AUC equaled 440 μmol/L
and 4400 μmol·h/L, respectively, and taking fu into account,
fCmax and fAUC equaled 70 μmol/L and 700 μmol·h/L,
respectively. The fAUC represents a 50-fold and 30-fold
therapeutic index relative to the 3-log drop in CFU for the
mouse immunocompetent and neutropenic models, respec-
tively.
CONCLUSIONS
■
In summary, the therapeutic margin based on in vivo
correlations from rodent models bodes well for this novel
SPT chemotype and the continuation of optimization work
toward identifying a drug candidate. Efficient synthetic
procedures were devised to enable scale-up of large compound
quantities (ultimately over 500 g of (−)-1 were prepared for
the biological studies) as well as to enable variation of
substituents for SAR work. A novel mode-of-inhibition for
DNA gyrase was demonstrated by no loss of susceptibility to
bacteria resistant to fluoroquinolones and aminocoumarins, and
the generation of spontaneous resistant mutants to (−)-1 with
GyrB mutations map to the DNA binding site and do not
confer cross-resistance to fluoroquinolones. This is clearly
important as it mitigates issues with DNA gyrase based pre-
existing resistance. Activity against the Gram-positive bacteria S.
aureus (including methicillin and fluoroquinolone resistant
strains) and S. pyogenes bolstered the prospects for addressing
skin and skin structure infections as did the demonstration of in
vivo efficacy against S. aureus. Activity against S. pneumoniae and
H. influenzae brings in the additional possibility for the
treatment of respiratory tract infections such as community
acquired bacterial pneumonia. A next-generation SPT com-
pound would need improvements in the antibacterial activity
and spectrum relative to (−)-1, in particular improving the
activity against the Streptococci while maintaining favorable
physicochemical, PK, and tolerability properties.
1
C13H15F2NO3. H NMR (400 MHz, CDCl3) δ: 1.2 (s, 6H), 2.9 (d,
2H), 3.1 (d, 2H), 3.9 (m, 2H), 7.2 (s, 1H), 7.3 (t, 1H), 8.1 (m, 1H).
rel-[2-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-3,4-difluoro-phenyl]-
methanol. NaBH4 (12.6 g, 359 mmol) was added portionwise to a
stirred and ice-bath cooled solution of the previous compound in 250
mL of THF. Afterward, a solution of iodine (32.5 g, 139 mmol) in 250
mL of THF was added at a rate to maintain the internal temperature
10 °C. After the addition was complete, the mixture was brought to
room temperature and heated at reflux for 12 h. It was cooled to room
temperature and quenched with MeOH (250 mL). Solvents were
evaporated and the residue treated with 2 M NaOH (500 mL) for 2 h.
The mixture was extracted with EtOAc (3 × 150 mL), and the
combined organic phases were washed with water and brine before
being dried (Na2SO4) and concentrated to give the title compound as
a gummy solid. UPLC (ESI) m/z (M + H)+: 257.2 for C13H17F2NO2.
1H NMR (400 MHz, CDCl3) δ: 1.24 (s, 6H), 3.0 (d, 3H), 3.1 (d, 2H),
3.9 (m, 2H), 4.78 (s, 2H), 6.9 (d, 1H), 7.0 (t, 1H).
rel-(2R,6S)-4-[6-({[tert-Butyl(diphenyl)silyl]oxy}methyl)-2,3-di-
fluorophenyl]-2,6-dimethylmorpholine (14a). Imidazole (8.5 g, 126
mmol) followed by t-butyl chlorodiphenylsilane (30 mL, 115 mmol)
were added over a period of 15 min to an ice-cooled solution of the
H
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previous compound (27.0 g, 105 mmol) in CH2Cl2. The mixture was
brought to room temperature and stirred for 12 h. The reaction
mixture diluted with CH2Cl2 and washed successively with 1 N HCl (1
× 250 mL), water, and brine. The organic layer dried (Na2SO4),
filtered, and concentrated. The residue was purified over a silica gel
flash column using a gradient of EtOAc in petroleum ether to give the
title compound as a white solid. Yield: 45 g (94%). UPLC (ES) MH+:
495.6 for C29H35F2NO2Si. H NMR (400 MHz, CDCl3) δ 1.1 (s, 15
H), 2.6 (d, 2 H), 2.8 (m, 2H), 3.5 (t, 2H), 4.7 (s, 2H), 7.0 (q, 1H); 7.3
(t, 1H), 7.4 (m, 10H).
MeOH, 0.1% dimethylethylamine, 40 °C, 100 bar. The first eluting
compound (+)-1 (202 mg, 40%) was consistent with (2S,4R,4aR)-11-
fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-1H,1′H-spiro[isoxazolo[4,5-
g][1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione
(+)-1. UPLC RT = 0.90 min. (ES) MH+: 403.0 for C19H19FN4O5. 1H
NMR (300 MHz, DMSO-d6) δ 11.9 (s, 1H), 11.5 (s, 1H), 7.18 (s,
1H), 4.09 (d, J = 13.0 Hz, 1H), 3.94 (d, J = 8.9 Hz, 1H), 3.74−3.87
(m, 1H), 3.61−3.73 (m, 1H), 3.5−3.6 (m, 1H), 3.09 (t, J = 12.6 Hz,
1H), 2.96 (d, J = 14.0 Hz, 1H), 2.42 (s, 3H), 1.14 (d, J = 6.2 Hz, 3H),
0.89 (d, J = 6.4 Hz, 3H). 19F NMR (282 MHz, DMSO-d6) δ −156.1.
1
13C NMR (75 MHz, DMSO-d6) δ 170.9, 167.6, 154.6, 151.5 (d, JCF
=
rel-1-{5-({[tert-Butyl(diphenyl)silyl]oxy}methyl)-4-[(2R,6S)-2,6-di-
methylmorpholin-4-yl]-2,3-difluorophenyl}ethanone (15a). sec-Bu-
tyllithium (1.4 M in cyclohexane, 66.4 mL, 93 mmol) was added to a
stirred solution of 14a (15.0 g, 30.0 mmol) in THF (150 mL) at −78
°C under N2. After stirring for 1 h, N-methoxy-N-methyl-acetamide
(3.4 mL, 45 mmol) was added and, after stirring for 30 min at −78 °C,
the solution was allowed reach room temperature with stirring
continuing for 12 h. The reaction mixture treated with saturated
aqueous NH4Cl solution and the aqueous layer extracted with EtOAc
(2 × 100 mL). The organic phases were combined, dried (Na2SO4),
and concentrated. The residue was purified over a silica gel flash
column using a gradient of EtOAc in petroleum ether to give the title
compound as yellow solid. Yield: 13.2 g (82%). UPLC (ES) MH+:
12.1 Hz), 149.4, 134.5 (d, JCF = 2.7 Hz), 133.8 (d, JCF = 240 Hz),
122.6 (d, JCF = 1.6 Hz), 114.9 (d, JCF = 2.7 Hz), 114.3, 72.0, 71.7, 64.4,
56.4 (d, JCF = 9.3 Hz), 53.1, 38.7, 18.2, 18.1, 9.3. HRMS (ES) MH+
calcd for C19H20FN4O5 403.1412, found 403.1421; [α] = +296 (c =
0.1 in MeOH); >98% ee by SFC chiral analysis.
The second eluting compound (199 mg, 39%) was consistent with
(2R,4S,4aS)-11-fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-1H,1′H-
spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-
2′,4′,6′(3′H)-trione (−)-1. UPLC RT = 0.90 min. (ES) MH+: 403.0
1
for C19H19FN4O5. H NMR (300 MHz, DMSO-d6) δ 11.8 (s, 1H),
11.4 (s, 1H), 7.17 (s, 1H), 4.09 (d, J = 13.0 Hz, 1H), 3.94 (d, J = 8.9
Hz, 1H), 3.74−3.87 (m, 1H), 3.61−3.73 (m, 1H), 3.49−3.58 (m, 1H),
3.09 (t, J = 12.6 Hz, 1H), 2.96 (d, J = 14.0 Hz, 1H), 2.42 (s, 3H), 1.14
(d, J = 6.2 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H). 19F NMR (282 MHz,
DMSO-d6) δ −156.1. 13C NMR (101 MHz, DMSO-d6) δ 170.9, 167.6,
154.6, 151.5 (d, JCF = 11.7 Hz), 149.4, 134.5 (d, JCF = 2.2 Hz), 133.8
1
538.6 for C31H37F2NO3Si. H NMR (400 MHz, CDCl3) δ: 1.1 (s,
15H), 2.8 (m, 4H), 3.4 (m, 2H), 4.6 (s, 2H), 7.3 (t, 4H), 7.4 (t, 2H),
7.6 (d, 4H), 7.8 (s, 1H), 10.2 (s, 1H).
rel-1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6S)-2,6-dime-
thylmorpholino)-3-fluoro-2-(propan-2-ylideneaminooxy)phenyl)-
ethanone. Propan-2-one oxime (913 mg, 12.50 mmol) in 15 mL of
THF was treated with KOtBu (935 mg, 8.33 mmol) at room
temperature for 45 min. A solution of 15a (2.24 g, 4.17 mmol) in 10
mL of THF was added, and the mixture was stirred at rt for 3 h. After
quenching with satd aqueous NH4Cl, the mixture was extracted with
EtOAc. The EtOAc was washed with water and brine, dried (Na2SO4),
and concentrated to give the title compound (2.5 g, 100%). UPLC
(ES) MH+: 591.3 for C34H43FN2O4Si; used in the next step without
any further purification.
rel-1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6S)-2,6-dime-
thylmorpholino)-3-fluoro-2-(propan-2-ylideneaminooxy)phenyl)-
ethanone. A solution of the preceding compound (2.4 g, 4.06 mmol)
in 30 mL of EtOH was treated with 10 mL of 10% aq HCl at 75 °C for
2 h. After cooling to rt, the mixture was diluted with EtOAc and
washed wtih 10% aqueous NaHCO3. The organic layer was washed
with water, dried (Na2SO4), and concentrated. The residue was
purified by silica gel chromatography (35−55% EtOAc gradient in
hexanes) to afford 0.65 g (42%) of the title compound. UPLC (ES)
MH+: 295 for C15H19FN2O3. 1H NMR (300 MHz, CDCl3) δ 1.22 (d, J
= 6.4 Hz, 6H) 2.55 (s, 3H) 2.9−3.2 (m, 4H) 3.7−4.0 (m, 2H) 4.82 (s,
2H) 7.31 (s, 1H).
(d, JCF = 240 Hz), 122.6, 114.9, 114.3, 72.0, 71.7, 64.4, 56.4 (d, JCF
=
8.8 Hz), 53.1, 38.8, 18.2, 18.1, 9.3. HRMS (ES) MH+ calcd for
C19H20FN4O5 403.1412, found 403.1393; [α] = −291 (c = 0.1 in
MeOH), >98% ee by SFC chiral analysis.
(2S,4S,4aS)-11-Fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-
1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyri-
midine]-2′,4′,6′(3′H)-trione (−)-2. A mixture of (+)-1 (320 mg, 0.80
mmol) in 1.2 mL of EtOH and 0.8 mL of water was heated at 150 °C
in a microwave reactor for 5 min. After cooling to rt and then to 4 °C
overnight, precipitated white solids were collected, rinsed with cold
60% EtOH, and dried in vacuo at 80 °C to afford 175 mg of material
identical to starting (+)-1. The mother liquor was concentrated and
purified by reverse phase HPLC (Waters Xbridge C18 column, 45−
60% gradient of CH3CN/10 mM aqueous NH4OAc) to afford two
materials. The first eluting material afforded additional (+)-1 and the
second eluting material afforded 14 mg (11%) of the title compound
as a white solid. UPLC RT = 0.87 min. MH+: 403.0 for C19H19FN4O5.
