Design, synthesis, and structure-activity relationship studies of highly potent novel benzoxazinyl-oxazolidinone antibacterial agents

Article abstract of DOI:10.1021/jm200614t

A series of novel benzoxazinyl-oxazolidinones bearing nonaromatic heterocycle or aryl groups were designed and synthesized. Their in vitro and in vivo antibacterial activities were investigated. Most of the (3S, 3aS) biaryl benzoxazinyl-oxazolidinones exhibited potent activity against Gram-positive pathogens. SAR trends were observed; a pyridyl C ring was preferable to other 5- or 6-member aryl C rings, while fluorine substitution on the B ring generated derivatives with reduced activity. Various substituent group positions on the pyridyl ring were also evaluated. The resulting compounds displayed excellent activity against linezolid-resistant strains. Compound 45 exhibited excellent in vitro activity, with a MIC value of 0.25-0.5 μg/mL against MRSA and an activity against linezolid-resistant strains of 8-16-fold higher potency than linezolid. In a MRSA systemic infection model, compound 45 displayed an ED ₅₀ < 5.0 mg/kg, a potency that is nearly 3-fold better than that of linezolid. This compound also showed excellent pharmacokinetic profiles, with a half-life of more than 5 h as well as an oral bioavailability of 81% in rats.

Full text of DOI:10.1021/jm200614t

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and StructureꢀActivity Relationship Studies of
Highly Potent Novel Benzoxazinyl-Oxazolidinone Antibacterial
Agents
||
||
Qisheng Xin,†, Houxing Fan,†, Bin Guo,† Huili He,† Suo Gao,† Hui Wang,‡ Yanqin Huang,§
and Yushe Yang*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences,
Chinese Academy of Sciences, Shanghai 201203, China
‡Department of Microbiology, China Pharmaceutical University, Nanjing, Jiangsu Province 210009, China
§MicuRx Pharmaceuticals, Inc., Shanghai 201203, China
S Supporting Information
b
ABSTRACT:
A series of novel benzoxazinyl-oxazolidinones bearing nonaromatic heterocycle or aryl groups were designed and synthesized. Their
in vitro and in vivo antibacterial activities were investigated. Most of the (3S, 3aS) biaryl benzoxazinyl-oxazolidinones exhibited
potent activity against Gram-positive pathogens. SAR trends were observed; a pyridyl C ring was preferable to other 5- or 6-member
aryl C rings, while fluorine substitution on the B ring generated derivatives with reduced activity. Various substituent group positions
on the pyridyl ring were also evaluated. The resulting compounds displayed excellent activity against linezolid-resistant strains.
Compound 45 exhibited excellent in vitro activity, with a MIC value of 0.25ꢀ0.5 μg/mL against MRSA and an activity against
linezolid-resistant strains of 8ꢀ16-fold higher potency than linezolid. In a MRSA systemic infection model, compound 45 displayed
an ED50 < 5.0 mg/kg, a potency that is nearly 3-fold better than that of linezolid. This compound also showed excellent
pharmacokinetic profiles, with a half-life of more than 5 h as well as an oral bioavailability of 81% in rats.
’ INTRODUCTION
2,14,15 which display very weak antibacterial activity. During the
course of our initial efforts to develop novel oxazolidinones, we
chose to explore the novel [6,6,5] tricyclic fused benzoxazinyl-
oxazolidinone 3.16 Although 3 also exhibited only weak anti-
bacterial activity, it was a novel lead suitable for further structural
modifications. Initially, [6,6,5] tricyclic fused benzoxazinyl-ox-
azolidinones of the general structure 4 with heterocycles attached
and the (3S, 3aS) configuration were investigated, however, all
showed much lower potency compared with linezolid.
