Strategies for recognition of stem-loop RNA structures by synthetic ligands: Application to the HIV-1 frameshift stimulatory sequence

Article abstract of DOI:10.1021/jm100231t

Production of the Gag-Pol polyprotein in human immunodeficiency virus (HIV) requires a -1 ribosomal frameshift, which is directed by a highly conserved RNA stem-loop. Building on our discovery of a set of disulfide-containing peptides that bind this RNA, we describe medicinal chemistry efforts designed to begin to understand the structure-activity relationships and RNA sequence-selectivity relationships associated with these compounds. Additionally, we have prepared analogues incorporating an olefin or saturated hydrocarbon bioisostere of the disulfide moiety, as a first step toward enhancing biostability. The olefin-containing compounds exhibit affinity comparable to the lead disulfide and, importantly, have no discernible toxicity when incubated with human fibroblasts at concentrations up to 1 mM.

Full text of DOI:10.1021/jm100231t

6018 J. Med. Chem. 2010, 53, 6018–6027
DOI: 10.1021/jm100231t
Strategies for Recognition of Stem-Loop RNA Structures by Synthetic Ligands: Application to the
HIV-1 Frameshift Stimulatory Sequence
Prakash B. Palde,‡ Leslie O. Ofori,§ Peter C. Gareiss,‡ Jaclyn Lerea,† and Benjamin L. Miller*,†,‡
†Department of Dermatology, ‡Department of Biochemistry and Biophysics, and §Department of Chemistry, University of Rochester,
Rochester, New York 14642
Received February 22, 2010
Production of the Gag-Pol polyprotein in human immunodeficiency virus (HIV) requires a -1
ribosomal frameshift, which is directed by a highly conserved RNA stem-loop. Building on our
discovery of a set of disulfide-containing peptides that bind this RNA, we describe medicinal chemistry
efforts designed to begin to understand the structure-activity relationships and RNA sequen-
ce-selectivity relationships associated with these compounds. Additionally, we have prepared analo-
gues incorporating an olefin or saturated hydrocarbon bioisostere of the disulfide moiety, as a first step
toward enhancing biostability. The olefin-containing compounds exhibit affinity comparable to the lead
disulfide and, importantly, have no discernible toxicity when incubated with human fibroblasts at
concentrations up to 1 mM.
Introduction
Two cis-acting elements in viral mRNA are responsible for
the -1 ribosomal frameshift: a heptameric (UUUUUUA)
slippery sequence where the frameshift takes place, and a down-
stream stimulatory signal.13 Modification of the stimulatory
sequence (either via natural variation or via laboratory muta-
tion) in ways affecting frameshifting efficiency translates to a
decrease in viral replication.14 Thus, targeting this structure
with small molecules is a potential strategy for inhibiting viral
replication.15,16 Efforts by several laboratories, including three
NMR structural analyses,17 indicate that the stimulatory RNA
in HIV-1 forms a stem-loop structure. This in turn may be
divided into an upper stem-loop and lower stem, separated by
a purine bulge (Figure 1). It has been hypothesized that frame-
shift efficiency is dependent on the mechanical stability of the
downstream RNA regulatory element, with higher frameshift-
ing rates correlating with a greater force required to unfold the
downstream RNA.18 Recent studies by the Visscher and Four-
my groups suggest that the lower stem and bulge regions of the
HIV-1 FSS readily unfold, while unfolding of the upper stem-
loop requires more force.19 Thus, further stabilization (or, alter-
natively, destabilization) of the upper stem-loop may alter
frameshift efficiency, suggesting that this sequence may consti-
tute a useful target for drug discovery. We have previously re-
ported the discovery of a compound (1) able to selectively bind
the HIV-1 frameshift-stimulating RNA upper stem-loop
(hereafter abbreviated “HIV-1 FSS”a).20 Compound 1 was
identified via the synthesis and in situ screening of an 11325
member resin-bound dynamic combinatorial library (RBDCL)
designed based on a core structure inspired by nucleic acid
binding, bisintercalating natural products.21 This molecule
binds the HIV-1 FSS with a dissociation constant (KD) of 4.1
(2.4 μM as measured by surface plasmon resonance (SPR) and
The design and synthesis of compounds able to selectively
bind specific RNA sequences with high affinity represent a
signature challenge in chemistry.1 Unlike DNA, which to a
great extent has been rendered an “open book” for molecular
recognition,2 the problem of sequence-selective RNA recog-
nition remains largely unsolved. In addition to the funda-
mental importance of this challenge (a subset of the more
general challenge of function-oriented synthesis3), RNA tar-
gets of potential therapeutic value are being discovered at an
increasing rate. For example, a notable pathogen against
which RNA-targeted therapeutics can potentially have an
impact is the human immunodeficiency virus (HIV). The
causative agent of AIDS, HIV continues to be a major threat
to human life despite intensive ongoing research. While mod-
ern antiretroviral drugs have dramatically increased the life
spans of those infected with the virus, a number of factors
drive the need for new anti-HIV drugs.4 Key among these
include the complexity of current antiretroviral regimens and the
emergence of drug resistance.5-7 Screening and directed design
approaches to the development of small molecules interfering
with HIV at the RNA level have primarily focused on REV8
and TAR,9 and those efforts have yielded a number of impor-
tant insights into HIV biology, as well as strategies for RNA
recognition. Recently, however, another RNA has garnered
interest as a potential therapeutic target. This RNA’s impor-
tance derives from the fact that the structural and enzymatic
proteins of HIV are encoded in overlapping gag and pol open
reading frames, respectively.10 Since pol is always translated as
a Gag-Pol fusion protein, the ribosome is required to slip one
nucleotide backward to produce the desired polyprotein.
This -1 frameshift event occurs with a frequency of 5-10%,11
and disruption of this percentage has been demonstrated to
severely decrease viral replication and infectivity.12
a Abbreviations: HIV-1 FSS, frameshift-stimulating RNA sequence
of HIV-1; SPR, surface plasmon resonance; RBDCC, resin-bound
dynamic combinatorial chemistry; RBDCL, resin-bound dynamic com-
binatorial library; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide.