1H NMR (300 MHz, DMSO-d6) δ 10.80 (br s, 2 H), 7.23 (s, 1 H),
3.93−4.19 (m, 2H), 3.75 (d, J = 13.2 Hz, 1 H), 3.67 (d, J = 6.8 Hz, 1
H), 3.42−3.62 (m, 2 H), 3.16 (d, J = 15.3 Hz, 1 H), 2.44 (s, 3 H), 1.27
(d, J = 6.0 Hz, 3 H), 1.00 (d, J = 6.2 Hz, 3 H). 19F NMR (282 MHz,
DMSO-d6) δ −152.5. 13C NMR (101 MHz, DMSO-d6) δ 171.2, 169.2,
154.7, 150.7 (d, JCF = 12.4 Hz), 149.8, 135.4 (d, JCF = 2.2 Hz), 134.6
(d, JCF = 242.2 Hz), 122.7, 115.2 (d, JCF = 5.1 Hz), 66.2, 65.3, 64.8,
52.9 (d, JCF = 9.5 Hz), 50.9, 37.4, 18.7, 16.9, 9.3. HRMS (ES) MH+
calcd for C19H20FN4O5 403.1412, found 403.1411; [α] = −164 (c =
0.1 in MeOH).
2-((2R,6R)-2,6-Dimethylmorpholino)-3,4-difluorobenzoic Acid.
LHMDS (1M, 622 mL, 0.622 mol) in THF was added dropwise to
a solution of 2,3,4-trifluorobenzoic acid (100 g, 0.56 mol) in
anhydrous THF (500 mL) cooled to −78 °C, maintaining the
temperature below −65 °C, and the mixture was stirred at −78 °C for
45 min. In a separate flask, LHMDS (1M, 622 mL, 0.622 mol) in THF
was added dropwise to a solution of (2R,6R)-2,6-dimethylmorpholine
(67.0 g, 0.56 mol) in THF (210 mL) cooled to −78 °C, maintaining
the temperature below −65 °C, and the mixture was stirred at −78 °C
for 45 min. The contents of the second flask were then transferred via
cannula to the first flask over 30 min, stirring was continued for 1 h at
−78 °C, and the mixture was warmed to rt with stirring for 18 h. The
solvent was removed, and the residue was poured into 1 N HCl and
extracted with EtOAc (3 × 750 mL). The combined organic extracts
were washed with water (1 L) and brine (1 L), dried (Na2SO4), and
concentrated to give the title compound (131.4 g, 85%) as a solid.
rel-6-((2R,6S)-2,6-Dimethylmorpholino)-7-fluoro-3-methylbenzo-
[d]isoxazole-5-carbaldehyde (16a). A mixture of the preceding
compound (0.65 g, 2.21 mmol) and MnO2 (3.84 g, 44.2 mmol) in 30
mL of CH2Cl2 was stirred at rt for 3 d. The MnO2 was filtered and
rinsed through with CH2Cl2. The filtrate was concentrated and dried
in vacuo to give the title compound (0.547 g, 85%) that was used in
the next step without further purification. UPLC (ES) MH+: 293 for
1
C15H17FN2O3. H NMR (300 MHz, CDCl3) δ 1.22 (d, J = 6.2 Hz,
6H) 2.58 (s, 3H) 2.9−3.25 (m, 4H) 3.7−4.0 (m, 2H) 7.94 (s, 1H)
10.38 (s, 1H).
rel-(2R,4S,4aS)-11-Fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-
1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyri-
midine]-2′,4′,6′(3′H)-trione ( )-1. A solution of 16a (543 mg, 1.86
mmol) and barbituric acid (238 mg, 1.86 mmol) in 50 mL of i-PrOH
was heated at 80 °C for 16 h. Solvent was removed, and the residue
was chromatographed on silica gel (EtOAc eluent) to afford 528 mg
(71%) of the title compound as a white solid. UPLC RT = 0.90 min.
(ES) MH+: 403.0 for C19H19FN4O5. 1H NMR (400 MHz, DMSO-d6)
δ 11.84 (s, 1H), 11.44 (s, 1H), 7.17 (s, 1H), 2.90−4.2 (m, 7H), 2.41
(s, 3H), 1.13 (d, J = 6.1 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H).
The enantiomers of 528 mg of ( )-1 were separated by SFC on a
Chiralpak AD column (21 mm × 250 mm, 5 μ), 80% CO2, 20%
I
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1
UPLC (ES) MH+: 272 for C13H15F2NO3. 1H NMR (300 MHz,
DMSO-d6) δ 1.17 (d, J = 6.4 Hz, 6 H) 2.84 (dd, J = 11.5, 5.5 Hz, 2 H)
3.20 (d, J = 11.3 Hz, 2 H) 3.86−4.19 (m, 2 H) 7.14−7.40 (m, 1 H)
7.44−7.61 (m, 1 H) 14.14 (s, 1 H).
C15H19FN2O3. H NMR (300 MHz, DMSO-d6) δ 7.65 (s, 1H), 5.36
(t, J = 5.3 Hz, 1H), 4.71 (t, J = 4.5 Hz, 2H), 4.04 (dd, J = 5.8, 3.2 Hz,
2H), 3.18 (d, J = 0.75 Hz, 1H), 3.14 (br s, 1H), 2.80 (dd, J = 11.3, 5.65
Hz, 2H), 2.55 (m, 4H), 1.23 (d, J = 5.8 Hz, 6H). 19F NMR (282 MHz,
CDCl3) δ −145.1.
(2-((2R,6R)-2,6-Dimethylmorpholino)-3,4-difluorophenyl)-
methanol. NaBH4 (73.1 g, 1.93 mol) was added portionwise to a
solution of the previous compound (131.0 g, 0.483 mol) in 400 mL of
THF at 0 °C. A solution of iodine (183 g, 0.724 mol) in 1 L of THF
was added dropwise, keeping the temperature below 10 °C. The
reaction mixture was then heated at reflux for 18 h. After cooling to rt,
the reaction was quenched with MeOH (370 mL), the solvent was
removed, and the residue was suspended in 2N NaOH (1.5 L) with
stirring at rt for 1.5 h. The mixture was extracted with EtOAc (3 × 1
L). The combined organic extracts were washed with brine (1.5 L),
dried over anhydrous Na2SO4, filtered, and evaporated to yield the title
compound (107 g, 86%) as colorless liquid. UPLC (ES) MH+: 258 for
6-((2R,6R)-2,6-Dimethylmorpholino)-7-fluoro-3-methylbenzo[d]-
isoxazole-5-carbaldehyde (16b). A mixture of the preceding
compound (13.0 g, 44.2 mmol) and MnO2 (26.9 g, 309 mmol) in
400 mL of CH2Cl2 was stirred for 3 days at rt. The reaction was
incomplete by TLC, additional MnO2 (11.5 g) was added, and the
mixture was stirred at rt overnight. The reaction mixture was filtered
through Celite, rinsing through with CH2Cl2 and with 15% i-PrOH in
dichloromethane. The combined filtrate was concentrated to afford the
title compound as a yellow oil (11g, 85%). 1H NMR (300 MHz,
CDCl3) δ 10.51 (s, 1H), 7.96 (s, 1H), 5.31 (s, 1H), 4.23 (dt, J = 3.2,
6.1 Hz, 2H), 3.41 (d, J = 11.68 Hz, 2H), 3.04 (dd, J = 5.65, 11.7 Hz,
2H), 1.34 (d, J = 6.4 Hz, 6H).
1
C13H17F2NO2. H NMR (300 MHz, CDCl3) δ 1.32 (d, J = 5.8 Hz, 6
H) 2.73−3.41 (m, 4 H) 4.00−4.29 (m, 2 H) 4.60−4.89 (m, 2 H)
(2R,4S,4aS)-11-Fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-
1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyri-
midine]-2′,4′,6′(3′H)-trione (−)-1. A mixture of 16b (655 mg, 2.24
mmol) and barbituric acid (287 mg, 2.24 mmol) in 3 mL of EtOH and
1 mL of water was heated at 120 °C for 1 h in a microwave reactor.
UPLC analysis of the reaction mixture showed and 9:1 mixture of
materials. The resultant solution was chilled in a freezer at −10 °C
overnight, during which solids precipitated. The solids were filtered,
rinsed with cold 3:1 EtOH−water, and dried under vacuum at 50 °C
overnight to afford 647 mg (72%) of the title compound. The filtrate
was concentrated, and the residue was purified by SFC on an (S,S)
Whelk-O1 column (21 mm × 250 mm, 5 μ), 85% CO2, 15% MeOH,
40 °C, 150 bar to separate two diastereomers. The analytical data for
the major diastereomer (74 mg, 8%) and the precipitated solids was
consistent with (−)-1 isolated via the racemic route. UPLC RT = 0.91
min. (ES) MH+: 403.0 for C19H19FN4O5. 19F NMR (282 MHz,
DMSO-d6) δ −156.1. 13C NMR (75 MHz, DMSO-d6) δ 170.9, 167.6,
154.6, 151.5 (d, JCF = 12.1 Hz), 149.4, 134.5 (d, JCF = 2.2 Hz), 133.8
(d, JCF = 239.9 Hz), 122.6 (d, JCF = 1.1 Hz), 122.4, 114.9 (d, JCF = 3.3
Hz), 114.3, 72.0, 71.7, 64.4, 56.4 (d, JCF = 9.3 Hz), 53.1, 38.7, 18.2,
18.1, 9.3. HRMS (ES) MH+ calcd for C19H20FN4O5 403.1412, found
403.1422; [α] = −297 (c = 0.1 in MeOH); >98% ee by SFC chiral
analysis. The minor diastereomer (51 mg, 6%) was consistent with
(2R,4R,4aR)-11-fluoro-2,4,8-trimethyl-2,4,4a,6-tetrahydro-1H,1′H-
spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyrimidine]-
2′,4′,6′(3′H)-trione (+)-2. UPLC RT = 0.88 min. (ES) MH+: 403.0
for C19H19FN4O5. 1H NMR (400 MHz, DMSO-d6) δ 11.4 (br s, 2H),
7.23 (s, 1H), 4.05−4.15 (m, 1H), 4.00 (quin, J = 6.4 Hz, 1H), 3.74
(dd, J = 3.9, 13.2 Hz, 1H), 3.66 (d, J = 6.8 Hz, 1H), 3.45−3.6 (m, 2H),
3.17 (d, J = 14.8 Hz, 1H), 2.44 (s, 3H), 1.26 (d, J = 5.5 Hz, 3H), 1.01
(d, J = 6.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 171.4, 169.3,
154.7, 150.7 (d, JCF = 11.7 Hz), 150.0, 135.4 (d, JCF = 2.2 Hz), 134.5
(d, JCF = 242.2 Hz), 122.7, 115.2, 115.1 (d, JCF = 3.7 Hz), 66.2, 65.3,
64.8, 52.9 (d, JCF = 9.5 Hz), 50.8, 37.5, 18.7, 16.9, 9.3. 19F NMR (282
MHz, DMSO-d6) δ −152.3. HRMS (ES) MH+ calcd for C19H20FN4O5
403.1412, found 403.1411; [α] = +148 (c = 0.1 in MeOH).
6.78−7.19 (m, 2 H).
(2R,6R)-4-(6-((tert-Butyldiphenylsilyloxy)methyl)-2,3-difluoro-
phenyl)-2,6-dimethylmorpholine (14b). A solution of tert-butylchlor-
odiphenylsilane (125.3 g, 0.457 mol) in 100 mL of CH2Cl2 was added
dropwise to a solution of the preceding compound (107 g, 0.416 mol)
and imidazole (33.95 g, 0.499 mol) in 1 L of CH2Cl2 at 0 °C before
stirring at rt for 16 h. The reaction mixture was poured into 1 N HCl
(1 L), the layers were separated, and the organic layer was washed with
saturated aqueous NaHCO3 (500 mL), water (500 mL), and brine
(500 mL) before being dried (Na2SO4). Removal of solvent was
followed by column chromatography on silica gel (3:97 EtOAc:
petroleum ether) to afford the title compound (158 g, 83%) as a
yellow oil. UPLC (ES) MH+: 496 for C29H35F2NO2Si. 1H NMR (300
MHz, CDCl3) δ 7.57−7.76 (m, 4H), 7.3−7.5 (m, 7H), 6.9−7.1 (m,
1H), 4.6−4.9 (m, 2H), 3.7−4.0 (m, 2H), 2.8−3.15 (m, 2H), 2.44−
2.77 (m, 2H), 1.09 (s, 9H), 1.04 (d, J = 6.4 Hz, 6H).
1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6R)-2,6-dimethyl-
morpholino)-2,3-difluorophenyl)ethanone (15b). Under N2, sec-
butyllithium (1.3 M in cyclohexane/hexane, 69.8 mL, 90.89 mmol)
in 20 mL of MBTE was added via cannula to a solution of 14b (18 g,
36.31 mmol) in 80 mL of MTBE cooled to −75 °C at a rate to keep
the temperature below −70 °C. The resultant slurry was stirred for 1 h
before N-methoxy-N-methylacetamide (9.74 g, 94.4 mmol) was added
dropwise, keeping the internal reaction temperature above −60 °C.
