Bifunctional inhibition of human immunodeficiency virus type 1 reverse transcriptase: Mechanism and proof-of-concept as a novel therapeutic design strategy

Article abstract of DOI:10.1021/jm400160s

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is a major target for currently approved anti-HIV drugs. These drugs are divided into two classes: nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the synthesis and biochemical evaluation of a novel bifunctional RT inhibitor utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI) linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in a base-specific manner. Moreover, this inhibitor demonstrates low nanomolar potency that has 4.3-fold and 4300-fold enhancement of polymerization inhibition in vitro relative to the parent TMC-derivative and d4T, respectively. This study serves as a proof-of-concept for the development and optimization of bifunctional RT inhibitors as potent inhibitors of HIV-1 viral replication.

Full text of DOI:10.1021/jm400160s

Article
pubs.acs.org/jmc
Bifunctional Inhibition of Human Immunodeficiency Virus Type 1
Reverse Transcriptase: Mechanism and Proof-of-Concept as a Novel
Therapeutic Design Strategy
Christopher M. Bailey,∥,† Todd J. Sullivan,∥,† Pinar Iyidogan,∥,† Julian Tirado-Rives,‡ Raymond Chung,†
Juliana Ruiz-Caro,‡ Ebrahim Mohamed,§ William Jorgensen,‡ Roger Hunter,§ and Karen S. Anderson*,†
†Department of Pharmacology, School of Medicine, and ‡Department of Chemistry, Yale University, New Haven, Connecticut 06520,
United States
§Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
S
* Supporting Information
ABSTRACT: Human immunodeficiency virus type 1 reverse transcriptase
(HIV-1 RT) is a major target for currently approved anti-HIV drugs. These
drugs are divided into two classes: nucleoside and non-nucleoside reverse
transcriptase inhibitors (NRTIs and NNRTIs). This study illustrates the
synthesis and biochemical evaluation of a novel bifunctional RT inhibitor
utilizing d4T (NRTI) and a TMC-derivative (a diarylpyrimidine NNRTI)
linked via a poly(ethylene glycol) (PEG) linker. HIV-1 RT successfully
incorporates the triphosphate of d4T-4PEG-TMC bifunctional inhibitor in
a base-specific manner. Moreover, this inhibitor demonstrates low
nanomolar potency that has 4.3-fold and 4300-fold enhancement of
polymerization inhibition in vitro relative to the parent TMC-derivative and
d4T, respectively. This study serves as a proof-of-concept for the
development and optimization of bifunctional RT inhibitors as potent
inhibitors of HIV-1 viral replication.
NRTIs are nucleoside analogue prodrugs that are converted
intracellularly into their respective triphosphate forms and
incorporated into the growing viral DNA chain by HIV-1 RT.
Consequently, these nucleotide analogues cause chain termi-
nation due to the lack of a 3′-hydroxyl (3′-OH) group in the
sugar moiety that is necessary for incorporation of the next
incoming nucleotide by RT.10 During this process, NRTIs
compete with their natural counterparts, deoxyribonucleoside
triphosphates (dNTPs), for binding to the polymerase active
site of RT in order to be incorporated into viral DNA.
Unfortunately, these competitive inhibitors could also act as
substrates for host polymerases like human mitochondrial DNA
polymerase γ (pol γ), which is responsible for replication and
repair of mitochondrial DNA,11,12 as well as DNA polymerase β
(pol β) that is responsible for nuclear DNA repair in vivo.13
Previous studies illustrate NRTI-induced mitochondrial toxicity
that causes serious adverse effects ranging from lactic acidosis
to hepatic steatosis during long-term HAART treatment.14,15
On the other hand, NNRTIs bind to an allosteric hydrophobic
site, approximately 10 Å away from the polymerase active site,
which is unique to HIV-1 RT and absent in host cell
polymerases.16 Therefore, unlike NRTIs, NNRTIs specifically
target RT while excluding any binding interaction with other
INTRODUCTION
■
The human immunodeficiency virus (HIV) epidemic still
remains a major public health concern since more than 30
million people around the world continue to live with HIV and
2.7 million people become newly infected with HIV each year.1
There have been fundamental improvements in the treatment
of HIV infection through highly active antiretroviral therapy
(HAART), leading to significant reductions in HIV-1 related
morbidity and mortality.2 HAART is a combination of several
antiretroviral drugs from different drug classes, yet eradication
of HIV has not been possible due to the rapid emergence of
drug-resistant HIV variants.3 The majority of Food and Drug
