Design at the atomic level: Generation of novel hybrid biaryloxazolidinones as promising new antibiotics

Article abstract of DOI:10.1016/j.bmcl.2008.10.014

From the X-ray crystal structures of linezolid and the non-selective antibiotic sparsomycin, we have derived a new family of hybrid oxazolidinones. From this initial compound set we have developed a new biaryloxazolidinone scaffold that shows both potent

Full text of DOI:10.1016/j.bmcl.2008.10.014

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6179–6183
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design at the atomic level: Generation of novel hybrid biaryloxazolidinones
as promising new antibiotics
Jiacheng Zhou b,––, Ashoke Bhattacharjee b, Shili Chen b, Yi Chen b, , Erin Duffy a, Jay Farmer b,
,
Joel Goldberg b,à, Roger Hanselmann b, Joseph A. Ippolito a, Rongliang Lou b, Alia Orbin b,§, Ayomi Oyelere b,–
,
Joe Salvino b,||, Dane Springer b,à, Jennifer Tran b,àà, Deping Wang a,§§, Yusheng Wu b, Graham Johnson a,b,
*
a Department of Structure-Based Drug Design, Rib-X Pharmaceuticals Inc., 300 George Street, Suite 301, New Haven, CT 06511, USA
b Department of Medicinal Chemistry, Rib-X Pharmaceuticals Inc., 300 George Street, Suite 301, New Haven, CT 06511, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
From the X-ray crystal structures of linezolid and the non-selective antibiotic sparsomycin, we have
derived a new family of hybrid oxazolidinones. From this initial compound set we have developed a
new biaryloxazolidinone scaffold that shows both potent antimicrobial activity as well as selective inhi-
bition of ribosomal translation. The synthesis of these compounds is outlined.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 12 May 2008
Revised 30 September 2008
Accepted 2 October 2008
Available online 7 October 2008
Keywords:
Oxazolidinone
Biaryloxazolidinone
Structure-based drug design
Linezolid
Sparsomycin
Gram-negative bacteria
X-ray crystal structure
Ribosome
Oral antibiotics
Hybrid antibiotics
Bacterial resistance to antibiotics has increased in part because
of an extended ‘drought’ in antibacterial drug development. This
38-year innovation gap ended in 2000 with the introduction of lin-
ezolid (currently marketed under the tradename Zyvox).1 Linezolid
The design of new antibiotics that have a broader and more
potent spectrum of antibacterial activity is an important objec-
tive. For ribosomal inhibitors this objective can be accomplished
by boosting intrinsic affinity for the ribosome, thereby overcom-
ing resistance and enhancing the spectrum against both linezo-
lid-susceptible and -resistant Gram-positive organisms and
important Gram-negative community respiratory pathogens
such as Moraxella catarrahalis and Haemophilus influenzae. Our
represents
a new chemical class of oxazolidinone-containing
antibacterials.
Unfortunately, it took less than a year after its introduction for
case reports to appear detailing instances of vancomycin-resistant
Enterococcus faecium and methicillin-resistant Staphylococcus aur-
eus showing cross-resistance to linezolid.2,3
discovery program began with
a detailed X-ray structural
knowledge of the provocative juxtaposition of the linezolid-
binding site4 with that of the non-selective antibiotic sparsomy-
cin within the 50S ribosomal subunit (Fig. 1A and B). In Figure
1A we show sparsomycin (pink) and linezolid (blue) as space-
filling representations within the RNA bases (gray) of the pepti-
dyl transferase center. In this figure the overlapping region of
these two antibiotics is highlighted by the dotted circle. Fig-
ure 1B shows this same overlap without the ribosomal context.
This relationship had been revealed from previous crystallo-
graphic studies.5,6 Both compounds bind within the peptidyl
transferase center of the ribosome, an area that we have
defined as an important ribofunctional loci for targeting inhibi-
tion of ribosomal activity.
* Corresponding author. Tel.: +1 203 848 6934; fax: +1 203 624 5627.
E-mail address: gjohnson@rib-x.com (G. Johnson).
Present address: 1517 Caywood Road, Lodi, NY 14860, USA.
Present address: Wyeth Research, CN 8000, Princeton, NJ 08543, USA.
Present address: 4890 Briarwood Drive, Macungie, PA 18062, USA.
Present address: Georgia Tech, 3305 IBB Building, GA 30332, USA.
à
§
–
||
Present address: Cephalon Inc., 383 Phoenixville Pike, Malvern, PA 19355, USA.
Present address: Goodwin and Procter, 53 State Street, Boston, MA 02109, USA.
Present address: 91 Golden Hill Drive, Guilford, CT 06437, USA.
Present address: Biogen Idec, 14 Cambridge Center, Cambridge, MA 02142, USA.
Present address: Incyte Corporation, Experimental Station, E336, Route 141 and
àà
§§
––
Henry Clay Road, Wilmington, DE 19880, USA.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.014