Meanwhile, a number of studies suggested that an aryl C ring of
the oxazolidinone subtype would significantly improve potency.17ꢀ20
We therefore focused on structureꢀactivity relationship studies of
biaryl [6,6,5] tricyclic fused benzoxazinyl-oxazolidinone derivatives 4
(5- or 6-member aryl C rings). As anticipated, a pyridyl ring17,19 was
preferable to other C ring subtypes. Next, the effects of changes to the
substituent group positiononthepyridylringwas evaluated. Herein, we
Antibiotic resistance is an emerging threat to global health.1ꢀ3
Infections caused by resistant Gram-positive bacteria such as
methicillin-resistant Staphylococcus aureus (MRSA), penicillin-
resistant Streptococcus pneumoniae (PRSP), and vancomycin-
resistant Enterococcus faecalis (VRE) are the most serious pro-
blems. Linezolid 14,5 (Scheme 1), the first and only oxazolidinone
drug, was approved in 2000 by the Food and Drug Administra-
tion (FDA) for the treatment of multidrug resistant Gram-
positive bacterial infections, including the above-mentioned
strains. However, after its commercial release, linezolid-resistant
strains of Staphylococcus aureus6 and Enterococcus faecium7ꢀ9
began to appear in the clinic, underscoring the increasingly
urgent need for improved antibiotics with potent antimicrobial
activity and with activity toward linezolid-resistant strains. There-
fore, many research groups have attempted to improve the
potency and the antibacterial spectrum of this new drug.10ꢀ13
Previous reports have described the design and synthesis of a
series of [6,5,5] tricyclic fused oxazolidinones, exemplified by
Received: May 14, 2011
Published: September 28, 2011
r
2011 American Chemical Society
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Scheme 1
Scheme 2a
a Reagents and conditions: (a) K2CO3, 4 Å molecular sieves, DMF; 120 °C; (b) (i) SnCl2 2H2O, EtOH, reflux, (ii) CbzCl, pyridine, CH2Cl2; (c) KOH,
3
THF; (d) L-(+)-DET, TBHP, Ti(OiPr)4, 4 Å molecular sieves, CH2Cl2, ꢀ40 °C ; (e) TBDMSCl, imidazole, DMAP, DMF, 0 °C; (f) nBuLi, THF, ꢀ78
°C to room temp; (g) nBu4NF, THF, 0 °C; (h) meta-nitro benzene sulfonyl chloride, Et3N, CH2Cl2, 0 °C to room temp; (i) (i) 25% NH3 H2O,
3
acetonitrile/2-propanol, 55 °C, (ii) CH3COCl, Et3N, CH2Cl2, 0 °C; (j) 10% Pd-C/H2, CH2Cl2/MeOH.
disclose our efforts toward the design and synthesis of a novel class
of benzoxazinyl-oxazolidinones, which exhibit excellent activity against
Gram-positive bacteria, including linezolid-resistant strains, resulting in
the discovery of a promising antibacterial drug candidate 45.
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Scheme 3a
a Reagents and conditions: (a) ArCHO, HCO2H, DMF, 100 °C, (b) 2-chloromethyl-5-nitrofuran, DIEA, acetonitrile, 50 °C.
Scheme 4a
a Reagents and conditions: (a) cis-2-butene-1,4-diol, NaH, THF, 0 °C to room temp; (b) (i) Zn powder, NH4Cl, THF (9a) or CH3OH (9b), room
temp, (ii) CbzCl, NaHCO3, THF:H2O = 2:1 (9a) or acetone:H2O = 2:1 (9b), 0 °C to room temp; (c) L-(+)-DET, TBHP, Ti(OiPr)4, 4 Å molecular
sieves, CH2Cl2, ꢀ40 °C; (d) TBDMSCl, DMAP, imidazole, DMF, 0 °C; (e) nBuLi, dry THF, ꢀ78 °C to room temp; (f) nBu4NF, THF, 0 °C to room
temp; (g) meta-nitro benzene sulfonyl chloride, Et3N, CH2Cl2, 0 °C to room temp; (h) (i) 25% NH3 H2O, acetonitrile/2-propanol, 55 °C, (ii)
3
CH3COCl, Et3N, CH2Cl2; 0 °C; (i) 33a to 34: bis(pinacolato)diboron, PdCl2(dppf) CH2Cl2, KOAc, DMSO, 80 °C.
