Synthesis and stereospecificity of 4,5-disubstituted oxazolidinone ligands binding to T-box riboswitch RNA

Article abstract of DOI:10.1021/jm2006904

The enantiomers and the cis isomers of two previously studied 4,5-disubstituted oxazolidinones have been synthesized, and their binding to the T-box riboswitch antiterminator model RNA has been investigated in detail. Characterization of ligand affinities and binding site localization indicates that there is little stereospecific discrimination for binding antiterminator RNA alone. This binding similarity between enantiomers is likely due to surface binding, which accommodates ligand conformations that result in comparable ligand-antiterminator contacts. These results have significant implications for T-box antiterminator-targeted drug discovery and, in general, for targeting other medicinally relevant RNA that do not present deep binding pockets.

Full text of DOI:10.1021/jm2006904

ARTICLE
pubs.acs.org/jmc
Synthesis and Stereospecificity of 4,5-Disubstituted Oxazolidinone
Ligands Binding to T-box Riboswitch RNA
Crina M. Orac,† Shu Zhou,† John A. Means,‡ David Boehm,†,§ Stephen C. Bergmeier,† and
Jennifer V. Hines*,†
†Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, United States
‡School of Sciences, University of Rio Grande, Rio Grande, Ohio 45674, United States
§Department of Chemistry, Leipzig University, Leipzig, Germany
S Supporting Information
b
ABSTRACT: The enantiomers and the cis isomers of two previously studied 4,5-
disubstituted oxazolidinones have been synthesized, and their binding to the T-box
riboswitch antiterminator model RNA has been investigated in detail. Characterization of
ligand affinities and binding site localization indicates that there is little stereospecific
discrimination for binding antiterminator RNA alone. This binding similarity between
enantiomers is likely due to surface binding, which accommodates ligand conformations
that result in comparable ligand-antiterminator contacts. These results have significant
implications for T-box antiterminator-targeted drug discovery and, in general, for
targeting other medicinally relevant RNA that do not present deep binding pockets.
’ INTRODUCTION
and the antiterminator leads to stabilization of the antiterminator
and precludes the formation of an alternative terminator struc-
tural element.9 Without the extra stabilization provided by the
antiterminator-tRNA base pairing, the terminator forms prefer-
entially and transcription is terminated. While there are other
important RNA elements within the T-box 50-UTR, the interac-
tion between the tRNA acceptor end and the antiterminator is
critical for switching between antitermination of transcription
(the entire gene is transcribed) and termination of transcription
(transcription is terminated before reaching the protein encod-
ing sequences).9 This critical role, the high degree of sequence
conservation in the antiterminator, and its prevalence in many
pathogenic bacteria10,16 make it an intriguing RNA target for
drug discovery.
While there have been a variety of studies related to targeting
RNA with small molecules, few have involved an in-depth analysis
of the RNAꢀligand interactions.1ꢀ6 We previously reported
4,5-oxazolidinones as potential ligands for targeting T-box ribos-
witch antiterminatormodel RNA AM1A(Figure 1b).12,17 Further
studies identified lead compounds that significantly altered the
function of the riboswitch.11 One compound inhibited the
riboswitch (rac-1, Figure 1c), while another acted as an agonist
and promoted tRNA-independent antitermination (rac-2,
Figure 1c). Preliminary characterization of the binding sites
Designing medicinal agents that specifically bind and disrupt
the function of RNA is an evolving field of study1ꢀ6 whose
importance will only grow as our knowledge of the role of
noncoding RNAs continues to expand. Noncoding RNAs, as the
name implies, do not encode a protein product but instead are
often critical regulators of gene expression.7,8 One medicinally
relevant example of a noncoding RNA is the T-box transcription
antitermination riboswitch.9 The T-box riboswitch is located in
the 50-untranslated mRNA region (50-UTR) of many important
Gram-positive bacterial genes and controls transcription of the
protein-encoding region of the mRNA.10 We recently reported
lead oxazolidinone compounds that act as molecular effectors of
T-box riboswitch function.11,12 As part of a comprehensive effort
to design and develop potential medicinal agents for disrupting
the T-box riboswitch,11ꢀ15 we wished to investigate the details of
the binding interaction between these lead oxazolidinones and
their target, the T-box antiterminator RNA structural element.
During transcription of genes containing the T-box ribos-
witch, the 50-UTR is transcribed before the protein-encoding
region of the mRNA and the riboswitch responds to the presence
or absence of nonaminoacylated (uncharged) cognate tRNA.9
The anticodon of the tRNA base-pairs with a complementary
specifier sequence located in stem I near the beginning of the 50-
UTR while the tRNA acceptor end nucleotides base-pair with
four nucleotides in the bulge of the antiterminator (Figure 1a).
The base pairing between the tRNA acceptor end nucleotides
Received: May 31, 2011
Published: August 03, 2011
r
2011 American Chemical Society
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Figure 1. (a) Schematic of T-box riboswitch binding tRNA. (b) Antiterminator model RNA AM1A. Nucleotides that base-pair with the acceptor end of
tRNA are noted with boxes. (c) Structure of racemic trans-4,5-disubstituted oxazolidinones rac-1 and rac-2.
Scheme 1
Scheme 2a
indicated that the two molecules were likely binding in different
(but somewhat overlapping) regions of the antiterminator RNA.11
In this paper we report the enantiopure synthesis of these lead
compounds as well as their cis analogs and the characterization of
their RNA binding modes to the model antiterminator RNA,
AM1A (Figure 1). These studies better elucidate the specific
ligandꢀRNA interactions that may be critical for developing a
medicinal agent targeted to the T-box antiterminator.
a Reagents and conditions: (i) AcOH, H2O, 18 h, 20%; (ii) TrCl, Et3N,
DMAP, CH2Cl2, 24 h, 64%.
The required enantiomers of 5 (Scheme 2) were obtained by
the known hydrolytic kinetic resolution of commercially available
racemic butadiene monoepoxide 6 using the catalyst (R,R)-7 and
its enantiomer (S,S)-7.21 Prior to the determination of the
enantiomeric purity (er), both enantiomers of 5 were converted
to their corresponding trityl ether, (R)-8 and (S)-8, derivatives.
As determined by NMR, using (S)-(ꢀ)-R-methoxy-R-trifluor-
omethylphenylacetyl chloride22 as the chiral derivatizing agent,
the R enantiomer was obtained in 99:1 er and the S enantiomer
was obtained in 98:2 er.
With both enantiomers of 8 in hand we proceeded with the
synthesis as shown in Scheme 3. The (R)-8 was converted to its
corresponding azidoformate (R)-3 in very good yield by using p-
nitrophenyl chloroformate followed by sodium azide.18 Azido-
formate (R)-3 was then subjected to the thermal intramolecular
aziridination to provide the bicyclic aziridine (4S,5R)-4. Next, the
aziridine ring-opening with N-phenylpiperazine followed by the
acidic cleavage of trityl group afforded the (4S,5R)-10 oxazoli-
dinone in excellent yield. The (4R,5S)-10 enantiomer was
’ RESULTS AND DISCUSSION
Chemistry. For the synthesis of both enantiomers of the lead
compounds rac-1 and rac-2, we used a method previously
developed in our laboratories18ꢀ20 and reported for the prepara-
tion of these racemic lead compounds (Scheme 1).11 The key
step in this method is the intramolecular acylnitrene-mediated
aziridination that provides the bicyclic aziridine 4 with very good
diastereoselectivity.19 The bicyclic aziridine 4 can be further
converted to the desired final compounds in three simple steps:
nucleophilic aziridine ring-opening, trityl ether deprotection, and
reaction with phenylacetyl chloride or 4-acetylphenyl isocyanate.
