Structure-activity studies of RNA-binding oxazolidinone derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.05.130

The structure-activity relationship of a series of oxazolidinones binding to T-box riboswitch antiterminator RNA has been investigated. Oxazolidinones differentially substituted at C-5 were prepared and the ligand-induced fluorescence resonance energy tra

Full text of DOI:10.1016/j.bmcl.2011.05.130

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4524–4527
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity studies of RNA-binding oxazolidinone derivatives
⇑
Iwona Maciagiewicz, Shu Zhou, Stephen C. Bergmeier, Jennifer V. Hines
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship of a series of oxazolidinones binding to T-box riboswitch antitermina-
tor RNA has been investigated. Oxazolidinones differentially substituted at C-5 were prepared and the
ligand-induced fluorescence resonance energy transfer (FRET) changes in FRET-labeled antiterminator
model RNA were assayed. Both qualitative and quantitative analysis of the structure–activity relationship
indicate that hydrogen bonding and hydrophobic properties play a significant role in ligand binding.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 22 April 2011
Revised 27 May 2011
Accepted 31 May 2011
Available online 15 June 2011
Keywords:
RNA
Oxazolidinone
Antiterminator
T-box riboswitch
FRET
Targeting RNA with small molecules is an emerging field for
which much remains to be explored.1–7 In this paper we report
on key ligand–RNA recognition features as part of our comprehen-
sive drug discovery investigation for targeting and disrupting the
function of T-box antiterminator RNA. The T-box antiterminator
is a key component of the T-box transcription antitermination
riboswitch.8,9 Riboswitches control RNA transcription (or transla-
tion) by binding and structurally responding to metabolic effector
molecules to control whether transcription (or translation) pro-
ceeds.10 In the T-box transcription antitermination riboswitch,
the 50-untranslated mRNA region (50-UTR) binds and structurally
responds to uncharged (non-aminoacylated) tRNA. The anticodon
of the tRNA binds to a complementary specifier sequence in Stem
1 of the 50-UTR and the four nucleotides at the tRNA acceptor end
base pair with four nucleotides in the bulge of the antiterminator
structural element (Fig. 1).8 This latter base pairing does not occur
with charged tRNA and is required for transcription antitermina-
tion to occur. In the absence of uncharged tRNA, an alternative,
more stable, secondary structural element (the terminator) is
formed and leads to termination of transcription.8 The antitermi-
nator and terminator structural elements share common
nucleotides and are therefore mutually exclusive. Thus, the entire
50-UTR functions as a riboswitch that responds to the charging
ratio of cognate tRNA (i.e., uncharged, cognate tRNA is the meta-
bolic effector molecule).8
of the antiterminator element,9,11 the T-box antiterminator is an
important target for RNA drug discovery. Previous work in our labs
has identified 4,5-oxazolidinones as ligands with high affinity and
specificity for the antiterminator element.12,13 In this paper we re-
port a structure–activity relationship study of a series of oxazolid-
inones binding to functionally relevant antiterminator model RNA
(AM1A, Fig. 1B)14,15 and a reduced function variant (C11U).14 We
previously observed that oxazolidinones with an N-phenyl pipera-
zine group at the C-4 position had excellent binding properties
with the antiterminator model RNA.12 In those previous studies
we had examined only a limited number of acyl groups at the
C-5 position. Given that the acyl group is introduced in the final
synthetic step and the ready availability of a large number of acyl
Given the prevalence of T-box riboswitches in controlling the
transcription of vital genes in Gram-positive bacteria (including
many pathogenic examples)9 and the high sequence conservation
⇑
Corresponding author.
E-mail address: hinesj@ohio.edu (J.V. Hines).
Figure 1. T-box riboswitch (A) and antiterminator model RNA AM1A (B) with
sequence variant C11U indicated by an arrow.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.130

I. Maciagiewicz et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4524–4527
4525
Figure 2. Comparison of experimental %Frel for AM1A versus C11U.
Scheme 1.
are shown in Table 1. Our choices for acylating agents were guided
by the identity of previously identified molecules showing good
binding to the antiterminator RNA. Two R groups that had stood
out were R = CH2Ph (ANB-22) and R = 4-(CH3CO)–C6H4NH (ANB-
40). We planned to examine substitution on the aromatic ring of
both the ester and carbamate derivatives, the position of the aro-
matic ring relative to the carbonyl, isosteric replacements for the
benzene ring, and a few non-aromatic R groups.
The series of oxazolidinones were screened for binding to anti-
terminator model RNA using a previously described Fluorescence
Resonance Energy Transfer (FRET)-based assay.12,17 Data are re-
ported as the relative percentage change in the fluorescence
%Frel = [(F ꢀ F0)/F0] ꢁ 100, where F0 represents the fluorescence at
585 nm of the FRET-labeled RNA in the absence of ligand and F rep-
resents the fluorescence in the presence of the ligand.
donors, we decided to leave the phenyl piperazine moiety intact
and examine the effect of changing acyl groups.
