Discovery of isoxazolinone antibacterial agents. Nitrogen as a replacement for the stereogenic center found in oxazolidinone antibacterials

Article abstract of DOI:10.1016/j.bmcl.2004.06.076

A series of potential antimicrobial derivatives possessing bioisosteric replacements for the central oxazolidinone ring found in oxazolidinone antibacterials have been prepared. The design concept involved replacement of the requisite sp³-hybridized stereogenic center found at the 5-position of the oxazolidinone with a nitrogen atom. The synthesis and antibacterial activity of three such ring systems, the benzisoxazolinones, pyrroles, and isoxazolinones is described.

Full text of DOI:10.1016/j.bmcl.2004.06.076

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4735–4739
Discovery of isoxazolinone antibacterial agents. Nitrogen as
a replacement for the stereogenic center found in
oxazolidinone antibacterials
a
a
a
Lawrence B. Snyder,a, Zhaoxing Meng, Robert Mate, Stanley V. DÕAndrea,
*
Anne Marinier,b Claude A. Quesnelle,b Patrice Gill,b Kenneth L. DenBleyker,a
Joan C. Fung-Tomc,a MaryBeth Frosco,a Alain Martel,b John F. Barretta and
Joanne J. Bronsona
aBristol-Myers Squibb Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492, USA
b
´
Bristol-Myers Squibb Pharmaceutical Research Institute, 100, boul. de lÕIndustrie, Candiac, Quebec, Canada J5R 1J1
Received 29 April 2004; revised 21 June 2004; accepted 23 June 2004
Available online 30 July 2004
Abstract—A series of potential antimicrobial derivatives possessing bioisosteric replacements for the central oxazolidinone ring
found in oxazolidinone antibacterials have been prepared. The design concept involved replacement of the requisite sp3-hybridized
stereogenic center found at the 5-position of the oxazolidinone with a nitrogen atom. The synthesis and antibacterial activity of three
such ring systems, the benzisoxazolinones, pyrroles, and isoxazolinones is described.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
to make it to market, having been approved in April
2000 by the FDA for the treatment of bacterial infec-
tions due to Gram positive organisms. Linezolid has
good activity against Gram positive organisms including
those resistant to currently available antimicrobials, it is
100% orally bioavailable and since it is not derived from
a natural product, resistance has been slow to emerge.6
Herein, we detail our initial effortsto identify a new
and potentially improved antibacterial agent using the
oxazolidinone silhouette as a starting point.7 One such
approach involved searching for a bioisostere to replace
the central oxazolidinone heterocycle (Fig. 1).
The major impetusfor much of antibacterial drug dis-
covery in recent yearshasbeen the emergence of drug
resistant strains of pathogenic microoganisms. Of par-
ticular importance are methicillin resistant Staphylococ-
cus aureus (MRSA), penicillin resistant Streptococcus
pneumoniae (PRSP), and vancomycin resistant Entero-
coccus (VRE) species.1 In addition, there have been re-
cent reports of vancomycin resistant Staphylococcus
aureus isolated from patients in the United States.2
Oxazolidinone antibacterialswere firts identified by
researchers at E.I. du Pont de Nemours and were shown
to be potent antimicrobial agents.3 Scientists at UpJohn
were able to optimize thisimportant new chemotype
over the course of a decade culminating in the discovery
of linezolid (Zyvoxꢁ).4 Although many other research
organizations have pursued the oxazolidinone class of
antibacterials, and at least two have produced clinical
candidates,5 linezolid isthe first and only oxazolidinone
Prior to embarking on a search for a new oxazolidinone
bioisostere with improved properties, we consulted the
Keywords: Oxazolidinone; Isoxazolinone; Bioisostere; Antibacterial.
*
Corresponding author. Tel.: +1-203-677-6190; fax: +1-203-677-
7702; e-mail: lawrence.snyder@bms.com
Figure 1. Strategy for discovering new linezolid analogs.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.076

4736
L. B. Snyder et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4735–4739
Figure 3. Overlay of benzisoxazolinone with linezolid.