*To whom correspondence should be addressed. E-mail: Benjamin_
Miller@urmc.rochester.edu. Phone: 585-275-9805. Fax: 585-273-1346.
r
pubs.acs.org/jmc
Published on Web 07/30/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6019
altogether surprising, as immobilization of 1 on the SPR chip
reduces both the degrees of freedom available to the com-
pound and its overall charge. The measured binding affinity
of 1 for the HIV-1 FSS did not change in the presence of an
excess of unrelated competing RNAs (either yeast tRNA,
entry 2, or total yeast RNA, entry 3), although the total
change in fluorescence decreased. To confirm that this
reduction in the total change in fluorescence reflected a
nonspecific interaction between the Cy3-labeled HIV-1
FSS and competitor RNAs rather than off-target binding
by compound 1, we titrated an identical solution of yeast
tRNA into Cy3-labeled HIV-1 FSS. Consistent with our
hypothesis, the dilution-corrected Cy3 fluorescence decreased
in a manner dependent on the concentration of tRNA
(Supporting Information, p S32). Use of excess yeast tRNA
as a measure of selectivity was pioneered by Tor and collea-
gues;28 following Tor’s terminology, where “specificity” is
defined as the ratio of the KD to the sequence of interest
over the KD observed in the presence of a large excess of
competitor RNAs, our results suggest a specificity ratio of
approximately 1. As expected, the measured binding constant
could be altered by addition of unlabeled (competing) HIV-1
FSS (entry 4).
Figure 1. (Left) Secondary structure of the HIV-1 frameshift-indu-
cing RNA stem-loop. The sequence shown is that of the HIV group
M subtype D.25 The upper stem-loop sequence (or “HIV-1 FSS”)
used as the primary target for RBDCC screening and for the
experiments described herein is boxed. The slippery sequence where
the -1 frameshift occurs is underlined. (Right) Lead molecule 1,
previously identified via RBDCC.
Single mutation (entry 5) or complete sequence flipping
(entry 6) of the stem did not produce an experimentally
significant change in the binding constant. In contrast, the
introduction of single (entry 7) or multiple (entry 8) muta-
tions into the loop region caused a 2-fold and 4-fold reduc-
tion in the binding, respectively. No saturable binding was
observed to a DNA homologue of the HIV-1 FSS (entry 9) or
to unrelated RNA hairpins (entries 10 and 11; note that these
sequences have been examined in previous studies in our
laboratory29 and are known to be folded under the condi-
tions of the titrations). Taken together, these results confirm
that compound 1 binds the target HIV-1 FSS with high
affinity and good selectivity. Alterations to the tetraloop had
the greatest impact on binding.
Structure-Activity Relationships of the 2-Ethylquinoline-
3-carboxamide Moiety. With a more detailed understanding
of the solution-phase binding selectivity of 1 in hand, we next
turned toward modifications of the compound itself in order
to examine the structural features governing binding. On the
basis of prior reports of the ability of quinolines to act as
general (non-sequence-selective) RNA intercalators,1b one
would hypothesize intercalation as the primary function of
the 2-ethylquinoline-3-carboxamide moieties of 1. To test
that hypothesis, compounds 2-7 were synthesized on solid-
phase resin beads using methods previously described for the
synthesis of 1. Binding constants were measured by fluores-
cence titration (Table 2). Changing the 2-ethyl group to
methyl (2) or proton (3) yielded experimentally insignificant
(2) or only modest (3) reduction in affinity for the HIV-1
FSS. This is consistent with molecular mechanics calcula-
tions indicating that there is little difference in the conforma-
tional landscape of these three compounds. Decreasing the
available π-surface, exemplified in the 2-methyl-3-carboxy-
pyridine compound (4) and uncapped peptide (5), comple-
tely ablated binding. This stark difference in affinity between
1 and 5 was confirmed by a filter-binding assay (Supporting
Information, Figure S7). The introduction of a dioxolane
ring (6) resulted in a compound with roughly 4-fold lower
affinity than 1, while replacement of the quinoline with an
anthraquinone yielded a molecule (7) with affinity equivalent
to 1.
serves as an important lead for the development of high-affinity
ligands for the HIV-1 FSS. Other recent reports of compounds
targeting frameshifting in HIV include a description by Butcher
and Tor of the interaction of guanidinoneomycin with the HIV-
1 FSS upper stem-loop22 and a recent report of several mole-
cules targeting the bulge region.23 This latter study suggests that
the question of the “best” region of the FSS to target should not
yet be regarded as settled. Heveker and colleagues have de-
scribed peptides able to interfere with frameshifting in a two-
reporter bacterial system, but it is uncertain whether this inter-
ference involves a direct interaction with the FSS.24
This paper details our efforts to expand our understanding
of the structural factors governing recognition of the HIV-1
FSS by 1. We approached the problem via solution-phase
binding analysis and by the directed synthesis of several ana-
logues of 1 to explore structure-activity relationships with a
particular focus on modificationstothe quinolineheterocycle.
As part of the structure-activity analysis, we also report the
first steps toward enhancing the biostability of 1, via the syn-
thesis and binding analysis of analogues of 1 incorporating
hydrocarbon replacements (bioisosteres) for the disulfide.
Analysis of the Binding Selectivity of 1. Previously reported
SPR measurements on 1 were conducted with the compound
tethered to the SPR chip via one of its amino groups and
involved a relatively limited set of RNA sequences. As a first
step toward increasing our understanding of the interaction
of 1 and related compounds with the HIV-1 FSS, we carried
out a series of solution-phase fluorescence titration measure-
ments. In these experiments, the fluorescence of 50-Cy3-
labeled RNA was monitored as a function of added
compound.26,27
The RNA and DNA sequences employed and results of
titrations (alongside previously reported SPR values, where
available) are shown in Table 1; selected titration data and
associated curve fits are shown in Figure 2. All data were fit
to a 1:1 binding model incorporating ligand depletion.
Binding to the HIV-1 FSS derived from HIV group M
subtype D (entry 1) was found to be 0.35 ( 0.11 μM in
solution, roughly an order of magnitude tighter than the
binding constant obtained previously by SPR. This is not

6020 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Palde et al.
Table 1. Binding Constants (KD, µM) for 1 and Selected Oligonucleotidesa
a “Solution” measurements were carried out by titrating unlabeled compound into Cy3-labeled RNA and measuring changes in the fluorescence
spectrum, while SPR measurements were performed by immobilizing the compound on the sensor chip and flowing increasing concentrations of
unlabeled RNA. ND = not determined. Right: Butcher et al. NMR structure of the HIV-1 FSS RNA (PDB code 1PJY)17a color-coded by binding
constant change in altered sequences (blue = least; red = most).
pyridines have insufficient π-surface area to support inter-
calation. As discussed below, the (Cys-Pro-Phe)2 peptide is
not a passive player in binding, however, despite the lack of
RNA-binding affinity displayed by 4 and 5. Further efforts
are underway to explore the ability of other heterocycles with
larger π-surface area to enhance affinity.
Replacement of the Disulfide with Nonlabile Bioisosteres.
