A distal methyl substituent attenuates mitochondrial protein synthesis inhibition in oxazolidinone antibacterials

Article abstract of DOI:10.1016/j.bmcl.2007.07.022

Oxazolidinone analogs bearing substituted piperidine or azetidine C-rings are described. Analogs with a methyl group at the 3-position of the azetidine ring or the 4-position of the piperidine ring exhibited reduced mitochondrial protein synthesis inhibition while retaining good antibacterial potency.

Full text of DOI:10.1016/j.bmcl.2007.07.022

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5036–5040
A distal methyl substituent attenuates mitochondrial
protein synthesis inhibition in oxazolidinone antibacterials
Adam R. Renslo,a,* Andy Atuegbu,a Prudencio Herradura,a Priyadarshini Jaishankar,a
Mingzhe Ji,a Karen L. Leach,b Michael D. Huband,b Michael R. Dermyer,b Luping Wu,b
J. V. N. Vara Prasadb and Mikhail F. Gordeeva
aPfizer Global Research and Development, 34790 Ardentech Ct., Fremont, CA 94555, USA
bPfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, MI 48105, USA
Received 6 June 2007; revised 3 July 2007; accepted 6 July 2007
Available online 13 July 2007
Abstract—Oxazolidinone analogs bearing substituted piperidine or azetidine C-rings are described. Analogs with a methyl group at
the 3-position of the azetidine ring or the 4-position of the piperidine ring exhibited reduced mitochondrial protein synthesis inhi-
bition while retaining good antibacterial potency.
Ó 2007 Elsevier Ltd. All rights reserved.
Linezolid (1) is the first member of the oxazolidinone class
of antibacterial protein synthesis inhibitors to reach the
market.1 The oxazolidinones bind to 23S rRNA in the
50S subunit of the bacterial ribosome, near the peptidyl
transferase center.2 Showing no significant cross-resis-
tance with the existing antibacterial classes, linezolid has
become an important addition to the clinician’s arma-
mentarium. Although linezolid is generally well tolerated,
prolonged courses of therapy are sometimes complicated
by reversible myelosuppression that can require cessation
of treatment. It has been suggested3 that mitochondrial
protein synthesis (MPS) inhibition is responsible for this
effect and in a recent report4 the oxazolidinone eperezolid
was shown to slow the proliferation of various mamma-
lian cell lines via inhibition of MPS.
The identification of oxazolidinones with reduced MPS
inhibitory activity could expand the utility of the class
to include the treatment of deep-seated infections that
require extended courses of therapy. In connection with
this objective, we explored a series of oxazolidinone ana-
logs bearing hydroxy-azetidine or hydroxy-piperidine
C-rings (e.g., 2). These heterocycles bear an obvious
resemblance to the morpholine C-ring of linezolid but
offer the option to introduce an additional substituent
in proximity to the oxygen atom (i.e., R in 2). In the
course of our investigations, we made the unexpected
and surprising observation that MPS inhibition is signif-
icantly affected by the nature of the alkyl substituent in
these analogs. In particular, MPS inhibition was attenu-
ated in the case where, R = methyl and furthermore, this
modification only marginally affected antibacterial po-
tency. Herein, we describe the synthesis and biological
properties of a series of substituted azetidine and piper-
idine-containing oxazolidinone analogs.
R
F
O
F
HO
C N
N
F
O
O
NA O
B
N
O
1 linezolid
2
The preparation of the azetidine and piperidine analogs
followed standard synthetic protocols for oxazolidinone
synthesis5 and is summarized in Schemes 1–5. Scheme 1
illustrates the synthesis of azetidine analogs 6a–c bearing
a single substituent (OH, OMe, or F, respectively) in the
3-position of the azetidine ring. Hydrogenolysis of the
known azetidine 36 was followed by a nucleophilic aro-
matic substitution reaction with 3,4,5-trifluoronitroben-
zene to yield 4a. Alcohol 4a could then be converted to
the methyl ether 4b or the fluoro azetidine 4c. Reduction
NHAc
NHAc
Keywords: Antibacterials; Oxazolidinones; Selectivity; Mitochondrial
protein synthesis inhibition; Azetidines; Piperidines.