After stirring cold for 1 h, the reaction slurry was quenched with 200
mL of saturated aqueous NH4Cl. The mixture was warmed to rt and
extracted with EtOAc. The EtOAc was washed with brine, dried
(Na2SO4), and concentrated to give a an oil that was chromatographed
on silica gel (5% EtOAc in hexanes) to afford the title compound as an
oil (17.4 g, 89%). 1H NMR (300 MHz, CDCl3) δ 7.85 (d, J = 7.7 Hz,
1H), 7.53−7.64 (m, 4H), 7.26−7.39 (m, 6H), 4.7 (d, J = 13.9 Hz,
1H), 4.62 (d, J = 13.9 Hz, 1H), 3.74−3.9 (m, 2H), 2.98 (d, J = 11.3
Hz, 2H), 2.61 (dd, J = 5.4, 11.4 Hz, 2H), 2.55 and 2.53 (2s, 3H), 1.03
(s, 9H), 0.98 (d, J = 6.4 Hz, 6H).
(6-((2R,6R)-2,6-Dimethylmorpholino)-7-fluoro-3-methylbenzo[d]-
isoxazol-5-yl)methanol. Potassium t-butoxide (1.0 M in THF, 61.3
mL, 61.3 mmol) was added under N2 to a solution of acetone oxime
(4.48 g, 61.3 mmol) in 40 mL of THF at 0 °C. The resulting slurry
was warmed to rt and stirred for 30 min. After cooling to 0 °C, a
solution of the 15b (23.6 g, 43.8 mmol) in 10 mL of THF was added,
and the resulting yellow slurry was warmed to rt with stirring over 3.5
h. The reaction mixture was quenched with aqueous NH4Cl and
extracted with EtOAc (2×). The EtOAc was washed with brine, dried
(Na2SO4), and concentrated to give dark-orange oil. The oil was
dissolved 180 mL of EtOH, and 65.7 mL of 1 M HCl was added. The
mixture was heated at 70 °C for 3 h, and after cooling to rt, EtOH
solvent was removed. The aqueous residue neutralized with saturated
aqueous NaHCO3 and extracted twice with EtOAc. The combined
organic extract were washed with brine, dried (Na2SO4), and
concentrated to give an oil that was chromatographed on silica gel
(5−25% gradient of EtOAc in hexanes) to afford the title compound
as yellow solid (7.7 g, 60%). UPLC (ES) MH+: 295.03 for
rel-3-Chloro-2-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-4-fluoroben-
zoic Acid. The title compound was prepared as described for 2-
((2R,6S)-2,6-dimethyl-morpholin-4-yl)-3,4-difluoro-benzoic acid using
4.4 g (22.8 mmol) of 3-chloro-2,4-difluorobenzoic acid and 2.8 mL
(22.8 mmol) of cis-2,6-dimethylmorpholine and 2 × 25 mL (25.1
mmol) LHMDS to afford 5.2 g (80%) of product. MS (ES) MH+:
1
288.0, 289.8 for C13H15ClFNO3. H NMR (300 MHz, DMSO-d6) δ
13.7 (br s, 1H), 7.60 (t, J = 7.54 Hz, 1H), 7.23 (t, J = 8.57 Hz, 1H),
3.60−3.93 (m, 2H), 2.74−3.03 (m, 4H), 1.08 (d, J = 6.22 Hz, 6H). 19F
NMR (282 MHz, DMSO-d6) δ −107.7.
rel-Methyl 3-Chloro-2-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-4-
fluorobenzoate. A solution of the preceding compound (10.0 g,
34.8 mmol) and 1 mL of conc H2SO4 in 50 mL of methanol was
heated at reflux for 12 h. The reaction mixture was concentrated and
diluted with EtOAc. The organic layer was washed with H2O (2 × 20
mL), dried (Na2SO4), and concentrated. The residue purified by silica
gel chromatography (EtOAc−petroleum ether gradient) to obtain the
J
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Journal of Medicinal Chemistry
Article
title compound as a white solid. Yield: 9.0 g (86%). MS (ES) MH+:
301.9, 302.8 for C14H17ClFNO3. 1H NMR (300 MHz, CD2Cl2) δ 7.36
(dd, J = 6.2, 8.7 Hz, 1H), 6.85 (t, J = 8.4 Hz, 1H), 3.81 (s, 3H), 3.73
(ddd, J = 2.5, 6.4, 9.3 Hz, 2H), 2.81−2.96 (m, 2H), 2.62−2.80 (m,
2H), 1.04 (d, J = 6.2 Hz, 6H). 19F NMR (282 MHz, DMSO-d6) δ
−107.8.
rel-Methyl 5-Acetyl-3-chloro-2-[(2R,6S)-2,6-dimethylmorpholin-
4-yl]-4-fluorobenzoate. A solution of the preceding compound (1.5
g, 4.96 mmol) in 10 mL of anhydrous THF was added dropwise to a
stirred solution of LDA (3.1 equiv) at −50 °C in THF, and the
solution was stirred for 1 h at −50 °C. N-Methoxy-N-methylacetamide
(1.66 g, 14.9 mmol) in THF (5 mL) was added dropwise, and stirring
was continued for 1 h. The reaction mixture was quenched with
saturated aqueous NH4Cl and extracted with EtOAc. The organic layer
was washed with brine and dried (Na2SO4). Solvent was removed, and
the residue was purified by silica gel chromatography (EtOAc−
petroleum ether gradient) to obtain 800 mg (47%) of the title
compound. MS (ES) MH+: 344 for C16H19ClFNO4.
rel-Methyl-3-chloro-2-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-4-flu-
oro-5-[N-hydroxyethanimidoyl]benzoate. Hydroxylamine hydro-
chloride (223 mg, 3.48 mmol) was added to a solution of the
preceding compound (800 mg, 2.32 mmol) in 6 mL of
methanol:pyridine (1:1), and the mixture was stirred at rt for 12 h.
Solvents were removed under vacuum, and the residue was
chromatographed on silica gel (EtOAc−petroleum ether gradient) to
give 700 mg (87%) of the title compound. MS (ES) MH+: 359 for
C16H20ClFN2O4.
rel-7-Chloro-6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-3-methyl-
1,2-benzoxazole-5-carboxylic Acid. A solution of the preceding
compound (700 mg, 1.94 mmol) and Cs2CO3 (1.9 g, 5.84 mmol) in
DMF (4 mL) was heated at 130 °C for 14 h. The reaction mixture was
cooled to rt and filtered through Celite rinsing through with EtOAc.
The solvent was removed under vacuum, and the residue was purified
by silica gel chromatography (EtOAc−petroleum ether gradient) to
afford the title compound. Yield: 250 mg, (41%). MS (ES) MH+: 335
for C15H17ClN2O4.
rel-[7-Chloro-6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-3-methyl-
1,2-benzoxazol-5-yl]methanol. BF3 (1.6 mL, 2.59 mmol) was added
to a stirred solution of NaBH4 (78 mg, 2.43 mmol) in 1 mL of
diglyme, and the generated diborane gas was purged into solution of
the preceding compound (250 mg, 0.8 mmol) in THF (1 mL). The
mixture was stirred at rt for 30 min before being quenched with
methanol (1 mL) and concentrated. The residue was purified by silica
gel chromatography (ethyl acetate−petroleum ether gradient) to give
the title compound as a solid. Yield: 130 mg (52%). MS (ES) MH+:
311 for C15H19ClN2O3.
rel-7-Chloro-6-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-3-methyl-
1,2-benzoxazole-5-carbaldehyde. NMO (103 mg, 8.8 mmol) and
TPAP (31 mg, 0.88 mmol) were added to a solution of the preceding
compound (130 mg, 4.4 mmol) in 5 mL of anhydrous CH2Cl2 was
added at 0 °C, and the mixture was warmed to rt with stirring for 1 h.
The reaction mixture was filtered, the solvents were removed under
vacuum, and the residue was purified by silica gel chromatography
(EtOAc−petroleum ether gradient) to give the title compound as a
yellow solid. Yield: 35 mg, (27%). MS (ES) MH+: 309 for
C15H17ClN2O3.
18.1, 9.5. HRMS (ES) MH+ calcd for C19H20ClN4O5 419.1117, found
419.1130.
rel-1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6S)-2,6-dime-
thylmorpholino)-2,3-difluorophenyl)-2,2,2-trifluoroethanone. sec-
Butyllithium (1.3 M in hexane, 11.5 mL, 15.0 mmol) was added to
a stirred solution of 14a (2.478 g, 5 mmol) in 10 mL of THF at −78
°C under N2. After stirring for 1 h at −78 °C, 2,2,2-trifluoro-N-
methoxy-N-methylacetamide (3.14 g, 20.0 mmol) was added. After
stirring for 30 min at −78 °C, the solution was warmed to rt. The
reaction was quenched with satd aqueous NH4Cl and extracted with
EtOAc. The EtOAc was dried (MgSO4) and concentrated. The residue
was chromatographed on silica gel (30−70% CH2Cl2 in hexanes
gradient) to afford the title compound. MS (ES) MH+: 610 for
C31H34F5NO3Si·H2O (hydrate form). 1H NMR (300 MHz, CDCl3) δ
7.64−7.70 (m, 4H), 7.4−7.5 (m, 7H), 4.65 (s, 2H), 3.4−3.6 (m, 2H),
2.8−2.9 (m, 4H), 1.12 (s, 9H), 1.10 (s, 6H). 19F NMR (282 MHz,
CDCl3) δ −73.7, −134.0, −146.1.
1-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-dime-
thylmorpholin-4-yl]-2,3-difluoro-phenyl]-2,2,2-trifluoro-ethanone
Oxime. A mixture of the preceding compound (1.9 g, 3.2 mmol),
pyridine (2.6 mL, 32 mmol), and hydroxylamine hydrochloride (223
mg, 3.21 mmol) in 50 mL of ethanol was heated at 80 °C for 40 h. The
mixture was concentrated, diluted with water, and extracted with
EtOAc. The organic phases were dried (Na2SO4) and concentrated to
give the title compound (1.95 g, 98%), which was used in the next step
without further purification. MS (ES) MH+: 607 for C31H35F5N2O3Si.
1H NMR (300 MHz, CDCl3) δ 1.03−1.15 (m, 15H), 2.56−2.91 (m,
4H), 3.33−3.59 (m, 2H), 4.58−4.80 (m, 2H), 7.27−7.51 (m, 7H),
7.57−7.74 (m, 4H).
rel-6-((2R,6S)-2,6-Dimethylmorpholino)-7-fluoro-3-
(trifluoromethyl)benzo[d]isoxazol-5-yl)methanol. A mixture of the
preceding compound (1.9 g, 3.13 mmol) and CsCO3 (5.10 g, 15.66
mmol) in 20 mL of DMF was heated at 60 °C for 4 h. Solids were
filtered off and rinsed through with EtOAc. The filtrate was diluted
with water, and the organic layer was separated, washed with water,
dried (MgSO4), and concentrated. The residue was purified by
chromatography on silica gel (20−35% EtOAc in hexanes) to afford
the title compound (0.460 g, 42.2%). MS (ES) MH+: 349 for
1
C15H16F4N2O3. H NMR (300 MHz, CDCl3) δ 7.58 (s, 1H), 4.86 (s,
2H), 3.73−3.96 (m, 3H), 3.04−3.14 (m, 2H), 2.92−2.99 (m, 2H),
1.26 (d, J = 6.2 Hz, 6H). 19F NMR (282 MHz, CDCl3) δ −62.3,
−140.8.
rel-6-((2R,6S)-2,6-Dimethylmorpholino)-7-fluoro-3-
(trifluoromethyl)benzo[d]isoxazole-5-carbaldehyde. A mixture of
the preceding compound (460 mg, 1.32 mmol) and MnO2 (4.59 g,
52.8 mmol) in 50 mL of CH2Cl2 was stirred at rt for 3 d. The MnO2
was filtered off and rinsed through with CH2Cl2. The filtrate was
concentrated and chromatographed on silica gel (20% EtOAc in
hexanes) to give the title compound (260 mg, 56.9%). MS (ES) MH+:
1
347 for C15H14F4N2O3. H NMR (300 MHz, CDCl3) δ 10.3 (s, 1H),
8.06 (s, 1H), 3.7−4.0 (m, 2H), 3.0−3.3 (m, 4H), 1.23 (d, J = 6.2 Hz,
6H).
