Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA

Article abstract of DOI:10.1021/acs.jmedchem.6b01450

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemist

Full text of DOI:10.1021/acs.jmedchem.6b01450

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Article
Development of Small Molecules with a Non-Canonical
Binding Mode to HIV-1 Trans Activation Response (TAR) RNA
Fardokht A Abulwerdi, Matthew D Shortridge, Joanna Sztuba-Solinska, Robert
Wilson, Stuart F.J. Le Grice, Gabriele Varani, and John S Schneekloth, Jr.
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01450 • Publication Date (Web): 16 Nov 2016
Downloaded from http://pubs.acs.org on November 16, 2016
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Development of Small Molecules with a Non-Canonical Binding Mode to HIV-1 Trans Activation
Response (TAR) RNA
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Fardokht A. Abulwerdi,†,‡ Matthew D. Shortridge,# Joanna Sztuba-Solinska,‡ Robert Wilson,† Stuart F. J.
Le Grice,‡ Gabriele Varani,# and John S. Schneekloth, Jr.*,†
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†Chemical Biology Laboratory, National Cancer Institute, Frederick, Maryland 21702, USA
‡Basic Research Laboratories, National Cancer Institute, Frederick, Maryland 21702, USA
#Department of Chemistry, University of Washington, Seattle, Washington 98195, USA
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Keywords:
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HIV-1 transactivation response RNA (TAR), RNA-binding small molecules, thienopyridine compounds,
NMR, SHAPE.
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Abstract
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Small molecules that bind to RNA potently and specifically are relatively rare. The study of
molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic
element, has long been an important model system for the chemistry of targeting RNA. Here we report
the synthesis, biochemical and structural evaluation of a series of molecules that bind to HIV-1 TAR
RNA. A promising analog, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-
derived peptide with an IC50 of 40 µM. NMR characterization of a soluble analog, 2, revealed a non-
canonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by Selective 2’-
hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within
the context of an in vitro-synthesized 365-nt HIV-1 5’- untranslated region (UTR). Thus, these
compounds exhibit a novel and specific mode of interaction with TAR, providing important implications
for RNA ligand design.
Introduction
RNA-binding small molecules are an important and highly challenging area of therapeutic
research. The discovery of non-coding RNAs and their widespread role in human disease strongly
suggests that opportunities to redress disease conditions by targeting RNA should be plentiful.1
Structural and analytical techniques over the last few decades have provided information about RNA
structure and function, yet examples of small drug-like molecules that interact with RNA are still rare
relative to proteins. The polyanionic nature of RNA, coupled with its highly flexible and dynamic
structure and repetitive nature of its surface, present challenges for inhibitor design. Many known RNA-
binding small molecules are polycationic, binding tightly (with Kds in the mid nM range) but with limited
specificity. Furthermore, they tend to be larger than desirable for typical drugs and do not satisfy the
characteristic physicochemical properties of modern successful drug candidates.
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A well-characterized system to study small molecule RNA antagonists is transactivation
response (TAR) element from HIV type 1 (HIV-1), a 59-nucleotide stem-loop RNA located in the 5’-
untranslated region (UTR) of the viral genome. TAR plays a pivotal role in HIV replication via its
interaction with Tat, a virus-encoded protein that recruits the host transcriptional machinery to facilitate
efficient transcription of the viral DNA.2-5 While the TAR structure is evolutionary conserved, sequence
heterogeneity exists among various virus isolates.6 However, in the case of HIV-1 subtypes, base pair
co-variations, e.g., A-U conversion into G-C has been shown to maintain TAR structural integrity.7 In
addition to being a transcriptional activator, TAR has also been shown to play a role in initiation of
reverse transcription.8-10 More recently, several groups have demonstrated that TAR also functions as a
miRNA that downregulates cellular apoptotic genes and leads to extended cellular proliferation and
continued virus production.11-13 In sum, The central role of TAR in HIV-1 biology underscores its
potential as a therapeutic target.14 Thus, inhibition of the Tat-TAR interaction has been pursued as a
potential anti-HIV therapeutic strategy. While numerous classes of small molecules have been
reported15-17, the lack of anti-HIV drugs targeting TAR in the clinic highlights the challenges in
developing small molecule RNA binders. TAR RNA remains both an important anti-viral target and a
model system to develop RNA-targeting molecules.
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Previously, we utilized a small molecule microarray (SMM) screening platform to rapidly screen
immobilized small molecules against nucleic acid targets.18 In one example, we reported the discovery
of 1, a thienopyridine compound that interacts with the TAR hairpin.19 However, at that time little was
known about the mode of interaction between the compound and RNA. Here, we have developed a
chemical synthesis of 1 to generate a focused library of 25 compounds derived from the initial hit
structure. Biochemical and biophysical analyses of this series of compounds, including affinity
measurements, NMR analysis, and SHAPE, provided information about structure-activity relationships
as well as insights into the mode of binding of this class of compounds to HIV-1 TAR.
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Results and Discussion
Chemistry
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To prepare a focused compound library, we developed a modular and convergent synthetic
route to 1 and its analogs. For preparation of 1, 2-chloro-6-methylnicotinonitrile (24) was treated with
thiourea in refluxing n-butanol to provide 2-mercapto-6-methylnicotinonitrile (25) in essentially
quantitative yield (Scheme 1).20 In parallel, reaction of 3-(trifluoromethyl)aniline (26) with bromoacetyl
bromide in the presence of catalytic amounts of 4-N,N-dimethylamino-pyridine (DMAP) provided 2-
bromo-N-(3-(trifluoromethyl)phenyl)acetamide (27) in high yield (Scheme 1).21 Reaction of
mercaptopyridine 25 with bromoacetamide 27 in refluxing ethanol in the presence of excess sodium
ethoxide promoted bromide displacement by the thiol group of the mercaptopyridine intermediate
followed by cyclization to afford the thienopyridine ring structure, providing 1 in 58% yield (Scheme 1).22
We next used this route to design and synthesize several analogs of 1 to evaluate how
structural changes would affect affinity for TAR. 2-4, containing a second pyridine ring, were prepared
by reaction of the associated aminopyridines (28g-i) with bromoacetyl bromide to provide the
bromoacetamide as an HBr salt (29g-i) (Scheme 2A).23 Treatment of bromoacetamides 29g-i with 2-
mercapto-6-methylnicotinonitrile (25) in refluxing ethanol in the presence of excess base yielded the
assembled thienopyridine ring system (Scheme 2A). 5-9 were prepared via a similar synthetic route to
1 (Scheme 2B). In the case of 10, with a bulkier isopropyl group at the 6-position of the pyridine core,
reaction of 2-chloro-6-isopropylnicotinonitrile (30c) with thiourea failed to yield the desired
mercaptopyridine. Therefore, Lawesson’s reagent was used to synthesize the desired mercaptopyridine
(31c) in modest yield.24 The reaction of mercaptopyridine 31c with the desired bromoacetamide (27)
proceeded as described for 1 to provide 10 (Scheme 2B). Preparation of 11, lacking an amine, was
achieved by a modified Sandmeyer reaction, employing NaNO2/H2SO4/Cu2O with hypophosphorous
acid (Scheme 3).25 Additionally, reaction of 1 with triphosgene at room temperature provided cyclized
12 (Scheme 3).26 Analogs containing a sulfonamide linker were synthesized by the reaction of desired
anilines (26 and 34) and chloromethanesulfonyl chloride in the presence of triethylamine followed by a
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basic workup27 to provide the sulfonamide intermediate (35-36) (Scheme 3). Sulfonamides 35-36 were
treated with the desired mercaptopyridine (25) in the presence of excess base in N,N-dimethylformaide
(DMF) at 130 oC to afford 13 and 14 (Scheme 3).27
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We also prepared 15-19, containing oxadiazolyl linkers between the thienopyridine core and the
aryl group (Scheme 4). For 15 and 16, which contain 1,3,4-oxadiazol-2,5-yl and 1,2,4-oxadiazol-3,5-yl
rings (respectively), commercially available chloromethyl phenyl substituted oxadiazoles (37I-m) were
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treated with 2-mercapto-6-methylnicotinonitrile (25) in the presence of excess base in DMF at 130 C,
producing the thienopyridine scaffold in good yield. 17-23 were synthesized similar to 16 (Scheme 4
and Scheme S1). In the case of 24-26, the desired chloromethyloxadiazoles were conveniently
prepared as reported previously28 and further treated with 25 to yield the desired products (Scheme
S2). Collectively, the convergent route reported here furnished a library of 25 analogs of the initial hit
structure.