3
substitution of bromobenzene 516 afforded compound 6, before
’ CHEMISTRY
reduction of the nitro functional group using SnCl2. The aniline
produced was protected with benzyl chloroformate (CbzCl) to
Compound 14 and the key intermediate 16 were prepared using
the same synthetic route, as described in Scheme 2. Nucleophilic
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ARTICLE
afford compound 7. After cleavage of the propionic ester, Sharpless
asymmetric epoxidation of 8 at ꢀ40 °C, gave the desired com-
pound 9 (2R,3S). The hydroxyl group of 9 was then protected
using tert-butyldimethylsilyl chloride (TBDMSCl). Next, a tandem
cyclization was used to construct the core [6,6,5] tricyclic ring
system,14 to give 11 (3R, 3aS) from 10, using nBuLi as the base, at
ꢀ78 °C. meta-Nitrobenzene sulfonyl chloride was used to form an
aryl sulfonate in 13, to provide a good leaving group, after cleavage
of the protecting TBDMS group. Amination of 13 with aqueous
ammonia, followed by acetylation with acetyl chloride, gave
compounds 14 and 15. The key intermediate 16 was produced
from 15 by further deprotection.
The synthesis of benzoxazinyl-oxazolidinones bearing non-
aromatic heterocycles is outlined in Scheme 3. Compounds
17ꢀ23 were obtained from key intermediate 16 by reaction
with aromatic aldehydes. A nucleophilic substitution between
key intermediate 16 and 2-(chloromethyl)-5-nitrofuran yielded
compound 24.
The synthesis of 33a and its fluorine substituted analogue 33b
was initiated from nitrofluorobenzene 25 (Scheme 4). Nucleo-
philic substitution of 25 with cis-2-butene-1,4-diol gave com-
pound 26. Because the reduction of the nitro group of 26 using
SnCl216 resulted in a low yield, zinc powder was instead chosen as
the reductant. The yield obtained over two steps (nitro reduction
and protection of the aniline with Cbz) was increased to 73%
(27a) and 56% (27b), respectively, by adopting this alternative
reducing agent. The synthetic procedures used subsequently
were similar to those used in the synthesis of compound 14
(Scheme 2). The absolute configuration of 30a, which is a good
representative of this novel [6,6,5] tricyclic ring system, was
confirmed by X-ray crystallography (Figure 1).
Scheme 5a
a Reagents and conditions: (a) ArB(OH)2, Pd(PPh3)4, 2 N Cs2CO3,
THF, reflux; (b) ArB(pin), Pd(PPh3)4, 2 N KF, dioxane, 80 °C; (c) 10%
Pd-C, H2, room temp.
Figure 1. X-ray structure of compound 30a.
Scheme 6a
a Reagents and conditions: (a) ArBr, PdCl2(dppf) CH2Cl2, K2CO3, dioxane/EtOH/H2O, reflux; (b) ArBr, Pd(PPh3)4, 2 N Cs2CO3, dioxane, 80 °C;
3
(c) 10% Pd-C, H2, room temp; (d) CF3COOH, CH2Cl2, room temp.
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Scheme 7a
a Reagents and conditions: (a) ArBr, Pd(PPh3)4, 2 N Cs2CO3, dioxane, 80 °C.
Scheme 8a
a Reagents and conditions: (a) (i) 2-chloroethyl carbonochloridate, K2CO3, CH3CN, 0 °C to room temp, (ii) reflux.
Scheme 9a
a Reagents and conditions: (a) methyl 4-bromocrotonate, K2CO3, KI, acetone, reflux; (b) DIBAL-H, toluene, 0 °C; (c) (i) Zn powder, NH4Cl, THF,
room temp, (ii) CbzCl, NaHCO3, THF:H2O = 2:1, 0 °C to room temp; (d) L-(+)-DET, TBHP, Ti(OiPr)4, 4 Å molecular sieves, CH2Cl2, ꢀ40 °C; (e)
TBDMSCl, DMAP, imidazole, DMF, 0 °C; (f) nBuLi, dry THF, ꢀ78 °C to room temp; (g) nBu4NF, THF, 0 °C to room temp; (h) MsCl, Et3N, CH2Cl2,
Et3N, CH2Cl2, 0 °C to room temp; (i) NaN3, DMF, 80 °C; (j) PPh3, H2O, THF, 45 °C to room temp; (k) CH3COCl, Et3N, CH2Cl2; 0 °C; (l)
bis(pinacolato)diboron, PdCl2(dppf) CH2Cl2, KOAc, DMSO, 80 °C; (m) 5-bromo-2-pyridinecarbonitrile, Pd(PPh3)4, Cs2CO3, dioxane/H2O, 80
3
°C, 82%.
