Syntheses and antibacterial activity studies of new oxazolidinones from nitroso Diels-Alder chemistry

Article abstract of DOI:10.1016/j.bmcl.2009.10.018

A series of novel oxazolidinone antibiotics having [2.2.1] and [2.2.2] bicyclic oxazine moieties at the C-5 side chain of the A-ring was synthesized by nitroso Diels-Alder reactions, from three linezolid analogs containing morpholine, piperazine and thiomorpholine, respectively, as the C-ring components. Subsequent N-O bond cleavage generated oxazolidinones with 4-amino cyclo-2-en-1-ol substituents. The in vitro antibacterial activities of these oxazolidinone analogs were evaluated.

Full text of DOI:10.1016/j.bmcl.2009.10.018

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1302–1305
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and antibacterial activity studies of new oxazolidinones from nitroso
Diels–Alder chemistry
a
b
Shanshan Yan a, Marvin J. Miller a, , Timothy A. Wencewicz , Ute Möllmann
*
a Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
b Leibniz Institute for Natural Product Research and Infection Biology—Hans Knöll Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel oxazolidinone antibiotics having [2.2.1] and [2.2.2] bicyclic oxazine moieties at the C-5
side chain of the A-ring was synthesized by nitroso Diels–Alder reactions, from three linezolid analogs
containing morpholine, piperazine and thiomorpholine, respectively, as the C-ring components. Subse-
quent N–O bond cleavage generated oxazolidinones with 4-amino cyclo-2-en-1-ol substituents. The
in vitro antibacterial activities of these oxazolidinone analogs were evaluated.
Received 29 August 2009
Revised 2 October 2009
Accepted 5 October 2009
Available online 8 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Oxazolidinone antibiotic analogs
Nitroso cycloaddition
Oxazolidinones are a new class of synthetic antimicrobial
agents which are now clinically useful. They inhibit bacterial pro-
tein synthesis by binding to the 50S ribosomal subunit at the trans-
lation step.1 The most promising feature of these compounds is
their oral activity against multidrug-resistant Gram-positive bacte-
ria, including methicillin-resistant Staphylococcus aureus (MRSA) as
well as select anaerobic organisms.2 Linezolid (Fig. 1) is the first
oxazolidinone drug approved for the treatment of Gram-positive
bacterial infections in humans.2b,3 Unfortunately, resistance to lin-
ezolid has already been observed in Gram-positive bacteria such as
S. aureus and Enterococcus faecium.4 More recently, resistant strains
of MRSA, E. coli and other bacteria have also been identified.5 Thus,
there is significant need for the rapid development of novel anti-
bacterial agents. Four types of chemical modifications of linezolid
and oxazolidinone-type antibiotics have been reported, including
modifications on each of the A-, B- and C-rings as well as the C-5
side chain of the A-ring substructure.2b,6 The structure–activity-
relationship studies of oxazolidinone antibiotics show that the ste-
reochemistry (S) at the C-5 position of the A-ring is critical in terms
of biological activity. While an acetamide substituent on the 5-
methyl group is found to produce optimal antibacterial activity,
modification of the C-5 side chain would still benefit the develop-
ment of new oxazolidinone antibiotics. In this regard, a frequently
employed strategy involves the addition of heterocycles, such as
triazoles,7 and pyridones.8 However, to the best of our knowledge,
the incorporation of bicyclic oxazines into this class of antibiotics
has not been disclosed.
Syntheses of interesting biologically relevant molecules using
nitroso Diels–Alder chemistry (NDA) is of growing interest. As a
synthetic tool for the formation of 4-amino cyclopent-2-en-1-ol
derivatives in one step, acylnitroso Diels–Alder reactions are useful
for the creation of unique structural and functional diversity.9 We
and others have demonstrated that the nitroso bicyclic oxazine 3
derived from NDA reaction between in situ generated acylnitroso
2 and 1,3-cyclopentadiene can undergo various chemical transfor-
mations (Scheme 1).9b We envisioned that modification at the A-
ring C-5 side chain of linezolid-type oxazolidinone antibiotics
using nitroso Diels–Alder chemistry would generate novel analogs
with incorporation of bicyclic oxazine functionalities. These new
O
C
N
O
B
O
A
F
N
H
H
N
5
1
O
C-5 side chain
Figure 1. Linezolid antibiotic (1).
O
O
O
[O]
N
O
R
OH
O
R
N
H
NDA
R
N
3
2
* Corresponding author.
E-mail address: mmiller1@nd.edu (M.J. Miller).
Scheme 1. Acylnitroso Diels–Alder (NDA) reaction.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.018