The ability of the disulfide to undergo exchange with other
thiols in solution30 was a critically important design feature
of the RBDCL used to identify 1. However, its susceptibility
to reduction or exchange in a biological environment would
present an obvious obstacle to investigation of this class of
molecules in a cellular context. Reduction is particularly a
concern in the cytosol, where the ratio of reduced:oxidized
glutathione (GSH:GSSG) ranges from 30:1 to 100:1.31 Pre-
vious work has shown that in most “biologically active”
peptides the disulfide linkage serves only a structural role
and replacing it with thioether (-S-CH2-) or all carbon
Figure 2. Selected fluorescence titration data (dilution-corrected
fluorescence change as a function of ligand concentration) and
curve fits for the experiments summarized in Table 1.
(olefin, -CH2-CH2-) bioisosteres generally enhances bio-
stability of the molecule without affecting its function.32
A
particularly striking example of the interchangeability of
disulfide and olefin linkages was reported in 2000 by Nico-
laou and co-workers, who found similar selectivities in the
receptor-accelerated synthesis of disulfide- and olefin-linked
Observation that 2-methylpyridine derivative 4 is unable
to bind supports an intercalative binding mechanism for 1, as
it is well-known (at least in a DNA-binding context) that

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6021
Table 2. Binding Constants (KD) for Heterocycle Analogues of 1 Binding to the HIV-1 FSS RNA, As Measured by Fluorescence Titrationa
a All values are an average ( standard deviation of three replicate titrations. Compounds 1-7 all likely carry a charge of þ2.
vancomycin dimers.33 Saturated hydrocarbon analogues of
disulfides have also been reported, for example, in the
enkaphalins.32d Therefore, in order to build toward com-
pounds suitable for cellular studies, we next synthesized
dicarba (olefin and hydrocarbon) analogues of compound 1.
Ruthenium-catalyzed olefin metathesis is an attractive
and powerful tool for the formation of carbon-carbon
double bonds.34 Grubbs’ catalysts (first and second gener-
ation) have been previously used in the synthesis of cyclic
peptides via ring-closing metathesis.32,35 Likewise, olefin
cross-metathesis of amino acid derivatives36 and more com-
plex peptides is well established. Therefore, to substitute for
the cysteine amino acid in 1, the olefin was introduced in the
form of L-allylglycine.37 Following preparation of the me-
tathesis precursor peptide 8, initial efforts focused on carry-
ing out olefin self-metathesis of this compound in solution.
However, we were unable to remove all traces of catalyst
from the product olefin-containing peptide despite the ap-
plication of various catalyst removal strategies.38 To avoid
this issue, we turned to the use of on-bead cross-metathesis
between resin-bound and solution-phase 8. As shown in
Scheme 1, cleavage of the resin-bound adduct following
cross-metathesis gave a mixture of geometrical isomers (9
and 10) in a 2:3 ratio and 64% yield. The mixture was then
separated by preparative HPLC to give the purified E and Z
isomers. Preparation of the saturated analogue 11 was
accomplished by on-bead hydrogenation of metathesis ad-
duct (Wilkinson’s catalyst; 40 psi H2), followed by TFA-
mediated cleavage.
2-fold difference in their binding affinity, suggesting that the
linker is sufficiently flexible to access a favorable conforma-
tion for binding regardless of olefin geometry. The 4-fold
decreased affinity of the saturated analogue (11) relative to 1
may be either a reflection of the increased flexibility of the
compound relative to the olefin bioisostere or potentially a
result of its increased hydrophobicity. As with compound
1, the binding affinity of dicarba analogues to the stem-
loop was unchanged in the presence of excess yeast tRNA,
evidence of selectivity for binding to the HIV-1 RNA stem-
loop. Interestingly, monomeric compound 8 also bound to
the HIV-1 RNA stem-loop with similar affinity to that of
both the parent compound (1) and analogues 9 and 10, but
binding was completely ablated by the presence of excess
yeast tRNA. This is similar to the behavior shown by
2-ethylquinoline-3-carboxylic acid, which binds RNA non-
specifically. Thus, both the full-length peptide and quinoline
are required for selective binding to the HIV-1 FSS.
Study of Binding Kinetics Using SPR. While the equilibri-
um dissociation constant (KD) is obviously an important
measure of ligand quality in vitro, kinetic parameters (and, in
particular, the dissociation rate koff) have been proposed
as more effective predictors of in vivo selectivity.39 Slower
values for koff mean longer target-bound residence times40
and higher target selectivity. In contrast, increases in koff
have been correlated with decreases in inhibitor activity.41
Therefore, to assess the kinetics of binding by dicarba analo-
gues, we turned to surface plasmon resonance.
For binding studies using SPR, biotinylated HIV-1 FSS
RNA was immobilized on a Biacore CM5 SPR sensor chip
coated with streptavidin. Compounds 1 and 9-11 were then
flowed over the HIV-1 FSS RNA functionalized SPR chips
in the running buffer (1ꢀ PBS buffer equipped with 5 mM
MgCl2 and 0.005% Tween-20). Representative sensorgrams
As measured by fluorescence titration, both 9 and 10
bound to the HIV-1 FSS with an affinity comparable to that
of compound 1. Thus, bioisosteric replacement of the disul-
fide linkage in 1 with an olefin does not interfere with RNA
recognition (Table 3). Olefin isomers 9 and 10 show only a

6022 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Palde et al.
Scheme 1. Synthesis of Olefin and Saturated Hydrocarbon Analogues of 1
Table 3. Binding Affinity of Dicarba Analogues to the HIV-1 FSS RNA (4 ꢀ 10-7 M) in Comparison to 1 As Measured by Fluorescence Titrations in
1ꢀ PBS Buffer (pH 7.2) at 25 °Ca
compound
MW
charge
binding affinity (KD) (μM)
binding affinity (KD) in the presence of 20ꢀ yeast tRNA (μM)
1
2-ethylquinoline-3-carboxylic acid
2
-1
1
0.35 ( 0.11
0.29 ( 0.03
0.47 ( 0.04
0.33 ( 0.02
0.18 ( 0.02
1.27 ( 0.11
0.20 ( 0.03
no binding
no binding
0.39 ( 0.02
0.26 ( 0.04
1.41 ( 0.20
201
598
8
9
1168
1168
1170
2
10
11
2
2
a The reported KD values are an average of two separate titration experiments in each case.