Biochemical evaluation of compound library
a. Affinity measurement
To evaluate the TAR binding properties of analogs described above, we utilized a series of
biochemical assays that provide information on both compound affinity for TAR and its ability to
displace a Tat-derived peptide from TAR, revealing a clear picture of structure-activity relationship. To
probe direct binding of compounds to TAR, we utilized two different 2-aminopurine (2-AP) assays. 2-
AP is an environmentally-sensitive fluorophore that reports on the local environment of the base.29
Conformational changes in the RNA upon binding of a ligand result in changes in fluorescence
intensity, which can be used to derive accurate affinities. Two distinct constructs were used where
either U25 or U23 (both of which are located in the bulge region of TAR hairpin) was replaced with a 2-
AP fluorophore (Figure 1A). U25 is a flexible residue30 and its replacement by 2AP has been previously
utilized in probing TAR-ligand interactions29. To evaluate larger numbers of compounds, we
miniaturized the 2-AP assay to be compatible with plate reader-based analysis. This format required a
higher concentration of RNA, but enabled a significantly reduced assay volume of 100 µL per sample in
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a 96 well plate. Once optimized, this assay generated reproducible apparent binding constants (Kdapp),
albeit with slightly higher values than obtained under previous assay conditions.19 Values obtained
under these conditions were both internally consistent and comparable to recently published Kds of
several TAR-binding small molecules.31
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Kdapp obtained from 2-AP assays with both constructs were in good agreement (Table 1).
Synthetic 1 showed an apparent Kdapp 25-2AP of 47.7 11.6 µM. Next, the trifluoromethylphenyl group
was replaced by different pyridine isomers (2-4) which dramatically improved compound solubility,
making them usable at high concentrations in NMR experiments (see below). Interestingly, analog 2,
containing 4-pyridyl substitution, exhibited a similar binding affinity (Kdapp 25-2AP = 40.0 0.7 µM) to 1
(Figure 1B). Analogs 3, containing 3-pyridyl (Kdapp 25-2AP = 68.0 26.0 µM) and 4 with 2-pyridyl (Kdapp 25-
2AP = 50.3 10.3 µM) substituents showed ~2-fold weaker binding affinity compared to 2. Changing the
position of the meta-trifluoromethyl group in 1 to para (7, Kdapp 25-2AP = 75.3 9.2 µM) or ortho (8, Kdapp 25-
= 90.1 4.7 µM) decreased binding affinity approximately 2-fold. Additionally, extending the meta-
2AP
trifluoromethyl phenyl by a methylene linker (9, Kdapp 25-2AP = 66.4 0.9 µM) maintained a similar binding
affinity to 1.
We further investigated the role of substitution on the pyridyl ring of thienopyridine scaffold.
Moving the methyl group to the 5-position (6, Kdapp 25-2AP = 48.5 5.4 µM) maintained a similar binding
affinity as 1 while the 4-methyl substituent (5, Kdapp 25-2AP = 66.5 4.5 µM) showed ~2-fold weaker
binding affinity. Replacing the methyl substituent with a bulkier isopropyl group in 10 (Kdapp 25-2AP = 43.3
13.3 µM) maintained similar affinity compared to 1. Next, Analog 11, a desamino thienopyridine,
showed inferior binding affinity, highlighting the importance of the amino group. Additionally, replacing
amide for a sulfonamide in analog 13 and 14 or a cyclized analog, 12, completely abolished binding.
Next, we evaluated the oxadiazole series. Interestingly, isosteric replacement of the amide
linker in 1 with 1,3,4-oxadiazol-2,5-yl in 15 (Kdapp 25-2AP = 49.1 7.8 µM) (Figure 1B) as well as 1,2,4-
oxadiazol-3,5-yl in 16 (Kdapp 25-2AP = 47.7 17.8 µM) retained binding affinity as observed for 1. Analogs
17-19, containing the 1,3,4-oxadiazol-2,5-yl but with different substitution pattern, retained the binding
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affinity of 15. However, evaluation of 1,3,4-oxadiazol-2,5-yl analogs 20-26 either decreased solubility or
abolished binding affinity (Table S1).
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b. Competitive binding assay measurements
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In addition to the 2-AP direct binding assays, we utilized an orthogonal FRET-based assay to
evaluate disruption of the Tat-TAR interaction.32 In this assay, a 16-mer peptide derived from Tat
protein is labeled with fluorescein at its N-terminus and TAMRA at its C-terminus. In solution, the two
fluorophores in the free peptide are in close proximity and quench each other. Upon binding to TAR,
the peptide adopts an extended conformation, inducing FRET. In this assay, a compound capable of
disrupting the Tat-TAR interaction will cause a loss of FRET signal. All synthesized analogs were
evaluated in FRET assay. While many, including 2, did not display activity in this assay up to 400 µM, it
is notable that oxadiazole analogs 15 (Figure 1C) and 16 (IC50 = 40.1 5.6 µM and IC50 = 45.1 25.8
µM respectively) displaced the Tat peptide with IC50 values similar to their apparent Kds (Table 1).
Additionally, while removal of meta-trifluoromethyl group on the phenyl ring in 17 or its replacement with
meta-chloro in 18 abolished FRET signal, the absence of 6-methyl substituent in 19 maintained FRET
activity similar to 15 (Table 1) suggesting the importance of phenyl substituent on the ability of the
oxadiazole analogs to displace the Tat peptide.
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Taken together, our thienopyridine analogs displayed mid micromolar binding to the TAR hairpin
under the assay conditions. While the majority of the structural changes in this study resulted in subtle
effects, isosteric replacement of the amide with oxadiazoles provided analogs which not only
maintained binding affinity to TAR but displaced the Tat peptide in the FRET assay at concentrations in
the mid micromolar range. It is also noteworthy that the compounds in this series with similar binding
affinity show differing behavior in the FRET-based assay. However, these observations are consistent
with a report in which peptides with similar TAR binding affinity also exhibited very different IC50 values
in displacing a Tat peptide.33 This seemingly contradictory observation suggests that the determinants
of binding may differ from those required for disrupting an associated RNA/peptide interaction, at least
for some ligands.
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NMR studies
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To better understand the mode of interaction of the compounds with TAR, we investigated the
small molecule-RNA complex by NMR. Many compounds in this series exhibited poor aqueous
solubility, which limited high-resolution structure analysis. Compound 2 displayed increased solubility
relative to 1 and could be studied at concentrations amenable for ligand-detected NMR experiments
and some limited RNA-detected experiments. 2 was first evaluated using line broadening methods
(Figure 2A and Figure S1A-C).34 In this experiment, its proton spectra was recorded in the presence
and absence of 10µM TAR. Upon binding, the ligand resonances broaden in response to the increase
in rotational correlation time while bound to target RNA.34 In the case of 2, the two doublets
corresponding to protons on the pyridine ring of the thienopyridine scaffold (protons a and b in Figure
2A) are completely broadened in presence of TAR suggesting the pyridine ring directly interacts with
the hairpin while other proton peaks do not change (Figure S1A). In contrast, ligand-detected NMR of a
non-binding analog 14 shows no line broadening in neither the aromatic (Figure 2B) nor aliphatic region
(Figure S1B). The line-broadening pattern in the 1D-1H NMR experiments is consistent with the 2-AP
fluorescent-based affinity measurements indicating mid micromolar binding.
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Ligand-detected NMR experiments require lower ligand concentration to measure binding,
which is useful for low solubility molecules. However, these methods are also susceptible to non-
specific aggregation effects, which generate false positive results. Therefore, to identify and verify
binding of 2, we evaluated ligand-induced chemical shift perturbations in total correlation spectroscopy
(TOCSY) experiments. Here, free TAR hairpin was evaluated upon the addition of 2 (Figure 3A-B and
Figure S1D-E). Specific changes in the TOCSY spectrum suggest single site binding with the largest
changes occurring at bases C39, U40 and C41. These residues are located directly below the -UCU-
bulge residues U23, C24 and U25. Resonances within residues U23 and C24 also show significant
chemical shift changes. However, unlike many TAR ligands, U25 does not show significant changes in
chemical shift, nor do any of the remaining pyrimidines, indicating a new binding mode compared to
previously reported TAR-binding ligands such as argininamide and acridine, which disrupt stacking of
A22 and U23 and bind to the major groove of TAR similar to Tat-derived peptides.35-37 On the other
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hand, the solution NMR structure of Neomycin B bound to TAR indicates that this aminoglycoside binds
in the minor groove instead.38 However TAR residues G21, U42 and G26 that are shifted in presence of
Neomycin B are not the same residues perturbed by 2. Additionally, Hoescht 33258 has been reported
to bind to the TAR hairpin, though it binds to the major groove/upper bulge region primarily at and
around bases G36-U40.39
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The concentration-dependent chemical shift perturbations of TAR residues induced by 2
suggest that complex formation occurs in the fast exchange regime of the NMR time scale. However,
residues U40 and C41 also experience broadening in addition to chemical shift changes specific to
these residues. These results are entirely consistent with binding in the mid micromolar range, in
agreement with binding affinity determined by biochemical assays. The perturbed residues are
concentrated in the bulge and lower bulge region of the TAR hairpin, suggesting that 2 may be a lower
bulge binder.