(2R,4S,4aS)-11-Fluoro-2,4-dimethyl-8-(trifluoromethyl)-2,4,4a,6-
tetrahydro-1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]-
quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione ( )-4. A mixture of
the preceding compound (255 mg, 0.74 mmol) and barbituric acid
(104 mg, 0.81 mmol) in 10 mL of i-PrOH was heated at reflux for 3
days. Solvent was removed, and the solid residue was triturated with
MeOH. The solids were collected by filtration to afford give 220 mg of
the title compound. The mother liquor concentrated and purified on
silica gel (35−50% EtOAc gradient in CH2Cl2) to get afford additional
(122 mg) material. Total yield: 322 mg, 96%. MS (ES) MH+: 457 for
rel-(2R,4S,4aS)-11-Chloro-2,4,8-trimethyl-1,2,4,4a-tetrahydro-
2′H,6H-spiro[1,4-oxazino[4,3-a][1,2]oxazolo[4,5-g]quinoline-5,5′-
pyrimidine]-2′,4′,6′(1′H,3′H)-trione ( )-3. A solution of the preced-
ing compound (35 mg, 0.12 mmol) and barbituric acid (16 mg, 0.113
mmol) in i-PrOH was heated at reflux for 16 h. Solvents were
evaporated, and the residue was chromatographed on silica gel
(gradient of MeOH in CHCl3) to give the title compound as a solid.
Yield: 10 mg (25%). UPLC RT = 0.93 min. (ES) MH+: 419.3, 421.3
1
C19H16F4N4O5. H NMR (300 MHz, DMSO-d6) δ 11.9 (s, 1H), 11.6
(s, 1H), 7.35 (s, 1H), 3.54−4.28 (m, 5H), 2.86−3.24 (m, 2H), 1.14 (d,
J = 6.0 Hz, 3H), 0.89 (d, J = 6.4 Hz, 3H)
1
for C19H19ClN4O5. H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H),
The (2S,4R,4aR) and (2R,4S,4aS) enantiomers were separated by
SFC using a Chiralpak AD, 21 mm × 250 mm, 5 μ column (elution
with 25% MeOH, 75% CO2 at 60 mL/min, 40 °C, and 100 bar with
detection at 220 nm), providing (+)-4 and (−)-4:
(2S,4R,4aR)-11-Fluoro-2,4-dimethyl-8-(trifluoromethyl)-2,4,4a,6-
tetrahydro-1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]-
11.45 (s, 1H), 7.30 (s, 1H), 4.49 (d, J = 12.80 Hz, 1H), 3.9−4.05 (m,
2H), 3.6−3.7 (m, 1H), 3.54 (d, J = 14.3 Hz, 1H), 3.0−3.10 (m, 2H),
2.42 (s, 3H), 1.17 (d, J = 6.02 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H). 13C
NMR (151 MHz, DMSO-d6) δ 170.7, 167.6, 160.8, 155.2, 149.5,
144.1, 122.4, 118.0, 113.0, 97.7, 72.6, 72.5, 65.8, 56.4, 52.8, 40.1, 18.2,
K
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Journal of Medicinal Chemistry
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quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione (+)-4. First eluting
compound, 129 mg (38%). UPLC RT = 1.12 min. (ES) MH+:
457.0 for C19H16F4N4O5. 1H NMR (300 MHz, DMSO-d6) δ 0.89 (d, J
= 5.5 Hz, 3H), 1.14 (d, J = 5.1 Hz, 3H), 2.83−3.21 (m, 2H), 3.57−
4.23 (m, 5H), 7.34 (s, 1H), 11.72 (s, 2H). 19F NMR (282 MHz,
DMSO-d6) δ −61.6, −155.9; >98% ee by chiral HPLC; [α] = +285 (c
= 0.1 in MeOH).
(2R,4S,4aS)-8-(Difluoromethyl)-11-fluoro-2,4-dimethyl-2,4,4a,6-
tetrahydro-1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]-
quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione (−)-5. A solution of
the previous compound (2.42 g, 7.36 mmol) and barbituric acid (0.94
g, 7.36 mmol) in 100 mL of ethanol was heated at 90 °C for 4 days.
Solvent was removed and the residue was purified by reverse phase
HPLC (CH3CN/water gradient) to isolate the title compound as a
solid (4.66 g, 63%). UPLC RT = 1.02 min. MS (ES) MH+: 439.1 for
C19H17F3N4O5. 1H NMR (300 MHz, CDCl3) δ 1H NMR (300 MHz,
DMSO-d6) δ 11.8 (br s, 1H), 11.5 (br s, 1H), 7.3−7.7 (t, J = 36 Hz,
1H), 7.24 (s, 1H), 4.05 (d, J = 13.6 Hz, 1H), 3.9 (d, J = 8.85 Hz, 1H),
3.55−3.8 (m, 3H), 3.0−3.14 (m, 1H), 2.87 (d, J = 14.3 Hz, 1H), 1.08
(d, J = 6.0 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H). 19F NMR (282 MHz,
DMSO-d6) δ −117.1, −155.9. 13C NMR (101 MHz, DMSO-d6) δ
170.8, 167.6, 152.9 (d, JCF = 12.4 Hz), 152.5 (t, JCF = 29.3 Hz), 149.4,
135.6, 133.4 (d, JCF = 241.5 Hz), 125.1, 114.6, 110.1 (t, JCF = 237 Hz),
109.1, 72.1, 71.7, 64.5, 56.3 (d, JCF = 9.5 Hz), 52.9 (s), 38.2 (s), 18.2
(s), 18.1 (s); [α] = −285 (c = 0.1 in MeOH). HRMS (ES) MH+ calcd
for C19H18F3N4O5 439.1224, found 439.1241.
rel-1-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-di-
methylmorpholin-4-yl]-2,3-difluoro-phenyl]-2-methoxy-ethanone.
Prepared from 14b and N,2-dimethoxy-N-methyl-2-methylacetamide
as described for 15b. MS (ES) MH+: 568.8 for C32H39F2NO4Si.
rel-1-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-di-
methylmorpholin-4-yl]-2,3-difluoro-phenyl]-2-methoxy-ethanone
Oxime. A mixture of the preceding compound (8.85 g, 15.4 mmol),
pyridine (12.5 mL, 154 mmol), and hydroxylamine hydrochloride
(1.07 g, 15.4 mmol) in 100 mL of EtOH was heated at 90 °C for 8 h.
The reaction mixture was concentrated, diluted with water, and
extracted with EtOAc. The EtOAc was dried (MgSO4) and
concentrated. The residue was purified by silica gel chromatography
(15% EtOAc in hexanes) to give the title compound (8.45 g, 93%).
MS (ES) MH+: 583.8 for C32H40F2N2O4Si.
rel-[6-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-7-fluoro-3-(methoxy-
methyl)-1,2-benzoxazol-5-yl]methanol. A mixture of the preceding
compound (8.4 g, 14.3 mmol) and CsCO3 (18.6 g, 57.1 mmol) in 60
mL of DMF heated at 60 °C for 2 h. The mixture was filtered and
solids rinsed through with EtOAc. The EtOAc was washed with water,
dried (MgSO4), and concentrated. The residue was chromatographed
on silica gel (20−30% EtOAc gradient in hexanes) to afford 2.59 g
(55%) of the title compound. MS (ES) MH+: 325.4 for C16H21FN2O4.
rel-6-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-7-fluoro-3-(methoxy-
methyl)-1,2-benzoxazole-5-carbaldehyde. A mixture of the preced-
ing compound (2.56 g, 7.75 mmol) and MnO2 (13.5 g, 155 mmol) in
150 mL was stirred at rt for 4 d. MnO2 was filtered off by rinsing
through with CH2Cl2. The filtrate was concentrated to afford the title
compound (2.44 g, 96%). MS (ES) MH+: 323.4 for C16H19FN2O4.
rel-(2R,4S,4aS)-11-Fluoro-8-(methoxymethyl)-2,4-dimethyl-
1,2,4,4a-tetrahydro-2′H,6H-spiro[1,4-oxazino[4,3-a][1,2]oxazolo-
[4,5-g]quinoline-5,5′-pyrimidine]-2′,4′,6′(1′H,3′H)-trione ( )-6. A
solution of the previous compound (2.42 g, 7.36 mmol) and barbituric
acid (0.94 g, 7.36 mmol) in 100 mL of ethanol was heated at 90 °C for
4 days. Solvent removed and the residue was purified by reverse phase
HPLC (CH3CN/water gradient) to isolate the title compound as a
solid (4.66 g, 63%). UPLC RT = 0.92 min. (ES) MH+: 433.3 for
C20H21FN4O6. 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 11.48
(s, 1H), 7.23 (s, 1H), 4.72 (s, 2H), 4.10 (d, J = 12.8 Hz, 1H), 3.95 (d, J
= 8.8 Hz, 1H), 3.74−3.86 (m, 1H), 3.66−3.73 (m, 1H), 3.63 (d, J =
14.0 Hz, 1H), 3.34 (s, 3H), 3.03−3.18 (m, 1H), 2.94 (d, J = 14.0 Hz,
1H), 1.15 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 3H). 19F NMR (282
MHz, DMSO-d6) δ −156.1. 13C NMR (101 MHz, DMSO-d6) δ 170.9,
167.6, 155.5, 152.0 (d, JCF = 12.4 Hz), 149.4, 134.7 (d, JCF = 2.0 Hz),
133.7 (d, JCF = 240.0 Hz), 123.3, 115.3 (d, JCF = 3.0 Hz), 112.8, 72.1,
71.7, 64.5, 64.4, 58.2, 56.4 (d, JCF = 8.8 Hz), 53.0, 38.5, 18.2, 18.1.
HRMS (ES) MH+ calcd for C20H22FN4O6 433.1518, found 433.1519.
rel-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-dime-
thylmorpholin-4-yl]-2,3-difluoro-phenyl]-cyclopropyl-methanol. sec-
Butyllithium (4.5 mL, 1.4 M in cyclohexane, 6.3 mmol) was added to a
stirred solution of 14a (1 g, 2.0 mmol) in 100 mL of THF at −78 °C,
and stirring was continued for 15 min. Cyclopropane carboxaldehyde
(735 mg, 7.0 mmol) in 5 mL of THF was added dropwise, and the
(2R,4S,4aS)-11-Fluoro-2,4-dimethyl-8-(trifluoromethyl)-2,4,4a,6-
tetrahydro-1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]-
quinoline-5,5′-pyrimidine]-2′,4′,6′(3′H)-trione (−)-4. Second eluting
compound, 124 mg (37%). UPLC RT = 1.12 min. (ES) MH+: 457.0
for C19H16F4N4O5. 1H NMR (300 MHz, DMSO-d6) δ 0.89 (d, J = 6.2
Hz, 3H), 1.14 (d, J = 6.0 Hz, 3H), 2.89−3.25 (m, 2H), 3.56−4.30 (m,
5H), 7.34 (s, 1H), 11.7 (s, 2H). 19F NMR (282 MHz, DMSO-d6) δ
−61.6, −155.9. 13C NMR (101 MHz, DMSO-d6) δ 170.7, 167.7, 153.6
(d, JCF = 12.4 Hz), 149.4, 148.5 (q, JCF = 37.6 Hz), 136.2, 133.1 (d, JCF
= 241.5 Hz), 126.3 (d, JCF = 2.2 Hz), 119.9 (q, JCF = 272.5 Hz), 113.8,
108.0, 72.1, 71.7, 64.5, 56.3 (d, JCF = 9.5 Hz), 52.7, 38.1, 18.1, 18.1;
>98% ee by chiral HPLC; [α] = −305 (c = 0.1 in MeOH). HRMS
(ES) MH+ calcd for C19H17F4N4O5 457.1130, found 457.1152.