Table 4. Evaluation of Binding Kinetics Using SPRa
compound
dissociation rate (koff) (s-1
)
association rate (kon) (M-1 s-1
)
dissociation constant (KD) (μM)
chi2
1
1.41 ꢀ 10-2
1.28 ꢀ 10-2
7.5 ꢀ 10-3
1.3 ꢀ 10-2
ND
1.79 ꢀ 103
1.15 ꢀ 103
1.62 ꢀ 103
1.05 ꢀ 103
ND
7.88
11.20
4.66
1.07
1.85
2.73
1.09
0.18
9
10
11
neomycin
12.30
2.6
a The kinetic parameters kon and koff were obtained from a 1:1 global fit to six sensorgrams generated by injecting different concentrations of
each ligand. The dissociation constant (KD) for neomycin was obtained from a steady-state analysis of seven sensorgrams; obtaining kinetic constants
for this compound was not possible.
are provided in Supporting Information, while kinetic and
equilibrium binding constants are summarized in Table 4.
The binding constant (KD = 7.88 μM) obtained for 1 (Table 4)
in this experimental setup (where HIV-1 FSS RNA is immo-
bilized onto the chip, or the reverse of our previously reported
method) is in conformity with the binding constant (KD =
4.1 ( 2.40 μM) measured with the earlier experiments. It is
interesting to note that immobilization of either binding
partner for SPR appears to cause an order-of-magnitude loss
in the affinity of these compounds relative to solution-phase
measurements. Likewise, measured dissociation constants for
9-11 were at least 10-fold weaker in this format than in the
fully solution phase measurements.
The kinetic data obtained from SPR studies (Table 4)
show that the koff of compounds 1 and 9-11 lie in the range
of 10-2 to 10-3 s-1, corresponding to a dissociative half-life
(t1/2 = 0.693/koff) of 50-90 s. Dissociative half-life is a para-
meter that determines the residence time of a ligand at the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6023
Figure 3. MTT assay for compound 10 (blue) vs mitomycin C (red).
Results suggest that compound 10 is nontoxic to human fibroblasts
at concentrations up to 1.0 mM.
target site, which in turn governs target selectivity. Indeed, it
has been hypothesized that the poor selectivity shown by
some aminoglycosides is due at least in part to their relatively
high koff (significant amount of dissociation in 30 s).42 To test
this in the context of the HIV-1 FSS sequence, we examined
the binding of neomycin by SPR. While steady-state meth-
ods could be used to derive a binding constant (2.6 μM), both
kon and koff were too rapid to determine accurately from the
data (Supporting Information, Figure S13). This is consis-
tent with the hypothesized relationship between the off rate
and sequence selectivity, as unlike our compounds neomycin
is known to bind with similar affinity to a diverse range of
RNAs.1 Hence, these data support the assertion that com-
pounds of the structural class defined by 1, 9, 10, and 11 are
promising candidates for further development as selective
RNA-binding ligands.
While the analyses described thus far suggest compounds
9-11 possess significant selectivity for target RNA seque-
nces, a more stringent test is to examine their compatibility
with cells, since a lack of selectivity in RNA-binding ability
can manifest as toxicity.43 We therefore determined the
tolerance of human fibroblasts to compound 10 using the
widely employed MTT assay,44 which serves as a reporter for
mitochondrial activity. We were gratified to observe that
compound 10 caused no statistically significant change in cell
viability at concentrations up to 1.0 mM. In contrast, mito-
mycin C, a DNA cross-linking agent widely used in che-
motherapy, caused significant cell death at all concentrations
tested (Figure 3).
In conclusion, we have employed directed analogue synth-
esis to improve our understanding of the interaction of
RBDCC-derived compounds with the HIV-1 FSS RNA, a
critical regulatory element of HIV replication. The picture
that emerges based on the data obtained to date is that the
2-ethyl-3-carboxyquinoline moieties of 1 and dicarba analo-
gues 9-11 are the primary source of affinity, most likely via
intercalation, while selectivity of binding resides in the pep-
tide (Figure 4). Replacement of the disulfide moiety with an
olefin bioisostere does not diminish activity, a discovery that
is an important first step toward the production of com-
pounds suitable for cellular assays of frameshifting. Finally,
compounds are nontoxic to cells at relevant concentrations.
Neither compound 1 nor analogues 9-11 are sufficiently
conformationally rigid to permit generation of meaningful
hypotheses with regard to their RNA-bound conformation
in the absence of experimental data; thus, X-ray or NMR
structural analysis will be an important next step toward
Figure 4. Schematic of binding results for analogues described
herein: selective, high-affinity binding to the HIV-1 FSS requires
both peptide and heterocyclic portions of lead compounds. Affinity
is largely determined by the presence of the quinoline moiety, while
selectivity derives from the peptide. Binding appears insensitive to
olefin geometry.
understanding the behavior of these compounds. Efforts are
underway to employ this newfound understanding in the
design and synthesis of new compounds with further im-
proved affinity, selectivity, and biostability. In particular, as
substantial portions of 9 and 10 are still peptidic, conversion
to peptidomimetic structures is a primary current focus. It is
also not yet clear whether the full peptide is required for
selectivity, and therefore deletion studies will be important to
test that question. Completion of these experiments will set
the stage for in vitro studies designed to test the ability of such
RNA-binding compounds to interfere with frameshifting in
HIV.
Experimental Section
Materials and General Methods. Commercially available
reagents were obtained from Aldrich Chemical Co. (St. Louis,
MO), Fluka Chemical Corp. (Milwaukee, WI), and TCI America
(Portland, OR) and used as received unless otherwise noted.
Water used for reactions and aqueous workup was doubly
distilled. Reagent grade solvents were used for all nonaqueous
extractions. Reactions were monitored by analytical thin-layer
chromatography using EM silica gel 60 F-254 precoated glass
plates (0.25 mm). Compounds were visualized on the TLC plates
with a UV lamp (λ= 254 nm) and staining with I2/SiO2. Syn-
thesized compounds were purified using flashchromatography on
EM silica gel 60 (230-400) mesh or, alternatively, via preparative
reverse-phase HPLC.
Analysis. 1H NMR spectra were recorded at 25 °C on either a
Bruker Avance 400 (400 MHz) or a Bruker Avance 500 (500
MHz) instrument. Chemical shifts (δ) are reported in parts per
million (ppm) downfield from tetramethylsilane and referenced
to the residual protium signal in the NMR solvent (CDCl3, δ =
7.26). Data are reported as follows: chemical shift, multiplicity
(s = singlet, d = doublet, t = triplet, m = multiplet), integra-
tion, and coupling constant (J) in Hertz (Hz). 13C NMR spectra
were recorded at 25 °C on a Bruker Avance 400 (100 MHz) or
Bruker Avance 500 (125 MHz). Chemical shifts (δ) are reported
in parts per million (ppm) downfield from tetramethylsilane
and referenced to carbon resonances in the NMR solvent.

6024 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Palde et al.