The azidoformate precursor 3 is readily accessible from 3-bu-
tene-1,2-diol (5, Scheme 1). In order to apply this synthetic plan
for the synthesis of our target enantiomers, it was necessary to
first prepare both enantiomers of 5.
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Scheme 3a
Scheme 5a
a Reagents and conditions: (i) a) p-NO2PhOC(O)Cl, pyridine, CH2Cl2,
2 h; (b) NaN3, acetone/H2O, 4 days, (R)-3 (80% two steps), (S)-3 (80%
two steps); (ii) sealed tube, CH2Cl2, 109 °C, (4S,5R)-4 (55%), (4R,5S)-4
(55%); (iii) N-phenylpiperazine, CH2Cl2, 2 h, (4S,5R)-9 (91%), (4R,5S)-
9 (86%); (iv) HCl, EtOAc, 30 min, (4S,5R)-10 (93%), (4R,5S)-10 (92%).
Scheme 4a
a Reagents and conditions: (i) LiAlH4, THF, 18 h, 67%; (ii) mCPBA,
CH3CN, 4 °C, 4 days, 90%; (iii) NaN3, NH4Cl, EtOH, 17 h, 90%; (iv) p-
TsOH, acetone, 12 h, 67%; (v) TsCl, pyridine, CH2Cl2, 12 h, 70%; (vi)
N-phenylpiperazine, K2CO3, EtOH, 12 h, 60%; (vii) H2, 10 mol % Pd/
C, EtOAc, 97%; (viii) PhOCOCl, Et3N, DMAP, CH2Cl2, 15 min, 89%;
(ix) oxalic acid, THF/H2O, 60 °C, 8 h, 66%.
Scheme 6a
a Reagents and conditions: (i) PhCH2COCl, Et3N, DMAP, CH2Cl2, 5 h,
36%; (ii) 4-acetylphenyl isocyanate, Et3N, DMAP, CH2Cl2, 5 h, 50%.
a Reagents and conditions: (i) PhCH2COCl, Et3N, DMAP, CH2Cl2, 5 h,
(4S,5R)-1 (81%), (4R,5S)-1 (77%); (ii) 4-acetylphenyl isocyanate, Et3N,
DMAP, CH2Cl2, 5 h, (4S,5R)-2 (80%), (4R,5S)-2 (82%).
(11, Scheme 5). We first subjected compound 11 to a standard
reduction with lithium aluminum hydride23 followed by the
epoxidation of the resulting trans double bond using m-chlor-
operbenzoic acid in acetonitrile to provide the epoxy diol 12.24
The next step in the synthesis was the nucleophilic ring-opening
of the epoxide 12 with sodium azide,25 which afforded the azido
triol 1326 with the desired trans relative configuration. The
differentiation between the three hydroxyl groups of 13 was
accomplished by protecting the two adjacent groups as the
isopropylidene acetal.27 None of the six-membered ring aceto-
nide was observed.28 The free primary hydroxyl of acetonide 1429
was then tosylated, and the resulting tosyloxy group was sub-
stituted with N-phenylpiperazine, affording compound 16. The
catalytic hydrogenation of azide 16 followed by the reaction of
prepared in the same manner and with similar yields starting
from (S)-8.
The desired final compounds, (4S,5R)-1 and (4S,5R)-2, were
prepared from (4S,5R)-10 (Scheme 4) by reaction with pheny-
lacetyl chloride, providing (4S,5R)-1 and 4-acetylphenyl isocya-
nate, affording (4S,5R)-2 in good yields. Their enantiomers
(4R,5S)-1 and (4R,5S)-2 were obtained in the same manner
starting from (4R,5S)-10.
For the synthesis of cis isomers of rac-1 and rac-2 our synthetic
plan started from commercially available 2-butyne-1,4-diol
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Table 1
predicted binding location was similar between enantiomeric pairs
but differed between the two structural series (Figure 2). The
(4R,5S)-1/(4S,5R)-1 compounds bound along the major groove
of helix A1 (G3-G5) extending up into the 50 end of the bulge
(U6-G8), while the (4R,5S)-2/(4S,5R)-2compounds bound solely
to the bulge region (U6-G8 and the major groove of the G5:C25
closing base pair). This difference in predicted binding location is
consistent with the differing effects of rac-1 and rac-2 on T-box
transcription antitermination.11
A lack of stereospecific ligand binding with relatively simple
RNA structural motifs has been observed previously.34,35 Given
that the known structure of AM1A36 presents multiple binding
surfaces and few, if any, deep binding pockets, it was not a
foregone conclusion that enantiomers would bind with widely
differing affinities. Comparison of the energetically most favor-
able docked structures (Figure 2) indicates that each enantio-
meric pair likely binds to a similar region in AM1A with
comparable binding interactions (H-bonding, πꢀπ stacking)
but with different functional group partners within the antiter-
minator model RNA (Figure 3). With comparable electrostatics,
the primary interaction differences between the two enantio-
meric pairs are that the enantiomers of 2 form three hydrogen
bonds with AM1A while the enantiomers of 1 form two hydro-
gen bonds along with differential amounts of πꢀπ stacking.
Futher, more detailed, structural studies are needed to best
determine the role that these and other binding interactions
may play in ligand binding specificity.
There is always the possibility, however, that the rigid receptor
docking studies do not fully predict the structure of the
RNAꢀligand complex. It is conceivable that the ligand binds
via an induced fit and/or tertiary structure capture. In-line
probing37 experiments were conducted to determine if there
was a significant restructuring of the RNA upon ligand binding.
In-line probing monitors ligand-induced changes in RNA flex-
ibility. No dramatic secondary structural changes were detected
(Figure 4). Instead, the observed ligand-induced changes in
the in-line cleavage patterns were indicative of conformation/
flexibility changes in the RNA without a significant reordering of
secondary structure base pairing.
Each pair of enantiomers exhibited similar ligand-induced
changes, and the two structural series had very different ligand-
induced changes. These observations are consistent with the
binding and docking studies. For both (4R,5S)-1 and (4S,5R)-1
the ligand-induced cleavage changes observed were a slight
increase along the 50 side of the A1 helix (especially at nucleotide
A2) and a more significant decrease in cleavage for bulge
nucleotides U6-C11. These results indicate that ligand binding
leads to reduced conformational flexibility for U6-C11 and
increased flexibility at nucleotide A2. Both (4R,5S)-2 and
(4S,5R)-2 resulted in an increase in cleavage at the 30 end of
the bulge, especially at C11 and also an increase in cleavage
within the tetraloop (U17-G20). These results indicate that
ligand binding leads to increased flexibility at C11 and in the
tetraloop. Both effects could be due to the ligand binding to the 50
end of the bulge (as predicted in the docking studies), resulting in
a slight conformational change toward the 30 end of the bulge.