The oxazolidinones used in this study were prepared as previ-
ously reported.12 As shown in Scheme 1, the aziridine 1 was trea-
ted with N-phenyl piperazine to provide 2 in 85% yield. We had
previously converted 2 directly to 3 by reaction with an excess of
an acid chloride.16 As we needed to use a larger variety of acyl do-
nors such a reaction sequence would not be generally useful. The
trityl group was thus removed with HCl to provide the correspond-
ing alcohol. The alcohol could then be acylated with a wide variety
of acylating agents and reaction conditions. Depending upon the
availability and reactivity acid chlorides, carboxylic acid/DCC, car-
boxylic acid/polystyrene supported DCC (PS-DCC), carboxylic acid/
silica gel supported DCC (Si-DCC), or an isocyanate could be used
for this reaction. The yields and identities of the oxazolidinones
A comparison of ligand-induced FRET changes in the antitermi-
nator RNA for each compound is detailed in Table 1 and relative
Table 1
Oxazolidinones ligands: Synthetic yield and ligand-induced FRET RNA changes
Compound Yielda (%)
R
AM1A %Frel
C11U %Frel
Compound Yielda (%)
R
AM1A %Frel
C11U %Frel
c
c
c
c
ANB-22
ANB-40
IMB-7
IMB-8
IMB-9
NA
NA
53
27
52
65
26
18
52
68
58
82
58
68
14
40
90
87
C6H4CH2–
4-(CH3CO)–C6H4NH–
4-(MeO)–C6H4CH2–
8.5
7.7
13.7
18.1
9.5
15.0
15.5
20.6
9.5
15.3
ꢀ1.8
8.9
15.9
13.4
18.8
25.4
14.0
20.2
21.0
26.1
9.4
16.2
ꢀ4.9
14.7
7.7
IMB-55
IMB-56
IMB-57
IMB-58
IMB-59
IMB-60
IMB-61
IMB-62
IMB-63
IMB-64
IMB-65
IMB-66
IMB-67
IMB-68
IMB-69
IMB-71
IMB-72
IMB-73
70
56
76
3-(MeO)–C6H4–
3,4,5-(PhCH2O)3–C6H2–
2-Furan–
4-(NO2)–C6H4CH2–
3,4-Cl2–C6H3CH2–
3-(MeO)–C6H4–CH2–
4-Ph–C6H4–CH2–
3-(Me2N)–C6H4–
2-F–C6H4–
2-Cl–C6H4–
2-I–C6H4–
2-CN–C6H4–
2-(PhO)–C6H4–
2-(Me)–3-(NO2)–C6H4–
(2-Norbornyl)–CH2–
n-C3H7–
(c-C6H11)(CH2)3–
(Me)2CCH(CH2)2CH(Me)CH2– 38.9
23.8
14.3
6.4
24.7
18.6
14.7
16.5
28.9
19.6
22.3
27.1
20.4
28.8
22.4
16.7
26.5
24.5
27.4
19.7
29.0
42.4
n-C7H15
–
35a
64b
60b
48b
63b
68b
64b
45b
25b
60b
31b
51b
72b
75b
53b
9.1
(Me)2CHCH2–
(Me)2CH(CH2)2–
29.1
18.9
25.3
24.6
18.5
30.4
28.6
7.3
30.8
23.3
29.5
10.1
31.8
IMB-10
IMB-11
IMB-12
IMB-13
IMB-14
IMB-15
IMB-16
IMB-17
IMB-18
IMB-19
IMB-20
IMB-21
IMB-22
n-C5H11
–
Ph(CH2)2–
PhOCH2–
2,6-Cl2–C6H2–
4-(NO2)–C6H4NH–
3,4-Cl2–C6H4NH–
4-(MeO)–C6H4NH–
4-(n-C5H11)–C6H4NH–
4-(CH3CH2)–C6H4NH–
n-C6H13NH–
3-(NO2)–C6H4NH–
2-(t-Bu)–6-Me–
C6H3NH–
7.8
10.6
18.3
17.4
ꢀ1.1
14.8
11.1
19.1
23.7
0.2
15.4
IMB-23
IMB-24
IMB-25
IMB-49
IMB-50
IMB-51
IMB-52
IMB-53
IMB-54
73
25
39
76
16
57
79
76
78
PhNH–
n-C4H9NH–
PhCH2NH–
3-(Me)–C6H4NH–
(Me)3C–
Ph–
4-(NO2)–C6H4–
4-Cl–C6H4–
8.4
10.5
6.1
16.8
7.1
14.8
13.1
29.1
27.4
11.2
14.0
11.7
26.7
13.2
18.6
15.2
22.8
27.5
IMB-74
IMB-75
IMB-76
IMB-77
IMB-78
IMB-79
IMB-80
IMB-81
16
22
48b
83
32
53b
43
25
(Me)3CNH–
n-C3H7NH–
(Ph)(CH2)5–
c-C6H11–
PhO–
(BocNH)–(CH2)4–
(R)-PhCH(BocNH)–
(S)-PhCH(BocNH)–
8.9
10.3
32.6
19.5
12.4
29.4
39.6
26.7
8.7
9.6
30.9
20.7
21.3
27.7
42.6
19.4
4-(Me)–C6H4–
a
b
c
The reported yield is based on the two-step sequence of detritylation followed by acylation.