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Table 1. In Vitro Antibacterial Activity of Benzoxazinyl-
oxazolidinones Bearing Nonaromatic Heterocycles
Table 2. In Vitro Antibacterial Activity of Biaryl Benzoxazi-
nyl-oxazolidinones
a Abbreviations: S.a., Staphylococcus aureus. ATCC 29213. MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains. MRSE, methicillin-resistant
Staphylococcus epidermidis, 5 strains. PRSP, penicillin-resistant Streptococ-
cus pneumoniae, 4 strains. E.f., Enterococcus faecalis, 6 strains.
The boric acid ester 34 was synthesized by reacting 33a and
bis(pinacolato)diboron, mediated with PdCl2(dppf) CH2Cl2,
3
resulting in a high yield (87.9%).19 However, the fluorine
substituted analogue of boric acid ester 34 could not be obtained
in the same manner. The use of other catalytic systems (Pd(OAc)2/
biphenyl-2-yl di-tert-butylphosphine, Pd(dba)2/biphenyl-2-yl di-
tert-butylphosphine, and PdCl2(dppf) CH2Cl2/CuI), other bases
3
(tBuONa, tBuOK and Et3N), or longer reaction times (24ꢀ48 h)
did not result in any reaction.
The synthesis of the biaryl benzoxazinyl-oxazolidinones is
outlined in Schemes 5, 6, and 7. They were assembled by Suzuki
coupling of benzoxazinyl-oxazolidinones 33 with a series of
arylboronic acids or boric acid esters (Scheme 5) or key inter-
mediate boric acid ester 34 with bromide-substituted heteroaro-
matic fragments (Schemes 6 and 7).
Compounds 41 and 47 were generated from compounds 38
and 44 by catalytic hydrogenation of the nitro moieties to amines
(Schemes 6 and 7). Compounds 64 and 66 were obtained from
compounds 63 and 65 via cleavage of the N-Boc groups after
Suzuki coupling (Scheme 7).
a Abbreviations: S.a., Staphylococcus aureus. ATCC 29213. MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains. MRSE, methicillin-resistant
Staphylococcus epidermidis, 5 strains. PRSP, penicillin-resistant Streptococ-
cus pneumoniae, 4 strains. E.f., Enterococcus faecalis, 6 strains.
The novel fragments 72a, 73a, and 74a were generated
from aminopyridine bromides by one-pot acylation and nucleo-
philic substitution with commercially available 2-chloroethyl carbo-
nochloridate21 (Scheme 8).
Compound 93, a diastereomer of 45, was synthesized as outlined
in Scheme 9. 5-Bromo-2-nitrophenol (80) was reacted with the E-
isomer of alkene methyl 4-bromocrotonate in a basic solution, and
the product was then reduced with diisobutylaluminium hydride
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Table 3. In Vitro Antibacterial Activity of Substituted Pyridyl
Benzoxazinyl-oxazolidin-ones
Table 4. In Vitro Antibacterial Activity of Substituted Pyridyl
Benzoxazinyl-oxazolidinones
a Abbreviations: S.a., Staphylococcus aureus. ATCC 29213. MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains. MRSE, methicillin-resistant
Staphylococcus epidermidis, 5 strains. PRSP, penicillin-resistant Streptococ-
cus pneumoniae, 4 strains. E.f., Enterococcus faecalis, 6 strains.