Figure 2. Other potential oxazolidinone bioisosteres reported in the
literature.
literature and found many examplesof oxazolidinone
isosteres with varying levels of activity. Figure 2 illus-
tratesosme of the ÔactiveÕ (butenolide8,9 and isoxazo-
line10) aswell as ÔinactiveÕ isosteres.9,11 Asthere has
yet to appear a crystal structure of an oxazolidinone
bound to its prokaryotic target (the 50S ribosomal sub-
unit),4 one isleft to infer the critical interactions, which
differentiate active five membered ring heterocyclesfrom
those that are inactive. Although one of linezolids criti-
cal structural elements resulting in good antimicrobial
activity hasbeen shown empirically to be the 5-( S)-con-
figured stereocenter, we became intrigued by the idea of
replacing thisstereogenic carbon with a nitrogen atom.
With thisdesign element in mind, three potential oxazo-
lidinone bioisosteres wherein a nitrogen atom would re-
place the sp3-hybridized stereogenic center found in the
oxazolidinone were surveyed. Our work on the benzis-
oxazolinones, pyrroles, and isoxazolinones is described
below.12
Scheme 1. Reagents: (a) (1) Zn, NH4Cl (for 3h, Hydrazine/RaNi), (2)
NaOH; 31% for 3a, 66% for 3b, 21% for 3c, 66% for 3d, 3% for 3e, 67%
for 3f, 41% for 3g, 53% for 3h, 69% for 3i, 59% for 3j; (b) K2CO3,
AcOCH2NHAc, DMF; 21% for 4a, 57% for 4b, 68% for 4c, 72%
for 4d, 0% for 4e, 53% for 4f, 17% for 4g, 47% for 4h, 26% for 4i, 66%
for 4j.
order to avoid over-reduction when R = 4-F (3h). The
requisite acetamidomethyl side chain was appended by
making use of an acetamidomethyl acetate electrophile
described by Barnes et al.14 Unfortunately benzisoxazol-
inones 4a–j were found to be devoid of antibacterial
activity. It wasthought that tricyclic derivatives 9–11
might more closely mimic the spatial arrangement of
linezolid. The synthesis of these analogs is outlined in
Scheme 2. Disappointingly, 9–11 also showed a lack of
antibacterial activity.
2. Benzisoxazolinones
At an early stage of the program we became aware of a
disclosure by Wierenga et al. describing the antimicro-
bial properties of a select series of benzisoxazolinones.13
As these derivatives possessed primarily Gram negative
activity, we decided to investigate the possibility of
designing a hybrid structure, which might incorporate
the Gram negative activity of the benzisoxazolinones
with the Gram positive activity of the oxazolidinones.
3. Pyrroles
Asshown in Figure 3, the proposed benzisoxazolinone
(A) overlaysreaosnably well with the oxazolidinone
(B). A series of benzisoxazolinone analogs were synthe-
sized as shown in Scheme 1 starting from commercially
available nitrobenzoates. Partial reduction of the nitro
group led to the intermediate hydroxylamines, which
were cyclized in alkaline solution to the parent benzis-
oxazolinone. Hydrazine and Raney nickel were used in
Concurrent with our work exploring the feasibility of
benzisoxazolinones as antibacterial agents, we began
to explore pyrrolesasa potential oxazolidinone bioiso-
stere wherein the pyrrole nitrogen atom would replace
the sp3-hybridized stereogenic center found in the oxa-
zolidinone ring. Commercially available N-TIPS pyrrole
waseslectively iodinated at the C-3 poistion and con-
verted to the boronic acid using known conditions15

L. B. Snyder et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4735–4739
4737
Scheme 2. Reagents: (a) BuLi, (MeS)2; 48%, (b) (1) AcOH/H2O; (2)
NaH, Me2CO3; 63%; (c) NH2OH; 40%; (d) K2CO3, AcOCH2NHAc,
DMF; 60%; (e) DDQ; 50%; (f) Peracetic acid; 99%.