In sum, the NMR studies indicate that 2 binds to TAR and is likely a lower bulge/minor groove
binder. This binding mode does not perturb many residues in the Tat binding site itself, which provides
insight into the varying ability of compounds in this series to displace the Tat peptide. Attempts to derive
a full structure of TAR bound to 2 by intermolecular NOESY experiments were hampered by the limited
solubility of the ligand under the required NMR conditions.
SHAPE
Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) can be used to establish
RNA secondary structure and identify sites of interaction of RNA with other molecules by examining
backbone flexibility.40 Electrophilic reagents, e.g. 1M7 (1-methyl-7-nitroisatoic anhydride) modify the 2’-
OH of less-structured RNA nucleotides, which causes reverse transcription (RT) to halt one nucleotide
before the modification. Modification frequencies are then used to estimate a ‘reactivity profile’ that
quantifies the propensity of each nucleotide to be modified by the chemical probe. High reactivity
reflects nucleotides that are flexible and unstructured, while low reactivity provides structural
constraining, such as formation of base pairing, stacking or RNA–ligand interactions.
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We applied SHAPE to probe RNA conformational changes that would be diagnostic of
compound binding in the context of an in vitro-synthesized 365-nt HIV-1 5’ UTR RNA. Changes in 1M7
reactivity at each nucleotide of the HIV-1 5’ UTR RNA in the presence of 2 and 15 are presented in
Figure 4 (relative to the analogous experiment performed in its absence). The decrease in nucleotide
reactivity upon analog addition is indicated in gradients of blue, while increase in reactivity is
represented in gradients of red; unchanged nucleotide reactivity is in gray. While the global topology of
the 5’ UTR was unaffected upon incubation with 2 and 15, substantial changes in chemical reactivity
were observed mostly within TAR (Figure 4A-B). In particular, addition of 2 caused a decrease in 1M7
reactivity for residue U25 within the TAR bulge and U31, G33 and A35 within the apical loop (Figure
4A). Reactivity changes observed in the apical loop residues for 2 could reflect ligand binding to the
lower bulge region (as discussed above) causing disruption of a direct interaction between the apical
loop and the bulge as recently demonstrated by Krawczyk et al.41 Addition of 15 to RNA showed a
similar 1M7 reactivity reduction throughout TAR compared to 2, and in particular with residues U13-14,
C19 and G32 (Figure 4B). Taken together, reduced 1M7 reactivity suggests that analog binding causes
structural perturbations to TAR due to a direct or, for loop residues, indirect interaction with 2 and 15.
Furthermore, these results are consistent with our observations for 1, which we showed also bound
selectively to the TAR hairpin.19 Although the global topology of the 5′ UTR was unchanged upon
compound binding, subtle changes in chemical reactivity were observed downstream of the TAR
hairpin. These “off target” effects may reflect the influence of compound binding on long-range tertiary
interactions42, 43 and/or indirect structural rearrangements in the 5’ UTR.44 In conclusion, the SHAPE
data suggest specific binding of both 2 and 15 to the TAR region of HIV-1 UTR in the presence of other
structured motifs.
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Conclusions
We have characterized the interaction of a series of thienopyridine compounds to the HIV-1
TAR RNA hairpin. The initial structure, 1, was discovered in a screen of immobilized small molecules,
which identified several structures binding to TAR specifically over other RNA stem-loops. Compound 1
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and a focused library of 25 analogs were synthesized using a concise and modular synthetic route.
These compounds were then evaluated in several biochemical assays for binding to the TAR hairpin as
well as to disrupt the Tat-TAR interaction. A clear structure-activity relationship was observed in the
binding assays. A number of analogs, but not all those that bound to TAR, were able to displace a Tat-
derived peptide from the RNA. Replacing the amide linker with either of two oxadiazolyl groups yielded
analogs with similar affinity to the original hit that were also able to disrupt the Tat/TAR interaction. The
most potent analog, 15, showed a similar binding affinity to 1 and exhibited an IC50 of 40 µM for
displacement of Tat-derived peptide. SHAPE studies showed that both 2 and 15 retained global
specificity for binding to the TAR motif within the 365-nt HIV-1 5’ UTR RNA. NMR studies further
confirmed that 2 binds to TAR and causes chemical shift perturbations exclusively within and below the
bulge region. These results suggest that 2 binds to the lower bulge region, possibly within the minor
groove. The perturbed residues differ from those interacting with a Tat-derived peptide, neomycin,
Hoechst 33258 or argininamide, which could provide a basis for the varying ability of compounds in this
class to displace the peptide. Future efforts will focus on improving solubility of analogs for cell-based
studies and further development of a bifunctional molecule to tether our lower bulge binder with an
apical loop binder such as 5-(N,N)-dimethyl amiloride31 to achieve both increased specificity and
enhanced affinity through synergistic binding.
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Experimental Section
Biochemical assays
Nucleic acids were purchased from Dharmacon with the following sequences:
TAR
RNA:
5’-GGCAGAUCUGAGCCUGGGAGCUCUCUGCC-3’
and
and
25-2AP
TAR:
5’-
5’-
GGCAGAUC(2AP)GAGCCUGGGAGCUCUCUGCC-3
23-2AP
TAR:
GGCAGA(2AP)CUGAGCCUGGGAGCUCUCUGCC-3. TAR and 2AP TAR constructs were
o
reconstituted in 1x PBS pH 7.4 to a concentration of ~100 µM and annealed by heating to 95 C for 3
min. The constructs were allowed to cool to room temperature for at least 30 min. Tat FRET peptide, a
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16-mer peptide labeled at its N-terminus by FAM and C-terminus by TAMRA, was purchased from
AnaSpec (catalog #: AS-64346-05). A fluorescence-based assay employing TAR constructs labeled
with 2-AP either at bulge residues U23 or U25 was used. 2-AP TAR constructs were used at 1 µM. 5
µL of the tested compound in DMSO and 95 µL of 2-AP TAR in the assay buffer (1x PBS pH 7.4) were
added to assay plate (Nunc black plates) and incubated at room temperature with rocking for 30 min.
Fluorescence values were measured at an excitation wavelength of 310 ± 9 nm and an emission
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wavelength of 365 ± 9 nm using a microplate reader and values were normalized to free 2-AP TAR.
Most analogs were not fluorescent at this wavelength, and in the case of fluorescent compounds, the
inherent fluorescence was subtracted from that of the complex (TAR and compound) and then
normalized to free 2-AP TAR. Apparent Kd values were determined by non-linear regression by fitting
the measured fluorescence (GraphPad Prism 6.0 software). FRET studies were performed as
previously reported32 with modification. Unlabeled TAR was used at 100 nM and FAM-Tat-TAMRA at
12 nM. 5 ꢀL of the tested compound (in DMSO) and 95 ꢀL of unlabeled RNA/peptide complex in assay
buffer (50 mM Tris pH 7.4; 20 mM KCl; 0.01% Triton X-100) were added to Nunc black assay plates
and incubated at room temperature for 30 min with rocking. It should be noted that each plate
contained four wells for negative control (equivalent to 0% inhibition and highest fluorescent/FRET)
which included 100 nM unlabeled TAR and 12 nM FAM-Tat-TAMRA in presence of 5% DMSO.
Additionally, three wells were assigned for the positive control (equivalent to 100% inhibition and
lowest fluorescence/FRET) which included 12 nM of FAM-Tat-TAMRA in presence of 5% DMSO.
Fluorescence values were measured at an excitation wavelength of 485 ± 20 nm and an emission
wavelength at 590 ± 35 nm using a microplate reader. Most analogs were not fluorescent at this
wavelength; however for analogs 2, 3, 4, 16, 23 and 25, the fluorescence of complex (unlabeled
TAR/labeled Tat) in presence of compound exceeded the maximum fluorescence as indicated by the
negative control wells. Therefore, the inherent fluorescence of compound was subtracted from that of
the complex plus compounds. IC50 values were determined by non-linear regression fitting of the
competition curves.