a Abbreviations: S.a., Staphylococcus aureus. ATCC 29213. MRSA, methi-
cillin-resistant Staphylococcus aureus, 6 strains. MRSE, methicillin-resistant
Staphylococcus epidermidis, 5 strains. PRSP, penicillin-resistant Streptococ-
cus pneumoniae, 4 strains. E.f., Enterococcus faecalis, 6 strains.
summarized in Tables 1ꢀ6. Most target compounds displayed
excellent in vitro antibacterial activity. Compound 45, which was
the most potent compound tested, exhibited activity several
times more potent than linezolid, with minimum inhibitory
concentration (MIC) values of 0.125ꢀ0.5 μg/mL against S.
aureus ATCC 29213, 0.25ꢀ0.5 μg/mL against MRSA, MRSE,
and PRSP and 0.5 μg/mL against E. faecalis.
As shown in Table 1, benzoxazinyl-oxazolidinones bearing
nonaromatic heterocycles showed very weak in vitro activity.
Compound 14, an analogue of linezolid, was the most potent of
these and exhibited MIC values of only 4ꢀ16 μg/mL against all
strains. This compound is therefore less potent than linezolid.
Exchanging the substituted piperazine for aromatic rings
produced biaryl benzoxazinyl-oxazolidinones. To our delight, a
significant improvement in potency was observed. As shown in
(DIBAL-H) to yield (E)-4-(5-bromo-2-nitrophenoxy)but-2-en-1-
ol(82). AfterasimilarreactionschemetothatdepictedinSchemes2
and 4, the desired product 93 was obtained.
’ RESULTS AND DISCUSSION
In Vitro and in Vivo Activities. The target compounds
were evaluated for their antibacterial activity against a panel of
susceptible and resistant Gram-positive organisms, includ-
ing linezolid-resistant strains. The results of these studies are
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Table 2, most of the unsubstituted 6-membered aryl analogues
(37, 42, 43, and 49) exhibited activity superior to linezolid, while
2-thienyl 35 and 3-thienyl 36 displayed lower activity, similar to
that of linezolid. In previous SAR studies5 for linezolid and
similar molecules, a meta fluorine on the phenyl B ring has often
been found to improve antibacterial activity. By contrast, in our
[6,6,5] study, the fluorinated benzoxazinyl-oxazolidinones 53,
54, and 55 were less active than their unsubstituted analogues 42,
43, and 45.
Analogues substituted on the phenyl ring with ꢀNO2, ꢀCN,
CH3-(CO)ꢀ, and ꢀNH2 moieties (38ꢀ41) exhibited similar
activities to those observed for the unsubstituted parent 37. The
replacement of the pyridyl group of 43 with substituted pyridyls
(44ꢀ47) resulted in more active compounds than 43. Pyridyl
rings substituted with both electron-withdrawing groups (ꢀNO2
and ꢀCN, compounds 44, 45 vs 43) and electron-donating
groups (ꢀNH2, 47) led to compounds with significantly in-
creased activity. However, compounds containing pyrimidyl
rings substituted with either electron-donating groups (OMe,
ꢀNH2, compounds 50 and 51) or an electron-withdrawing
group (ꢀCN, compound 52) had activity that was significantly
reduced. Therefore, these SAR studies have established that the
substituents follow the trend pyridyl > phenyl > pyrimidyl >
thienyl with respect to in vitro activity.
Accordingly, the 3-pyridyl core structure was chosen as a
model for further modifications, including substitution with
heteroaromatic rings, simple alkyl groups, and nonaromatic
rings. The results are summarized in Table 3. All the derivatives
tested displayed similar activity to 43, with the exception of 67,
68, and 70. Compounds 59, 61, 64, and 69 showed 2- to 4-fold
lower MICs than that of linezolid. Compound 72 was equipotent
with compound 45 and exhibited an activity that is 4- to 8-fold
higher than that of linezolid.