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J. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6179–6183
balance the resultant compounds for properties consistent with
both intravenous and oral administration.
Results and discussion: The atomic coordinates for the structure
of the 50S-sparsomycin complex have been previously determined
by Hansen and colleagues.5 The structure shows the thymine head
group of sparsomycin to be involved in an extensive network of
interactions involving key residues within the core peptidyl trans-
ferase region as well as the CCA end of the P-site tRNA substrate
(Fig. 2). The fact that these interacting elements are very highly
conserved across both prokaryotic and eukaryotic ribosomes may
explain why sparsomycin lacks bacterial selective activity.
Although our primary target molecules would maintain the
essential molecular elements of both linezolid and sparsomycin,
our ultimate plan was to design into our new structures bacterial
specificity by selectively modifying portions of the sparsomycin
thymine head group involved in making the non-discriminatory
interactions with the ribosome. To jump-start the program, a pool
of potential bridging elements between the two antibiotics, linezo-
lid, and sparsomycin, were identified via an in-house implementa-
tion of Caveat8 (data not shown). We prioritized the synthesis of
several of the best combinations featured therein, based on opti-
mized interactions with the ribosome. Three chemically-distinct
templates (Fig. 3, compounds 1–3) were prepared retaining the
key structural features of the phenyloxazolidinone fragment of lin-
ezolid and the thymine ring of sparsomycin with different bridge
elements. The first of these molecules (1) incorporated a piperidine
ring bridge element. The second construct (2) utilized a simplified
linear alkenyl bridge that was designed to maintain the relative po-
sition of the thymine head group of sparsomycin and the phen-
yloxazolidinone of linezolid in an extended conformation. The
third composite molecule (3) incorporated an aromatic-rich phenyl
bridge element. This third molecule was expected to take advan-
Figure 1. Initial design for enhanced oxazolidinones (sparsomycin is shown in pink,
linezolid in blue, and ribosomal RNA bases in gray).
As reviewed previously,7 our discovery strategy for the design
of these enhanced oxazolidinones can be summarized by the fol-
lowing five steps: (I) establish that different ribofunctional loci de-
fined by adjacent or overlapping antibiotics binding to the 50S can
be exploited simultaneously to deliver a potent antibacterial
compound; (II) identify molecular features important for driving
bacterial selectivity within the sparsomycin-binding region; (III)
optimize the bridge elements spanning these loci and identify
the minimal core and optimal bridging element required for
increasing potency (Fig. 1C); (IV) identify the features necessary
for enhancing potency against Gram-negative bacteria; and (V)
tage of
p stacking to key ribosomal bases, a feature frequently
found within the ribosome.
The ribosomal selectivity for these constructs was evaluated
through inhibition of translation of ribosomes derived from both
Escherichia coli and rabbit reticulocyte9 (Table 1). Pursuing the no-
tion that simplifying the thymine head group of sparsomycin and
hence reducing its network of hydrogen bonds with the RNA back-
bone might improve selectivity away from eukaryotes, we also
prepared compounds 4 and 5. As can be seen in Table 1 compounds
1 and 2 were potent inhibitors of E. coli translation and were as ex-
pected, non-selective. Compound 3 showed greater selectivity
probably reflecting the influence of the additional aryl ring on
enhancing binding to the bacterial ribosome. Nonetheless, given
the large size of these hybrid structures the observed level of trans-
lation activity was reassuring. We were also gratified to obtain a
crystal structure of compound 2 (Fig. 4) bound to the ribosome
which clearly confirmed both our computational projection and
overall structural hypothesis.10
Compounds 4 and 5, where the thymine group had been simpli-
fied to a pyridine ring, were less potent against E. coli but clearly
demonstrated improved selectivity for bacterial ribosomes over
those of rabbit reticulocyte (Table 1). Of these first five compounds,
the two which incorporated a phenyl linking element (compounds
3 and 5), demonstrated both superior translation and antimicrobial
inhibition. In addition to reducing entropy, this result confirmed
our hypothesis that the biaryloxazolidinone scaffold (Fig. 5) could
take advantage of an energetically favorable
p stacking interaction
with the ribosome. Their refinement into a new family of bia-
ryloxazolidinones is described in the companion letter.18 Although
derived here from first principles by use of the ribosomal structure,
several examples of substituted biaryl oxazolidinones have been
previously reported.12–15
Figure 2. Web of interactions made by the thymine head group of sparsomycin
(shown in orange) with RNA bases (shown as white stick figures). A bound P-site
tRNA analog is represented as a space-filling surface. The thymine head group is to
the left of the sparsomycin structure.
Synthesis: Our initial hybrid probe molecules 1–5 (Fig. 3) were
prepared as shown in Schemes 1–3. The common thymine acrylic

J. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6179–6183
6181
OH
O
O
O
Bridge-
N
HN
NH
Element
O
NH O
F
NHAc
Bridging Element
Modification
HN
HN
O
O
O
NH
O
O
O
O
O
O
HN
NH
O
O
O
O
N
N
N
HN
O
N
NH
HN
O
F
F
O
F
F
NHAc
NHAc
NHAc
NHAc
NH
1
2
3
O
NH
O
O
O
NH
O
N
N
N
F
NHAc
N
4
5
Figure 3.
Table 1
Initial biological results
Linezolid
Sparsomycin
1
2
3
4
5
Intrinsic affinity
E. coli translation IC50
Selectivity
(lM)
4.6
Y
6 0.02
N
0.26
N
0.03
N
0.03
Y
16
Y
0.58
Y
MIC (lg/ml)
S. pneumoniae 02J1175 mef(A)
2
1
32
16
8
2
4
4
>128
>128
8
1
1
32
60.25
60.25
16
>128
60.25
2
2
32
>128
8
0.5
0.5
16
S. pyogenes Msr 610 erm(B)
2
>128
8
E. faecalis P5 (linezolidÒ
)
H. influenzae parent strain (RD1)
H. influenzae 895 (AcrB-KO)
>128
>128
8
1
2
All minimal inhibitory concentration (MIC) determinations were carried out under NCCLS conditions.11
O
O
N
F
HN
O
Figure 5. Biaryloxazolidinone scaffold.
acid head group was prepared by a literature method.16 In each case
this element was attached to the target scaffold via an EDCI
(1-ethyl-3-(30-dimethylaminopropyl)carbodiimide) mediated amide
coupling. 4-Aminomethylpiperidine substituted 3-fluorophenyl
oxazolidinone, the precursor for compound 1 was generated in a
multistep synthesis as shown in Scheme 1. Compounds 2 and 4
were prepared as shown in Scheme 2 starting with carbobenzyloxy
(Cbz)-protected 4-iodo-3-fluoroaniline. Compounds 3 and 5 were
generated from the versatile intermediate 4-iodo-3-fluorophenyl
oxazolidinone acetamide (6)17 as shown in Scheme 3. The more ad-
vanced intermediates 7, 8, and 9 generated in this scheme were la-
Figure 4. Crystal structure of Compound 2 (cyan) bound within the 50S rRNA
pocket (depicted as white van der Waals surface). The tRNA mimetic (CCA)
(magenta) is also shown bound within the ribosomal P Site. The crystal structure of
sparsomycin (orange) is superimposed for reference.
ter incorporated into the synthesis of more advanced molecules18
.