The coupling was carried out using Si-DCC or PS-DCC and the corresponding carboxylic acid.
Each ligand binding reaction contained 100 nM 30Fl-AM1A-18-Rhd17 or 30Fl-C11U-18-Rhd17 and 10
lM of ligand (from DMSO stock solution) in 100
lL binding buffer
(50 mM sodium phosphate pH 6.5, 50 mM NaCl, 5 mM MgCl2 and 0.01 mM EDTA). Following excitation at 467 nm, the normalized percent change in fluorescence was
calculated as %Frel = [(F/F0) ꢀ 1] * 100 where F is the fluorescence at 585 nm in the presence of ligand and F0 in the absence of ligand (i.e., DMSO control). Data represent the
average of duplicate experiments.

4526
I. Maciagiewicz et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4524–4527
Figure 3. Comparison of experimental versus calculated %Frel for AM1A and C11U with 90% prediction bands shown as dashed lines.
RNA specificity is summarized in Figure 2. The ligand-induced ef-
fects were, on average, 23% lower for AM1A compared to C11U.
This is consistent with the known small structural differences be-
tween AM1A and C11U14,15 and likely indicates slight ligand bind-
ing mode differences between the two RNA.
Interestingly, two compounds led to a negative %Frel (IMB-15
and IMB-21). Both IMB-15 and IMB-21 contain a strongly elec-
tron-withdrawing nitro group on the phenyl carbamate. The fact
that related nitro-substituted phenyl esters (IMB-52, IMB-58,
IMB-68) do not show this effect suggests that the decrease in the
FRET may be due to ligand-specific binding interactions resulting
from the enhanced acidity of the carbamate N–H rather than a
non-specific electrostatic repulsion with the phosphodiester back-
bone of the RNA helices. The hypothesis that H-bonding may play a
role in binding is further supported by the fact that in the series of
4-substituted phenyl carbamates the %Frel increases as the electro-
negativity of the substituent decreases.
compounds with the greatest extent of ligand-induced FRET
changes (IMB-73 and IMB-80). Other compounds with high QPlog-
Po/w values included IMB-61, IMB-67, IMB-69, IMB-76, and IMB-
81. This trend was overshadowed by other factors in the carbamate
series. For example, IMB-22, a phenyl carbamate substituted by a
t-butyl group and methyl group, did not show this higher %Frel even
though it has a high QPlogPo/w value. This hydrophobic trend,
along with the significance of hydrogen bonding, is consistent with
previous studies which indicated that non-ionic interactions,
including hydrophobic interactions, likely play a role in oxazolidi-
none binding.12,13
With regards to RNA specificity, the globularity descriptor20
was significant in the AM1A fit (0.01 < P < 0.05), but not in the
C11U fit. This is consistent with the known structural differences
between the two antiterminator model RNA.14,15 The ligand globu-
larity may contribute to the observed ligand-induced FRET differ-
ences between AM1A and C11U.
While the relative magnitude of ligand-induced %Frel change
does not necessarily correlate with RNA affinity, previous studies
have demonstrated that FRET changes are indicative of ligand
binding.12,13,17 Consequently, the oxazolidinone %Frel data were
correlated with ligand descriptors in order to begin to develop a
quantitative structure–activity relationship (QSAR) for ligands
binding the T-box antiterminator RNA. Physico-chemical descrip-
tors of the compounds were computed using QikProp 2.1 (Schrö-
dinger) and utilized in a multiple linear regression analysis of the
data with QikFit 2.1 (Schrödinger). The correlation of experimental
versus calculated %Frel data are shown in Figure 3 and the fit
parameters are listed in the Supplementary data. Previous studies
indicated the significance of protonated amines and ionic interac-
tions in RNA ligand binding.12,17,18 In this study, the library was
specifically designed to investigate non-ionic interactions at the
C-5 position.