High-resolution mass spectra (HRMS) were acquired at the
University of Buffalo mass spectrometry facility, Buffalo,
NY, or at the mass spectrometry facility of the University of
California, Riverside.
reverse-phase preparative HPLC using a water-acetonitrile
gradient with 0.1% TFA. Purified compounds were verified as
being >95% purity by analytical HPLC.
Compound 2. FTIR (neat): 3065.52, 3018.87, 2953.30, 2920.99,
2915.69, 1664.45, 1643.24, 1634.56, 1538.12, 1433.38, 1312.95,
1296.08, 1241.50, 1198.68, 1173.60, 1125.87, 1021.72, 952.29
cm-1. 1H NMR (400 MHz, CD3OD): δ 8.6 (s, 1H), 8.58 (s, 1H),
8.06 (d, 1H, J = 8.3), 8.03-7.83 (m, 6H), 7.71-7.61 (t, 1H, J =
7.34, J = 7.83), 7.30-7.7.12 (m, 10H), 7.09 (d, 4H, J = 6.85),
5.33 (q, 1H, J = 4.88), 4.5-4.3 (m, 4H), 4.01-3.91 (m, 1H),
3.91-3.78 (m, 2H), 3.59-3.50 (m, 1H), 3.5 (dd, 1, J = 9.23, J =
4.88), 3.40-3.30 (m, 1H), 3.27-2.96 (m, 2H), 2.90-2.70 (m,
10H), 2.71-2.65 (t, 2H, J = 1.95, J = 2.93), 2.66-2.56 (s, 3H),
2.22-2.04 (m, 4H), 2.05-1.91 (m, 4H), 1.92-1.79 (m, 4H),
1.80-1.67 (m, 6H), 1.66-1.46 (m, 4H). 13C NMR (100 MHz,
CD3OD): δ 172.5, 169.6, 167.7, 161.45, 161.1, 156.4, 143.9,
136.9, 132.6, 129, 128.8, 128.2, 127.8, 127.7, 126.5, 125.9,
124.3, 60.7, 56.6, 55.1, 51.1, 39, 36.9, 36.6, 35.5, 28.9, 27.1,
24.5, 20.9. HRMS m/z calculated for C62H75N12O8S2 [M þ H] þ:
1179.5267; found: 1179.5299.
Compound 3. FTIR (neat): 3068.12, 2960.53, 2940.76, 2450.23,
2270.34, 1651.43, 1634.08, 1538.12, 1435.90, 1199.16, 1127.32
cm-1. 1H NMR (400 MHz, CD3OD): δ 9.08 (s, 1H), 8.68 (d, J =
18.2, 1H), 8.06-7.75 (m, 6H), 7.62 (dd, J = 17.0, J = 10.1, 2H),
7.32-7.07 (m, 10H), 5.35-5.21 (m, 2H), 4.53-4.32 (m, 3H),
4.23 (d, J = 8.4, 1H), 4.07 - 3.91 (m, 2H), 3.80 (s, 2H),
3.55-2.89 (m, 22H), 2.76 (dd, J = 14.5, J = 7.2, 4H), 2.10
(dd, J = 19.8, J = 8.1, 2H), 2.00-1.63 (m, 9H), 1.67-1.35 (m,
2H). 13C NMR (100 MHz, CD3OD): δ 172.6, 170.0, 161.2,
147.7, 137.2, 136.8, 132.0, 128.8, 126.9, 126.5, 60.7, 59.3, 55.1,
54.1, 50.1, 39.1, 39.8, 36.5, 35.4, 28.8, 27.1, 25.6. HRMS
m/z calculated for C60H71N12O8S2 [M þ H]þ: 1151.4954; found:
1151.4931.
Compound 4. FTIR (neat): 3150.13, 3100.92, 2950.45, 2834.56,
1651.92, 1635.04, 1553.55, 1539.09, 1516.91, 1435.90, 1419.03,
1199.16, 1173.60, 1127.80, 1021.24, 951.81 cm-1. 1H NMR (400
MHz, CD3OD): δ 8.48-8.3 (m 1H), 8.38 (d, 1H, J = 4.89),
7.75-7.67 (m, 2H), 7.66 (d, 1H, J = 7.83), 7.38 (dd, 2H,
J = 4.40, J = 4.40), 7.35-7.15 (m, 12H), 7.05 (dd, 2H, J =
1.96, J = 1.46), 5.22-5.16 (m, 2H), 4.91 (d, 1H, J = 3.91), 4.81
(d, 1H, J = 3.91), 4.42 (q, 2H, J = 6.84, J = 1.95, J = 6.35), 4.34
(q, 2H, J = 4.89, J = 3.42, J = 4.89), 3.94 (m, 2H), 3.75 (m, 2H),
3.62 (d, 1H, J = 2.44), 3.26-3.17 (m, 4H), 2.86-2.75 (m, 4H),
2.64 (s, 3H). 13C NMR (100 MHz, CD3OD): δ 18.59, 24.45,
24.38, 27.05, 28.78, 35.45, 36.64, 38.98, 50.57, 55.11, 60.89,
125.86, 126.51, 128.16, 128.82, 134.63, 136.96, 140.43, 145.59,
147.31, 166.74, 170.23, 172.50. HRMS m/z calculated for
C54H71N12O8S2 [M þ H]þ: 1079.4954; found: 1079.4957
Compound 5. FTIR (neat): 3068.39, 3029.00, 2995.31, 2967.15,
2950.40, 2932.08, 2929.67, 1655.29, 1650.95, 1645.171575.25,
1524.14, 1488.46, 1444.10, 1435.42, 1388.17, 1350.08, 1320.18,
1199.64, 1176.50, 1131.65, 1001.95, 959.05 cm-1. 1H NMR (400
MHz, CD3OD): δ 7.35-7.27 (m, 10H), 4.64-4.49 (tt, 6H, J =
3.66, J= 5.79, J = 5.49, J= 6.71), 3.86-3.80, (m, 2H), 3.79-3.70
(m, 2H, 3.55-3.49 (tt, J = 4.57, J = 7.62, J = 5.19, J = 5.79, J =
8.55) 3.28-3.09 (m, 10H), 2.90-2.71 (m, 6H), 2.29-2.24 (m, 2H),
2.14-2.06 (m, 7H), 1.99-1.73 (m, 5H). 13C NMR (125 MHz,
CD3OD): δ 173.33, 172.97, 166.88, 162.37, 137.67, 129.94, 129.10,
129.03, 127.48, 61.31, 56.19, 51.57, 49.00, 48.83, 48.66, 48.49,
48.32, 48.15, 47.98, 39.91, 38.19, 37.79, 37.49, 36.37, 29.92, 27.99,
25.60. HRMS m/z calculated for C40H61N10O6S2 [M þ H]þ:
841.4211; found: 841.4208.