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NMR
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HIV-1 TAR RNA hairpin was transcribed and purified as described previously.45-47 Ligand detected
NMR experiments were collected on a Bruker DRX 500MHz equipped with TCI cryoprobe or Bruker
Avance III 600 MHz spectrometer equipped with TCI cryoprobe. NMR screening buffer consisted of 20
mM d19-Bis-Tris at pH6.5, 10 mM sodium chloride, and 11.1 µM DSA as chemical shift reference
(integrates to 100 µM)48, prepared in 99.99% D2O or 95%H2O/5%D2O. Each ligand was first dissolved
in DMSO to a 10 mM stock concentration in d6-DMSO. Free ligand screening samples were prepared
at 150 µM in NMR screening buffer with final DMSO concentration of 15%. We used this concentration
as the safe upper limit beyond which the RNA structure might be affected. For RNA detected
experiments, the lyophilized RNA pellets were dissolved in DMSO-free NMR screening buffer and
heated for 4 min at 90oC, then snapped cooled for 5 min at -20 oC. 1D-1H experiments for both ligand
and RNA were collected using the Bruker “zgesgp” excitation sculpting pulse sequence to suppress
background water signal. Data was collected with 64 scans and 16K data points. Data was processed
using 16K zero points, Fourier transformed followed by manual phase and baseline correction. 2D-1H
TOCSY experiments were collected using the Bruker “mlevesgpph” pulse sequence with excitation
sculpting water suppression, data collected with 8 scans, 2K x 512 data points, a recycle delay of 1.2 s
and spin-lock mixing time of 80ms. Data was processed using NMRPipe and visualized using SPARKY.
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SHAPE
RNA preparation for SHAPE analysis - DNA template for in vitro transcription were generated by
PCR amplification of the HIV-1 molecular clone pNL4-3 using the following primers:
T7L 5’- TAATACGACTCACTATAGGTCTCTCTG - 3’ (containing T7 promoter, underlined)
369R 5’- GCTTAATACCGACGCTCTCGC - 3’
All PCR experiments were performed using Invitrogen Platinum® Taq DNA polymerase High Fidelity.
Transcripts were synthesized with the MEGAscript T7 transcription kit (Thermo Fisher Scientific)
following the manufacturer’s protocol, and RNAs were purified from denaturing 8 M urea/6%
polyacrylamide gels, followed by electro-elution and ethanol precipitation. Purified RNAs were dissolved
in sterile water and stored at −20 ºC.
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Renaturation and 1M7 treatment of RNA - 5 pmoles of RNA were heated to 90 ºC for 3 min, and
gradually cooled to 4 ºC (0.1 ºC/sec). The volume was adjusted to 10 µL in a final buffer of 50 mM Tris-
HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2. Samples were then divided into 5 µl experimental (+) and
control (-) aliquots (2.5 pmoles each) and incubated at 37 ºC for 15 min. Analog 2 and 15 were added
to both (+) and (-) RNA solutions to a final concentration of 5 µM and 0.5 µM respectively, and
incubated for 10 min at room temperature. Chemical modification was initiated by addition of 1 µL of
1M7 (in anhydrous DMSO) to the (+) RNA solution to a final concentration of 3.5 mM, or DMSO alone
to the (-) RNA reaction, followed by incubation at 37 ºC for 5 min. Completed reactions were
precipitated at −20 ºC with 10 ng/µL glycogen, 0.3M sodium acetate pH 5.2 and 2.5 volumes of cold
ethanol. RNA was collected by centrifugation, and resuspended in 10 µL of water. A parallel experiment
lacking analogs was performed as a control.
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Primer extension - For detection of 2′-O-adducts, 2.5 pmoles of modified and unmodified RNAs were
mixed, respectively, with 5 pmoles of Cy5-labelled (for 1M7-modified samples) or 6 pmoles of Cy5.5-
labelled (for unmodified samples) DNA primer complementary to their 3’ end. Samples were incubated
at 85 ºC for 1 min, 60 ºC for 5 min, 35 ºC for 5 min and 50 ºC for 2 min to anneal primers to initiate
reverse transcription. RNA was reverse transcribed at 50 ºC for 50 min with 100U RT (Invitrogen
Superscript III), 1x RT buffer (Invitrogen), 5 mM DTT and 500 mM dNTPs (Promega). RNA was
hydrolyzed with 200 mM NaOH for 5 min at 95 ºC and samples were neutralized with an equivalent
volume of HCl. Sequencing ladders were prepared using the Epicentre cycle sequencing kit according
to the manufacturer’s instructions and primers labeled with WELLRed D2 and LICOR IR-800 dyes.
Modified and control samples were mixed with the sequencing ladders, precipitated as above, dried
and resuspended in 30 µL deionized formamide. Primer extension products were analyzed on a
Beckman CEQ8000 Genetic Analysis System programmed with the separation method described
previously.49
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SHAPE data analysis - Electropherograms were processed using the program SHAPEfinder program,
following the software developer’s protocol and included the required pre-calibration for matrixing and
mobility shift for each set of primers as described.40, 50 The area under each negative peak was
subtracted from that of the corresponding positive peak. The resulting peak area difference at each
nucleotide position was then divided by the average of the highest 8% of peak area differences,
calculated after discounting any results greater than the 3rd quartile plus 1.5x the interquartile range.
RNAstructure software version 5.7 was used to predict RNA secondary structure(s) on the basis of
pseudo-free energy constraints derived from SHAPE reactivity values. All reactivity data used in 2D
structure analysis was averaged from three independent experiments. Varna (ver. 3-9), the
visualization applet for RNA secondary structure, was used to produce high-quality images.51
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Chemistry
Chemicals were purchased from commercial sources and used without further purification unless
otherwise noted. Anhydrous solvents were prepared by passage over activated alumina. Thin-layer
chromatography was conducted with E. Merck silica gel 60 F254 precoated plates (0.25 mm) and
visualized by exposure to UV light (254 nm). Flash column chromatography was performed using
1
normal phase or reverse phase on a CombiFlash® Rf 200i (Teledyne Isco Inc). H NMR spec
tra
were recorded at 400 or 500 MHz, and are reported relative to deu
1H NMR spectra are reported as follows: chemical shift (δ ppm)
and integration. 1 3 C NMR spectra were recorded
t
erated solvent signals. Data for
,
multiplicity, coupling constant (Hz),
100 or 125 MHz. Data for 13C NMR spectra are
t
a
reported in terms of chemical shift. All NMR spectra were standardized to the NMR solvent signal
Analytical LC/MS was performed using a Shimadzu LCMS-2020 Single
as reported previously 52
.
Quadrupole utilizing a Kinetex 2.6 ꢀm C18 100 Å (2.1 x 50 mm) column obtained from
Phenomenex Inc. Runs employed a gradient of 0→90% ACN/0.1% aqueous formic acid over
4
minutes at a flow rate of 0.2 mL/min. Purities of final compounds were assessed by analytical reverse-
phase HPLC using Agilent Proshell 120 EC-C18 (4.6 mm x 50 mm; particle size 2.7 µm). Runs
employed a gradient of 10% ACN/water (1 min), 10-90% ACN/water (6 min), and 90% ACN/water (2
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min) flow = 1 mL/min. High-resolution LC/MS analyses acquired on an Agilent 6520 Accurate-Mass
Q-TOF LC/MS System, (Agilent Technologies, Inc., Santa Clara, CA) equipped with a dual electro-
spray source, operated in the positive-ion mode. Separation was performed on Zorbax 300SB-C18
Poroshell column (2.1 mm x 150 mm; particle size 5 µm). All final compounds were purified to >95%
purity unless otherwise stated.
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Representative procedure for thienopyridine carboxamide preparation
3-Amino-6-methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide (1).
Synthesized using reported procedure with modification.22 To a suspension of 25 (51 mg, 0.34 mmol)
in anhydrous EtOH (2 mL) was added 27 (92 mg, 0.32 mmol) and sodium ethoxide (54 mg, 0.75 mmol).
The mixture was heated to reflux for 1 h 15 min. The mixture was diluted with EtOAc (10 mL) and
washed with H2O (10 mL x 2) and brine (10 mL). The organic layer was dried (Na2SO4), filtered, and
concentrated in vacuo. Crude was purified by flash column chromatography (hexanes/EtOAc) on silica
gel to give 1 (65 mg, 58%) as a light yellow solid. Purity 100%.1H NMR (500 MHz, DMSO-d6) δ 9.70 (s,
1H), 8.41 (d, J = 8.3 Hz, 1H), 8.23 (s, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.47 (s,
2H), 7.41 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 2.61 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ
164.23, 159.90, 158.54, 147.83, 140.00, 131.15, 129.58, 129.14 (q, J = 31.5 Hz), 125.31 (q, J = 272.4
Hz), 124.20, 123.60, 119.61, 119.41 (q, J = 3.8 Hz), 116.81 (q, J = 4.0 Hz), 94.75, 24.29. ESI HRMS:
m/z 352.07332 (M+H)+.