Table 5. In Vitro Activity of Selected Pyridyl Benzoxazinyl-
oxazolidinones against Linezolid-Resistant Bacteria
MIC (μg/mL)a
Because of the very potent in vitro activity of compounds 45,
46, and 72, a more in-depth survey of the effects of substituents
on the various pyridines was investigated (Table 4). Compounds
73, 75, and 78, which feature meta-substitution on the meta-
pyridyl ring, exhibited reduced activity. Compounds 74, 76, and
79, which have meta-substitution on the ortho-pyridyl ring,
exhibited dramatically reduced activity. While compound 77,
which has para-substitution on the ortho-pyridyl ring, showed
improved activity (77 vs 75, 76), it remained less potent than 45.
Inverting one chiral center of 45 gave the (3S, 3aR) diaster-
eomeric compound 93, and this compound was found to be
virtually inactive (Table 4). This result indicates that a (3S, 3aS)
absolute configuration for the [6,6,5] tricyclic ring (45) system is
required for potency.
The in vitro activities of selected pyridyl benzoxazinyl-oxazo-
lidinones against linezolid-resistant strains are shown in Table 5.
All compounds tested exhibited more potent activity against
common linezolid-resistant pathogens than linezolid. Com-
pounds 45, 46, and 72 displayed 8ꢀ16-fold more potency than
linezolid against all the linezolid-resistant strains tested.
Selected compounds were evaluated for in vivo efficacy in a
mouse systemic infection model via oral dosing. As shown in
Table 6, compound 45 displayed an efficacy about 4-fold (ED50:
2.5 mg/kg) and 3-fold (ED50: <5.0 mg/kg) higher than linezolid
against S. aureus and MRSA, respectively. Compound 72 demon-
strated oral efficacy comparable (ED50: 9.5 mg/kg) with linezolid
(ED50: 14.1 mg/kg) in the MRSA infection model. It is inter-
esting to note that compound 46 exhibited disappointingly low
efficacy in this in vivo study (ED50 > 20.0 mg/kg) against S.
aureus despite a good in vitro antibacterial profile.
compd
S.a.
LRSA
LRSE
LREF
LREFA
1
44
45
46
47
56
59
62
64
69
72
1
0.125
0.125
0.5
2ꢀ4
2
2
1
1ꢀ2
1
0.5ꢀ1
2
1
1
1
2ꢀ4
1ꢀ2
1
0.5
16
4
4
2ꢀ4
2
0.25
0.25
0.5
2
4
2
1ꢀ2
1
8
1
1
0.25
0.5
16
4
4
4ꢀ8
1
4
1
1
0.25
1
2
1
1ꢀ4
8ꢀ16
0.5ꢀ1
8ꢀ16
>16
16
a Abbreviations: S.a., Staphylococcus aureus. ATCC 29213. LRSA, line-
zolid-resistant Staphylococcus aureus, 3 strains. LRSE, linezolid-resistant
Staphylococcus epidermidis, 1 strain. LREF, linezolid-resistant Enterococ-
cus faecalis, 3 strains. LRFEA, linezolid-resistant Enterococcus faecium, 3
strains.
Table 6. Antibacterial Efficacies (ED50) of Selected Com-
pounds in Mice Systemic Infection Models
compd
strain
ED50 (mg/kg)a
45
S. aureus (ATCC 19636)
MRSA(ATCC 33591)
S. aureus (ATCC 19636)
S. aureus (ATCC 19636)
MRSA (ATCC 33591)
S. aureus (ATCC 19636)
MRSA (ATCC 33591)
2.5
<5.0
>20.0
11.3
9.5
46
72
1
9.5
Pharmacokinetic (PK) Profiles of the Selected Antibacter-
ial Agents. Next, the highly potent compounds 45 and 72 were
subjected to pharmacokinetic performance assessment in rats
(Table 7). Both compounds exhibited favorable pharmacokinetic
14.1
a ED50 is the dose at which 50% of infections had been successfully
treated after oral administration.
Table 7. Pharmacokinetic Parameters of Compounds 45 and 72
compd
route
dose (mg/kg)
Cmaxa (μg/mL)
Tmax(h)
t1/2
AUC0ꢀ∞ (μ g h/mL)
clearanceb (mL/h/kg)
F (%)
3
45
po
iv
15
15
50
10
5 ( 1
7
4.0 ( 1.4
2.0
5.5 ( 0.7
5.6 ( 0.2
4.7 ( 0.7
3.7 ( 0.1
66 ( 10
82 ( 8
229 ( 31
184 ( 18
nc
81
72
po
iv
9 ( 1
na
6.0 ( 1.2
na
123 ( 23
136 ( 33
18
77 ( 21
a na = not applicable. b nc = not calculated.