1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6R)-2,6-dimethyl-
morpholino)-2,3-difluorophenyl)-2,2-difluoroethanone. sec-Butyl-
lithium (1.3 M in hexane, 4.7 mL, 6.1 mmol) was added to a stirred
solution of 14b (1.5 g, 3.03 mmol) in 15 mL of THF at −70 °C under
N2. After stirring for 1.5 h at this temperature, 2,2-difluoro-N-methoxy-
N-methylacetamide (1.26 g, 9.08 mmol) was added. After stirring for
30 min at −78 °C, the reaction was quenched with satd aqueous
NH4Cl and warmed to rt. The mixture was extracted with EtOAc,
which was dried (MgSO4) and concentrated. The residue was
chromatographed on silica gel (10% EtOAc in hexanes) to afford
1.54 g (89%) of the title compound. UPLC (ES) MH+: 574 for
1
C31H35F4NO3Si. H NMR (300 MHz, CDCl3) δ 7.60−7.74 (m, 4H),
7.36−7.52 (m, 6H), 6.38 (td, J = 54,3 Hz 1H), 4.66 (s, 2H), 3.4−3.6
(m, 2H), 2.8−2.9 (m, 4H), 1.08−1.14 (m, 15H). 19F NMR (282 MHz,
CDCl3) δ −127.0, −134.8, −146.6.
1-(5-((tert-Butyldiphenylsilyloxy)methyl)-4-((2R,6R)-2,6-dimethyl-
morpholino)-2,3-difluorophenyl)-2,2-difluoroethanone Oxime. A
mixture of the preceding compound (8.85 g, 15.4 mmol), pyridine
(12.5 mL, 154 mmol), and hydroxylamine hydrochloride (1.07 g, 15.4
mmol) in 100 mL of EtOH was heated at 90 °C for 8 h. The reaction
mixture was concentrated, diluted with water, and extracted with
EtOAc. The EtOAc was dried (MgSO4) and concentrated. The residue
was purified by silica gel chromatography (15% EtOAc in hexanes) to
give the title compound (8.45 g, 93%). UPLC (ES) MH+: 589.3 for
1
C31H36F4N2O3Si. H NMR (300 MHz, CDCl3) δ 7.65−7.7 (m, 4H),
7.3−7.5 (m, 7H), 7.13 (t, J = 57 Hz, 1H), 4.7−4.9 (m, 2H), 3.8−4.0
(m, 2H), 3.0 (m, 2H), 2.7 (m, 2H), 1.1 (s, 7H), 1.07 (d, J = 6.8 Hz,
6H). 19F NMR (282 MHz, CDCl3) δ −117.1, −124.4, −135.4,
−138.9.
(3-(Difluoromethyl)-6-((2R,6R)-2,6-dimethylmorpholino)-7-
fluorobenzo[d]isoxazol-5-yl)methanol. A mixture of the preceding
compound (8.4 g, 14.3 mmol) and Cs2CO3 (18.6 g, 57.1 mmol) in 60
mL of DMF heated at 60 °C for 2 h. The mixture was filtered and
solids rinsed through with EtOAc. The EtOAc was washed with water,
dried (MgSO4), and concentrated. The residue was chromatographed
on silica gel (20−30% EtOAc gradient in hexanes) to afford 2.59 g
(55%) of the title compound. UPLC (ES) MH+: 331.0 for
1
C15H17F3N2O3. H NMR (300 MHz, CDCl3) δ 7.66 (s, 1H), 7.0 (t,
J = 53 Hz, 1H), 4.7−5.1 (m, 2H), 4.0−4.3 (m, 2H), 2.79−3.41 (m,
4H), 1.34 (d, J = 6.0 Hz, 6H). 19F NMR (282 MHz, CDCl3) δ −115.8,
−140.4.
3-(Difluoromethyl)-6-((2R,6R)-2,6-dimethylmorpholino)-7-
fluorobenzo[d]isoxazole-5-carbaldehyde. A mixture of the preceding
compound (2.56 g, 7.75 mmol) and MnO2 (13.5 g, 155 mmol) in 150
mL was stirred at rt for 4 d. MnO2 was filtered off by rinsing through
with CH2Cl2. The filtrate was concentrated to afford the title
compound (2.44 g, 96%). MS (ES) MH+: 329.2 for C15H15F3N2O3.
1H NMR (300 MHz, CDCl3) δ 10.4 (s, 1H), 8.16 (s, 1H), 7.01 (t, J =
53 Hz, 1H), 4.1−4.4 (m, 2H), 2.9−3.5 (m, 4H), 1.32 (d, J = 6.6 Hz, 6
H). 19F NMR (282 MHz, CDCl3) δ −115.9, −142.8.
L
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solution was stirred for an additional 1 h. The reaction mixture was
quenched with satd aqueous NH4Cl and extracted with EtOAc. The
organic layer was washed with brine and dried (Na2SO4), and solvent
was removed. The residue was chromatographed over silica gel
(EtOAc−petroleum ether gradient), providing the title compound.
Yield: 1.04 g, (92%). MS (ES) MH+: 566.2 for C33H41F2NO3Si.
rel-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-dime-
thylmorpholin-4-yl]-2,3-difluoro-phenyl]-cyclopropyl-methanone.
PCC (1.07 g, 5 mmol) was added to a solution of the preceding
compound (1.0 g, 1.77 mmol) in 15 mL of CH2Cl2 at 0 °C, and the
mixture was allowed to stir for 1h at rt. The reaction mixture was
filtered through Celite, and solvents were removed from the filtrate.
The residue was purified by silica gel chromatography (EtOAc−
petroleum ether gradient), providing the title compound. Yield: 828
mg (83%). MS (ES) MH+: 564.2 for C33H39F2NO3Si.
rel-Cyclopropyl-[4-[(2R,6S)-2,6-dimethylmorpholin-4-yl]-2,3-di-
fluoro-5-(hydroxymethyl)phenyl]methanone. TBAF (181 mg, 1.46
mmol) was added in small portions to a stirred solution of the
preceding compound (820 mg, 1.46 mmol) in 10 mL of THF at 0 °C,
and stirring was continued for 1 h with warming to rt. The solvent was
removed and the residue was purified by chromatography on silica gel
(EtOAc−petroleum ether gradient), providing the title compound.
Yield: 395 mg, (75%). MS (ES) MH+: 362.2 for C20H21F2NO3.
rel-Cyclopropyl-[4-[(2S,6R)-2,6-dimethylmorpholin-4-yl]-2,3-di-
fluoro-5-(hydroxymethyl)phenyl]methanone. PCC (234 mg, 1.08
mmol) was added to a solution of the previous compound (390 mg,
1.08 mmol) in 5 mL of CH2Cl2 at 0 °C, and the mixture was stirred
with warming to room temperature for 1 h. The reaction mixture was
filtered through Celite rinsing through with CH2Cl2. The filtrate was
concentrated, and the residue was purified by silica gel chromatog-
raphy column (EtOAc−petroleum ether gradient), providing the title
compound. Yield: 244 mg, (70%). MS (ES) MH+: 324.2 for
C17H19F2NO3.
72.2, 64.8, 56.8 (d, JCF = 9.2 Hz), 53.6, 39.2, 18.7, 18.6, 8.2, 7.8, 6.8.
HRMS (ES) MH+ calcd for C21H22FN4O5 429.1569, found 429.1580.
rel-1-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-di-
methylmorpholin-4-yl]-2,3-difluoro-phenyl]-2,2-dimethyl-propan-
1-ol. sec-Butyllithium (4.5 mL, 1.4 M in cyclohexane, 6.3 mmol) was
added to a stirred solution of 14a (1 g, 2.0 mmol) in 100 mL of THF
at −78 °C, and stirring was continued for 15 min. Pivalaldehyde (0.76
mL, 7.0 mmol) in 5 mL of THF was added dropwise, and the solution
was stirred for an additional 1 h. The reaction mixture was quenched
with satd aqueous NH4Cl and extracted with EtOAc. The organic layer
was washed with brine and dried (Na2SO4), and solvent was removed.
The residue was chromatographed over silica gel (EtOAc−petroleum
ether gradient), providing the title compound. Yield: 1.1 g, (94%). MS
(ES) MH+: 582.8 for C34H45F2NO3Si.
rel-1-[5-[[tert-Butyl(diphenyl)silyl]oxymethyl]-4-[(2R,6S)-2,6-di-
methylmorpholin-4-yl]-2,3-difluoro-phenyl]-2,2-dimethyl-propan-
1-one. NMO (439 mg, 3.8 mmol) and TPAP (61 mg, 0.19 mmol)
were added to a stirred solution of the preceding compound (1.1 g, 1.9
mmol) in CH2Cl2:CH3CN (1:1, 10 mL) 0 °C. The mixture was
allowed to warm to room temperature with stirring for 12 h. The
solvents were removed, and the residue was purified by silica gel
chromatography (EtOAc−petroleum ether gradient) to provide 727
mg (66%) of the title compound. MS (ES) MH+: 580.8 for
C34H43F2NO3Si.
rel-1-[4-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-2,3-difluoro-5-
(hydroxymethyl)phenyl]-2,2-dimethyl-propan-1-one. TBAF (3265
mg, 1.25 mmol) was added in small portions to a stirred solution of
the preceding compound (725 mg, 1.25 mmol) in 10 mL of THF at 0
°C and stirring was continued for 1h with warming to rt. The solvent
was removed and the residue was purified by silica gel chromatography
(EtOAc−petroleum ether gradient), providing the title compound.
Yield: 320 mg, (75%). MS (ES) MH+: 342.4 for C18H25F2NO3.
rel-2-[(2R,6S)-2,6-Dimethylmorpholin-4-yl]-5-(2,2-dimethylpro-
panoyl)-3,4-difluorobenzaldehyde. NMO (213 mg, 1.84 mmol) and
TPAP (40 mg, 0.09 mmol) were added to a stirred solution of the
preceding compound (320 mg, 0.94 mmol) in 10 mL of
CH2Cl2:CH3CN (1:1) at 0 °C, and the mixture was stirred for 12 h
with warming to rt. The solvents were removed, and the residue was
purified by silica gel chromatography (EtOAc−petroleum ether
gradient) to give title compound. Yield: 242 mg, (76%). MS (ES)
MH+: 340.4 for C18H23F2NO3.
rel-(2R,4S,4aS)-8-(2,2-Dimethylpropanoyl)-9,10-difluoro-2,4-di-
methyl-1,2,4,4a-tetrahydro-2′H,6H-spiro[1,4-oxazino[4,3-a]-
quinoline-5,5′-pyrimidine]-2′,4′,6′(1′H,3′H)-trione. A solution of the
preceding compound (240 mg, 0.71 mmol) and barbituric acid (100
mg, 0.78 mmol) in i-PrOH was heated at reflux for 12 h. The solvent
was removed, and the residue was chromatographed on neutral
alumina (MeOH gradient in CH2Cl2) to give 258 mg (81%) of the
title compound as a white solid. MS (ES) MH+: 450.4 for
C22H25F2N3O5.
rel-(2R,4S,4aS)-8-(Cyclopropanecarbonyl)-9,10-difluoro-2,4-di-
methyl-spiro[2,4,4a,6-tetrahydro-1H-[1,4]oxazino[4,3-a]quinoline-
5,5′-hexahydropyrimidine]-2′,4′,6′-trione. A solution of the preced-
ing compound (244 mg, 0.76 mmol) and barbituric acid (106 mg, 0.83
mmol) in i-PrOH was heated at reflux for 12 h. The solvent was
removed, and the residue was chromatographed on neutral alumina
(MeOH gradient in CH2Cl2) to give 245 mg (75%) of the title
compound as an off-white solid. MS (ES) MH+: 434.1 for
C21H21F2N3O5.
rel-(2R,4S,4aS)-8-Cyclopropyl-11-fluoro-2,4-dimethyl-1,2,4,4a-
tetrahydro-2′H,6H-spiro[1,4-oxazino[4,3-a][1,2]oxazolo[4,5-g]-
quinoline-5,5′-pyrimidine]-2′,4′,6′(1′H,3′H)-trione ( )-7. A solution
of the previous compound (212 mg, 0.49 mmol) and hydroxylamine
hydrochloride (51 mg, 0.73 mmol) in 3 mL of MeOH:pyridine was
heated to 90 °C for 12 h. Solvents were removed, and the residue was
chromatographed on silica gel (EtOAc gradient in petroleum ether) to
give 92 mg (42%) of rel-(2R,4S,4aS)-8-[C-cyclopropyl-N-hydroxy-
carbonimidoyl]-9,10-difluoro-2,4-dimethyl-spiro[2,4,4a,6-tetrahydro-
1H-[1,4]oxazino[4,3-a]quinoline-5,5′-hexahydropyrimidine]-2′,4′,6′-
trione as a solid. UPLC RT = 1.00 min. (ES) MH+: 429.2 for
rel-(2R,4S,4aS)-9,10-Difluoro-8-[N-hydroxy-2,2-dimethylpropani-
midoyl]-2,4-dimethyl-1,2,4,4a-tetrahydro-2′H,6H-spiro[1,4-
oxazino[4,3-a]quinoline-5,5′-pyrimidine]-2′,4′,6′(1′H,3′H)-trione. A
solution of preceding compound (250 mg, 0.57 mmol) and
hydroxylamine hydrochloride (60 mg, 0.86 mmol) in 3 mL 1:1
MeOH:pyridine was heated to 90 °C for 12 h. Solvents were removed,
and the residue was chromatographed on silica gel (EtOAc−petroleum
ether gradient) to give title compound. Yield: 0.225 mg (85%). MS
(ES) MH+: 465.5 for C22H26F2N4O5.
rel-(2R,4S,4aS)-8-tert-Butyl-11-fluoro-2,4-dimethyl-1,2,4,4a-tetra-
hydro-2′H,6H-spiro[1,4-oxazino[4,3-a][1,2]oxazolo[4,5-g]quinoline-
5,5′-pyrimidine]-2′,4′,6′(1′H,3′H)-trione ( )-8. A mixture of the
preceding compound (220 mg, 0.47 mmol) and Cs2CO3 (155 mg,
0.47 mmol) in 1.5 mL of DMF was heated at 100 °C for 14 h. The
reaction mixture was filtered through Celite rinsing through with
EtOAc. The solvent was removed, and the residue was purified by
reverse phase HPLC (10 mM ammonium acetate in water, CH3CN
gradient), providing 45 mg (22%) of the title compound as a white
solid. UPLC RT = 1.10 min, MS (ES) MH+: 445.2 for C22H25FN4O5.