This change could induce a long-range topological strain that
affects the tetraloop nucleotides.
compd
Kd a (μM)
Emodel b (kcal/mol)
rac-1
13 ( 411
0.9 ( 0.411
12 ( 5
rac-2
(4R,5S)-1
(4S,5R)-1
(4R,5S)-2
(4S,5R)-2
cis-1
ꢀ101.0
ꢀ98.9
ꢀ117.4
ꢀ117.4
16 ( 5
3 ( 1
1.6 ( 0.5
NBc
cis-2
1.8 ( 0.7
a Kd values determined using FRET-derived binding assay with 100 nM
labeled AM1A RNA. See Experimental Section for details and Support-
ing Information for the binding isotherms. All R2 values are g0.8. b Glide
calculated Emodel of the most energetically favorable docked proton-
ated ligand. c NB: No apparent binding was observed at 130 min
(the standard incubation time for all other compounds), Kd > 100 μM.
At 70 min of incubation time, however, a binding isotherm was observed
(Kd = 5.1 ( 2.2, R2 = 0.78). This apparent time-dependence will be
investigated in future work.
the resulting amine with phenyl chloroformate afforded the
carbamate 17. The cleavage of the isopropylidene acetal with
trifluoroacetic acid in water30 afforded only a low yield of the
unprotected diol. A more suitable method for our substrate
proved to be the use of an aqueous solution of oxalic acid in
tetrahydrofuran that directly provided the cis-10 oxazolidinone
after a basic workup.
The final compounds cis-1 and cis-2 (Scheme 6) were prepared
from cis-10 in the same manner as their corresponding trans
isomers. Treating cis-10 with phenylacetyl chloride afforded cis-1,
while the reaction with 4-acetylphenyl isocyanate provided cis-2.
Biological Results. Binding Affinity. The binding affinity for
each compound was determined using the previously described
fluorescence resonance energy transfer (FRET) antiterminator
RNA binding assay.11,13 The observed Kd values are summarized
in Table 1.
For the racemic cis compounds, cis-1 did not bind AM1A while
cis-2 bound with affinity similar to the corresponding racemic
trans compound rac-2. The (4R,5S)-1/(4S,5R)-1 affinities for
binding AM1A were approximately 7-fold weaker than those for
(4R,5S)-2/(4S,5R)-2, consistent with results from previous
binding studies of the corresponding racemic compounds.11
Interestingly, however, the enantiomeric pair (4R,5S)-1 and
(4S,5R)-1 had similar RNA affinities. The (4R,5S)-2 and
(4S,5R)-2 enantiomeric pair also had similar RNA affinities.
Ligand Binding Site. Ligand docking studies using the Glide
module of First Discovery 2.7 (Schr€odinger) were conducted to
begin to determine possible binding modes for the pairs of
enantiomers. Glide performs flexible ligand docking to a rigid
receptor by utilizing an OPLS-AA31 derived molecular mechanics
potential function, a grid-based docking method, and a discretized
version of the ChemScore empirical scoring function32 followed by
energy minimization of the best-refined poses.33 The results of
these docking studies were consistent with results from the binding
studies. The most energetically favorable Emodel (an indicator of
best docked structure) was similar between enantiomeric pairs (see
Table 1). Between the two different structural series, however,
Emodel was ∼17% more stable for (4R,5S)-2 or (4S,5R)-2 with
AM1A compared to (4R,5S)-1 or (4S,5R)-1, consistent with the
∼7-fold difference in the observed Kd values. In addition, the Glide
These studies reveal that the lack of a deep binding pocket and
redundancy of similar functional groups within the relatively
simple structural motif of the antiterminator can lead to a lack of
stereospecific discrimination in ligand binding. In the context of
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Figure 2. Lowest energy docked structure of monoprotonated oxazolidinones binding to AM1A.
Figure 3. Summary of H-bonding and π-stacking interactions between docked oxazolidinones and AM1A.
the entire T-box riboswitch, there is a significant likelihood that a
ligand functional group that did not interact with the antitermi-
nator could be in a position to stereospecifically disrupt tRNA
binding. Studies are currently in progress to investigate this
possibility. The finding that the oxazolidinones likely bind
AM1A via surface binding has significant implications for RNA
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Figure 4. In-line probing of enantiomeric ligands binding to AM1A: (a) (4R,5S)-1 and (4S,5R)-1; (b) (4R,5S)-2 and (4S,5R)-2. Significant changes are
summarized on secondary structure of AM1A for increased (shaded circle) and decreased (open circle) in-line cleavage. Significant changes were
identified by comparing the slopes of the ligand-induced relative band intensity changes (see Supporting Information).
drug discovery that is focused on targeting other medicinally
relevant RNAs.
running reactions involving azides. p-Nitrophenyl chloroformate (2.4 g,
12 mmol) was added to a solution of allylic alcohol (R)-8 (2 g, 6 mmol)
in CH2Cl2 (20 mL). The solution was cooled to 0 °C, and pyridine
(1.46 mL, 18 mmol) was added dropwise. The reaction mixture was
allowed to warm to room temperature and stirred for 1 h. Then it
was washed with saturated aqueous NaHCO3 solution (2 ꢁ 20 mL) and
brine (2 ꢁ 20 mL). The organic layer was dried over MgSO4, filtered,
and concentrated to dryness, and the residue was dissolved in acetone
(22 mL). A solution of NaN3 (2.73 g, 42 mmol) in H2O (13 mL)
was then added to this solution, and the reaction mixture was stirred at
room temperature for 72 h when it was diluted with H2O and the
aqueous portion extracted with EtOAc (3 ꢁ 20 mL). The combined
organic layers were washed with 10% K2CO3 aqueous solution (2 ꢁ
20 mL) and then dried over MgSO4, filtered, and concentrated by rotary
evaporation. The residue was purified by flash chromatography (5%
’ EXPERIMENTAL SECTION
General. Reagents and starting materials were obtained from
Aldrich unless otherwise stated. All RNA experiments were conducted
using RNase-free conditions and molecular biology grade reagents.
Fluorescently labeled RNA, 30-Fl-AM1A-(18)-Rhd was prepared as
previously described.11,13 Racemic butadiene monoepoxide 6 was
purchased from Alfa Aesar. Both enantiomers of the (salen)Co(II)
complex 7 are commercially available from Aldrich. CH2Cl2 and THF
were dried using a SOLV-TEK solvent purification system. Acetone was
dried over 4 Å molecular sieves and distilled immediately before use.
Et3N was dried over calcium hydride and distilled immediately before use.