In summary, we have investigated the structure–activity rela-
tionship for a series of oxazolidinone ligands binding to T-box anti-
terminator model RNA. The overall QSAR highlighted the
importance of hydrogen bonding and hydrophobic properties in li-
gand binding. Further studies on binding affinity, effects of chiral-
ity, 3D descriptors and binding location will be reported in the
future.
Acknowledgments
We thank the National Institutes of Health (GM073188) for sup-
port of this work. We also thank Ohio University for support of the
BioMolecular Innovation and Technology project.
Supplementary data
In the multiple linear regression fit for AM1A (R2 = 0.8) and the
fit for C11U (R2 = 0.8), the descriptors with greatest contribution to
the fit that were the most statistically significant (P <0.01) were di-
pole (computed dipole moment), FISA (hydrophilic component of
total solvent accessible surface area), donorHB (number of hydro-
gen bond donors), QPlogPo/w (predicted octanol/water partition
coefficient), and index of cohesive interaction in solids.19,20 That
the dipole moment and hydrogen bonding were significant is con-
sistent with the observations discussed above with respect to the
acidity of the carbamate nitrogen in IMB-15 and IMB-21. Substitu-
tion of the phenyl of benzoate esters showed a clear impact of this
with halogen, and methoxy substitution (IMB-53, IMB-55, IMB-64,
and IMB-65) leading to higher %Frel values. The significance of the
QPlogPo/w descriptor highlights the importance of hydrophobic
groups. For example, ligands with higher QPlogPo/w values
typically correlated with higher %Frel values, including the two
Supplementary data (multiple linear regression data as well as
alternate versions of Table 1 containing the data organized by
functional group) associated with this article can be found, in the
online version, at doi:10.1016/j.bmcl.2011.05.130.
References and notes
1. Duca, M.; Malnuit, V.; Barbault, F.; Benhida, R. Chem. Commun. 2010, 46, 6162.
2. Ramisetty, S. R.; Baranger, A. M. Bioorg. Med. Chem. Lett. 2010, 20, 3134.
3. Meyer, S. T.; Hergenrother, P. J. Org. Lett. 2009, 11, 4052.
4. Liu, Y.; Peacey, E.; Dickson, J.; Donahue, C. P.; Zheng, S. X.; Varani, G.; Wolfe, M.
S. J. Med. Chem. 2009, 52, 6523.
5. Manfroni, G.; Gatto, B.; Tabarrini, O.; Sabatini, S.; Cecchetti, V.; Giaretta, G.;
Parolin, C.; Del Vecchio, C.; Calistri, A.; Palumbo, M.; Fravolini, A. Bioorg. Med.
Chem. Lett. 2009, 19, 714.
6. Tor, Y. ChemBioChem 2003, 4, 998.
7. Thomas, J. R.; Hergenrother, P. J. Chem. Rev. 2008, 108, 1171.
8. Green, N. J.; Grundy, F. J.; Henkin, T. M. FEBS Lett. 2010, 584, 318.

I. Maciagiewicz et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4524–4527
4527
9. Gutierrez-Preciado, A.; Henkin, T. M.; Grundy, F. J.; Yanofsky, C.; Merino, E.
Microbiol. Mol. Biol. Rev. 2009, 73, 36.
10. Smith, A. M.; Fuchs, R. T.; Grundy, F. J.; Henkin, T. M. RNA Biol. 2010, 7, 104.
11. Grundy, F. J.; Moir, T. R.; Haldeman, M. T.; Henkin, T. M. Nucleic Acids Res. 2002,
30, 1646.
12. Anupam, R.; Nayek, A.; Green, N. J.; Grundy, F. J.; Henkin, T. M.; Means, J. A.;
Bergmeier, S. C.; Hines, J. V. Bioorg. Med. Chem. Lett. 2008, 18, 3541.
13. Means, J.; Katz, S. J.; Anupam, R.; Nayek, A.; Hines, J. V.; Bergmeier, S. C. Bioorg.
Med. Chem. Lett. 2006, 16, 3600.
14. Gerdeman, M. S.; Henkin, T. M.; Hines, J. V. Nucleic Acids Res. 2002, 30, 1065.
15. Gerdeman, M. S.; Henkin, T. M.; Hines, J. V. J. Mol. Biol. 2003, 326, 189.
16. Bergmeier, S. C.; Arason, K. M. Tetrahedron Lett. 2000, 41, 5799.
17. Means, J. A.; Hines, J. V. Bioorg. Med. Chem. Lett. 2005, 15, 2169.
18. Anupam, R.; Denapli, L.; Muchenditsi, A. M.; Hines, J. V. Bioorg. Med. Chem.
2008, 16, 4466.
19. Jorgenson, W. J.; Duffy, E. M. Bioorg. Med. Chem. Lett. 2000, 10, 1155.
20. QikProp 2.1 Operating Manual; Schrödinger Press, 2004.