Representative procedure for pyridinylthienopyridine carboxamide preparation
3-Amino-6-methyl-N-(pyridin-4-yl)thieno[2,3-b]pyridine-2-carboxamide (2). Synthesized using a
similar procedure used to prepare 1 except using 29g. Mixture was heated to reflux overnight. Upon
cooling to room temperature and addition of H2O, yellow solid precipitated. Solid was filtered and
washed with cold EtOH. Crude was purified by flash column chromatography (hexanes/EtOAc) on silica
gel to give 2 (18 mg, 22% over two steps) as a yellow solid. Purity 100%.1H NMR (400 MHz, DMSO-d6)
δ 9.72 (s, 1H), 8.44 – 8.38 (m, 3H), 7.76 (d, J = 6.3 Hz, 2H), 7.53 (s, 2H), 7.35 (d, J = 8.3 Hz, 1H), 2.60
(s, 3H).13C NMR (100 MHz, DMSO-d6) δ 164.49, 160.15, 158.63, 149.99, 148.33, 146.11, 131.24,
123.44, 119.65, 114.19, 94.63, 24.29. ESI HRMS: 285.08048 (M+H)+.
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6-Methyl-2-(pyridin-3-ylcarbamoyl)thieno[2,3-b]pyridin-3-aminium
2,2,2-trifluoroacetate
(3).
3
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7
8
9
Synthesized using a similar procedure used to prepare 1 except using 29h. Mixture was heated to
reflux overnight. Upon cooling to room temperature and addition of H2O, yellow solid precipitated. Solid
was filtered and washed with H2O to give the title compound (26 mg, 46%) as a light yellow solid. Purity
98%.1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 8.88 (d, J = 2.2 Hz, 1H), 8.39 (d, J = 8.3 Hz, 1H),
8.27 (dd, J = 4.7, 1.4 Hz, 1H), 8.11 – 8.06 (m, 1H), 7.43 (s, 2H), 7.38 – 7.32 (m, 2H), 2.60 (s, 3H).13C
NMR (100 MHz, DMSO-d6) δ 164.21, 159.84, 158.52, 147.61, 144.13, 142.59, 135.73, 131.12, 127.95,
123.60, 123.27, 119.59, 94.87, 24.27. ESI HRMS: 285.08043 (M+H)+.
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3-Amino-6-methyl-N-(pyridin-2-yl)thieno[2,3-b]pyridine-2-carboxamide 2,2,2-trifluoroacetate (4).
Synthesized using a similar procedure used to prepare 1 except using 29i. Mixture was heated to reflux
overnight. Upon cooling to room temperature and addition of H2O, yellow solid precipitated. Solid was
filtered and washed with cold EtOH. Crude was purified by flash column chromatography
(hexanes/EtOAc) on silica gel to give 4 (18 mg, 32%) as a yellow solid. Purity 100%.1H NMR (400 MHz,
DMSO-d6) δ 9.54 (s, 1H), 8.39 (d, J = 8.3 Hz, 1H), 8.35 (d, J = 4.4 Hz, 1H), 8.05 (d, J = 8.3 Hz, 1H),
7.83 – 7.77 (m, 1H), 7.42 (s, 2H), 7.34 (d, J = 8.3 Hz, 1H), 7.12 (dd, J = 6.8, 5.2 Hz, 1H), 2.60 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 163.99, 159.86, 158.62, 151.96, 147.88, 147.62, 137.86, 131.15,
123.66, 119.51, 119.35, 114.70, 95.67, 24.28. ESI HRMS: 285.08095 (M+H)+.
3-Amino-4-methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(5).
Synthesized using a similar procedure used to prepare 1 except using 31a. Crude was purified by flash
column chromatography (hexanes/EtOAc) on silica gel to give 5 (44 mg, 37%) as a light yellow solid.
Purity 96%. 1H NMR (500 MHz, DMSO-d6) δ 9.78 (s, 1H), 8.49 (d, J = 4.7 Hz, 1H), 8.17 (s, 1H), 8.00 (d,
J = 8.3 Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.21 (d, J = 5.4 Hz, 1H), 7.08 (s, 2H),
2.81 (s, 3H).13C NMR (125 MHz, DMSO-d6) δ 164.40, 159.28, 150.00, 149.70, 145.16, 139.76, 129.64,
129.19 (q, J = 31.0 Hz), 125.28, 124.93, 124.49, 123.12, 122.01, 119.69, 117.08, 96.93, 20.00. ESI
HRMS 352.07318 (M+H)+.
3-Amino-5-methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(6).
Synthesized using a similar procedure used to prepare 1 except using 31b. Crude was purified by silica
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gel chromatography (hexanes/EtOAc) to give 6 (36 mg, 30%) as a light yellow solid. Purity 99%. H
NMR (500 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.55 (s, 1H), 8.36 (s, 1H), 8.21 (s, 1H), 7.99 (d, J = 8.1 Hz,
1H), 7.56 (t, J = 7.9 Hz, 1H), 7.46 – 7.39 (m, 3H), 2.43 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ
164.17, 156.13, 151.54, 147.34, 139.93, 130.86, 129.60, 129.16 (q, J = 31.4 Hz), 128.89, 125.80,
124.24, 124.22 (q, J = 272.2 Hz), 119.49 (d, J = 3.5 Hz), 116.85 (q, J = 3.8 Hz), 96.12, 17.96. ESI
HRMS: 352.07272 (M+H)+.
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3-amino-6-methyl-N-(4-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(7).
Synthesized using a similar procedure used to prepare 1 except using 33d. Crude was purified by silica
1
gel chromatography (hexanes/EtOAc) to give 7 (28 mg, 23%) as a light yellow solid. Purity 97%. H
NMR (400 MHz, DMSO-d6) δ 9.72 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 7.96 (d, J = 8.5 Hz, 2H), 7.67 (d, J =
8.6 Hz, 2H), 7.47 (s, 2H), 7.35 (d, J = 8.3 Hz, 1H), 2.60 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ
164.23, 159.94, 158.57, 147.91, 142.90, 131.14, 125.62 (q, J = 3.5 Hz), 123.55, 123.21, 120.48,
119.61, 94.91, 24.27. ESI HRMS: 352.07336 (M+H)+.
3-Amino-6-methyl-N-(2-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(8).
Synthesized using a similar procedure used to prepare 1 except using 33e. Crude was purified by silica
1
gel chromatography (hexanes/EtOAc) to give 8 (32 mg, 38%) as a light yellow solid. Purity 96%. H
NMR (500 MHz, DMSO-d6) δ 9.20 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.71 (t, J =
8.0 Hz, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 8.3 Hz, 1H), 7.26 (s, 2H),
2.60 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 164.60, 159.64, 158.42, 146.95, 135.82, 132.89, 131.09,
130.77, 126.92, 126.40 (q, J = 4.9 Hz), 125.90 (q, J = 29.2 Hz), 123.74, 123.71 (q, J = 273.3 Hz),
119.56, 95.28, 24.29. ESI HRMS: 352.07291 (M+H)+.
3-Amino-6-methyl-N-(3-(trifluoromethyl)benzyl)thieno[2,3-b]pyridine-2-carboxamide (9).
Synthesized using a similar procedure used to prepare 1 except using 33f. Crude was purified by silica
1
gel chromatography (hexanes/EtOAc) to give 9 (106 mg, 60%) as a light yellow solid. Purity 100%. H
NMR (500 MHz, DMSO-d6) δ 8.35 – 8.30 (m, 2H), 7.66 (s, 1H), 7.64 – 7.62 (m, 1H), 7.61 – 7.55 (m,
2H), 7.31 (d, J = 8.3 Hz, 1H), 7.18 (s, 2H), 4.48 (d, J = 5.9 Hz, 2H), 2.58 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ 165.27, 159.50, 158.22, 145.89, 141.75, 131.65, 131.07, 129.53, 129.11 (q, J = 31.5 Hz),
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124.47 (q, J = 272.2 Hz), 124.06, 123.90 (q, J = 3.7 Hz), 123.63 (q, J = 3.8 Hz), 119.69, 95.79, 42.03,
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24.35. ESI HRMS: 366.08889 (M+H)+.
6
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3-Amino-6-isopropyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(10).
8
9
Synthesized using a similar procedure used to prepare 1 except using 31c. Crude was purified by flash
column chromatography (hexanes/EtOAc) on silica gel to give 10 (44 mg, 78%) as a light yellow oil
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which solidified upon standing. Purity 93%. H NMR (500 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.44 (d, J =
8.4 Hz, 1H), 8.22 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.9 Hz, 1H), 7.47 (s, 2H), 7.43 – 7.38 (m,
2H), 3.15 (p, J = 6.9 Hz, 1H), 1.29 (d, J = 6.9 Hz, 6H). 13C NMR (125 MHz, DMSO-d6) δ 168.50, 164.23,
158.51, 147.78, 139.97, 131.47, 129.58, 129.15 (q, J = 31.7 Hz), 124.27, 124.23 (q, J = 272.8 Hz),
124.03, 119.43 (q, J = 3.8 Hz), 117.37, 116.86 (q, J = 4.0 Hz), 94.92, 39.85, 35.91, 22.41. ESI HRMS:
380.10396 (M+H)+.