Melting points were determined with a MEL-TEMP II melting point
apparatus and are reported uncorrected. Specific rotations were mea-
sured on an AUTOPOL IV (Rudolph Research Analytical) polarimeter
with a sodium (λ = 589 nm) lamp and are reported as follows: [R]λT,°C
(c in g/100 mL, solvent). The capillary GC analyses were performed on a
Shimadzu GC-17A gas chromatograph employing the Rtx-5 (15 m ꢁ
0.25 mm i.d. ꢁ 0.25 μm df; Restek) column. The retention times are
reported in minutes as follows: initial T °C to final T °C, rate, duration of
run. The purities of tested compounds were determined via HPLC
analysis on a Shimadzu LC-10AT machine equipped with a UV detector
employing a Discovery-C8 (15 cm ꢁ 4.6 mm ꢁ 5 μm; Supelco) column,
eluting with MeOH in H2O at 1 mL/min flow rate (gradient started at
8 min with 50% MeOH/H2O and ended after 5 min with 90% MeOH/
H2O, run time of 22 min). The compounds showed >95% purity unless
otherwise stated. 1H NMR and 13C NMR spectra were recorded with a
Br€uker AVANCE (300 MHz) spectrometer. Chemical shifts are re-
ported in ppm on the δ scale relative to deuterated chloroform as an
internal standard. Data are reported as follows: chemical shift, multi-
plicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
coupling constant in Hz, integration.
EtOAc in hexanes) to provide 1.9 g (80%) of product as a colorless oil.
1
Rf = 0.36 (5% EtOAc in hexanes); H NMR (CDCl3, 300 MHz) δ
7.45ꢀ7.26 (m, 15H, Ar), 5.82 (ddd, J = 6.5, 10.6, 17.2 Hz, 1H,
HCdCHH), 5.47ꢀ5.42 (m, 1H, OCH), 5.37 (d, J = 17.2 Hz, 1H,
HCdCHH), 5.29 (d, J = 10.6 Hz, 1H, HCdCHH), 3.32 (dd, J = 7.2,
10.2 Hz, 1H, TrOCHH), 3.23 (dd, J = 3.9, 10.2 Hz, 1H, TrOCHH); 13C
NMR(CDCl3, 75 MHz) δ 156.9, 143.5, 132.1, 128.6, 127.9, 127.2, 119.4,
86.8, 78.5, 64.8; [R]D24 +21.1 (c 1.04, CHCl3). For (S)-3: [R]2D4 ꢀ20.8
(c 1.0, CHCl3).
(4S,5R)-4-Trityloxymethyl-3-oxa-1-azabicyclo[3.1.0]hexan-
2-one ((4S,5R)-4).18 CAUTION: Reactions carried out in pressure tubes
are potentially explosive. While we did not experience any problems, the
reactions should be carried out behind a protecting shield. A solution of the
azidoformate (R)-3 (1.8 g, 4.5 mmol) in CH2Cl2 (70 mL) was placed in
an Ace sealedtube(catalogno. 8648-79). The tubewas cooled to ꢀ78 °C,
evacuated, sealed, and heated to 109 °C for 14 h. Then the tube was
allowed to cool to room temperature and the solvent removed by rotary
evaporation. The residue was washed with 5% EtOAc in hexanes and a
precipitate was formed that was isolated by filtration to afford 0.93 g of
(4S,5R)-4 (55%). Rf = 0.24 (30% EtOAc in hexanes); 1H NMR (CDCl3,
300 MHz) δ 7.36ꢀ7.25 (m, 15H, Ar), 4.68 (t, J = 3.2 Hz, 1H,
C(O)OCH), 3.56 (dd, J = 3.8, 10.5 Hz, 1H, TrOCHH), 3.25 (dd,
J = 3.4, 10.5 Hz, 1H, TrOCHH), 3.05 (t, J = 4.1 Hz, 1H, NCH), 2.56 (d,
(R)-1-(Triphenylmethoxy)-2-[(azidocarbonyl)oxy]-3-butene
((R)-3).18 CAUTION: Azides are potentially explosive especially when heated.
While we did not experience any problems, precaution should be taken when
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J = 4.2 Hz, 1H, NCHH), 2.18 (d, J = 4.2, Hz, 1H, NCHH); [R]2D4 ꢀ21.1
(c 1.04, CHCl3). For (4R,5S)-4: [R]2D4 +20.9 (c 1.04, CHCl3).
of cis-10. No further purification was necessary. Rf = 0.22 (95% EtOAc in
1
hexanes); H NMR (CDCl3, 300 MHz) δ 7.27ꢀ7.22 (m, 2H, Ph),
(R)-1-(Triphenylmethoxy)-3-buten-2-ol ((R)-8).18 To a solu-
tion of (R)-521 (0.24 g, 2.72 mmol) in CH2Cl2 (5 mL) was added trityl
chloride (0.909 g, 3.26 mmol) and DMAP (40 mg, 0.332 mmol)
followed by Et3N (0.76 mL, 5.44 mmol). The reaction mixture was
stirred under an argon atmosphere at room temperature for 24 h. The
reaction mixture was diluted with Et2O (20 mL) and washed with H2O
(10 mL), cold 1 M HCl (10 mL), saturated NaHCO3 (10 mL), H2O
(10 mL), and brine. The organic phase was dried over MgSO4, filtered,
and concentrated by rotary evaporation. The residue was purified by
flash chromatography (10% EtOAc in hexanes) to provide 0.575 g
(64%) of (R)-8 as a colorless liquid. Rf = 0.25 (10% EtOAc in hexanes);
1H NMR (CDCl3, 300 MHz) δ 7.45 (m, 15H, Ph), 5.82 (ddd, J = 5.6,
10.6, 17.2 Hz, 1H, CHdCH2), 5.35 (d, J = 17.2 Hz, 1H, dCHH), 5.20
(d, J = 10.6 Hz, 1H, dCHH), 4.27ꢀ4.32 (m, 1H, CHOH), 3.25 (dd, J =
3.8, 9.4 Hz, 1H, TrOCHH), 3.15 (dd, J = 7.4, 9.4 Hz, 1H, TrOCHH), 2.4
(d, J = 3.9 Hz, 1H, OH); 13C NMR (CDCl3, 75 MHz) δ 143.8, 137.0,
128.7, 127.9, 127.2, 116.4, 86.8, 72.1, 67.5; [R]2D4 +27.8 (c 1.0, i-PrOH).
For (S)-8: [R]2D4 ꢀ28.0 (c 1.01, i-PrOH).
6.90ꢀ6.84 (m, 3H, Ph), 5.73 (s, 1H, NH), 5.36 (br s, 1H, OH), 4.81 (ddd,
J = 5, 7.7, 9 Hz, 1H, OCH), 4.16 (ddd, J = 4, 7.7, 11 Hz, 1H, NHCH),
3.90ꢀ3.77 (m, 2H, HOCH2), 3.25ꢀ3.12 (m, 4H, PhN(CH2)2), 2.90
(dd, J = 11, 13 Hz, 1H, NCHH), 2.83ꢀ2.76 (m, 2H, NCH2), 2.69ꢀ2.62
(m, 2H, NCH2), 2.42 (dd, J = 4, 13 Hz, 1H, NCHH); 13C NMR (CDCl3,
75 MHz) δ 158.7, 150.9, 129.4, 120.7, 116.6, 78.3, 59.3, 57.8, 53.7,
51.2, 49.2.
2-Azido-2-(2,2-dimethyl[1,3]dioxolan-4-yl)ethanol (13). To
a solution of epoxide 1224 (0.52 g, 5 mmol) in EtOH (8 mL) at room
temperature were added successively NH4Cl (0.615 g, 11.5 mmol) and
NaN3 (0.747 g, 11.5 mmol). The mixture was heated to reflux for 17 h.