*
Corresponding author at present address: Department of Pharma-
ceutical Chemistry, University of California, San Francisco, USA.
E-mail: adam.renslo@ucsf.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.022

A. R. Renslo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5036–5040
5037
X
O
F
F
Me3SiO
a, b
N
F
4a
4b
4c
O
a, b
N
F
NBn
NBoc
14
NO2
R
3
X = OH
X = OMe
X = F
c
15
16
R = NO2
R = NHCbz
d
c, d
e-g
e, f, or g
R
HO
R
HO
X
N
X
F
F
F
F
N
F
N
N
F
h,i
O
O
h, i, or j
F
N
NHCbz
5a-c
F
NHCbz
N
O
O
6a X = OH
6b X = OMe
6c X = F
17a R = H
17b R = Me
17c R = Et
18a-c
NHAc
NHAc
Scheme 1. Reagents and conditions: (a) H2, Pd(OH)2/C, MeOH; (b)
3,4,5-trifluoronitrobenzene, DIEA, DMF, 45 °C, 95% for two steps; (c)
NaH, MeI, DMF, 94%; (d) DAST, CH2Cl2, À78 °C to rt; 72%; (e) Fe,
NH4Cl, EtOH, H2O, 80 °C; (f) CbzCl, pyridine, CH2Cl2, 50–90% for
two steps; (g) TBS–Cl, Et3N, DMF (for 5a only), 98%; (h) t-BuOLi,
(S)-ClCH2CH(OAc)CH2NHAc, DMF, 50–70%; (i) Et3N–HF, THF,
76% (for 6a only).
Scheme 3. Reagents and conditions: (a) TFA, ClCH2CH2Cl; (b) 3,4,5-
trifluoronitrobenzene, DIEA, DMF, 50 °C, 12 h, 90% for two steps; (c)
H2 10% Pd/C, EtOAc, 18 h; (d) CbzCl, pyridine, CH2Cl2, 23 °C, 1 h,
80% for two steps; (e) DIBAL, CH2Cl2, À78 °C, 80% then TBSCl,
Et3N, DMF, 23 °C, 15 h, 52%; (for 17a); (f) MeMgBr, THF, À78 °C,
79% (for 17b); (g) EtMgBr, THF, À78 °C, 61% (for 17c); (h) 3.0 equiv
LiOt-Bu, MeOH, THF, DMF, 2.0 equiv (S)-ClCH2CH(OAc)CH2N-
HAc, then HF–Et3N, THF, 23 °C, 27% for two steps (for 18a);
(i)
3.0 equiv
LiOt-Bu,
MeOH,
THF,
DMF,
2.0 equiv
of the nitro group in 4a–c was followed by protection of
the resulting amine as a benzyl carbamate (Cbz) to yield
5a–c (in the case of 5a, the hydroxyl was protected as a
tert-butyldimethylsilyl ether). Finally, the oxazolidinone
ring was installed by reaction with lithium tert-butoxide
and (1S)-2-(acetylamino)-1-(chloromethyl)ethyl acetate
according to the established procedure5 to provide 6b
and 6c. For 6a, a final deprotection step was required
(HF–Et3N).
(S)-ClCH2CH(OAc)CH2NHAc, 44% (for 18b); (j) 2.5 equiv LiOt-Bu,
1.3 equiv (S)-ClCH2CH(OH)CH2NHBoc, DMF, 20 h then 4 M HCl/
dioxane; then Ac2O, Et3N, CH2Cl2, 17 h, 34% over three steps.
Trifluoromethyl azetidine intermediate 9 was prepared
in two steps from 4a via oxidation to the azetidinone fol-
lowed by reaction with trimethylsilyl trifluoromethane.7
Compounds 8b and 9 were converted to the oxazolidi-
nones 12 and 13 as described above for the preparation
of 6a–c.