6-Methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-carboxamide
(11).
Synthesized
using reported procedure with modification.25
was added a solution of 50% H PO (259 µL
To a solution of 1 (56 mg, 0.16 mmol) in MeOH (6 mL)
, 2.4 mmol) at 0 °C. To the reaction mixture was added
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conc. H2SO4 (655 µL), Cu2O (14 mg, 0.10 mmol),1 M NaNO2 aqueous solution (239 µL) and the
reaction mixture was stirred for 30 min at 0 °C. After stirring for 2 h and 30 min at room temperature,
the reaction mixture was diluted with H2O (30 mL) and the aqueous layer was extracted with EtOAc (20
mL x 2). The combined organic layer was washed with brine, dried over Na SO , filtered and
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concentrated in vacuo. Crude was purified by flash column chromatography (hexanes/EtOAc) on silica
gel to give 11 (12 mg, 22%) as a white solid. Purity 98%.1H NMR (500 MHz, DMSO-d6) δ 10.83 (s, 1H),
8.36 – 8.32 (m, 2H), 8.21 (s, 1H), 8.05 (d, J = 8.2 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 7.8 Hz,
1H), 7.41 (d, J = 8.2 Hz, 1H), 2.63 (s, 3H).13C NMR (125 MHz, DMSO-d6) δ 161.21, 160.54, 158.36,
139.31, 137.39, 133.71, 130.21, 130.09, 129.46 (q, J = 31.7 Hz), 124.53, 124.09 (q, J = 272.3 Hz),
123.78, 120.96, 120.34 (q, J = 3.7 Hz), 116.35 (q, J = 4.0 Hz), 24.29. ESI HRMS: 337.06096 (M+H)+.
7-Methyl-3-(3-(trifluoromethyl)phenyl)pyrido[3',2':4,5]thieno[3,2-d]pyrimidine-2,4(1H,3H)-dione
(12). Synthesized using reported procedure with modification.26 Triphosgene (13 mg, 0.043 mmol) was
added to a stirred solution of 1 (30 mg, 0.084 mmol) in THF (2.5 mL) at room temperature. The
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resulting mixture was heated under reflux for 1 h. The mixture was then cooled to room temperature
and diluted with THF (5 mL). The solid was collected by filtration and rinsed with cold THF to give a
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white solid (28 mg, 90%). Purity 98%. H NMR (500 MHz, DMSO-d6) δ 12.81 (s, 1H), 8.65 (d, J = 8.4
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Hz, 1H), 7.87 (s, 1H), 7.83 (d, J = 7.4 Hz, 1H), 7.78 – 7.72 (m, 2H), 7.55 (d, J = 8.4 Hz, 1H), 2.67 (s,
3H). 13C NMR (125 MHz, DMSO-d6) δ 161.09, 160.73, 159.08, 151.33, 138.79, 136.39, 133.62, 131.96,
130.16, 129.73 (q, J = 32.1 Hz), 126.28 (q, J = 3.9 Hz), 125.24 (q, J = 3.7 Hz), 124.98, 122.82, 121.07,
120.37, 107.68, 24.46.. ESI HRMS: 378.05243 (M+H)+.
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Representative procedure for thienopyridine sulfonamide preparation
3-Amino-6-methyl-N-(3-(trifluoromethyl)phenyl)thieno[2,3-b]pyridine-2-sulfonamide
(13).
Synthesized using reported procedure with modification.27 To a solution of 25 (32 mg, 0.21 mmol) in
anhydrous DMF (1.5 mL) was added 35 (63 mg, 0.22 mmol) and sodium ethoxide (33 mg, 0.46 mmol).
The mixture was heated to 130 oC for 1 h. The mixture was diluted with H2O (10 mL) and extracted with
EtOAc (10 mL x 2) and finally washed with brine (10 mL). The organic layer was dried (Na2SO4),
filtered, and concentrated in vacuo. Crude was purified by silica gel chromatography (hexanes/EtOAc)
to give 13 (27 mg, 33%) as a light yellow solid. Purity 96%.1H NMR (400 MHz, DMSO-d6) δ 10.75 (s,
1H), 8.35 (d, J = 8.3 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.45 – 7.38 (m, 2H), 7.37 – 7.30 (m, 2H), 6.83 (s,
2H), 2.55 (s, 3H).13C NMR (126 MHz, DMSO) δ 160.74, 158.96, 144.69, 139.28, 132.25, 130.93,
130.25 (q, J = 31.5 Hz), 125.34, 123.57, 123.16, 120.51, 115.43, 97.64, 24.68. ESI HRMS: 388.04044
(M+H)+.
3-Amino-N-(3-fluorophenyl)-6-methylthieno[2,3-b]pyridine-2-sulfonamide (14). Synthesized using
a similar procedure used to prepare 13 except using 36. Crude was purified by silica gel
chromatography (hexanes/EtOAc) to give 14 (96 mg, 72%) as an oil which was solidified to a beige
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solid upon standing. Purity 100%. H NMR (400 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.33 (d, J = 8.3 Hz,
1H), 7.33 (d, J = 8.4 Hz, 1H), 7.26 (td, J = 8.4, 6.9 Hz, 1H), 6.97 – 6.88 (m, 2H), 6.85 – 6.80 (m, 1H),
6.80 – 6.76 (m, 2H), 2.54 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 162.21 (d, J = 242.8 Hz), 160.13,
158.48, 143.89, 139.73 (d, J = 10.7 Hz), 131.72, 130.82 (d, J = 9.5 Hz), 123.13, 119.98, 114.88 (d, J =
2.7 Hz), 110.08 (d, J = 21.0 Hz), 105.70 (d, J = 25.4 Hz), 97.72, 24.17. ESI HRMS: 338.04340 (M+H)+.
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Representative procedure for oxadiazolyl thienopyridine preparation
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6-Methyl-2-(5-(3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)thieno[2,3-b]pyridin-3-amine (15).
To a stirring solution of 25 (42 mg, 0.28 mmol) in anhydrous DMF (2 mL) was added commercially
available 5-(chloromethyl)-3-[3-(trifluoromethyl)phenyl]-1,3,4-oxadiazole, 37I, (66 mg, 0.24 mmol) and
sodium ethoxide (50 mg, 0.63 mmol). The mixture was heated to 130 oC for 1 h. The mixture was then
cooled to room temperature and solid precipitated at the bottom of the flask. The solid was collected by
filtration and rinsed with cold MeOH and dried in vacuo to give a yellow solid (28 mg, 31% yield). Purity
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96%. H NMR (500 MHz, DMSO-d6) δ 8.50 (d, J = 8.3 Hz, 1H), 8.40 (d, J = 7.9 Hz, 1H), 8.35 (s, 1H),
8.01 (d, J = 7.9 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.26 (s, 2H), 2.61 (s, 3H). 13C
NMR (125 MHz, DMSO-d6) δ 162.36, 160.47, 159.33, 159.28, 143.73, 131.25, 130.80, 130.50, 130.18,
129.92, 128.06 (q, J = 3.3 Hz), 124.80, 124.45, 123.38, 122.77 (q, J = 3.7 Hz), 122.63, 119.88, 85.96,
24.21. ESI HRMS: 377.06832 (M+H)+.
6-Methyl-2-(3-(3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-5-yl)thieno[2,3-b]pyridin-3-amine (16).
Synthesized using a similar procedure used to prepare 15 except using commercially available 5-
(chloromethyl)-3-[3-(trifluoromethyl)phenyl]-1,3,4-oxadiazole, 37m. Precipitated solid was filtered,
rinsed with cold MeOH, and dried in vacuo to give the title compound (31 mg, 67% yield) as a red solid.
Purity 99%. 1H NMR (500 MHz, DMSO-d6) δ 8.55 (d, J = 8.3 Hz, 1H), 8.48 (d, J = 7.7 Hz, 1H), 8.43 (s,
1H), 8.00 (d, J = 7.6 Hz, 1H), 7.84 (t, J = 7.7 Hz, 1H), 7.63 (s, 2H), 7.41 (d, J = 8.3 Hz, 1H), 2.61 (s, 3H).