1H NMR (400 MHz, DMSO-d6) δ 0.9 (d, 3H), 1.1 (d, 3H), 1.4 (s,
1
C21H22F2N4O5. H NMR (400 MHz, DMSO-d6) δ: 11.1 (br s, 3H),
4.0 (d, 1H), 3.8 (d, 1H), 3.7 (m, 1H), 3.6 (m, 1H), 3.3 (m, 1H), 3.0
(m, 1H), 2.9 (d, 1H), 2.3 (m, 1H), 1.1 (d, 3H), 0.85 (d, 3H), 0.8 (d,
2H), 0.3 (m, 1H). The material was dissolved in 1.5 mL of DMF along
with Cs2CO3 (82 mg, 0.41 mmol), and the mixture was heated to 100
°C for 14 h. The reaction mixture was cooled to rt and filtered through
Celite. The filtrate was concentrated, and the residue was purified by
reverse phase HPLC (CH3CN gradient in water with 0.1% TFA).
1
UPLC RT = 1.00 min. MS (ES) MH: C21H21FN4O5. H NMR (400
MHz, DMSO-d6) δ 11.8 (br s, 1H), 11.5 (br s, 1H), 7.20 (s, 1H), 4.07
(d, J = 13.0 Hz, 1H), 3.93 (d, J = 8.8 Hz, 1H), 3.72−3.83 (m, 1H),
3.61−3.72 (m, 1H), 3.55 (d, J = 14.3 Hz, 1H), 3.08 (t, J = 12.4 Hz,
1H), 2.94 (d, J = 14.3 Hz, 1H), 2.12−2.27 (m, 1H), 1.13 (d, J = 6.3
Hz, 3H), 0.96−1.11 (m, 4H), 0.88 (d, J = 6.3 Hz, 3H). 19F NMR (282
MHz, DMSO-d6) δ −156.1. 13C NMR (126 MHz, DMSO-d6) δ 171.4,
168.2, 160.3, 152.4 (d, JCF = 11.9 Hz), 150.0, 135.0 (d, JCF = 1.8 Hz),
134.3 (d, JCF = 240.1 Hz), 123.2, 115.2 (d, JCF = 2.7 Hz), 113.7, 72.5,
9H), 2.9 (d, 1H), 3.1 (t, 1H), 3.6 (m, 1H), 3.7 (m, 2H), 3.9 (d, 1H),
M
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Journal of Medicinal Chemistry
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4.1 (d, 1H), 7.4 (s, 1H), 11.3 (bs, 2H). 19F NMR (282 MHz, DMSO-
d6) δ −156.3. 13C NMR (101 MHz, DMSO-d6) δ 172.4, 169.0, 164.7,
152.3 (d, JCF = 11.7 Hz), 151.4, 134.1, 133.7 (d, JCF = 238.6 Hz),
122.9, 115.9, 111.9, 72.3, 71.6, 64.4, 56.4, 56.3, 52.7, 33.0, 28.7, 18.2,
18.1. HRMS (ES) MH+ calcd for C22H26FN4O5 445.1882, found
445.1893.
(m, 2H), 7.9 (s, 1H), 10.3 (s, 1H. 19F NMR (282 MHz, DMSO-d6) δ
−144.2.
rel-(2S,4R,4aR)-11-Fluoro-2,4,8-trimethyl-1,2,3,4,4a,6-hexahydro-
1′H-spiro[isoxazolo[4,5-g]pyrido[1,2-a]quinoline-5,5′-pyrimidine]-
2′,4′,6′(3′H)-trione ( )-9. A solution of 72 mg (0.25 mmol) of the
preceding compound and 32 mg (0.25 mmol) barbituric acid in 3 mL
of i-PrOH was heated at refllux overnight. Solvent was removed, and
the title compound (80 mg, 81%) was isolated as a white solid by
crystallization from 3:1 ethanol−water. UPLC RT = 1.09 min, .MS
(ES) MH+: 401.1 for C20H21FN4O4. 1H NMR (300 MHz, DMSO-d6)
δ 0.6 (d, 3H), 0.8−1.1 (m, 1H), 0.9 (d, 3H), 1.8 (m, 3H), 2.4 (s, 3H),
2.6−3.1 (m, 2H) 3.5 (d,1H), 3.8 (d, 1H) 3.9−4.0 (m, 1H) 7.1 (s, 1H)
11.4 (br s, 2H). 19F NMR (282 MHz, DMSO-d6) δ −160.0. 13C NMR
(101 MHz, DMSO-d6) δ 171.4, 167.8, 154.5, 151.6 (d, JCF = 12.4 Hz),
149.6, 135.7 (d, JCF = 2.9 Hz), 133.9 (d, JCF = 240.0 Hz), 123.1 (d, J =
1.5 Hz), 114.5, 113.8, 66.2, 58.1(d, JCF = 8.8 Hz), 54.1, 43.0, 38.8, 32.4,
31.3, 18.6, 18.4, 9.3. HRMS (ES) MH+ calcd for C10H22FN4O4
401.1620, found 401.1626.
1-(5-((tert-Butyldiphenylsilyloxy)methyl)-2,3,4-trifluorophenyl)-
ethanone (21). A solution of s-butyllithium (1.4 M in cyclohexane,
6.42 mL, 9.0 mmol) was added slowly to a solution of the tert-
butyldiphenyl(2,3,4-trifluorobenzyloxy)silane29,49 (3 g, 7.49 mmol) in
30 mL of THF cooled in a dry ice−acetone bath to maintain a
temperature below −60 °C. After stirring in for 20 min, N-methoxy-N-
methylacetamide (1.035 mL, 9.74 mmol) was added dropwise,
maintaining a temperature below −60 °C. The mixture was stirred
cold for 40 min before being quenched with NH4Cl (aqueous) and
warmed to room temperature. The solution was partitioned between
EtOAc and water. The EtOAc was separated and washed with brine.
The combined aqueous layers were extracted with EtOAc, which was
washed with brine. The combined EtOAc layers were dried (MgSO4)
and concentrated to give an oil that was chromatographed on silica gel
(10% CH2Cl2 in hexanes followed by gradient elution to 70% CH2Cl2
in hexanes) to give two materials. The first eluting component (0.76 g)
was consistent with starting material, and the second eluting
component (2.05 g) was consistent with the title compound. MS
Methyl 3,4-Difluoro-2-hydroxybenzoate. A solution of 3,4-
difluoro-2-hydroxybenzoic acid (6.45 g, 37.05 mmol) and sulfuric
acid (6 mL, 113 mmol) in MeOH (25 mL) was heated at reflux
overnight. The mixture was diluted with water and extracted with
ether. The ether was washed with aqueous NaHCO3, water, and brine.
The combined aqueous layers were twice more extracted with ether,
which was washed with NaHCO3, water, and brine. The combined
ether extracts were dried (MgSO4) and concentrated to give 6.4 g of
1
(ES) MH+: 443.0 for C25H25F3O2Si. H NMR (300 MHz, CDCl3) δ
1.1 (s, 9H), 2.6 (d, 3H), 4.8 (s, 2H), 7.3−7.5 (m, 6H) 7.6−7.7 (m,
4H), 7.8−7.95 (m, 1H. 19F NMR (282 MHz, DMSO-d6) δ −131.4,
−132.9, −159.4.
1
product as a white solid. MS (ES) MH−: 187.1 for C8H6F2O3. H
NMR (300 MHz, DMSO-d6) δ 10.8 (s, 1H), 7.65 (ddd, J = 9.0, 6.3,
2.3 Hz 1H), 6.9−7.1 (m, 1H), 3.9 (s, 3H). 19F NMR (282 MHz,
DMSO-d6) δ −128.7, −160.5.
(6,7-Difluoro-3-methyl-1,2-benzisoxazol-5-yl)methanol (22). Po-
tassium t-butoxide (1 M solution in THF, 23.8 mL) was added to a
stirred solution of propan-2-one oxime (1.73 g, 23.7 mmol) in THF
(25 mL) at 0 °C under N2, and the mixture was stirred with warming
to rt for 1 h. The mixture was cooled to −78 °C and a solution of the
preceding compound (3.0 g, 6.8 mmol) in THF (15 mL) was added,
and stirring was continued for 20 min at −78 °C before warming to
−20 °C for 2 h. The reaction mixture was quenched with a saturated
aqueous NH4Cl solution and extracted with EtOAc. The organic layer
was dried (Na2SO4), filtered, and concentrated. The residue was
dissolved in 130 mL of ethanol and treated with 5 mL of 5% aqueous
HCl with heating at 45 °C for 8 h. Addition of 10% aqueous NaHCO3
was followed by extraction with EtOAc. The organic layer was washed
with water, dried (MgSO4), and concentrated. The residue was
purified over silica gel (15−20% EtOAc in hexanes gradient) to give
the title compound as yellow solid. Yield: 600 mg, (46%). MS (ES)
MH+: 200 for C9H7F2NO2. 1H NMR (400 MHz, DMSO-d6) δ: 2.6(s,
3H), 4.6 (d, 2H), 5.55 (s, 1H), 7.7 (d, 1H).
3,4-Difluoro-N,2-dihydroxybenzamide (25). A solution of hydrox-
ylamine (50% in water) (50 mL, 816 mmol) was added to a solution
of the preceding compound (7.75 g, 41.2 mmol) in dioxane (200 mL),
and the mixture was stirred at room temperature for 3 days. The
mixture was partitioned between water and EtOAc. The aqueous layer
was acidified with concentrated HCl and extracted with EtOAc twice
more. The EtOAc layers were washed with brine, combined, and
concentrated to give 7.9 g of a solid. LC-MS ES MH+: 190.0 for
C7H5F2NO3. 1H NMR (300 MHz, DMSO-d6) δ 13.14 (br s, 1H), 11.7
(s, 1H), 9.54 (br s, 1H), 7.37−7.72 (m, 1H), 6.96 (ddd, J = 7.4, 9.2,
10.0 Hz, 1H). 19F NMR (282 MHz, DMSO-d6) δ −131.6, −161.6.
6,7-Difluoro-1,2-benzisoxazol-3(2H)-one. A mixture of 25 (7.91 g,
41.8 mmol) and carbonyl diimidazole (13.6 g, 83.7 mmol) in 200 mL
of THF was heated at reflux for 90 min.The mixture was partitioned
between EtOAc and water and acidified with conc HCl. The solution
was extracted three times with EtOAc, each extract being washed with
water and brine. Drying (MgSO4) of the combined extracts and
removal of solvent gave 6.88 g of product as an off-white solid. MS
6,7-Difluoro-3-methylbenzo[d]isoxazole-5-carbaldehyde. A mix-
ture of the preceding compound (0.7 g, 3.51 mmol) and MnO2 (6.11
g, 70.30 mmol) in 70 mL of CH2Cl2 was stirred at rt for 3 days. TLC
shows conversion to a higher Rf spot with starting material. Additional
MnO2 (5 g) was added, and the mixture was heated at reflux for 24 h.