After cooling, the reaction mixture was diluted with EtOAc (20 mL) and
then filtered. The filtrate was concentrated by rotary evaporation to
R
provide 0.66 g (90%) of 13 as a yellowish oil. Rf = 0.3 (EtOAc); t = 1.1 min
(91%) (40ꢀ100 °C at 10 C°/min, then to 250 °C at 25°/min, 12 min);
1H NMR (D2O, 300 MHz) δ 3.93 (dd, J = 3, 11 Hz, 1H), 3.81ꢀ3.62
(m, 5H); 13C NMR (D2O, 75 MHz) δ 70.4, 64.2, 61.9, 60.5. Matched
analytical data previously reported.26
2-Azido-2-(2,2-dimethyl[1,3]dioxolan-4-yl)ethanol (14). To
a solution of triol 13 (0.6 g, 4 mmol) in dry acetone (110 mL/mmol) was
added p-toluenesulfonic acid (0.3 g, 1.6 mmol). The reaction mixture was
stirred at room temperature for 18 h. K2CO3 (0.77 g, 5.6 mmol) was added
to the reaction flask, and the mixture was stirred for another 2 h. Then the
reaction mixture was filtered and the filtrate was concentrated by rotary
evaporation. The residue was purified by flash chromatography (20%
EtOAc in hexanes) to provide 0.5 g (67%) of 14 as a colorless oil. Rf = 0.18
(20% EtOAc in hexanes); tR = 3.13 min (100%) (75ꢀ300 °C at 25 °C/
min, 12 min); 1H NMR (CDCl3, 300 MHz) δ 4.14ꢀ4.04 (m, 2H, OCH
and OCHH), 3.92 (dd, J = 5, 8 Hz, 1H, OCHH), 3.89ꢀ3.81 (m, 1H,
HOCHH), 3.68 (dd, J = 6, 12 Hz, 1H, HOCHH), 3.63ꢀ3.57 (m, 1H,
N3CH), 2.61 (t, 1H, OH), 1.44 (s, 3H, CH3), 1.34 (s, 3H, CH3); 13C
NMR (CDCl3, 75 MHz) δ 109.9, 75.5, 66.6, 64.8, 62.7, 26.5, 25.2.
Matched analytical data previously reported.29
(4S,5R)-4-(4-Phenylpiperazin-1-ylmethyl)-5-trityloxymethyl-
oxazolidin-2-one ((4S,5R)-9). To a solution of (4S,5R)-4 (0.78 g,
2.09 mmol) in CH2Cl2 (4 mL) was added freshly distilled N-phenylpiper-
azine (0.35 mL, 2.3 mmol) neat. The mixture was stirred at room
temperature until complete conversion shown by TLC (typically 2 h).
The solvent was removed under reduced pressure, and the residue was
purified by flash chromatography (30% EtOAc in CH2Cl2) to provide 1.02
g (91%) of (4S,5R)-7 as a white solid (mp 96.1ꢀ97.9). Rf = 0.30 (30%
1
EtOAc in CH2Cl2); H NMR (CDCl3, 300 MHz) δ 7.49 (d, 6H, Ar),
7.36ꢀ7.24 (m, 11H, Ar), 6.91 (d, 3H, Ar), 5.69 (s, 1H, NH), 4.28 (q, J =
4.5, 9.3 Hz, 1H, C(O)OCH), 3.87(q, J= 6, 13.3 Hz, 1H, NHCH), 3.49 (dd,
J = 4.5, 10.3 Hz, 1H, TrOCHH), 3.25 (dd, J = 4.2, 10.3 Hz, 1H, TrOCHH),
3.12ꢀ3.02 (m, 4H, PhNCH2), 2.64ꢀ2.42 (m, 6H, NCH2); 13C NMR
(CDCl3, 75 MHz) δ 158.7, 151.1, 143.4, 129.1, 128.6, 128.0, 127.2, 119.9,
116.1, 86.9, 79.3, 63.8, 62.4, 53.4, 51.5, 49.0; Anal. Calcd for C34H35N3O3:
C, 76.52; H, 6.61; N, 7.87. Found: C, 76.54; H, 6.21; N, 7.48; [R]2D3 ꢀ32.1
(c 1.04, CHCl3). For (4R,5S)-9: [R]2D3 +33.0 (c 1.02, CHCl3).
2-Azido-2-(2,2-dimethyl[1,3]dioxolan-4-yl)ethyl-4-methyl-
benzenesulfonate (15). To a solution of acetonide 14 (0.5 g,
2.67 mmol) in CH2Cl2 (5 mL) was added toluenesulfonyl chloride
(0.61 g, 3.2 mmol). The mixture was cooled to 0 °C, and pyridine
(0.65 mL, 8.01 mmol) was added. The reaction mixture was allowed to
warm to room temperature and stirred overnight. Then it was diluted with
EtOAc and washed with saturated aqueous NaHCO3 and brine. The
organic phase was dried over MgSO4, filtered and the solvent removed by
rotary evaporation. The residue was purified by flash chromatography
(10% EtOAc in hexanes) to afford 0.64 g (70%) of 15 as a colorless oil.
(4S,5R)-5-Hydroxymethyl-4-(4-phenylpiperazin-1-ylmethyl)-
oxazolidin-2-one ((4S,5R)-10). To a solution of (4S,5R)-9 (0.69 g,
1.3 mmol) in EtOAc (5 mL) was added a solution of HCl in EtOAc (1.5 M,
4.5 mL, 6.5 mmol). The mixture was allowed to stir at room temperature for
10 min when it was diluted with EtOAc (20 mL) and H2O (30 mL) and the
layers were separated. Saturated aqueous NaHCO3 solution (10 mL) was
added to the aqueous layer and extracted with EtOAc (4 ꢁ 10 mL). The
combined organic layers were washed with H2O (10 mL), dried over
MgSO4, filtered, and concentrated by rotary evaporation to afford 0.36 g
(93%) of (4S,5R)-10 as a white solid (mp 140.5ꢀ141.9). No further
purification was necessary. Rf = 0.16 (EtOAc);1H NMR(CDCl3, 300 MHz)
δ 7.31ꢀ7.26 (m, 2H, Ar), 6.94ꢀ6.87 (m, 3H, Ar), 5.98 (s, 1H, NH), 4.33
(q, J=4.3, 10.3 Hz, 1H, C(O)OCH), 3.96(q, J= 6.7, 13.3 Hz, 1H, NHCH),
3.87 (dd, J = 4.6, 12.0 Hz, 1H, HOCHH), 3.76 (dd, J = 4.0, 12.0 Hz, 1H,
HOCHH), 3.21 (m, 4H, PhNCH2), 2.70 (m, 4H, NCH2), 2.66 (dd, J = 7.2,
12.5 Hz, 1H, NCHH), 2.53 (dd, J = 6.7, 12.5 Hz, 1H, NCHH); 13C NMR
(CDCl3, 75 MHz) δ 157.3, 149.5, 127.7, 118.7, 114.8, 79.9, 61.3, 60.7, 52.3,
R
Rf = 0.35 (20% EtOAc in hexanes); t = 7.57 min (100%) (75ꢀ300 °C at
25 °C/min, 10 min); 1H NMR (CDCl3, 300 MHz) δ 7.83 (d, J = 8 Hz,
2H, Ph), 7.38 (d, J = 8 Hz, 2H, Ph), 4.33 (dd, J = 3, 10.7 Hz, 1H, OCH),
4.08ꢀ4.02 (m, 2H, OCH2), 3.99ꢀ3.87 (m, 2H, TsOCH2), 3.71ꢀ3.65
(m, 1H, N3CH), 2.47(s, 3H, PhCH3), 1.40(s, 3H, CH3), 1.31(s, 3H, CH3);
13C NMR (CDCl3, 75 MHz) δ 145.5, 132.6, 130.2, 128.2, 110.4, 74.4,
69.4, 66.7, 62.2, 26.7, 25.1, 21.9. HRMS: calcd for C14H19N3O5S Na+,
3
364.0938; found, 364.0956.