Binding Analysis. Fluorescence titrations were performed on
a Cary Eclipse fluorescence spectrophotometer using a 10 mm
path-length semimicro quartz fluorescence cell with 400 μL
sample holding capacity. The 50-Cy3-labeled HIV-1 FSS RNA
was purchased from Integrated DNA Technologies, Inc. Auto-
claved 1ꢀ phosphate-buffered saline (PBS) (4.3 mM Na2HPO4,
1.47 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl; pH = 7.2) was
used as a buffer to dissolve the RNA and the compounds to be
tested. A solution of HIV-1 FSS RNA in 1ꢀ PBS buffer (400 μL,
400 or 500 nM) was heated to 65 °C for 4 min, and 2 μL of a
MgCl2 solution (1 M) was added to it. The solution was then
allowed to cool slowly to room temperature in the presence of
MgCl2 in order to ensure that the RNA assumes its secondary
stem-loop structure. This solution of 50-Cy3-labeled HIV-1
FSS RNA was taken in the cell and excited with a wavelength
of 550 nm and a 2.5 nm slit width. The emission spectrum was
collected from 555 to 600 nm wavelength range at a PMT
voltage of 715 V and a 5 nm slit width. The compound in 1ꢀ
PBS buffer was added to the cell in either 2 or 4 μL increments
from the stock solution (either 20, 50, or 200 μM) leading to a
concentration range starting from 100 nM to 10 μM. After each
addition, the solution was allowed to equilibrate for a minimum
of 10 min, and fluorescence emission spectra were taken.
Equilibrium was determined to be established after obtaining
three similar fluorescence spectra taken at 1 min intervals. The
decrease in fluorescence of 50-Cy3-labeled HIV-1 FSS RNA was
then noted at 564.1 nm for each added concentration of the
compound. The fluorescence units (FU) were then dilution
corrected, and the FU after each addition was subtracted from
the FU at zero compound concentration to give ΔFU. This
ΔFU was plotted against compound concentration using Origin
7 (OriginLab Corp.). The data were fit to a one-site binding
model accounting for ligand depletion.45 In this analysis, the
free ligand concentration term is substituted with total ligand
concentration minus the bound ligand concentration (LT - B) as
RTðLT - BÞ
B ¼
KD þ ðLT - BÞ
Solving the equation for B (real solution for the quadratic
equation)
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðLT þ KD þ RTÞ - ð- LT - KD - RTÞ - 4LTRT
B ¼
2
where B = bound ligand:receptor complex concentration, LT =
total ligand concentration, KD = dissociation constant, and RT =
total receptor concentration.
Titrations were carried out with each compound at least two
times. Buffer control titrations were performed by titrating
similar amounts of PBS, pH 7.2, into the Cy-3 RNA and con-
firmed a concentration-dependent linear fluorescence change
matching what is theoretically expected. For the competition
experiments with yeast total tRNA, the competing RNA was
added to the 50-Cy3-labeled HIV-1 FSS RNA at a concentration
of 8 or 16 μM. This mixture of RNA was then titrated with the
compounds in a similar manner described above. Titrations into
other labeled sequences (entries 5 through 11, Table 1) were
carried out analogously to the procedure employed for the HIV-
1 FSS RNA. Surface plasmon resonance (SPR) experiments
were conducted using a Biacore-X instrument (GE Healthcare);
procedures are described in the main text and in greater detail in
the Supporting Information.
Compound 6. FTIR (neat): 3021.78, 2985.12, 2966.80, 2920.51,
1668.31, 1651.92, 1634.56, 1538.61, 1468.69, 1464.35, 1298.00,
1265.70, 1231.46, 1198.88, 1173.60, 1050.34 cm-1 1H NMR
.
(400 MHz, CD3OD): δ 8.46 (d, 1H, J = 16.62), 8.37 (s, 2H),
7.29-7.20 (m, 18H), 7.06 (d, 6H, J = 7.83), 6.22 (d, 4H, J =
4.40), 5.54 (s, 2H), 5.28 (q, 1H, J = 4.48, J = 4.40, J = 4.48),
4.44, (m, 2H), 3.89 (q, 1H, J = 7.34, J = 6.85), 3.89 (q, 1H, J =
7.34, J = 6.85), 3.79 (q, 3H, J = 8.80, J = 6.35), 3.43 (dd, 1H,
Synthesis of Analogues 2-7. Compounds 2-7 were synthe-
sized on resin using methods previously described for com-
pound 1. Following completion of the synthesis, compounds
were cleaved from resin, ether precipitated, and purified by

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6025
J = 8.91, J = 8.91), 3.29-2.95 (m, 10H), 2.74 (q, 2H, J = 6.85,
J = 6.85), 2.68, (d, 1H, J = 1.96), 2.64 (q, 2H), 2.06 (m, 2H),
1.94 (q, 2H, J = 6.35, J = 6.84, J = 8.31), 1.72 (t, 3H, J =
6.84), 1.27 (t, 3H, J = 7.82). 13C NMR (100 MHz, CD3OD): δ
172.5, 171.12, 169.52, 167.55, 157.77, 149.46, 143.96, 136.89,
128.89, 129.02, 125.86, 103.39,102.95, 60.59, 56.63, 55.142,
38.99, 36.61, 35.466, 28.92, 27.05, 24.51, 13.45. HRMS m/z
calculated for C66H79N12O12S2 [M þ H]þ: 1295.5376; found:
1295.5350.
preparative RP-HPLC (isocratic elution, 30% acetonitrile in
water with 0.1% TFA).
Compound 8. FTIR (neat): 3249, 2934, 2817, 1672, 1659, 1643,
1634, 1529, 1517, 1201, 1177, 1128, 1026 cm-1. 1H NMR (400
MHz, CDCl3): δ 8.90 (s, 1H), 8.30 (t, 1H, J = 8.8), 8.09 (d, 1H,
J = 8.4), 8.03 (t, 1H, J = 7.8), 7.82 (t, 1H, J = 7.8), 7.71-7.69
(m, 3H), 7.31-7.27 (m, 3H), 7.12 (d, 2H, J = 6.8), 5.88-5.84 (m,
1H), 5.34-5.24 (m, 2H), 4.49-4.88 (m, 1H), 4.59-4.5 (m, 1H),
4.32-4.28 (m, 1H), 3.98 (s, 1H), 3.68-3.62 (m, 1H), 3.39-3.21
(m, 4H), 3.11-3.10 (m, 2H), 2.94-2.90 (m, 2H), 2.46-2.42 (m,
2H), 2.22-2.13 (m, 1H), 2.07-2.01 (m, 1H), 1.96-1.90 (m, 1H),
1.85-1.80 (m, 3H), 1.35 (t, 3H, J = 7.4), 1.25 (s, 2H). 13C NMR
(100 MHz, CDCl3): 172.2, 171.9, 171.4, 166.2, 161.4, 136.4,
132.6, 129.7, 129.0, 128.6, 128.4, 127.1, 126.1, 119.3, 61.4, 54.5,
51.4, 47.9, 37.0, 36.7, 35.9, 35.8, 28.7, 27.4, 26.8, 24.9, 15.2, 14.2.