Scheme 2 illustrates the synthesis of analogs 11a–c, 12,
and 13 in which a 3-alkyl substituent has been intro-
duced adjacent to the hydroxy, methoxy, or fluoro sub-
stituents of the azetidine ring. The 3-methyl and 3-ethyl
intermediates 8a and 8b were prepared by reaction of the
appropriate Grignard reagent with the azetidinone 7
which was prepared in three steps from 4a. Intermediate
8a was converted to 11a–c in a manner analogous to
that described above for the synthesis of 6a–c from 4a.
The synthesis of piperidine-containing oxazolidinones is
described in Schemes 3–5. The preparation of analogs
18a–c originated with the commercially available piperi-
done 14, which was converted in two steps to the nitro-
aniline 15. Reduction of the nitro function and
conversion of the resulting amine to a benzyl carbamate
provided 16. At this stage, the ketone was reduced with
R
F
HO
O
F
F
HO
N
F
a-c
N
F
N
d
NO2
NHCbz
F
NHCbz
8a
8b
R = Me
R = Et
4a
7
a, g
e, f
R
X
F3C
HO
F3C
HO
F
F
F
N
N
F
N
F
b, c
f
O
F
N
NHCbz
NO2
O
9
10
NHAc
12 (R = Et) 13 (R = CF3)
R = Me
11a X = OH
11b X = OMe
11c X = F
Scheme 2. Reagents and conditions: (a) Swern, 65–80%; (b) H2, Pd/C, EtOAc; (c) CbzCl, pyridine, CH2Cl2, 75% for two steps; (d) MeMgBr or
EtMgBr, THF, 0 °C, 75%; (e) DAST, CH2Cl2, À78 °C to rt, 30% (for 11c only); (f) LiOt-Bu, MeOH, THF, DMF, (S)-ClCH2CH(OAc)CH2NHAc,
30–60%; (g) CF3–SiMe3, TBAF, THF, 16%.

5038
A. R. Renslo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5036–5040
sequence of reactions analogous to those used for the
synthesis of 18 from 14.
CN
O
a, b
NBoc
NBoc
19
c,d
The 4-alkyl analog 27 was prepared using a different
synthetic route starting with the commercially available
amino ester 23 (Scheme 5). Following Boc-protection of
the amine function in 23, the 4-methyl substituent was
introduced by reaction with LDA and iodomethane. Re-
moval of the Boc group (affording 24) was followed by
coupling to 3,4,5-trifluoronitrobenzene as before. The
methyl ester was then reduced and the resulting alcohol
converted to the mesylate 25. The oxazolidinone ring
was installed, and in a final step, the mesylate group
was displaced with cyanide to furnish the desired
4-methyl analog 27.
14
CN
CN
F
F
N
g-i
N
F
O
F
N
R
O
20
21
R = NO2
R = NHCbz
22
e, f
NHAc
Scheme 4. Reagents and conditions: (a) diethyl(cyanomethyl) phos-
phonate, LiBr, Et3N, THF, 23 °C, 8 h, 99%; (b) H2, 10% Pd/C, MeOH,
23 °C, 20 h, 98%; (c) 4.0 M HCl/dioxane, 23 °C, 25 h, 100%; (d) 3,4,5-
trifluoronitrobenzene, DIEA, DMF, 70 °C, 82%; (e) Fe, NH4Cl,
EtOH, H2O, 95 °C, 4 h, 87%; (f) CbzCl, pyridine, CH2Cl2, 0–23 °C,
65%; (g) 2.5 equiv LiOt-Bu, 1.3 equiv (S)-ClCH2CH(OH)CH2NHBoc,
DMF, 20 h; (h) 20% TFA in CH2Cl2, 0–23 °C, 3 h; (i) Ac2O, pyridine,
CH2Cl2, 23 °C, 17 h, 38% over three steps.