1-Azido-1-(2,2-dimethyl[1,3]dioxolan-4-yl)-2-(4-phenylpi-
perazine-1-yl)ethane (16). To a solution of 15 (0.51 g, 1.5 mmol) in
EtOH (5 mL) was added K2CO3 (0.41 g, 3 mmol) followed by N-
phenylpiperazine (0.25 mL, 1.65 mmol). The reaction mixture was
heated to reflux overnight. After cooling to room temperature, the
reaction mixture was transferred into a separatory funnel and diluted
with EtOAc and water. The aqueous layer was extracted with EtOAc
(3 ꢁ 10 mL), and the combined organic layers were dried over MgSO4
and filtered. The solvent was removed by rotary evaporation. The residue
was purified by means of flash chromatography (10% EtOAc in toluene)
to provide 0.3 g (60%) of 16 as a colorless oil. Rf = 0.25 (10% EtOAc in
50.6, 47.7. HRMS: calcd for C15H21N3O3 Na+, 314.1475; found, 314.1476.
3
[R]2D8 ꢀ55.4 (c1.05, CHCl3). For(4R,5S)-10:[R]D28 +54.5 (c1.05, CHCl3).
5-Hydroxymethyl-4-(4-phenylpiperazin-1-ylmethyl)-
oxazolidin-2-one (cis-10). The acetonide 17 (1.1 g, 2.62 mmol) was
placed in a flask followed by oxalic acid (1.65 g, 18.34 mmol), H2O
(16.5 mL), THF (33 mL), and 12 N HCl (0.2 mL), and the reaction
mixture was stirredat 60°C for 8 h. The reaction mixture was poured into a
5% aqueous K2CO3 and stirred for 30 min and then was extracted with
CH2Cl2. The combined organic layers were dried over MgSO4, filtered,
and the solvent was removed by rotary evaporation to provide 0.5 g (66%)
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Journal of Medicinal Chemistry
ARTICLE
toluene); tR = 8.09 min (100%) (75ꢀ300 °C at 25 °C/min, 10 min); IR
2100 cmꢀ1 (s, N3); 1H NMR (CDCl3, 300 MHz) δ 7.31ꢀ7.26 (m, 2H,
Ph), 6.96ꢀ6.85 (m, 3H, Ph), 4.11ꢀ4.04 (m, 2H, OCH2), 3.97ꢀ3.91 (m,
1H, OCH), 3.77ꢀ3.71 (m, 1H, N3CH), 3.23ꢀ3.18 (m, 4H, PhN-
(CH2)2), 2.79ꢀ2.72 (m, 2H, NCH2), 2.70ꢀ2.63 (m, 3H, NCH2 and
NCHH), 2.56 (dd, J = 9, 13.3 Hz, 1H, NCHH), 1.49 (s, 3H, CH3), 1.38
(s, 3H, CH3); 13C NMR (CDCl3, 75 MHz) δ 151.4, 129.3, 119.9, 116.3,
109.9, 76.4, 66.5, 61.0, 59.7, 53.8, 49.4, 26.6, 25.4; HRMS: calcd for
7.09ꢀ7.04 (m, 2H, Ar), 6.96 (s, 1H, NH), 6.72ꢀ6.65 (m, 3H, Ar), 5.26 (s,
1H, NH), 4.32 (q, J = 4.5, 8.0 Hz, 1H, C(O)OCH), 4.20 (m, 2H,
C(O)OCH2), 3.66 (q, J = 5.6, 13.0 Hz, 1H, HNCH), 2.98 (t, J = 4.6 Hz,
4H, PhNCH2), 2.54ꢀ2.30 (m, 6H, NCH2), 2.37 (s, 3H, COCH3); 13
C
NMR (CDCl3, 75 MHz) δ 196.9, 158.4, 152.7, 151.3, 142.2, 132.9, 130.1,
129.4, 120.3, 118.1, 116.4, 78.3, 65.2, 62.4, 53.9, 51.7, 49.4, 26.5. Anal. Calcd
for C24H28N4O5: C, 63.70; H, 6.24; N, 12.38. Found: C, 63.71; H, 6.23; N,
12.21. [R]2D2 ꢀ41.7 (c 1.05, CHCl3). For (4R,5S)-2: [R]2D2 +42.4 (c 1.02,
CHCl3).
C17H25N5O2 Na+, 354.1900; found, 354.1899 .
3
N-[1-(2,2-Dimethyl[1,3]dioxolan-4-yl)-2-(4-phenylpipera-
zine-1-yl)ethyl]benzamide (17). To a solution of 16 (0.7 g,
02.11 mmol) in EtOAc (4 mL) was added 10% Pd/C (w/w) (0.225 g,
0.211 mmol). The reaction flask was equipped with a three-way stopper
with a H2 balloon attached. The flask was evacuated three times using an
aspirator, and then the reaction mixture was stirred under a hydrogen
atmosphere for 24 h. The reaction mixture was then filtered through a
short pad of Celite. The solvent was removed by rotary evaporation, and
the residue (0.625 g, 97%) was dissolved in CH2Cl2 (3 mL). To this
solution DMAP (25 mg, 0.211 mmol) was added followed by Et3N
(1.7 mL, 12.6 mmol). The mixture was cooled to 0 °C, and phenyl
chloroformate (1 mL, 3.1 mmol) was added neat. The reaction mixture
was allowed to warm to room temperature. After 15 min of being stirred,
the reaction mixture was diluted with EtOAc and transferred into a
separatory funnel. The reaction mixture was washed with H2O, saturated
aqueous NaHCO3, and brine. The organic layer was dried over MgSO4,
filtered, and concentrated down. The solid residue was washed with
hexanes and filtered to afford 0.774 g (89%) of 17. No further
2-Oxo-4-(4-phenylpiperazin-1-ylmethyl)oxazolidin-5-yl-
methylphenyl Acetate (cis-1). Compound cis-1 was prepared from
cis-10 on a 0.34 mmol scale following the general procedure to provide
50 mg (36%) of product as colorless oil. Rf = 0.21 (50% EtOAc in
1
hexanes); tR = 3.4 min (88%, isocratic 60% MeOH/H2O); H NMR
(CDCl3, 300 MHz) δ 7.29ꢀ7.16 (m, 7H, Ar), 6.84ꢀ6.77 (m, 3H, Ar),
5.56 (s, 1H, NH), 4.73 (ddd, J = 4.5, 6.5, 8 Hz, 1H, C(O)OCH), 4.35
(dd, J = 4.5, 12 Hz, 1H, C(O)OCHH), 4.19 (dd, J = 6.5, 12 Hz, 1H,
C(O)OCHH), 3.94 (ddd, J = 5, 8, 9.5 Hz, 1H, NHCH), 3.60 (s, 2H,
PhCH2), 3.08ꢀ3.05 (m, 4H, PhN(CH2)), 2.58ꢀ2.51 (m, 2H, NCH2),
2.41ꢀ2.34 (m, 3H, NCH2 and NCHH), 2.25 (dd, J = 5, 12 Hz, 1H,
NCHH); 13C NMR (CDCl3, 75 MHz) δ 171.1, 158.3, 151.2, 133.6, 129.5,
129.3, 128.9, 127.5, 120.2, 116.3, 75.4, 61.9, 57.5, 53.6, 51.2, 49.3, 41.4.