S. Yan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1302–1305
1303
X
X
X
N
F
N
F
e
c or d
NO2
O
b
O
N
F
X
NH
O
N
N
H
OBn
4: X = NH
OH
a
14: X = NBoc
15: X = O
8: X = NBoc
9: X = O
11: X = NBoc
12: X = O
5: X = NBoc
6: X = O
16: X = S
10: X = S
13: X = S
7: X = S
Scheme 2. Reagents and conditions: (a) Boc2O, DCM, 50%; (b) 3,4-difluoronitrobenzene, Hunig’s base, CH3CN, 49% (for 8), 84% (for 9), 70% (for 10); (c) (i) Pd/C, H2, MeOH, (ii)
CbzCl, NaHCO3, THF/H2O, 89% (for 11), 92% (for 12); (d) (i) In, NH4Cl, EtOH, reflux, (ii) CbzCl, NaHCO3, THF/H2O, 62% (for 13); (e) (i) nBuLi, THF, À78 °C, (ii) R-glycidyl butyrate,
71% (for 14), 70% (for 15), 80% (for 16).
analogs were anticipated to not only enhance the available set of
oxazolidinone antibiotics, but also provide new scaffolds for fur-
ther transformations. Herein we report the syntheses and biologi-
cal evaluation of a series of oxazolidinone antibiotics in which the
C-5 side chain has been modified by nitroso Diels–Alder reactions
and subsequent chemical transformations.
Diels–Alder reactions without further purification. Toward this
end, N-hydroxy carbamate 20 was chosen as the model substrate.
The cycloaddition reaction was carried out by in situ oxidation of
20 in the presence of 1,3-cyclohexadiene. Among several oxidants
attempted, including sodium periodate (NaIO4), Dess–Martin and
FeCl3/H2O2,12 we found that use of sodium periodate gave cycload-
duct 23b in the best yield (56%) as one regioisomer after chromatog-
raphy purification. For the thiomorpholine series, in order to again
avoid catalyst poisoning during the subsequent deprotection
step, O-p-methoxybenzyl hydroxylamine hydrochloride (PMBO
NH2ÁHCl)13 instead of OBHAÁHCl was used in the coupling reaction
with 16. The PMBgroup was removedunder acidic conditionsto give
N-hydroxy carbamate 22 in good yield. Using the optimized NDA
reaction conditions, a series of new oxazolidinones 23a–25b with
bicyclic oxazines at the C-5 side chain were synthesized. Among
them, cycloadducts 25a and 25b were obtained with the sulfur atom
in the thiomorpholinyl ring oxidized. N–O bond cleavage reactions
of these cycloadducts were also carried out, with intent to generate
the corresponding oxazolidinones with additional 1,4-amino alco-
To begin our investigation, three types of oxazolidinone precur-
sors 14–16 were synthesized from commercial piperazine (4), mor-
pholine (6) and thiomorpholine (7), respectively, as shown in
Scheme 2. The syntheses began by separate SNAr reactions of 3,4-
difluoronitrobenzene with mono Boc-protected piperazine, 5, mor-
pholine, 6, and thiomorpholine, 7, under basic conditions to afford