Scheme 4. Reagentsand conditions: (a) (1) n-BuLi, THF, À78ꢂC; (2)
I2; 99%; (b) 4-MeOPh(BOH)2, Pd(Ph3P)4, CsCO3, Tol/MeOH; 78%; (c)
(1) TBAF, (2) K2CO3, AcOCH2NHAc, DMF; 50% for 18, 67% for 20,
60% for 22; (d) (1) t-BuLi, THF, À78ꢂC, (2) MeN(OMe)COCF3; 47%;
(e) (1) t-BuLi, THF, À78ꢂC, (2) DMF; 64%.
ring carbonyl18 all of which have been amply docu-
mented.19 Examination of the overlay between the two
heterocycles( Fig. 4) however, wascompelling and taken
together with the relative simplicity of the chemistry re-
quired to test our hypothesis, the preparation of repre-
sentative examples was undertaken.
Scheme 3. Reagents: (a) ArX, Pd(Ph3P)4, Na2CO3, MeOH, reflux;
90% for 6a, 86% for 6b, 34% for 6c, 70% for 6d, 61% for 6e, 40% for 6f;
(b) TBAF; (c) K2CO3, AcOCH2NHAc, DMF; Yield for steps b and c
combined: 12% for 8a, 19% for 8b, 24% for 8c, 64% for 8d, 4% for 8e,
20% for 8f.
In the event, commercially available phenylacetic acids
were converted to their corresponding esters, formylated
by treatment with sodium hydride in ethyl formate and
cyclodehydrated with hydroxylamine20 (Scheme 5). The
acetamidomethyl side chain was appended to provide
the final isoxazolinone derivatives 25 required for micro-
biological testing. Since there was concern about the sta-
bility of the acetamidomethyl side chain, retro-amide
derivatives 26 were also prepared via alkylation with
iodoacetamide. It isnoteworthy that the ysntheissof
these analogs compares favorably with the correspond-
ing oxazolidinones, starting with readily available esters
and providing the final isoxazolinone analogs in only
three steps.21,22
(Scheme 3). Cross-coupling followed by deprotection
and appendage of the acetamidomethyl side chain re-
sulted in analogs 14a–f. As in the case of the benzisoxa-
zolinones, no antibacterial activity was detected. Since
pyrroles 14a–f lack a carbonyl or a ring oxygen found
in the oxazolidinones, synthesis of pyrrole derivatives
20 and 22, which posses potential hydrogen bond accep-
torsin the form of a formyl and trifluoromethyl group
were prepared asoutlined in Scheme 4. Again, a lack
of antibacterial activity was observed for these analogs.
Possibilities for the lack of activity of the pyrroles and
benzisoxazolinones include decreased binding to the
ribosomal target, decreased permeability or increased
efflux relative to the oxazolidinones.16 Since the oxazo-
lidinone literature suggests a ring oxygen as well as a
carbonyl group are essential elements for antibacterial
activity, it wasthought that an iosxazolinone might
more closely meet these requirements.
4. Isoxazolinones
The isoxazolinone ring system has found limited use in
medicinal chemistry presumably due to inherent insta-
bilities associated with the labile N–O bond,17 potential
Michael accepting properties, and hydrolytically labile
Figure 4. Overlay of isoxazolinone (top) and linezolid (bottom).

4738
L. B. Snyder et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4735–4739
a result of this initial data, the majority of analogs pre-
pared henceforth were 4-substituted. The serum effect
for selected analogs was in some cases significant (e.g.,
25h). In other cases, however, incubation with serum re-
sulted in a more modest 2–4-fold increase in MIC (e.g.,
25k and 25n). To address concerns about the stability of
the acetamidomethyl side chain appended to the isoxaz-
olinone ring nitrogen, the antibacterial activity of the
corresponding N–H isoxazolinones 24j, 24k, 24l, and
24n was investigated. As these unalkylated isoxazolinon-
eswere devoid of antibacterial activity while their
respective fully elaborated derivatives 25i, 25k, 25l,
and 25n possessed good potency it can be surmised that
the final products 25a–s have good stability23 under the
assay conditions.24 The retro-amide present in 26a and
26b results in inactive isoxazolinones. Lastly, a series
of related isoxazolinones not described in this disclosure
were evaluated against a human carcinoma HEP2 cell
line. In general, a 200-fold spread between MIC and
CC50 was found suggesting that antibacterial activity is
not due to cytotoxicity.