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profiles with excellent half-lives, areas under the concentration-
time curve (AUCs). The oral bioavailability of compound 45 is
81% in rats.
Pharmacokinetic Studies. Compounds 45 and 72 were tested in
pharmacokinetic studies on SpragueꢀDawley rats weighing 200ꢀ250 g,
with four mice (male) in each group. The tested compounds were
administered orally at a dose of 15 and 50 mg/kg or were administered
intravenously at a dose of 15 mg/kg and 10 mg/kg. Serial specimens
(0.3 mL) were collected via the retrobulbar vein, and quantification was
performed by LC-MS. Pharmacokinetic parameters were calculated
from the mean plasma concentration by noncompartmental analysis.
’ CONCLUSIONS
In conclusion, we have identified a novel class of antibacterial
agent, the benzoxazinyl-oxazolidinones. Nonaromatic hetero-
cycles or aromatic rings attached to the [6,6,5] tricyclic fused
core structure significantly improved the potency. The SAR
profile of this new class is quite different from the profiles found
in previous SAR studies on related compounds in that the
fluorination of this core structure led to a reduction in activity.
A remarkable diversity of functionality was tolerated at C-2 of the
pyridyl ring. Most of the target compounds exhibited potent
activity against Gram-positive pathogens, and particularly note-
worthy was the excellent activity shown by compounds 45, 46,
and 72 against linezolid-resistant strains. Compound 45 exhib-
ited an excellent PK profile as well as activity that was 3- to 4-fold
higher than linezolid in vivo. The favorable in vitro and in vivo
activities of this compound, combined with its excellent PK
profile, make it a very promising drug candidate. Evaluation of
the safety of this drug candidate is currently under investigation.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental details, elemen-
b
tal analysis, HPLC data, and X-ray structure of 30a are available
(PDF and CIF). This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail: ysyang@
mail.shcnc.ac.cn.
Author Contributions
These authors contributed equally to this work.
’ EXPERIMENTAL SECTION
’ ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China
(no. 20672125) and the National Science and Technology Major
Project of China (no. 2009ZX09301-001) for financial support.
Minimum Inhibitory Concentration Testing. The minimum
inhibitory concentrations of the novel compounds against Gram-posi-
tive bacteria were tested using linezolid as a positive control. Minimum
inhibitory concentration (MIC) values were determined using an agar
dilution method according to the methods of National Committee for
Clinical Laboratory Standards (NCCLS).22 Compounds were dissolved
in 50% water in DMSO to prepare a stock solution that had a concen-
tration of 320 μg/mL. Serial 2-fold dilutions were prepared from the
stock solution with sterile water and then 10-fold diluted with Muel-
lerꢀHinton (MH) agar medium to provide concentration ranges of
16ꢀ0.03125 μg/mL. The tested organisms were grown in MH broth
medium at 35 °C for 8 h and were adjusted to the turbidity of the 0.5
McFarland standard. The bacterial suspensions were inoculated onto the
drug-supplemented MH agar plates with a multipoint inoculator and
incubated at 35 °C for 16 h.
’ ABBREVIATIONS USED
MRSA, methicillin-resistant Staphylococcus aureus; PRSP, peni-
cillin-resistant Streptococcus pneumoniae; VRE, vancomycin-resis-
tant Enterococcus faecalis; FDA, Food and Drug Administra-
tion;CbzCl, benzyl chloroformate;TBDMSCl, tert-butyldi-
methylsilyl chloride; DIBAL-H, diisobutylaluminium hydride;
MIC, minimum inhibitory concentration;SAR, structureꢀactivity
relationship;AUCs, areas under the concentratione-time curve;
NCCLS, National Committee for Clinical Laboratory Standards
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