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J. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6179–6183
F
F
CbzCl (1.05 eq)
NaHCO3
H2/Pd(C)
HO
N
F
HO
N
F
NO2
HO
HO
HCO2NH4 (4.0 eq)
THF/MeOH (1:1)
0-25 °C
95%
N
H
THF/MeOH, 0-5 °C
NO2
NH2
Hunig's base
ACN, RT, 1h
99%
1. nBuLi (2.2eq)
O
TBDMSCl (1.1eq)
TBDMSO
N
F
TBDMSO
N
N
F
O
2.
O
O
TEA (1.5eq)
CH2Cl2, 0-25 °C
N
F
NHCbz
O
NHCbz
THF, -78 °C - 25 °C
89%
HO
NaN3 (2 eq)
DMF, 70 - 80 °C, 12 h
74%
1.
MsCl (1.1 eq)
TEA (2.0 eq)
TBDMSO
N
F
Ac2O, DMAP
O
TBDMSO
N
F
O
2. H2/Pd (C)
MeOH
96%
CH2Cl2, TEA
0-5 °C, 2h
85%
N
O
N
CH2Cl2, 0 - 25 °C, 6 h
94%
O
MsO
H2N
1. MsCl (1.1 eq)
Hunig's base (1.1 eq)
CH2Cl2, 0 - 25 °C, 2 h
TBDMSO
N
F
HO
O
N
F
TBAF (1.2 eq)
O
N3
N
O
O
N
THF, 0-25 °C
95%
NaN3 (4.0 eq)
2.
O
N
O
F
N
O
DMF, 60 - 70 °C, 4 h
74%
AcHN
AcHN
AcHN
O
OH
H
N
O
HN
H
H2N
N
F
N
N
F
HN
O
O
N
H
Me
H2/Pd (C)
MeOH, RT, 12h
O
O
O
N
EDCI (1.5 eq)
TEA, DMF, RT, 8h
61%
N
O
O
1
AcHN
AcHN
Scheme 1.
1. MsCl (1.1 eq)
TEA (2.0 eq)
CH2Cl2, 0 - 25 °C, 0.5h
N3
HO
1. nBuLi (2.2eq)
I
OH
O
2.
F
NHCbz
O
O
TEA(2.0eq)
F
F
NaN3 (2 equiv)
2.
Pd(dppf)Cl2 (4%)
ACN, reflux, 12h
40%
DMF, 70 - 80 °C, 12 h
74% for 2 steps
NHCbz
BocHN
NHCbz
BocHN
THF, -78 °C - 25 °C
64%
N3
1. MsCl (1.1 eq)
TEA (2.0 eq)
CH2Cl2, 0 - 25 °C, 0.5h
1. Ph3P
THF/H2O, 50 °C
F
F
F
NaN3 (4.0 eq)
2.
2. Boc2O(1.1eq)
K2CO3, THF/H2O
O
O
O
N
O
DMF, 70 - 80 °C, 4 h
76% for 2 steps
N
N
O
O
N3
HO
HO
BocHN
BocHN
H2/Pd(C)
4M HCl in dioxane
Ac2O, DMAP
HCO2NH4 (4.0 eq)
F
F
CH2Cl2, TEA
25 °C, 4h
94%
CH2Cl2, 2 h
97%
THF/MeOH, 0-5 °C
91%
O
O
N
N
O
O
H2N
AcHN
O
O
O
O
OH
HN
HN
N
O
H2N
H
N
H
O
O
N
H
F
N
O
EDCI (1.5 eq)
F
2
4
TEA (2.0eq), DMF, RT, 6h
82%
AcHN
O
N
O
O
O
N
AcHN
N
H
O
OH
N
F
N
O
EDCI (1.5 eq)
TEA (2.0eq),DMF, RT, 6h
51%
AcHN
Scheme 2.

J. Zhou et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6179–6183
6183
1.
HO
(1.3 eq)
B(OH)2
I
Cl
N3
NaN3 (1.5 eq)
O
K2CO3 (3.0 eq), Pd(PPh3)4 (5%)
toluene/EtOH/H2O (3/1/1),reflux, 8 h
O
O
F
N
O
H
F
N
F
N
O
O
DMF, 40 - 45 °C, 8 h
95%
6
7
2.
MsCl (1.1 eq),DIEA (2.0 eq)
DMF, 0 - 25 °C 6 h
H
H
8
AcHN
AcHN
AcHN
95% for two steps
O
O
O
O
NH
O
OH
HN
N
H
N
H
O
N
H
O
H2N
3
PPh3 (1.2 eq)
H2O (10 eq)
F
N
O
H
O
F
N
O
THF, 25 °C, 12 h
94%
O
AcHN
H
N
H
AcHN
9
N
O
F
N
O
5
O
H
OH
N
AcHN
Scheme 3.
MgCl, 60 mM KCl, 1 mM Dithiothreitol), optimal amounts of S100 extract from
E. coli, and mRNA (200–800 nM, final concentration) encoding firefly luciferase
in a final assay volume of 10 ml. A Victor2V Multilabel Reader (Perkin-Elmer)
was used to read luminescence. To obtain compounds selective for prokaryotic
ribosomes, we also tested the compounds to inhibit translation fueled by a
rabbit reticulocyte lysate (nuclease-treated) system (Promega part number
L4960) following the protocol in Promega’s Technical Manual 232 and using
mRNA (200–800 nM, final concentration) encoding firefly luciferase. IC50
Acknowledgments
We thank Laura Lawrence, Timothy McConnell, Bradford King,
and Xiang Luo, for the biological data incorporated in this paper.
References and notes
values were calculated using MDL Assay Explorer with
a one-site
competition model of binding.
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9. To test the inhibition of protein synthesis by these novel oxazolidinones we
developed an E. coli in vitro translation-only assay. We developed this assay by
modifying a previously described transcription/translation assay fueled with
an E. coli S30 extract (Promega part number: L1020 and Promega’s Technical
Bulletin No. 092). The translation-only assay uses purified E. coli 70S ribosomes
(20 nM final concentration) in TMK buffer (10 mM Tris–HCl, pH 7.4, 6 mM