2-Oxo-4-(4-phenylpiperazin-1-ylmethyl)oxazolidin-5-yl-
methyl (4-Acetylphenyl)carbamate (cis-2). Compound cis-2
was prepared from cis-10 on a 0.68 mmol scale following the general
procedure to provide 0.15 g (50%) of product as a white solid (mp
102.5ꢀ104.0). Rf = 0.23 (80% EtOAc in hexanes); 1H NMR (CD2Cl2,
300 MHz) δ 7.92 (dt, J = 8.8 Hz, 2H, Ar), 7.50 (dt, J = 8.8 Hz, 2H, Ar),
7.26ꢀ7.21 (m, 3H, Ar and NH), 6.92ꢀ6.89 (dd, 2H, Ar), 6.84 (t, 1H,
Ar), 5.54 (s, 1H, NH), 4.90 (ddd, J = 3.5, 7.5, 11.5 Hz, 1H, C(O)OCH),
4.56 (dd, J = 3.5, 12 Hz, 1H, OCHH), 4.35 (dd, J = 7.5, 12.0 Hz, 1H,
OCHH), 4.16 (ddd, J = 4.8, 10.0, 12.0 Hz, 1H, C(O)NHCH),
3.22ꢀ3.12 (m, 4H, PhNCH2), 2.77ꢀ2.70 (m, 2H, NCH2),
2.67ꢀ2.50 (m, 7H, C(O)CH3 and N(CH2)2); 13C NMR (CD2Cl2,
75 MHz) δ 197.0, 158.5, 152.9, 151.8, 142.6, 133.1, 130.2, 129.6, 120.2,
118.3, 116.5, 76.2, 63.2, 57.9, 51.5, 49.7, 30.3, 26.8.
General Method for In-Line Probing of RNA. AM1A was
prepared via in vitro transcription and 32P-50-end-labeled as previously
described.38 Labeled RNA (1 μL) was mixed with 5 μL of 2ꢁ in-line
probing buffer (100 mM Tris-HCl, 200 mM KCl, 40 mM MgCl2, pH
8.3) and 1 μL of DMSO ligand stock solution. For the control
experiment 1 μL of DMSO was added instead. After the total volume
was brought to 10 μL with H2O, the in-line probing experiments were
incubated at room temperature (∼25 °C) for 40 h. The resulting
cleavage products were separated via 20% denaturing polyacrylamide
gel electrophoresis (19:1 acrylamide/bis-acrylamide) and the bands
visualized via autoradiography. The relative normalized band intensities
were determined using Nucleo Vision (NucleoTech) and plotted
against ligand concentration to determine the slope for ligand-induced
relative band intensity changes.
RNA Ligand Binding Assay. Ligand RNA affinities were deter-
mined using the previously described FRET-derived ligand binding
assay and 100 nM 30-Fl-AM1A-(18)-Rhd or 30-Fl-C11U-(18)-Rhd.11,13
All RNAs were dialyzed and renatured prior to use. The FRET-labeled
RNA was mixed with a series of different concentrations of the ligand
(previously dissolved in DMSO) up to a final ligand concentration of 28
μM ((4S,5R)-2), 50 μM ((4R,5S)-2, cis-2), or 100 μM ((4R,5S)-1,
(4S,5R)-1, cis-1) in 50 mM NaH2PO4, pH 6.5, 50 mM NaCl, 5 mM
MgCl2, and 0.01 mM EDTA. Binding reactions were incubated for
130 min at 25 °C prior to analysis. By use of a Molecular Devices
FlexStation 96-well plate reader (Sunnyvale, CA), the FRET-labeled RNA
wasexcitedat467 nmwitha 515 nm cutoff filterwhilethe emissionspectra
were obtained over the range 515ꢀ640 nm. The FRET was determined
using the equation Qrel = |Q ꢀ Q0|/Q0 where Q is the fluorescence (F)
R
purification was necessary. Rf = 0.3 (30% EtOAc in hexanes); t =
8.3 min (100%) (75ꢀ300 °C at 25 °C/min, 10 min); 1H NMR (CDCl3,
300 MHz) δ 7.30ꢀ7.02 (m, 7H, Ph), 6.86ꢀ6.75 (m, 3H, Ph), 5.38 (d,
J = 5 Hz, 1H, NH), 4.22ꢀ4.16 (m, 1H, OCH), 4.03 (dd, J = 6, 8.7 Hz,
1H, OCHH), 3.92 (dd, J = 6, 8.7 Hz, 1H, OCHH), 3.87ꢀ3.78 (m, 1H,
NHCH), 3.13ꢀ3.10 (m, 4H, PhN(CH2)2), 2.69ꢀ2.54 (m, 6H, N-
(CH2)3), 1.38 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C NMR (CDCl3, 75
MHz) δ 154.8, 151.2, 150.9, 129.3, 129.1, 125.4, 121.6, 119.7, 116.0,
109.6, 76.8, 66.8, 57.7, 53.7, 51.2, 49.3, 29.7, 26.4, 25.1. HRMS: calcd for
C24H31N3O4 Na+, 448.2207; found, 448.2199.
3
General Method for the Synthesis of Enantiomers and Cis
Isomers of 1 and 2. To a solution of oxazolidinone alcohol 10
(1 equiv) in CH2Cl2 (0.1 M) were added DMAP (0.12 equiv), Et3N
(2 equiv), and phenylacetyl chloride or 4-acetylphenyl isocyanate
(1.2 equiv). The mixture was stirred at room temperature for 5 h. The
solvent was removed by rotary evaporation and the residue purified by
flash chromatography.