HRMS m/z calculated for C34H42N6O4 [M þ H]þ: 599.3346;
found: 599.3357.
Compound 9. FTIR (neat): 2957.1, 2920.2, 2853.5, 1673.1,
1667.8, 1457.1, 1377.0, 1201.1, 1179.3, 1134.1 cm-1. 1H NMR
(500 MHz, CD3OD): δ 8.63 (s, 2H), 8.08 (d, 2H, J = 8.0), 8.04
(d, 2H, J = 9.0), 7.89 (t, 2H, J = 8), 7.68 (t, 2H, J = 8.0),
7.21-7.11 (m, 10H), 5.78 (m, 2H), 4.48-4.42 (m, 2H), 4.36 (t,
2H, J = 7.5), 3.92-3.88 (m, 2H), 3.66-3.60 (m, 4H), 3.55-3.51
(m, 1H), 3.17-3.10 (m, 4H), 3.02-2.90 (m, 4H), 2.81 (s, 1H),
2.17-2.62 (m, 6H), 2.54-2.52 (m, 1H), 2.15-2.08 (m, 2H),
1.91-1.88 (m, 4H), 1.83-1.79 (m, 2H), 1.68-1.63 (m, 5H),
1.34-1.21 (m, 8H), 0.87-0.80 (m, 2H). 13C NMR (100 MHz,
CD3OD): 173.9, 171.9, 168.8, 162.3, 138.0, 134.2, 130.7, 130.2,
130.1, 129.9, 129.8, 129.5, 129.4, 129.3, 127.8, 127.4, 61.5, 56.5,
53.3, 38.4, 37.9, 36.7, 35.0, 29.1, 28.4, 25.9, 14.5. HRMS m/z
calculated for C66H80N12O8 [M þ H]þ: 1169.6300; found:
1169.6296
Compound 10. FTIR (neat): 2957.6, 2920.0, 2900.7, 2851.1,
1664.3, 1634.3, 1557.4, 1538.6, 1201.1, 1180.3, 1133.1 cm-1. 1H
NMR (500 MHz, CD3OD): δ 8.67 (s, 2H), 8.11-8.05 (m, 4H),
7.98-7.93 (m, 2H), 7.77-7.71 (m, 2H), 7.25-7.17 (m, 10H),
5.82-5.79 (m, 2H), 4.97-4.94 (m, 1H), 4.48-4.45 (m, 2H),
3.97-3.92 (m, 2H), 3.72-3.65 (m, 4H), 3.21-3.16 (m, 8H),
3.05-3.20 (m, 4H), 2.76-2.69 (m, 6H), 2.18-2.12 (m, 2H),
1.97-1.93 (m, 3H), 1.87-1.83 (m, 2H), 1.72-1.69 (m, 4H),
1.39-1.28 (m, 10H). 13C NMR (100 MHz, CD3OD): δ 172.6,
172.5, 170.3, 167.7, 161.0, 136.8, 132.8, 129.5, 128.7, 128.5,
128.1, 127.9, 127.6, 126.5, 126.1, 124.2, 69.8, 60.3, 55.2, 51.9,
37.1, 36.6, 35.4, 29.0, 27.6, 24.6, 13.2. HRMS m/z calculated for
C66H80N12O8 [M þ H]þ: 1169.6300; found: 1169.6297
Compound 7. FTIR (neat): 3100.23, 3050.10, 2950.66, 1690.89,
168.31, 1668.28, 1652.40, 1635.04, 1567.89, 1234.99, 1200.61,
1
1174.09, 1129.24 cm-1. H NMR (400 MHz, CD3OD): δ 8.64
(d, 2H, J = 1.96), 8.33-8.18 (m, 9H), 7.85 (m, 4H), 7.32-5.15 (m,
14H), 5.16 (q, 2H, J = 6.36, J = 2.44, J = 5.87), 4.46 (m, 4H),
3.95 (q, 2H, J = 9.78, J = 7.34, J = 6.85), 3.78 (m 2H), 3.27 (m,
4H), 3.24 (d, 4H), 3.20-3.08 (m, 6H), 3.04-2.95 (m, 4H), 2.7 (m,
5H), 2.6 (d, 4H, J = 4.40), 2.15 (m, 2H), 2.01-1.85 (m, 2H), 1.69
(t, 4H, J = 6.84), 1.29 (d, 4H, J = 3.913). 13C NMR (100 MHz,
CD3OD): δ 182.07, 182.02, 172.53, 170.42, 166.74, 138.36,
136.89, 135.20, 134.19, 133,40, 133.23, 132.50, 128.80, 128.72,
28.16, 127.02, 126.70, 126.54, 125.81, 60.84, 55.09, 51.96, 40.41,
38.98, 36.83, 36.61, 35.45, 28.89, 28.76, 27.07, 24.44. HRMS
m/z calculated for C70H73N10O12S2 [Mþ H]þ: 1309.4845; found:
1309.4823.
Synthesis of Dicarba Analogues. Resin-bound 8 was synthe-
sized using standard Fmoc methodology for peptide synthesis.
Wang resin (1.0 g, 100-200 mesh, 1 mmol/g loading) was
activated with 1,10-carbonyldiimidazole (DIC, 3.3 g, 10 mmol)
in 12 mL of DMF for 12 h on a LabQuake rotator. The resin was
then washed three times each with DMF, CH2Cl2, and DMF
again, followed by reaction with 1,3-diaminopropane (0.72 mL,
10 mmol) in DMF (12 mL) for another 12 h. The wash cycle was
then repeated. The coupling of the first amino acid was carried
out by adding Fmoc-L-Phe-OH (1.16 g, 3 mmol), HBTU (1.14 g,
3 mmol), and DIPEA (0.85 mL, 5 mmol) in 12 mL of DMF
to the resin and rotating the reaction mixture for 1 h. Following
a wash cycle, Fmoc deprotection was accomplished using 20%
piperidine in CH2Cl2 for 0.5 h followed again by the wash
cycle. Similarly, Fmoc-L-Pro-OH, Fmoc-L-allylglycine, and
2-ethylquinoline-3-carboxylic acid were coupled to synthesize
resin-bound monomeric compound (8).