Scheme 5. Reagents: (a) (1) NaH, ethyl formate (2) hydroxylamine;
35–49% over two steps; (b) K2CO3, AcOCH2NHAc, DMF; 14–65%;
(c) K2CO3, iodoacetamide; 45–50%.
5. Biological activity of isoxazolinones
Table 1 illustrates in vitro antibacterial activity of the
initial set of isoxazolinones. The initial unsubstituted iso-
xazolinone 25a showed promising antibacterial activity.
Substitution of the phenyl ring at the C-3 position by a
variety of functional groups( 25–f) resulted in analogs
with marginal Gram positive activity and no detectable
Gram negative potency. Substitution of the 4-position
however, provided isoxazolinones with much improved
Gram positive and measurable Gram negative antibac-
terial activity asillustrated by examples 25–m. Disubsti-
tution, in general, produced analogswith dratsically
reduced antibacterial activity (25o–s; 25n appeared to
be a lone exception to thisempirical obesrvation). As
In conclusion, we have outlined the discovery of a new
class of antibacterial agents based on the oxazolidinone
antibacterial chemotype. The design concept involved
replacement of an sp3-hybridized asymmetric carbon
atom with a nitrogen atom, thereby rendering the result-
ant analogs achiral and greatly simplifying the synthetic
sequence. Initial efforts to prepare benzisoxazolinones
and pyrrolesprovided compoundsdevoid of antibacte-
Table 1. Antibacterial activity of selected isoxazolinones
MIC without and (with 50% calf serum) (lg/mL)
Compound
R
S. aureusa
MRSAb
E. faecalisc
H. influenzaed
24j
24l
4-MeS
4-Ph
>128 (>64)
>128 (>64)
>128 (>64)
>128 (>64)
8 (16)
>128 (>64)
>128 (>64)
>128 (>64)
>128 (>64)
4 (8)
>128 (>64)
>128 (>64)
>128 (>64)
>128 (>64)
16 (16)
>64
>64
>64
>64
8
24k
24n
25a
25b
25c
25d
25e
25f
4-MeO
3,4-Methylenedioxy
H
3-F
8 (ND)
32 (64)
32 (64)
8 (>64)
8 (ND)
>128 (>64)
4 (>64)
8 (ND)
2 (16)
4 (ND)
16 (>64)
16 (64)
16 (ND)
32 (>64)
64 (>64)
16 (>64)
32 (ND)
>128 (>64)
8 (>64)
8 (ND)
1 (16)
>64
>64
>64
32
3-MeO
3-CF3
3-Cl
4 (16)
3-Me
4-F
4 (ND)
>128 (>64)
2 (>64)
4 (ND)
1 (8)
>64
32
25g
25h
25i
4-Br
4-Cl
8
32
25j
4-MeS
8
16
25k
25l
4-MeO
4-Ph
4-iPr
2 (4)
1 (4)
1 (64)
2 (8)
4 (64)
4 (>64)
1 (4)
2 (4)
16
25m
25n
25o
25p
25q
25r
2 (8)
1 (8)
3,4-Methylenedioxy
3,4-Di-MeO
3,4-Di-F
3,5-Di-MeO
3,5-Di-F
3,5-Bis-CF3
H
1 (4)
16
>64
>64
64
128 (>64)
128 (>64)
>128
64 (>64)
>128 (>64)
>128
>128 (>64)
>128 (>64)
>128
8 (ND)
>128
>128
8 (ND)
>128
>128
32 (ND)
>128
>128
>64
>64
>64
>64
8
25s
26a
26b
Linezolid
4-Br
>128
1 (1)
>128
1 (1)
>128
0.5 (0.5)
a Staphylococcus aureus A15090.
b Methicillin resistant Staphylococcus aureus A27223.
c Enterococcus faecalis A20688.
d Haemophilus influenzae A20191.

L. B. Snyder et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4735–4739
4739
AcrB:KANA H. influenzae (A2935) efflux pump mutants.