We evaluated the antibacterial activities of the new oxa-
zolidinone analogs by determining MIC90 values against
11 strains each of Staphylococcus aureus, Streptococcus
pneumoniae, and Enterococcus faecalis using standard
broth microdilution assay methods.9 An in vitro Esche-
richia coli transcription and translation (TnT) assay10
was used to provide a measure of intrinsic binding to
bacterial ribosomes. Inhibition of MPS was assessed
using an assay that measures [35S]methionine incorpora-
tion into mitochondrial proteins.4
MeO2C
MeO2C
a-c
NH
NH•HCl
24
23
d-f
CN
Table 1 summarizes the data for azetidine analogs 6a–c
and the corresponding 3-alkyl substituted derivatives
11a–c, 12, and 13. These analogs displayed antibacterial
potencies similar and in some cases superior to those of
linezolid. The hydroxy-, methoxy-, and fluoro-azetidine
analogs 6a–c had identical MIC90 values against the
three bacterial organisms examined, and were twofold
more potent than linezolid against S. aureus and E. fae-
calis strains. The eight azetidine analogs in Table 1
exhibited very similar activities in the TnT assay, sug-
gesting comparable intrinsic binding affinities to the
ribosome target. Antibacterial activities were minimally
impacted by the introduction of a 3-alkyl substituent in
the azetidine ring; the alkylated analogs were equipotent
or at most one dilution less potent than the correspond-
ing unsubstituted analogs (cf. 6a–c vs. 11a–c).
MsO
g,h
F
F
N
N
F
i-l
O
F
N
R
O
25
26
R = NO2
R = NHCbz
27
NHAc
Scheme 5. Reagents and conditions: (a) Boc2O, THF, DIEA, 65 °C,
17 h, quant.; (b) LDA, MeI, THF, À78 to 23 °C, 25 h, 70%; (c) 4.0 M
HCl/dioxane, 23 °C, 25 h, 93%; (d) 3,4,5-trifluoronitrobenzene, DIEA,
DMF, 70 °C, 85%; (e) CaCl2, NaBH4, EtOH, 0–50 °C, 6 h, 94%; (f)
MsCl, Et3N, CH2Cl2, 0–23 °C, 20 h, 71%; (g) H2, 10% Pd/C, EtOAc,
23 °C, 20 h; (h) CbzCl, pyridine, CH2Cl2, 23 °C, 90% over two steps; (i)
2.5 equiv LiOt-Bu, 1.3 equiv (S)-ClCH2CH(OH)CH2NHBoc, 0–23 °C,
17 h, 51%; (j) 20% TFA in CH2Cl2, 0–23 °C, 6 h; (k) Ac2O, pyridine,
CH2Cl2, 23 °C, 17 h; (l) KCN, DMSO, 80 °C, 20 h, 50% over three
steps.
The MPS inhibitory activity of analogs 6a (hydroxy)
and 6c (fluoro) was within the range of values obtained
for linezolid while the methoxy analog 6b was a some-
what more potent inhibitor of MPS. Interestingly, the
introduction of a 3-methyl substituent had a significant
effect on MPS inhibition. Hence, the 3-methyl analogs
11a and 11c had more than threefold higher IC50 values
than the des-methyl comparators 6a and 6c. A more dra-
matic effect was observed in the case of methoxy analogs
6b and 11b, with 3-methyl-3-methoxy analog 11b exhib-
iting a 15- to 30-fold reduction in MPS inhibition. The
3-ethyl and 3-trifluoromethyl analogs (12 and 13) were
prepared to probe steric and electronic effects of the
3-alkyl substituent. Surprisingly, neither of these ana-
logs differed significantly from the parent des-alkyl ana-
log 6a in the MPS assay. Hence, from this limited survey
of 3-alkyl substituents, it appears that a simple methyl
group has the most favorable attenuating effect on
MPS inhibition.
DIBAL to provide 17a, or, alternatively, reacted with
Grignard reagents to provide the 4-methyl and 4-ethyl
intermediates 17b and 17c, respectively. The oxazolidi-
none pharmacophore was then installed as before5 to
provide analogs 18a–c.
Recently, it was disclosed that piperidine-containing
oxazolidinone analogs bearing a 4-cyanomethyl substi-
tuent exhibit excellent in vitro and in vivo properties.8
We therefore targeted the cyanomethyl analog 22 along
with the corresponding 4-methyl analog 27, in order to
evaluate the effect of a 4-alkyl substituent in this series.