13C NMR (125 MHz, DMSO-d6) δ 172.46, 166.38, 160.44, 159.78, 146.38, 131.88, 131.35, 130.51,
129.92 (q, J = 32.1 Hz), 128.09 (d, J = 3.3 Hz), 127.46, 123.64 (q, J = 3.7 Hz), 122.93, 122.76 (q, J =
272 Hz), 119.99, 85.79, 24.28.ESI HRMS: 377.06809 (M+H)+.
6-Methyl-2-(5-phenyl-1,3,4-oxadiazol-2-yl)thieno[2,3-b]pyridin-3-amine (17). Synthesized using a
similar procedure used to prepare 15 except using 25 and commercially available 2-(chloromethyl)-5-
phenyl-1,3,4-oxadiazole, 37n. Precipitated solid was filtered, rinsed with cold MeOH, and dried in vacuo
to give the title compound (24 mg, 65% yield) as a yellow solid. Purity 96%. 1H NMR (500 MHz, DMSO-
d6) δ 8.46 (d, J = 8.3 Hz, 1H), 8.11 – 8.08 (m, 2H), 7.65 – 7.61 (m, 3H), 7.40 (d, J = 8.3 Hz, 1H), 7.18 (s,
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2H), 2.61 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 161.93, 161.45, 159.19, 159.17, 143.37, 131.70,
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131.12, 129.41, 126.50, 123.40, 123.28, 119.87, 86.23, 24.21.ESI HRMS: 309.08079 (M+H)+.
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2-(5-(3-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-6-methylthieno[2,3-b]pyridin-3-amine
(18).
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Synthesized using a similar procedure used to prepare 15 except using 25 and commercially available
2-(chloromethyl)-5-(3-chlorophenyl)-1,3,4-oxadiazole, 37o. Precipitated solid was filtered, rinsed with
cold MeOH, and dried in vacuo to give the title compound (26 mg, 42% yield) as a yellow solid. Purity
96%. 1H NMR (500 MHz, DMSO-d6) δ 8.46 (d, J = 8.3 Hz, 1H), 8.11 (t, J = 1.7 Hz, 1H), 8.06 (dt, J = 7.6,
1.2 Hz, 1H), 7.70 (ddd, J = 8.1, 2.0, 1.1 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.20
(s, 2H), 2.61 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 162.22, 160.43, 159.29, 159.27, 143.58, 134.03,
131.44, 131.41, 131.16, 125.93, 125.25, 125.20, 123.38, 119.87, 86.08, 24.21. ESI HRMS: 343.04216
(M+H)+.
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2-(5-(3-(Trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-yl)thieno[2,3-b]pyridin-3-amine
(19).
Synthesized using a similar procedure used to prepare 15 except using commercially available 2-
Mercaptopyridine-3-carbonitrile, 38, and 37m. Precipitated solid was filtered, rinsed with cold MeOH,
and dried in vacuo to give the title compound (55 mg, 76% yield) as a yellow solid. Purity 99%. 1H NMR
(500 MHz, DMSO-d6) δ 8.69 (dd, J = 4.6, 1.6 Hz, 1H), 8.61 (dd, J = 8.2, 1.6 Hz, 1H), 8.41 (d, J = 7.9 Hz,
1H), 8.36 (s, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 7.54 (dd, J = 8.1, 4.6 Hz, 1H), 7.29
(s, 2H). 13C NMR (125 MHz, DMSO-d6) δ 162.22, 160.66, 159.56, 149.93, 143.49, 131.17, 130.80,
130.55, 129.94 (q, J = 32 Hz), 128.14 (q, J = 3.3 Hz), 125.69, 124.79, 124.41, 122.84 (q, J = 3.9 Hz),
122.62, 119.86, 87.02. ESI HRMS: 363.05232 (M+H)+.
Representative procedure for 2-mercaptopyridine preparation
2-Mercapto-6-methylnicotinonitrile (25). Synthesized using reported procedure with modification.20 A
mixture of 2-chloro-6-methylnicotinonitrile, 24, (222 mg, 1.44 mmol) and thiourea (348 mg, 4.57 mmol)
was heated at reflux (118 °C) in n-butanol for 3 h. A yellow suspension was obtained. After cooling to
room temperature, the yellow solid was precipitated. The solids were collected by filtration, rinsed with
cold n-butanol, and dried in vacuo to give the title compound (217 mg, quantitative) as a light yellow
solid. 1H NMR (500 MHz, DMSO-d6) δ 14.07 (s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 6.73 (d, J = 7.7 Hz, 1H),
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2.39 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 177.70, 155.00, 145.03, 117.20, 113.40, 112.75, 19.14.
ESI MS: m/z 149.1 (M-H)-.
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Representative procedure for acetamide preparation
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2-Bromo-N-(3-(trifluoromethyl)phenyl)acetamide (27). Synthesized using reported procedure with
modification.21 A stirred solution of 3-(trifluoromethyl)aniline, 26, (170 µL, 1.36 mmol) and 4-
(dimethylamino)pyridine (70 mg, 42 mol%) in CH2Cl2 (8 mL) was treated dropwise with bromoacetyl
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bromide (140 µL, 1.61 mmol) at 0 C. The reaction mixture was allowed to warm up to room
temperature with good stirring overnight. The mixture was diluted with H2O (30 mL) and extracted with
EtOAc (20 mL x 2). The organic layer was dried (MgSO4), filtered, and concentrated in vacuo. Crude
was purified by flash column chromatography (hexanes/EtOAc) on silica gel to give 27 (339 mg, 88%)
as a light brown oil which solidified upon standing. 1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 7.83 (s,
1H), 7.75 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 7.9 Hz, 1H), 7.45 – 7.41 (m, 1H), 4.05 (s, 2H).13C NMR (125
MHz, CDCl3) δ 163.70, 137.55, 131.74 (q, J = 32.7 Hz), 129.87, 123.83 (q, J = 272.4 Hz), 123.18,
121.95 (q, J = 3.8 Hz), 116.88 (q, J = 3.9 Hz), 29.40. ESI MS: m/z 281.55, 283.50 (M+H)+.
Representative procedure for pyridinium acetamido bromide preparation
4-(2-Bromoacetamido)pyridin-1-ium bromide (29g). Synthesized using reported procedure with
modification.23 To a stirred solution of 4-aminopyridine, 28g, (201 mg, 2.14 mmol) in dry CH2Cl2 (5 mL)
o
at 0 C was added bromoacetyl bromide (195 µL, 2.24 mmol) dropwise. As soon as bromoacetyl
bromide was added, the reaction mixture turned cloudy and a white precipitate formed. The mixture
was allowed to warm to room temperature with good stirring overnight. Mixture was filtered and the
solid was washed with cold CH2Cl2 to give 29g as a white solid which was used in the next step without
further purification. ESI MS: m/z 214.45, 216.45 (M+H)+.
3-(2-Bromoacetamido)pyridin-1-ium bromide (29h). Synthesized using a similar procedure used to
prepare 29g using 3-aminopyridine, 28h. The mixture was allowed to warm to room temperature with
good stirring over 3 h. Mixture was filtered and the solid was washed with cold CH2Cl2 to give the title
compound (609 mg, 95%) as a beige solid which was used in the next step without further purification.
1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 9.20 (d, J = 2.3 Hz, 1H), 8.65 (d, J = 5.4 Hz, 1H), 8.50
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(d, J = 8.6 Hz, 1H), 7.99 (dd, J = 8.5, 5.5 Hz, 1H), 4.19 (s, 2H).13C NMR (100 MHz, DMSO-d6) δ 166.22,
137.81, 137.58, 134.35, 132.52, 127.48, 29.53. ESI MS: m/z 214.45, 216.40 (M+H)+.
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2-Bromo-N-(pyridin-2-yl)acetamide hydrobromide (29i). Synthesized using a similar procedure used
to prepare 29g except using 2-aminopyridine, 28i. The mixture was allowed to warm to room
temperature with good stirring over 3 h. Mixture was filtered and the solid was washed with cold CH2Cl2
to give the title compound (342 mg, 55%) as a light purple solid which was used in the next step without
further purification. 1H NMR (500 MHz, CDCl3) δ 8.81 (s, 1H), 8.32 (d, J = 3.7 Hz, 1H), 8.17 (s, 1H),
7.74 (t, J = 7.6 Hz, 1H), 7.13 – 7.06 (m, 1H), 4.01 (s, 2H).13C NMR (125 MHz, CDCl3) δ 164.23, 150.72,
148.15, 138.66, 120.72, 114.02, 29.20. ESI MS: m/z 214.45, 216.45 (M+H)+.
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2-Mercapto-4-methylnicotinonitrile (31a). Synthesized using a similar procedure used to prepare 25
except using 2-chloro-4-methylnicotinonitrile, 30a. Precipitated solid was filtered, rinsed with cold n-
butanol, and dried in vacuo to give the title compound (237 mg, quantitative) as a light yellow solid. 1H
NMR (400 MHz, DMSO-d6) δ 7.81 (d, J = 6.4 Hz, 1H), 6.82 (d, J = 6.4 Hz, 1H), 2.40 (s, 3H).13C NMR
(100 MHz, DMSO-d6) δ 177.05, 157.57, 140.65, 116.31, 115.93, 114.61, 20.99. ESI MS: m/z 149.1 (M-
H)-.