It istherefore likely that the lack of antibacterial activity
observed with the pyrrole and benzisoxazolinones is not
due to increased efflux.
rial activity. The isoxazolinone ring system however, has
proven itself to be an effective bioisostere of the
oxazolidinone heterocycle found in oxazolidinone anti-
bacterials. The pharmacological profiles of the isoxazol-
inone versions of linezolid and eperezolid4 aswell as
details of our efforts to design an improved version
linezolid will be the subject of future reports.
17. Ang, K. H.; Prager, R. H.; Williams, C. M. Aust. J. Chem.
1995, 48, 567.
18. De Sarlo, F.; Dini, G. J. Hetrocycl. Chem. 1967, 4, 533.
19. Batra, S.; Bhaduri, A. P. J. Indian Inst. Sci. 1994, 74, 213.
20. Beccalli, E. M.; La Rosa, C.; Marchesini, A. J. Org. Chem.
1984, 49, 4287.
References and notes
21. Experimental procedure for compound 25a: To a stirred
solution of ethyl phenylacetate (30mL; 0.19mol) in ethyl
formate (600mL) wasadded sodium hydride (60% in oil;
30g; 0.75mol) portionwise over 45min under a constant
stream of nitrogen. After 15 additional minutes LC
indicated consumption of starting material. The reaction
waspoured into a mixture of 1N HCl/ice, extracted with
ether, washed with brine, and dried over MgSO4 providing
49g of an amber liquid, which wasuesd without purifi-
cation. The amber liquid was dissolved in methanol/water
(600mL/60mL), hydroxylamine hydrochloride (25g) was
added and the solution was heated to reflux for 1.5h. The
solution was concentrated to near dryness, and residual
water wasazeotroped further from methanol (3 ·). The
solid was transferred to a Buchner funnel, washed with
water (200mL) and ether (250mL) and dried in vacuo to
provide 24a (18.4g; 59% over two steps) as a colorless
solid.18 To a stirred solution of AcOCH2NHAc13 (8.1;
62.1mmol) in dichloromethane (120mL) wasadded 24a
(2.0g; 12.4mmol). After 18h, 1N HCl wasadded until the
reaction mixture wasacidic, the aqueouswasextracted
with chloroform, and the organicswere dried over MgSO 4
providing a pale liquid. Sonication with ether provided
1.9g (67%) 25a as a colorless solid. 1H NMR (DMSO-d6):
d 8.95 (s, 1H), 8.93 (t, J = 6.1Hz, 1H), 7.76 (d, J = 7.7Hz,
2H), 7.38 (t, J = 9.4Hz, 2H), 7.25 (t, J = 8.3 Hz, 1H), 5.03
(d, J = 6.2Hz, 2H), 1.84 (s, 3H); 13C NMR (DMSO-d6): d
170.6, 168.7, 150.4, 129.4, 128.6, 126.8, 124.6, 102.3, 55.4,
22.3; IR (KBr pellet) 3288, 3057, 1722, 1667cmÀ1; LC
purity >99%; HRMS (FAB) calcd for C12H12N2O3:
233.093005, found: 233.09300.
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23. Additional stability tests in acidic and basic media were
done. Acidic stability (TFA/CH2Cl2; or 1N HCl/THF)
wasexcellent. Storage in citrate buffer for 18h (pH4.5)
resulted in <1% decomposition. Storage in basic media
(pH8.5) showed loss of the acetamidomethyl side chain in
a time and temperature dependent fashion (a maximum of
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degradation evident after storage at ambient temperature
for >3years.
24. The minimum inhibitory concentration (MIC) of the
compound wasobtained uins g a microbroth dilution
susceptibility test in accordance to the method recom-
mended by the National Committee for Clinical Labora-
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to test S. aureus and E. faecalis. Haemophilustest medium
broth was used to test H. influenzae. The final bacterial
inoculum contained approximately 5·105 CFU/well in a
microtiter plate. The volume of each well was100 lL and
the plateswere incubated at 35 ꢂC for 18h in ambient air.
The MIC wasdefined asthe lowets drug concentration
that prevented visible growth of the bacterium.
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16. Compounds 10, 18, 20, and 22 exhibited no antibacterial
activity against either AcrA:KANA E. coli (A2891) or