The synthesis of 22 began with a Horner–Wadsworth–
Emmons reaction between piperidone 14 and
diethyl(cyanomethyl)phosphonate (Scheme 4). Hydro-
genation of the resulting cyanoacrylate then provided
intermediate 19, which was carried on to 22 using a

A. R. Renslo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5036–5040
5039
Table 1. Mitochondrial protein synthesis inhibition (IC50, lM), E. coli in vitro transcription and translation assay (IC50, lM), and antimicrobial
activity (MIC90, lg/mL) of azetidine-containing oxazolidinones
R
F
X
N
O
F
N
O
NHAc
Compound
R
X
MPS IC50 (lM)
EC TnT IC50 (lM)
MIC90 S.a.
MIC90 S.p.
MIC90 E.f.
1
6a
—
H
—
11–26
18
68
4
2
4
4
4
1
1
1
2
2
4
2
4
2
2
OH
OH
OH
OH
1.9
3.4
2.7
2.4
11a
12
Me
Et
15–34
19
13
CF3
6b
11b
H
Me
OMe
OMe
4
65–112
2.6
3.7
2
4
1
1
2
4
6c
11c
H
Me
F
F
13
42
1.4
2.3
2
4
1
1
2
2
S.a., Staphylococcus aureus; S.p., Streptococcus pneumoniae; E.f. Enterococcus faecalis.
Table 2. Mitochondrial protein synthesis inhibition (IC50, lM), E. coli in vitro transcription and translation assay (IC50, lM), and antimicrobial
activity (MIC90, lg/mL) of piperidine-containing oxazolidinones
R
F
X
N
F
O
N
O
NHAc
Compound
R
X
MPS IC50 (lM)
EC TnT IC50 (lM)
MIC90 S.a.
MIC90 S.p.
MIC90 E.f.
1
—
H
—
11–26
11
4
2
4
4
1
1
1
1
4
2
2
2
18a
18b
18c
OH
OH
OH
1.7
2.1
2.1
Me
Et
34–56
13–23
22
27
H
Me
CH2CN
CH2CN
6
26
1.6
nt
2
2
1
1
1
1
S.a., Staphylococcus aureus; S.p., Streptococcus pneumoniae; E.f., Enterococcus faecalis; nt, not tested.
The antibacterial, TnT, and MPS inhibitory activities of
piperidine-containing oxazolidinones are presented in
Table 2. The larger piperidine ring places the distal sub-
stituents (i.e., R and X) further from the aromatic B-ring
than is the case in the azetidine series. For this reason, it
was unclear whether the effects observed for the
3-methyl azetidine analogs would hold also for 4-methyl
piperidine analogs. In fact, a similar trend was observed.
All of the piperidine analogs exhibited comparable IC50
values in the TnT assay. Likewise, antibacterial activities
(MIC90) were minimally affected by the introduction of
4-methyl or 4-ethyl substituents (cf. 18a vs. 18b–c and
22 vs. 27). In the MPS assay, however, 4-methyl analogs
18b and 27 exhibited between three and fivefold lower
levels of inhibition. The effect was again most significant
with a methyl substituent; the 4-ethyl analog 18c was
only marginally less active than des-alkyl analog 18a.
To further establish the significance of these effects, we
evaluated analogs such as 11a and 18b in two different
cell proliferation assays (data not shown). The trends
observed in the MPS assay were also apparent in the
cell-based assays, with methyl substituents conferring a
favorable effect.
The data presented here suggest that subtle structural
modifications far removed from the oxazolidinone phar-
macophore can significantly impact MPS inhibition in
oxazolidinone antibacterials. Importantly, attenuation
of MPS inhibition can be achieved with little or no im-
pact on the desired (antibacterial) bioactivity. At least
four compounds presented in the current work exhibit
significantly reduced MPS inhibition as compared to lin-
ezolid. These findings are suggestive of structural differ-
ences between the bacterial and mitochondrial
ribosomes that might be exploited to design safer and
more selective oxazolidinones.
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