The mixture was filtered through Celite rinsing through with CH2Cl2.
Solvent was removed from the filtrate to give 640 mg of the title
1
(ES) (M − H)−: 170 for C7H3F2NO2. H NMR (300 MHz, DMSO-
d6) δ 7.3−7.5 (m, 1H), 7.6 (m, 1H), 12.9 (s, 1H). 19F NMR (282
MHz, CDCl3) δ −137.6, −162.4.
3-Chloro-6,7-difluorobenzo[d]isoxazole (26). TEA (5.6 mL, 40.2
mmol) was added to an ice bath cooled mixture of the preceding
compound (6.88 g, 40.2 mmol) and POCl3 (13.1 mL, 141 mmol) in a
microwave reactor vessel (exothermic), and the mixture was heated at
140 °C for 6 h in a microwave reactor. Solvent was removed, and the
mixture was taken up in ether and washed with Na2CO3 (2×) and
brine. The combined aqueous layers were twice more extracted with
ether, which was washed with aqueous Na2CO3 and brine. The
combined ether layers were dried (MgSO4) and concentrated to give
an oil that slowly solidified. The material was chromatographed on
silica gel (hexanes followed by gradient elution to 50% CH2Cl2 1in
hexanes) to afford 5.64 g (71%) of product as an off-white solid. H
NMR (CDCl3) δ 7.2−7.35 (m, 1H), 7.4−7.5 (m, 1H). 19F NMR (282
MHz, CDCl3) δ −133.4, −157.9.
1
compound as a tan solid. MS (ES) MH+: 198 for C9H5F2NO2. H
NMR (300 MHz, CDCl3) δ 2.6 (s, 3H), 8.0 (d, 1H), 10.4 (s, 1H. 19F
NMR (282 MHz, DMSO-d6) δ −145.2, −157.1.
rel-6-((3S,5R)-3,5-Dimethylpiperidin-1-yl)-7-fluoro-3-
methylbenzo[d]isoxazole-5-carbaldehyde (23). A solution of the
previous compound (110 mg, 0.56 mmol), (3S,5R)-rel-3,5-dimethyl-
piperidine (HCl salt) (167 mg, 1.12 mmol), and DIEA (0.292 mL,
1.67 mmol) in 7 mL of CH3CN was heated at reflux overnight. The
mixture was diluted with EtOAc and washed with water and brine. The
combined aqueous layers were extracted with EtOAc, which was
washed with brine. The combined EtOAc layers were dried (MgSO4)
and concentrated to give an oil that was chromatographed on silica gel
(50% hexanes in CH2Cl2 followed by gradient elution to 100%
CH2Cl2) to give the title compound as yellow solid. MS (ES) MH+:
3-Chloro-6,7-difluorobenzo[d]isoxazole-5-carbaldehyde (27). A
solution of n-butyllithium (2.5 M in hexanes) (16.7 mL, 42 mmol) was
added slowly to a solution of 2,2,6,6-tetramethylpiperidine (7.6 mL, 45
mmol) in THF (50 mL) cooled in a dry ice−acetone bath. The
solution was warmed to 0 °C and recooled in a dry ice−acetone bath
1
291 for C16H19FN2O2. H NMR (300 MHz, CDCl3) δ 0.7−0.9 (q,
1H), 0.94 (d, 6H), 1.9 (m, 4H), 2.6 (s, 3H), 2.8−3.0 (m, 2H), 3.2−3.3
N
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Journal of Medicinal Chemistry
Article
before transferring via syringe to a solution of 26 (5.64 g, 30 mmol) in
THF (50 mL). After 1 h stirring, DMF (1.0 mL, 13.2 mmol) was
added all at once and the mixture was stirred with cooling in a dry ice−
acetone bath for 45 min. The mixture was transferred via cannula to a
solution of acetic acid (6.8 mL, 119 mmol) in 100 mL of Et2O, and the
mixture was warmed to rt. The mixture was diluted with EtOAc and
washed with brine. The combined aqueous layers were extracted again
with EtOAc, which was washed with brine. Drying (MgSO4) of the
combined extracts and removal of solvent gave a brown oil that was
chromatographed on silica gel (10% CH2Cl2 in hexanes followed by
gradient elution to 100% CH2Cl2) to give 3.32 g (51%) of product as a
white solid. 1H NMR (300 MHz, CDCl3) δ 10.4 (s, 1H), 8.1 (dd, J =
5.3, 1.7 Hz, 1H). 19F NMR (282 MHz, CDCl3) δ −142.3, 155.9.
3-Chloro-6-((2R,6R)-2,6-dimethylmorpholino)-7-fluorobenzo[d]-
isoxazole-5-carbaldehyde (28). A solution of 27 (10 g, 46 mmol),
(2R,6R)-2,6-dimethylmorpholine (6.35 g, 55 mmol), and K2CO3 (12.7
g, 92 mmol) in butyronitrile (100 mL) and water (10 mL) was heated
at reflux for 2 h. The mixture was cooled to room temperature before
being partitioned between EtOAc and water. The EtOAc was
separated and washed with brine. The combined aqueous layers
were extracted with EtOAc, which was washed with brine. The
combined EtOAc was dried (MgSO4) and concentrated to give an oil
that slowly solidified (15.3 g, 100% yield) and was sufficiently clean to
be used in subsequent steps. For characterization purposes, material
was chromatographed on silica gel (100% CH2Cl2 followed by
gradient elution to 5% EtOAc in CH2Cl2) to give the title compound.
MS (ES) MH+: 312.9, 314.7 for C14H14ClFN2O3. 1H NMR (300
MHz, CDCl3) δ 10.45 (s, 1H), 8.0 (d, J = 1.1 Hz, 1H), 4.24 (m, 2H),
3.43 (dm, J = 11.9 Hz, 2H), 3.0−3.1 (m, 2H), 1.33 (d, J = 6.4 Hz, 6H).
19F NMR (282 MHz, DMSO-d6) δ −143.4.
washed with brine. The combined EtOAc layers were dried (MgSO4)
and concentrated to give an the title compound (430 mg, 87%) as an
oil that slowly solidified. LC-MS ES MH+ 353.0 for C17H21ClFN2O5.
1H NMR (400 MHz, CDCl3) δ 7.58 (s, 1H), 6.22 (s, 1H), 4.05−4.11
(s and m, total of 7H), 3.9−4.00 (m, 2H), 3.20 (br s, 2H), 2.88 (br s,
2H), 1.26 (d, J = 6.5 Hz, 6H). 19F NMR (282 MHz, DMSO-d6) δ
−143.5.
(2R,4S,4aS)-11-Fluoro-8-methoxy-2,4-dimethyl-2,4,4a,6-tetrahy-
dro-1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-
pyrimidine]-2′,4′,6′(3′H)-trione (−)-11. A mixture of 30 (430 mg,
1.22 mmol) and barbituric acid (156 mg, 1.22 mmol) in MeOH (4
mL) and water (0.5 mL) were heated at 120 °C for 1 h in a microwave
reactor. Solvent was removed, and the residue was purified by chiral
SFC (21 mm × 250 mm, 5 μ (S,S) Whelk-O1 column, 80% CO2, 20%
MeOH) to give 391 mg (77%) of the title compound was a white
solid. UPLC RT = 0.94 min. MS (ES) MH+: 419.2 for C19H19FN4O6.
1H NMR (300 MHz, DMSO-d6) δ 11.76 (br s, 1H), 11.49 (br s, 1H),
7.08 (s, 1H), 4.09 (m., 1H), 4.04 (s, 3H), 3.93 (d, J = 8.6 Hz, 1H),
3.77 (m, 1H), 3.62−3.72 (m, 1H), 3.57 (d, J = 14.1 Hz, 1H), 3.17 (d, J
= 4.7 Hz, 1H), 3.09 (t, J = 12.2, 1H), 2.92 (d, J = 14.3 Hz, 1H), 1.14
(d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.2 Hz, 3H). 19F NMR (282 MHz,
DMSO-d6) δ −157.6. 13C NMR (75 MHz, DMSO-d6) δ 170.9, 167.6,
166.4 (d, JCF = 2.7 Hz), 152.8 (d, JCF = 12.6 Hz), 149.5, 135.1, 133.8
(d, JCF = 239.9 Hz), 122.6, 113.8 (d, JCF = 2.7 Hz), 105.2, 72.0, 71.7,
64.4, 57.6, 56.3 (d, JCF = 9.3 Hz), 53.1, 48.6, 38.5, 18.1; [α] = −303 (c
= 0.1 in MeOH). HRMS (ES) MH+ calcd for C19H20FN4O6 419.1361,
found 419.1360.
3-Azido-6-((2R,6R)-2,6-dimethylmorpholino)-5-(1,3-dioxolan-2-
yl)-7-fluorobenzo[d]isoxazole (31). A mixture of 29 (0.1 g, 0.28
mmol) and NaN3 (0.055 g, 0.84 mmol) in DMSO (3 mL) was heated
at 100 °C for 6 h in a microwave reactor. The mixture was diluted with
Et2O and water. The Et2O was separated and washed with water three
more times. The combined aqueous layers were twice more extracted
with Et2O, and each extract was washed with water three more times.
The combined Et2O layers were dried (MgSO4) and concentrated to
give an oil that slowly solidified. The material was purified by
chromatography on silica gel (100% CH2Cl2 with gradient elution to
40% EtOAc in CH2Cl2) to afford 80 mg (79%) of the title compound
3-Chloro-6-[(2R,6R)-2,6-dimethylmorpholin-4-yl]-5-(1,3-dioxo-
lan-2-yl)-7-fluoro-1,2-benzoxazole (29). A suspension of 28 (37 g,
118 mmol), p-TsOH acid (1.12 g, 5.9 mmol), and ethylene glycol
(26.4 mL, 473 mmol) in 100 mL toluene was heated at reflux with
azeotropic removal of water via a Dean−Stark trap for 3 h. After
cooling to rt, solvent was removed and the residue was partitioned
between Et2O and aqueous Na2CO3. The Et2O was separated and
washed with water and brine. The Et2O was dried (MgSO4) and
concentrated to give an oil that slowly solidified under vacuum (42.5 g,
1
as a white solid. LC-MS ES MH+ 363.0 for C16H18FN5O4. H NMR
1
100%). LC-MS ES MH+ 357.0, 359.0 for C16H18ClFN2O4. H NMR
(300 MHz, CDCl3) δ 7.63 (s, 1H), 6.28 (s, 1H), 4.14−4.28 (m, 4H),
3.91−4.11 (m, 2H), 3.32 (br s, 2H), 2.98 (br s, 2H), 1.34 (br s, 6H).
19F NMR (282 MHz, CDCl3) δ −142.5.
(300 MHz, DMSO-d6) δ 7.71 (s, 1H), 6.20 (s, 1 H), 3.9−4.2 (m, 6H),
3.2−3.3 (m, 2H), 2.90 (dd, J = 11.1, 5.1 Hz, 2H), 1.22 (d, J = 6.2 Hz, 6
H).
(2R,4S,4aS)-8-Amino-11-fluoro-2,4-dimethyl-2,4,4a,6-tetrahydro-
1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyri-
midine]-2′,4′,6′(3′H)-trione (−)-12. Ph3P (57.7 mg, 0.22 mmol) was
added to a suspension of 3-azido-6-((2R,6R)-2,6-dimethylmorpholi-
no)-5-(1,3-dioxolan-2-yl)-7-fluorobenzo[d]isoxazole (80 mg, 0.22
mmol) in 2 mL of acetic acid and 200 μL of water, during which
gas evolution was observed and the mixture became homogeneous.