para-substituted nitrobenzene derivatives 8–10 in moderate to
good yields. Palladium catalyzed hydrogenolyses of 8 and 9, fol-
lowed by reactions with CbzCl gave protected anilines 11–12. The
synthesis of thiomorpholine analog 13 required modification to
avoid poisoning of the catalyst by the sulfur atom during the hydro-
genation step. Thus, use of indium powder and ammonium chlo-
ride10 successfully reduced the nitro group of 10 and gave
compound 13 in 62% yield after Cbz protection. Then, as reported,11
compounds 11–13 were treated with nBuLi and R-glycidyl butyrate
sequentially at –78 °C to form oxazolidinone analogs 14–16 in 70–
80% yields with defined stereochemistry at the A-ring C-5 position.
With the desired aza-, oxa-, and thia-analogs, 14–16, in hand,
introduction of the N-hydroxy carbamate moiety at the C-5 side
chain and subsequent nitroso Diels–Alder reactions were explored
(Scheme 3). Reactions of 14 and 15 with O-benzylhydroxylamine
hydrochloride (OBHAÁHCl) and carbonyldiimidazole (CDI) gener-
ated protected N-hydroxy carbamates 17 and 18 in 96% and 94%
yields, respectively. Hydrogenolyses afforded the desired free N-hy-
droxy carbamates, 20 and 21, which were directly used in nitroso
14
hol substituents. Thus, Mo(CO)6 successfully reduced the N–O
bond of compounds 23a–25b to generate the corresponding oxazo-
lidinone analogs, 26a–28b, that contain aminocycloalkenol func-
O
O
N
F
N
F
O
a
O
O
N
O
N
15
OH
29
OAc
Scheme 4. Reagents and conditions: (a) Ac2O, pyridine, DMAP, DCM, 75%.
X
X
X
N
N
F
N
F
O
O
O
a or b
c or d
O
N
O
N
O
F
N
H
N
NHOR
O
OH
O
OH
14: X = NBoc
15: X = O
20: X = NBoc
21: X = O
17: X = NBoc, R = Bn
18: X = O, R = Bn
O
O
16: X = S
22: X = S
19: X = S, R = PMB
X
X
N
N
O
e or f
O
g
( )n
O
F
N
O
F
N
H
N
( )n
O
N
OH
O
26a: X = NBoc, n = 1
26b: X = NBoc, n = 2
27a: X = O, n = 1
27b: X = O, n = 2
28a: X = S, n = 1
28b: X = S, n = 2
23a: X = NBoc, n = 1
23b: X = NBoc, n = 2
24a: X = O, n = 1
O
O
O
24b: X = O, n = 2
25a: X = S=O, n = 1
25b: X = S=O, n = 2
Scheme 3. Reagents and conditions: (a) (i) CDI, CH3CN (ii) OBHAÁHCl, imidazole, 96% (for 17), 94% (for 18); (b) CDI, CH3CN, (ii) PMBONH2ÁHCl, imidazole, 92% (for 19); (c) Pd/
C, H2, MeOH; (d) (i) 10% TFA, DCM, (ii) satd Na2CO3; (e) NaIO4, 1,3-cyclopentadiene, MeOH/H2O, 0 °C, 50% (for 23a, from 17), 49% (for 24a, from 18), 40% (for 25a, from 19); (e)
NaIO4, 1,3-cyclopentadiene, MeOH/H2O, 0 °C, 50% (for 23a, from 17), 49% (for 24a, from 18), 40% (25a, from 19); (f) NaIO4, 1,3-cyclohexadiene, MeOH/H2O, 0 °C, 56% (for 23b,
from 17), 47% (for 24b, from 18), 42% (25b, from 19); (g) Mo(CO)6, CH3CN/H2O, 80 °C, 50% (for 26a), 43% (for 26b), 42% (for 27a), 62% (for 27b), 54% (for 28a), 55% (for 28b).