(4S,5R)-2-Oxo-4-(4-phenylpiperazin-1-ylmethyl)oxazolidin-
5-ylmethylphenyl Acetate ((4S,5R)-1). Compound (4S,5R)-1 was
prepared from (4S,5R)-10 on a 0.22 mmol scale following the general
procedure to afford 73 mg (81%) of product. Rf = 0.21 (50% EtOAc in
hexanes); tR = 14.9 min (89%); 1H NMR (CDCl3, 300 MHz) δ
7.30ꢀ7.17 (m, 7H, Ar), 6.85ꢀ6.78 (m, 3H, Ar), 5.34 (s, 1H, NH),
4.31 (q, J = 4.6, 9.1 Hz, 1H, C(O)OCH), 4.22 (d, J = 4.3 Hz, 2H,
C(O)OCH2), 3.60 (s, 2H, PhCH2), 3.58ꢀ3.51 (m, 1H, HNCH), 3.07 (t,
J = 4.8 Hz, 4H, PhNCH2), 2.56ꢀ2.41 (m, 5H, NCH2 and NCHH), 2.30
(dd, J = 5.5, 12.5 Hz, 1H, NCHH); 13C NMR (CDCl3, 75 MHz) δ 171.2,
158.0, 151.3, 133.7, 129.5, 129.4, 128.9, 127.5, 120.2, 116.4, 77.6, 64.4,
62.3, 53.7, 51.7, 49.4, 41.4. HRMS: calcd for C23H27N3O4 Na+,
3
432.1894; found, 432.1887; [R]2D5 ꢀ51.7 (c 1.00, CHCl3). For (4R,5S)-
1: [R]2D5 +52.8 (c 1.04, CHCl3); tR = 15.0 min (87%).
(4S,5R)-2-Oxo-4-(4-phenylpiperazin-1-ylmethyl)oxazolidin-
5-ylmethyl (4-Acetylphenyl)carbamate ((4S,5R)-2). Compound
(4S,5R)-2 was prepared from (4S,5R)-10 on a 0.41 mmol scale following
the general procedure to provide 0.15 g (80%) of product as a white solid
(mp 102.3ꢀ103.9). Rf = 0.3 (95% EtOAc in hexanes); 1H NMR (CDCl3,
300 MHz) δ 7.74 (d, J = 8.5 Hz, 2H, Ar), 7.29 (d, J = 8.5 Hz, 2H, Ar),
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Journal of Medicinal Chemistry
ARTICLE
ratio F585nm/F525nm in the presence of ligand (or absence for Q0). Binding
isotherms were constructed from duplicate Qrel data, and the dissociation
constant for each ligand was determined using Graphpad Prism,
version 4. Single-site versus two-site binding models were compared,
and in all cases where binding was observed, thesingle-site bindingwas the
preferred model.
Ligand Docking. Monoprotonated ligands were constructed in
Spartan 04 (Wavefunction), and their energy was minimized using
molecular mechanics with MMFF.39 The subsequent structures were
then exported to Macromodel (Schr€odinger) and docked to the antitermi-
nator model RNA structure 1N5336 using the Glide module of First
Discovery 2.7 (Schr€odinger). Parameters were set such that the enclosing
box encompassed the entire 1N53 structure (bulge region, A1 and A2
helices of AM1A), while the bounding box (for placing the ligand center)
encompassed the bulge region and the first 1ꢀ2 adjacent base pairs for each
helix. Images were rendered from the resulting structural files using PMV.40
(10) Gutierrez-Preciado, A.; Henkin, T. M.; Grundy, F. J.; Yanofsky,
C.; Merino, E. Biochemical Features and Functional Implications of the
RNA-Based T-Box Regulatory Mechanism. Microbiol. Mol. Biol. Rev.
2009, 73, 36–61.
(11) Anupam, R.; Nayek, A.; Green, N. J.; Grundy, F. J.; Henkin,
T. M.; Means, J. A.; Bergmeier, S. C.; Hines, J. V. 4,5-Disubstituted
Oxazolidinones: High Affinity Molecular Effectors of RNA Function.
Bioorg. Med. Chem. Lett. 2008, 18, 3541–3544.
(12) Means, J. A.; Katz, S.; Nayek, A.; Anupam, R.; Hines, J. V.;
Bergmeier, S. C. StructureꢀActivity Studies of Oxazolidinone Analogs
as RNA-Binding Agents. Bioorg. Med. Chem. Lett. 2006, 16, 3600–3604.
(13) Means, J. A.; Hines, J. V. Fluorescence Resonance Energy
Transfer Studies of Aminoglycoside Binding to a T Box Antiterminator
RNA. Bioorg. Med. Chem. Lett. 2005, 15, 2169–2172.
(14) Anupam, R.; Denapoli, L.; Muchenditsi, A.; Hines, J. V. Identi-
fication of Neomycin B-Binding Site in T-Box Antiterminator Model
RNA. Bioorg. Med. Chem. 2008, 16, 4466–4470.
(15) Acquaah-Harrison, G.; Zhou, S.; Hines, J. V.; Bergmeier, S. C.
Library of 1,4-Disubstituted 1,2,3-Triazole Analogs of Oxazolidinone
RNA-Binding Agents. J. Comb. Chem. 2010, 12, 491–496.
(16) Grundy, F. J.; Moir, T. R.; Haldeman, M. T.; Henkin, T. M.
Sequence Requirements for Terminators and Antiterminators in the
T-Box Transcription Antitermination System: Disparity between Con-
servation and Functional Requirements. Nucleic Acids Res. 2002, 30,
1646–1655.
’ ASSOCIATED CONTENT
S
Supporting Information. Binding isotherms, in-line
b
probing of AM1A binding with the enantiomers, H and 19F
1
NMR spectra for Mosher ester of 6, and H and 13C NMR
1
spectra for all compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(17) Maciagiewicz, I.; Zhou, S.; Bergmeier, S. C.; Hines, J. V.
Structure Activity Studies of RNA-Binding Oxazolidinone Derivatives.
Bioorg. Med. Chem. Lett. 2011, 4524–4527.
’ AUTHOR INFORMATION
(18) Bergmeier, S. C.; Katz, S. J. A Method for the Parallel Synthesis
of Multiply Substituted Oxazolidinones. J. Comb. Chem. 2002, 4,
162–166.
Corresponding Author
*Phone: 740-593-9464. E-mail: hinesj@ohio.edu.
(19) Bergmeier, S. C.; Stanchina, D. M. Synthesis of Vicinal Amino
Alcohols via a Tandem Acylnitrene AziridinationꢀAziridine Ring Opening.
J. Org. Chem. 1997, 62, 4449–4456.
’ ACKNOWLEDGMENT
(20) Bergmeier, S. C.; Stanchina, D. M. Acylnitrene Route to Vicinal
Amino Alcohols. Application to the Synthesis of (ꢀ)-Bestatin and
Analogues. J. Org. Chem. 1999, 64, 2852–2859.
(21) Schaus, S. E.; Brandes, B. D.; Larrow, J. F.; Tokunaga, M.;
Hansen, K. B.; Gould, A. E.; Furrow, M. E.; Jacobsen, E. N. Highly
Selective Hydrolytic Kinetic Resolution of Terminal Epoxides Catalyzed
by Chiral (salen)Co-III Complexes. Practical Synthesis of Enantioen-
riched Terminal Epoxides and 1,2-Diols. J. Am. Chem. Soc. 2002, 124,
1307–1315.
We thank the NIH (Grant GM073188) and Ohio University,
through the BioMolecular Innovation and Technology (BMIT)
project, for support of this work. We thank the DAAD for
fellowship support (DB).
’ ABBREVIATIONS USED
UTR, untranslated region
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