The resin was then split into two equal parts of 0.50 g. One
part was treated with 30% TFA/1% TEA in CH2Cl2 for 0.5 h to
obtain a cleaved product 8 (0.23 g) to be used as the solution
olefin component for the metathesis reaction. The other part of
the resin was dried in a desiccator under vacuum for 12 h and
then allowed to swell in dry CH2Cl2 (12 mL) for 20 min. The
resin was washed with CH2Cl2 (3 ꢀ 10 mL) followed by 0.8 M
LiCl in DMF (10 mL) for 10 min. The resin was then washed
with DMF (10 mL), and the 0.8 M LiCl wash was repeated for
two more times. Finally, the resin was washed with dry, degassed
1,2-dichloroethane (10 mL) and then suspended in the same
solvent (5 mL). To this suspension were added Grubbs’ second
generation catalyst (0.14 g, 0.17 mmol) in 5 mL of 1,2-dichlor-
oethane and the cleaved 8 (0.25 g, 0.42 mmol) in 10 mL of a 1:4
mixture of CH2Cl2 and 1,2-dichloroethane. The reaction mix-
ture was refluxed for 24 h. The reaction was then cooled to room
temperature, and additional Grubbs’ second generation catalyst
(0.07 g, 0.09 mmol) was added to the reaction mixture and
refluxed for another 24 h. After repeating this cycle a third time,
the reaction mixture was cooled to room temperature and
transferred to a standard solid-phase reaction vessel with filter-
ing. The resin was then washed with CH2Cl2 (3 ꢀ 10) and DMF
(3 ꢀ 10 mL) and suspended in 10 mL of DMF. DMSO (0.2 mL)
was added to the suspension and rotated for 12 h, in order to
remove colored ruthenium impurities.46 The resin was then
washed and the product cleaved off the resin with 25% TFA
in CH2Cl2 (10 mL) for 0.5 h to obtain a crude mixture of olefins 9
and 10 (0.25 g, 64%). The isomers were then separated using
Compound 11. Hydrogenation was performed on resin. The
resin-bound olefin metathesis product (resin: 0.25 g, 0.25 mmol)
was suspended in 6 mL of 10% MeOH in CH2Cl2 in a 16 mL
glass vial. Wilkinson’s catalyst (0.06 g, 0.06 mmol) was added to
this suspension, and the mixture was agitated on a Parr hydro-
genator under 40 psi H2 gas pressure at room temperature for
5 h. The resin was then washed with 10% MeOH in CH2Cl2 (3 ꢀ
10 mL), MeOH (3 ꢀ 10 mL), and CH2Cl2 (3 ꢀ 10 mL). The resin-
bound reduced product was then cleaved using 30% TFA/1%
TES in CH2Cl2 (6 mL). The cleaved product was collected, and
the solvent was removed in vacuo. The crude product was then
precipitated using cold ether and centrifuged (5000g, 5 min), and
the ether was decanted to obtain pure 11 in 60% yield (0.08 g).
FTIR (neat): 3300.3, 2959.5, 2910.2, 1675.4, 1652.4, 1641.2,
1621.5, 1535.7, 1199.2, 1190.5, 1177.0, 1124.9 cm-1. 1H NMR
(500 MHz, CD3OD): δ 8.67 (s, 2H), 8.10-8.05 (m, 4H),
7.95-7.91 (m, 2H), 7.74-7.69 (m, 2H), 7.24-7.20 (m, 10H),
4.50-4.40 (m, 4H), 4.20 (dd, 2H, J1 = 7.5, J2 = 2.5), 4.03-3.98
(m, 2H), 3.82-3.64 (m, 3H), 3.48 (q, 2H, J = 17.5), 3.27-3.14
(m, 10H), 3.05-3.02 (m, 4H), 2.98 (br s, 2H), 2.78-2.67 (m, 4H),
2.22-2.09 (m, 3H), 1.73-1.65 (m, 4H), 1.39-1.28 (m, 6H),
0.99-0.89 (m, 4H), 0.59 (m, 2H). 13C NMR (100 MHz,
CD3OD): δ 172.6, 172.5, 161.2, 161.0, 138.3, 136.8, 131.9,
129.6, 128.9, 128.1, 127.3, 126.4, 125.9, 125.3, 60.2, 55.2, 51.8,
36.9, 36.5, 35.3, 28.9, 28.3, 27.0, 25.1, 24.6, 13.2. HRMS m/z

6026 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Palde et al.
calculated for C66H82N12O8Na [M þ Na]þ: 1193.6271; found:
1193.6243
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blast cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM), containing 5% fetal bovine serum (Invitrogen) and
5% pen-strep. Cell cultures were incubated in a humidified
atmosphere containing 5% CO2 at 37 °C. The viability of human
fibroblasts in the presence of compound 10 was tested using the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. Cells were plated in a 96-well sterile plate at con-
centrations of 3.2 ꢀ 104 cells per well in a volume of 100 μL of
culture media. The cells were then allowed to grow to approxi-
mately 80% confluence by incubating at 37 °C for 48 h. The old
media were removed and replaced with 100 μL of different
concentrations (up to 1 mM) of compound 10 or mitomycin C
(as a control) in DMEM. Cells were exposed to compounds for
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Acknowledgment. The authors gratefully acknowledge fi-
nancial support of the NIH/NIAID P30AI078498, via the
University of Rochester Developmental Center for AIDS
Research (DCFAR), and NIH T32AR007472 (training grant
support to P.C.G.). We thank Prof. Thomas Foster for the use
of his fluorometer and Prof. Lisa DeLouise for guidance with
the MTT assays.
(13) (a) Dulude, D.; Baril, M.; Brakier-Gingras, L. Characterization of
the frameshift stimulatory signal controlling a programmed -1
ribosomal frameshift in the human immunodeficiency virus type 1.
Nucleic Acids Res. 2002, 30, 5094–5102.
(14) (a) Telenti, A.; Martinez, R.; Munoz, M.; Bleiber, G.; Greub, G.;
Sanglard, D.; Peters, S. Analysis of natural variants of the human
immunodeficiency virus type 1 gag-pol frameshift stem-loop struc-
ture. J. Virol. 2002, 76, 7868–7873. (b) Baril, M.; Dulude, D.;
Gendron, K.; Lemay, G.; Brakier-Gingras, L. Efficiency of a pro-
grammed -1 ribosomal frameshift in the different subtypes of the
human immunodeficiency virus type 1 group M. RNA 2003, 9, 1246–
1253.
Supporting Information Available: Spectral data for com-
pounds 2-11, selected fluorescence titration data for com-
pounds 2-11, SPR data and controls for compounds 9 -11,
and dynamic light scattering data for compounds 9 -11. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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