1304
S. Yan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1302–1305
Table 1
Antimicrobial activity in the agar diffusion assay (diameter of inhibition zone, measured in mm)
Compds
Gram-positive bacteria
Gram-negative bacteria
Mycobacterium vaccae
Bacillus subtilis Micrococcus luteus Staphylococcus aureus
Enterococcus faecalis
Escherichia coli Pseudomonas aeruginosa
ATCC 6633
ATCC 10240
SG 511a 134/93 (MRSA) ATCC 49532 1528 (VRE) ATCC 25922
K799/WTa
K799/61b
IMET 10670
1
29
14
15
31/35P
26.5
38
27/36P NT
26/35P NT
24/33P NT
23/32P NT
24/35P NT
13/19p 19
25
24
21
21
21.5
19
NT
NT
NT
19
NT
NT
NT
NT
NT
16
12P
19
16
NT
NT
NT
NT
12P
12P
NT
NT
15
0
0
0
0
0
0
NT
NT
NT
0
0
0
0
NT
NT
0
0
0
0
0
10P
17h
10P
11P
11P
0
0
0
0
11P
10P
10P
12P
0
42
43
NT
43
43
19p
13
30
15
35
18
12h
0
38/46p
34.5
38
41
16
NT
NT
NT
22/32P
15p
12P
0
NT
NT
15h
14
0
23/26P
26/29P
26/29P
12/18P
18P
17/30P
19P
21/28P
17P
13h
0
14P
13P
16
0
23a
23b
24a
24b
25a
25b
26a
26b
27a
27b
28a
28b
Cipro
14/20p
NT
NT
NT
0
0
0
0
NT
NT
0
0
35
16P
14p
15/21p 25
16P
18p
18/22p NT
15p
14p
0
15P
14P
12.5p
0
NT
NT
NT
14p
14p
NT
NT
0
17
12.5
12p
NT
NT
12.5
12
18
0
18
0
0
14P
12h
29.5
10P
10P
39
0
24
13
31/40P
19
p, partially clear inhibition zone/colonies in the inhibition zone.
P, unclear inhibition zone/many colonies in the inhibition zone.
h, faint indication of inhibition zone.
Exactly 50
lL of a 2.0 mM solution of each compound dissolved in 1:9 DMSO/MeOH was filled in 9 mm wells in agar media (Standard I Nutrient Agar, Serva or Mueller Hinton
II Agar, Becton, Dickinson and Company). Inhibition zones read after incubation at 37 °C for 24 h. Cipro (ciprofloxacin) was dissolved in H2O to give 5
lg/mL solution.
a
Wild type.
b
Permeability mutant.
tional groups, in moderate yields. In addition, under Mo(CO)6 condi-
tions, the sulfoxide group of 25a and 25b was also reduced. For com-
parison during the biological testing, an oxazolidinone analog 29
with a C-5 acetate substituent was synthesized from compound 15
(Scheme 4).
bacterial assays. We also thank the Lizzadro Magnetic Resonance
Research Center at Notre Dame for NMR facility and Nonka Sevova
for mass spectroscopic analyses. TAW acknowledges the University
of Notre Dame Chemistry–Biochemistry–Biology Interface (CBBI)
Program and NIH Training Grant T32GM075762 for a fellowship.
With the biological profile of parent linezolid, 1, known for
comparison, all the oxazolidinone analogs 23a–28b, 29, as well
as precursors 14–16, and ciprofloxacin, as a positive control, were
subjected to broad antibacterial studies against various strains of
Gram-positive and Gram-negative bacteria as well as Mycobacte-
rium vaccae, using agar diffusion assays (Table 1).15 Compound
29 was found to be comparable to linezolid (1) itself for all tested
organisms, as expected. Interestingly, all the oxazolidinone precur-
sors 14–16 were roughly equipotent in vitro with linezolid (1)
against several Gram-positive organisms, including Bacillus subtilis,
S. aureus, E. faecalis and M. luteus. They also exhibited antimycobac-
terial activity and could potentially be useful for treatment of M.
tuberculosis as they induced large inhibition zones against M. vac-
cae, a common model for M. tuberculosis. Most oxazolidinone ana-
logs with bicyclic oxazine or aminocycloalkenol moieties at the C-5
side chain, generated from NDA chemistry, had antimicrobial pro-
files similar to linezolid (1), but at a generally diminished level of
activity. In general, oxazolidinone analogs substituted with
[2.2.1] bicyclic oxazines (23a–25a) were more active than those
with [2.2.2] bicyclic oxazines (23b–25b). Among them, compound
25a derived from thiomorpholine exhibited the best activity. Ana-
logs 26a–28b, derived from reduction of the N–O bonds were rel-
atively inactive compared to their parent cycloadducts.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.10.018.
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In summary, we have synthesized a series of novel oxazolidi-
none antibiotics with [2.2.1] and [2.2.2] bicyclic oxazine as well
as aminocycloalkenol moieties at the C-5 side chain through nitro-
so Diels–Alder chemistry. These oxazolidinone analogs exhibited
in vitro antibacterial profiles similar to that of linezolid.
Acknowledgments
We gratefully acknowledge the NIH(GM 075855) for support of
this research. We thank Uta Wohlfeld (HKI) for performing anti-

S. Yan et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1302–1305
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