2-Mercapto-5-methylnicotinonitrile (31b). Synthesized using a similar procedure used to prepare 25
except using 2-chloro-5-methylnicotinonitrile, 30b. Precipitated solid was filtered, rinsed with cold n-
butanol, and dried in vacuo to give the title compound (121 mg, quantitative) as a light yellow solid. 1H
NMR (500 MHz, DMSO-d6) δ 14.10 (s, 1H), 8.04 (d, J = 1.9 Hz, 1H), 7.83 (s, 1H), 2.10 (s, 3H).13C NMR
(126 MHz, DMSO) δ 174.77, 147.03, 141.45, 122.24, 116.90, 116.08, 16.06. ESI MS: m/z 149.1 (M-H)-.
6-Isopropyl-2-mercaptonicotinonitrile (31c). Synthesized using reported procedure with
modification.24 A suspension of 2-chloro-6-isopropylnicotinonitrile, 30c, (53 mg, 0.29 mmol) and
Lawesson’s reagent (135 mg, >90% purity, 0.30 mmol) in toluene (2.5 mL) was refluxed at 110 oC for 4
h 30 min. The reaction mixture was cooled down to room temperature and filtered through a Celite plug
to remove any unreacted Lawesson’s reagent. The solvent was evaporated and crude was purified by
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silica gel chromatography (hexanes/EtOAc) to give 31c (22 mg, 42% yield) as a yellow sticky solid. H
NMR (400 MHz, DMSO-d6) δ 14.04 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 6.78 (d, J = 7.8 Hz, 1H), 3.06 (p, J
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= 6.9 Hz, 1H), 1.20 (d, J = 6.9 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 177.78, 163.81, 145.44,
117.06, 113.98, 109.04, 31.42, 21.17. ESI MS: m/z 179.1 (M+H)+, 177.1 (M-H)-.
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2-Bromo-N-(4-(trifluoromethyl)phenyl)acetamide (33d). Synthesized using a similar procedure used
to prepare 27 except using 4-(trifluoromethyl)aniline, 32d. The reaction mixture was allowed to warm up
to room temperature with good stirring over 8 h. The mixture was diluted with H2O (30 mL) and
extracted with EtOAc (20 mL x 2). The organic layer was dried (MgSO4), filtered, and concentrated in
vacuo. Crude (355 mg, 94%), a white solid, was used in the next step without further purification. 1H
NMR (500 MHz, CDCl3) δ 8.24 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.5 Hz, 2H), 4.05 (s,
2H).13C NMR (125 MHz, CDCl3) δ 163.69, 140.05, 127.18 (q, J = 33.0 Hz), 126.56 (q, J = 3.8 Hz),
124.04 (q, J = 271.8 Hz), 119.73, 29.44. ESI MS: m/z 281.50, 283.55 (M+H)+.
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2-Bromo-N-(2-(trifluoromethyl)phenyl)acetamide (33e). Synthesized using a similar procedure used
to prepare 27 except using 2-(trifluoromethyl)aniline, 32e, and letting the reaction to stir overnight at
room temperature. Crude (397 mg, 89%), a beige solid, was used in the next step without further
purification. 1H NMR (500 MHz, CDCl3) δ 8.60 (s, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H),
7.59 (t, J = 7.9 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 4.07 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 163.89,
134.59, 133.11, 126.38 (q, J = 5.3 Hz), 125.40, 124.17, 123.98 (q, J = 273.1 Hz), 120.79 (q, J = 29.9
Hz), 29.43. ESI MS: m/z 281.50, 283.50 (M+H)+.
2-Bromo-N-(3-(trifluoromethyl)benzyl)acetamide (33f). Synthesized using a similar procedure used
to prepare 27 except using 3-(trifluoromethyl)benzylamine, 32f, and letting the reaction to stir for 6h at
room temperature. Crude was purified by silica gel chromatography (hexanes/EtOAc) to give 33f (316
mg, 62%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 7.58 – 7.55 (m, 1H), 7.54 (s, 1H), 7.51 – 7.45
(m, 2H), 6.85 (s, 1H), 4.54 (d, J = 6.0 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 165.68, 138.54, 131.31 (q,
J = 33.5 Hz), 131.18, 129.49, 124.79, 124.54, 124.06 (q, J = 272.1 Hz), 43.80, 29.16. ESI MS: m/z
295.50, 297.55 (M+H)+.
Representative procedure for chloromethanesulfonamide preparation
1-Chloro-N-(3-(trifluoromethyl)phenyl)methanesulfonamide (35). Synthesized using reported
procedure with modification.27 To a stirring solution of 3-(trifluoromethyl)aniline, 26, (388 µL, 3.11 mmol)
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in CH2Cl2 (6 mL) was added Et3N (865 µL, 6.20 mmol) at room temperature. The reaction mixture was
cool down to 0 oC and chloromethanesulfonyl chloride (626 µL, 6.20 mmol) in CH2Cl2 (3 mL) was added
o
at 0 C. The reaction mixture was stirred at room temperature overnight. The reaction mixture was
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concentrated on vacuo then 2 N NaOH (15 mL) was added and the mixture was stirred at room
temperature for 2 h 30 min. The reaction mixture was neutralized by 1N HCl. The mixture was diluted
with H2O (10 mL) and extracted with EtOAc (20 mL x 2) and finally washed with brine (10 mL). The
organic layer was dried (Na2SO4), filtered, and concentrated in vacuo. Crude was purified by silica gel
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chromatography (hexanes/EtOAc) to give 35 (614 mg, 72%) as a beige solid. H NMR (400 MHz,
DMSO-d6) δ 10.77 (s, 1H), 7.62 – 7.56 (m, 1H), 7.56 – 7.51 (m, 2H), 7.49 (d, J = 7.6 Hz, 1H), 5.13 (s,
2H). 13C NMR (100 MHz, DMSO-d6) δ 138.25, 130.64, 129.96 (q, J = 31.8 Hz), 125.17, 123.64, 122.46,
120.71 (q, J = 3.6 Hz), 116.08 (q, J = 4.1 Hz), 54.87. ESI MS: m/z 272.0 (M-H)-.
1-Chloro-N-(3-fluorophenyl)methanesulfonamide (36). Synthesized using a similar procedure used
to prepare 35 except using 3-fluoroaniline, 34. Crude was purified by silica gel column chromatography
(hexanes/EtOAc) to give 36 (347 mg, 60%) as a yellow oil which was solidified upon standing. 1H NMR
(400 MHz, CDCl3) δ 7.35 (td, J = 8.2, 6.3 Hz, 1H), 7.11 – 7.03 (m, 2H), 6.97 (tdd, J = 8.3, 2.4, 0.8 Hz,
1H), 6.90 (s, 1H), 4.51 (s, 2H).13C NMR (100 MHz, CDCl3) δ 163.33 (d, J = 248.3 Hz), 136.96 (d, J =
10.2 Hz), 131.22 (d, J = 9.3 Hz), 117.68 (d, J = 3.2 Hz), 113.62 (d, J = 21.2 Hz), 109.71 (d, J = 25.2
Hz), 53.58. ESI MS: m/z 222.00 (M-H)-.
Supporting Information
Molecular formula strings and associated biochemical data, procedure for the synthesis of 20-26 and
biochemical data, ligand-detected full 1H NMR spectra of 2 and 14, full TOCSY spectra of free TAR and
1
in presence of different concentration of 2, H NMR and 13C NMR spectra for all compounds.
Corresponding Author
*Phone: +1 301-228-4620. Email:schneeklothjs@mail.nih.gov
Notes
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The authors declare no competing financial interest.
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Acknowledgements
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This work was supported by the Intramural Research Program of the National Institutes of Health,
Center for Cancer Research, the National Cancer Institute (NCI), and the IATAP program, National
Institutes of Health. Work at the University of Washington was supported by NIH-NIGMS GM110569.
M. Shortridge was supported by an American Cancer Society fellowship (PF-13-056-01-RMC). We
thank the Biophysics Resource in the Structural Biophysics Laboratory, NCI at Frederick for assistance
with Q-TOF LC-MS studies.
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Abbreviations Used
TAR, transactivation response element; 5’-UTR, 5’-untranslated region; SMM, small molecule
microarray; 2AP, 2-aminopurine; FRET, Forster resonance energy transfer; SHAPE, selective 2’-
hydroxyl acylation analyzed by primer extension. Solvent and reagent abbreviation used: 1M7, 1-
methyl-7-nitroisatoic anhydride; MgCl2, magnesium chloride; ACN, acetonitrile; MeOH, methanol;
EtOH, ethanol; EtOAc, ethylacetate; Na SO
H2SO4, sulfuric
4, sodium sulfate; NaOEt, sodium ethoxide;
2
acid; H3PO2, hypophosphorous acid; NaNO2, sodium nitrite; Cu2O, copper (I) oxide; Et3N,
trimethylamine.
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