After stirring for 10 min, barbituric acid (28.2 mg, 0.22 mmol) was
added and the mixture was heated at 120 °C for 1 h. Solvent was
removed, and the residue was purified by chiral SFC (21 mm × 250
mm, 5 μ (S,S) Chiralpak A1 column, 70% CO2, 30% MeOH) to give
37 mg (42%) of the title compound was a white solid. UPLC RT =
(2R,4S,4aS)-8-Chloro-11-fluoro-2,4-dimethyl-2,4,4a,6-tetrahydro-
1H,1′H-spiro[isoxazolo[4,5-g][1,4]oxazino[4,3-a]quinoline-5,5′-pyri-
midine]-2′,4′,6′(3′H)-trione (−)-10. A mixture of 28 (500 mg, 1.4
mmol) and barbituric acid (180 mg, 1.4 mmol) in 2 mL of EtOH and
2 mL of water was heated at 120 °C for 1 h. Solvent was removed, and
the residue was recrystallized from methanol, affording a methanol
solvate. The solids were dissolved in 1:1 CH3CN−water and
lyophilized to afford a white solid that was heated at 60 °C under
vacuum for 2 d to afford 422 mg (71%) of product consistent with the
title compound. UPLC RT = 1.03 min. MS (ES) MH+: 423.2, 425.3
for C18H17ClFN4O5. 1H NMR (300 MHz, DMSO-d6) δ 11.9 (s, 1H),
11.5 (s, 1H), 7.23 (s, 1H), 4.11 (d, J = 13.4 Hz, 1H), 3.98 (d, J = 8.85
Hz, 1H), 3.78 (d, J = 6.2 Hz, 1H), 3.6−3.7 (m, 2H), 3.13 (t, J = 12.0
Hz, 1H), 2.95 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 5.8 Hz, 3H), 0.89 (d, J
= 6.2 Hz, 3H). 19F NMR (282 MHz, DMSO-d6) δ −156.8. 13C NMR
(75 MHz, DMSO-d6) δ 170.7, 167.6, 152.8 (d, JCF = 13.2 Hz), 149.4,
148.5 (d, JCF = 2.7 Hz), 136.0 (d, JCF = 2.2 Hz), 133.2 (d, JCF = 241.5
Hz), 124.8 (d, JCF = 2.2 Hz), 113.7 (d, JCF = 2.7 Hz), 110.7, 72.1, 71.6,
64.5, 56.3 (d, JCF = 9.3 Hz), 52.8, 38.3, 18.2, 18.1. HRMS (ES) MH+
calcd for C18H17ClFN4O5 423.0866, found 423.0858; [α] = −273 (c =
0.1 in MeOH).
1
0.75 min. MS (ES) MH+: 404.3 for C18H18FN5O5. H NMR (400
MHz, DMSO-d6) δ 11.79 (br s, 1H), 11.44 (br s, 1H), 7.09 (s, 1H),
6.23 (s, 2H), 4.05 (d, J = 12.6 Hz, 1H), 3.90 (d, J = 8.8 Hz, 1H), 3.72−
3.83 (m, 1H), 3.59−3.71 (m, 1H), 3.45 (d, 14.3 Hz, 1H), 3.29 (s, 1H),
3.01−3.11 (m, 1H), 2.94 (d, J = 14.0 Hz, 1H), 1.13 (d, J = 6.0 Hz,
3H), 0.88 (d, JCF = 6.37 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ
172.1, 168.7, 159.0, 151.7 (d, JCF = 12.1 Hz), 150.8, 134.70, 134.67 (d,
JCF = 238.8 Hz), 121.3, 115.2, 109.6, 72.6, 72.2, 65.1, 56.9 (d, J = 8.8
Hz), 53.6, 40.9, 18.67, 18.66. 19F NMR (282 MHz, CDCl3) δ −157.3;
[α] = −158.9 (c = 0.1 in DMSO). HRMS (ES) MH+ calcd for
C18H18FN5O5 404.1370, found 433.1365.
Colorless crystals of a monohydrate form of (−)-1 were prepared
by recrystallization from ethanol/water (4:1). The diffraction data
were collected at 180 K on a Nonius KappaCCD diffractometer. The
crystal structure was solved and refined with the SHELXTL package.
All the hydrogen atoms attached to the C atoms and N atoms were
6-((2R,6R)-2,6-Dimethylmorpholino)-5-(1,3-dioxolan-2-yl)-7-fluo-
ro-3-methoxybenzo[d]isoxazole (30). A mixture of 29 (0.5 g, 1.40
mmol) and sodium methoxide (0.5 M in MeOH, 2.8 mL, 1.4 mmol)
was heated at 100 °C for 1.5 h in a microwave reactor. The mixture
was diluted with EtOAc, which was washed with water and brine. The
combined aqueous layers were extracted with EtOAc, which was
O
dx.doi.org/10.1021/jm501174m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
calculated. Absolute configuration was established by using anomalous
dispersion method, and the Flack parameter is found to be 0.1(3). The
diffraction data was deposited into the Cambridge Crystallographic
Data Centre (CCDC 1013311).
gene: Trp519Leu for the first strain (designated SAA4419) and
Ala438Ser for the second strain (designated SAB4419). All MIC values
were determined using CLSI methodology.50,51
In Vivo Efficacy. Mouse thighs were infected with a mid-log
culture of S. aureus (MSSA, ARC516 from the AstraZeneca Research
Collection) to achieve a target inoculum of 5 × 105 CFU. Groups of
five animals each received an intraperitoneal injection of test
compound at doses of 3−100 mg/kg/day on a q24h or q6h regimen
starting 2 h after infection. A group of 10 mice were sacrificed at 2 h
after infection. Two control groups of five mice received linezolid at
160 mg/kg q24 (positive control group) or vehicle alone (negative
control group). Efficacy and control groups were sacrificed 24 h after
the start of treatment. Thighs were removed, weighed, and
homogenized and aliquots plated onto tryptic soy agar plates and
incubated at 37 °C overnight for CFU/g thigh determination. The
experiment was repeated using mice that were rendered neutropenic
by injecting cyclophosphamide intraperitoneally 4 days (150 mg/kg of
body weight) and 1 day (100 mg/kg) before experimental infection.
In Vivo Safety. In the single dose phase, groups of three male rats
were given doses up to 1000 mg/kg/day (−)-1 once daily. The
animals were then observed for up to seven days before being killed for
scheduled necropsy. In the repeat dose phase, groups of three male
and three female rats were dosed once daily for 14 days at 0, 250, 500,
and 750 mg/kg/day and killed for scheduled necropsy on day 15.
Additional groups of two males and two females were dosed at 250,
500, and 750 mg/kg/day for 14 days for toxicokinetics. These animals
were killed without necropsy following completion of blood sampling.
The following were assessed in this study: clinical observations, body
weight, food consumption, body temperature, hematology, plasma
chemistry, toxicokinetics, and gross and microscopic pathology.
Biological Experimental Procedures. Compounds for oral, iv,
and ip dosing in mice and rats were formulated in 0.2 M meglumine/
20% hydroxypropyl-β-cyclodextrin in rats and DMA:TEG:saline 40/
40/20% in mice. Wistar Hannover rats used for pharmacokinetic and
toxicology studies were obtained from Charles River Laboratories
(Raleigh, NC). CD-1 mice were obtained from Charles River
Laboratories (Kingston, NY) and used for pharmacokinetic and
efficacy studies. All animals 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.
Inhibition of of DNA Gyrase Activity. IC50 values against E. coli
DNA gyrase and human topoisomerase IIα were determined using
literature procedures.35,48
Plasma Protein Binding Determination. Human, rat, and
mouse plasma protein binding was determined from a 10 μM
compound solution in a Dianorm plasma well incubating at 37 °C for
16 h. Free fractions were calculated from ratios of drug concentration
in buffer and plasma wells determined by LC-MS/MS.
Pharmacokinetic Studies. Pharmacokinetic properties of selected
compounds were studied in male rats and male mice. Plasma
pharmacokinetics were determined from 0 to 24 h following 15 min
iv infusions at 3 mg/kg or oral administration at 10 mg/kg. Serial 200
μL samples of whole blood were taken from the jugular vein of each
animal at time intervals. Concentration of compound in plasma was
determined by LC-MS/MS, and pharmacokinetic parameters were
estimated using a noncompartmental model in WinNonLin (Phar-
sight). Mean results were determined for each experiment with three
mice or three rats.
Minimum Inhibitory Concentration (MIC). MIC values were
determined by the broth microdilution method in accordance with the
Clinical and Laboratory Standards Institute (CLSI) guidelines,50,51 as
described previously.27 For more potent compounds with MIC values
<12.5 μM, the reported data represents the averages of three or more
replications.
ASSOCIATED CONTENT
■
S
* Supporting Information
Included are NMR NOESY spectra for (−)-1 and (+)-2 and a
model for (+)-2. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
DNA Biosynthesis Inhibition. Incorporation of radiolabeled
precursors (3H-thymidine, 3H-uridine, 14C-leucine, 14C-acetic acid,
and 14C−N-acetyl glucosamine) of biological macromolecules was
measured in S. aureus (MSSA) following previously described
methods.27,44
Corresponding Author
*Phone: 781-839-4689. Fax: 871-839-4220. E-mail: greg.
basarab@astrazeneca.com.
Present Addresses
Isolation and Characterization Resistant Bacteria. Suspen-
sions of cultures of methicillin-sensitive S. aureus (ARC516) were
transferred onto plates of Mueller−Hinton agar containing (−)-1 at
four concentrations: 0, 1.56, 3.12, and 6.25 μM. The frequency of
spontaneous resistance was determined in two independent studies
performed in triplicate on each occasion and was calculated from the
number of colonies on the compound-containing plates divided by the
number of colonies on compound-free plates after incubation for 48 h.
Values across studies were averaged. The incubations were extended to
4 d for isolation and analysis of colonies. When bacteria were plated on
agar plates containing a 2.12 and 6.25 μM of (−)-1, no resistant
colonies were recovered. One colony was selected from the 1.56 μM
plate, recovered on drug-free plates, and frozen in glycerol stocks.
Genomic DNA was prepared from this isolated resistant variant
(designated SAA4419), as well as from the original susceptible parent
strain, and the genes for the topoisomerase subunits (gyrA, gyrB, parC,
and parE) were amplified by PCR and sequenced using standard
protocols described previously.52 Gene sequences from the variant
strains were compared to those from the parent strain ARC516 and
found to be identical, Suspensions of cultures of SAA4419 were
transferred onto plates of Mueller−Hinton agar with containing (−)-1
at four concentrations: 0, 3.12, 6.25, and 12.5 μM as described above
for ARC516. Two colonies were selected from the 3.12 μM plate and
recovered on drug-free plates frozen in glycerol stocks. Genomic DNA
was prepared these isolated resistant variants and found to be identical
to ARC516 except for two different point mutations found in the gyrB
†For A.B.: Theravance Biopharma Inc., 901 Gateway Boulevard,
South San Francisco, California 94080, United States.
‡For H.B.H.: TASC, 8211 Terminal Road, Suite 1000, Lorton,
Virginia 22079, United States.
§For J.M.: Shire, 300 Shire Way, Lexington, Massachusetts
02451, United States.
∥For V.J.A.S.: Novartis Pharmaceuticals Corporation, Building
335, Office 3104B, One Health Plaza, East Hanover, New
Jersey 07936−1080, United States.
⊥For M.G.: Organix, Inc., 240 Salem Street, Woburn,
Massachusetts 01801, United States.
Notes
The authors declare the following competing financial
interest(s): The authors are or have been previously employees
of AstraZeneca.
ACKNOWLEDGMENTS
■
We thank the following individuals from AstraZeneca for their
valuable technical contributions and helpful advice: Natascha
Bezdenejnih, Nancy DeGrace, Dr. Camil Joubran, and Sharon
Tentarelli. Dedong Wu coordinated the small molecule X-ray
crystallography work. We also acknowledge contributions for
P
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Article
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ABBREVIATIONS USED
■
CDI, carbonyl dimidazole; CFU, colony-forming unit; Cl,
clearance; F, oral bioavailability; fu, fraction unbound; DIEA,
diisopropylaminoethylamine; GyrA, A-subunit of DNA gyrase;
GyrB, B-subunit of DNA gyrase; MRQR, methicillin resistant,
quinolone resistant S. aureus; MSSA, methicillin sensitive S.
aureus; NMM, N-methylmorpholine; ParC, C-subunit of
topoisomerase IV; ParE, E-subunit of topoisomerase IV; PPB,
plasma protein binding; SNAr, nucleophilic aromatic substitu-
tion; tolC, gene encoding outer membrane transport channel;
TBDPS, tert-butyldimphenylsilyl; TK, toxicokinetics
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