Administration (FDA) approved antiretroviral drugs target
HIV-1 reverse transcriptase (RT), the viral polymerase essential
for HIV-1 replication, and these drugs are classified into two
distinct classes: nucleoside reverse transcriptase inhibitors
(NRTIs) and non-nucleoside reverse transcriptase inhibitors
(NNRTIs). HIV-1 RT possesses a high mutation rate due to its
low fidelity caused by the lack of an intrinsic exonucleolytic
proofreading activity, therefore influencing the development of
rapid drug resistance against these inhibitors.4 In addition to
high mutation rate, multiple other factors may also give rise to
the high level of drug resistance, including the enormous
replication and recombination rates of HIV-1.5−9 As a result,
there is a continued need for novel anti-HIV drugs.
Received: January 30, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structures of d4T and various TMC-derivatives.1 For simplicity, we refer to [TMC-derivative] as TMC in text.
Figure 2. Structures of [d4T-4PEG-TMC] bifunctional inhibitor derivatives.
polymerases. In this paper, we utilized the high specificity of
NNRTIs as an anchor to design bifunctional inhibitors
consisting of an NRTI unit that is joined to an NNRTI moiety
via a noncleavable linker with the goal of achieving
complementary action at each distinct target site.
d4T continues to play a major role especially in resource-
limited settings, although d4T-induced mitochondrial toxicity
may cause serious health risks for HIV-positive patients after
chronic administration.29 In selecting a point of attachment for
the linker on the d4T, substitution at the C5 position of the
thymine ring was chosen due to an anticipated low interference
with base pairing, in addition to the established evidence
showing that addition of various linker entities at this position
does not interfere with antiviral activity.24,30−32 In previous
work, we described our initial efforts toward the design and
synthesis of novel bifunctional RT inhibitors utilizing
tetrahydroimidazobenzodiazepinone (TIBO), an early-gener-
ation NNRTI with nanomolar antiviral potency, as the NNRTI
portion attached to d4T via a PEG linker.33 Although we
demonstrated the incorporation of the triphosphate form of an
N3-substituted d4T-6PEG-TIBO bifunctional compound, the
use of 8-Cl-TIBO as a pharmacophore for our bifunctional
inhibitor was less than ideal since the addition of a 6PEG
alcohol at the 8-position substantially diminished the antiviral
activity.33
Our results from functionalizing 8-Cl-TIBO lead us toward
utilizing new flexible NNRTIs for the bifunctional inhibitor,
concomitant with the paradigm shift in NNRTI design. Not
only is the concept of flexibility important for overcoming
antiviral resistance mutations, but also one would anticipate
that this would allow the inhibitor to better accommodate the
addition of a linker moiety. Therefore, we selected the
diarylpyrimidine (DAPY) series because of their conforma-
tional flexibility and high potency against wild type (WT) and
various NNRTI-resistant HIV-1 viruses that resulted in two
FDA-approved drugs (Figure 1); TMC125 and TMC278.34
DAPY compounds present a common binding mode termed as
“butterfly-like” conformation, with the structural components
defined as wing I, wing II, and a body/linker.27 To ease
synthetic complexity for our bifunctional inhibitor, the bromine
and amino functionalities on the pyrimidine ring of TMC125
were omitted, rendering a TMC120-like compound that
incorporates several aspects of TMC125. Our TMC-derivative
The concept of joining the two drugs via a linker was
originally proposed by Smerdon et al.,17 based on observation
of the close proximity of the respective NRTI and NNRTI
binding sites. Further rationale includes mechanistic studies
establishing that the two sites could be simultaneously
occupied18,19 and studies on the mechanism of synergistic
inhibition of RT by NRTIs and NNRTIs.20−22 Earlier work on
developing chimeric NRTI/NNRTI inhibitors with a non-
cleavable linker showed limited success where there has been
no evidence for a pronounced synergistic antiviral effect in cell-
based assays.23−25 For interpretation of these results, one
important factor to be considered is the prerequisite
phosphorylation of the nucleoside portion of the bifunctional
inhibitor into the pharmacologically active triphosphate form.
Therefore, it is unclear whether the observed lack of activity
enhancement compared to the NNRTI alone in previous
reports was due to lack of phosphorylation by cellular kinases
or lack of binding affinity to RT. In the present study, we test
this issue directly by preparing the triphosphate form of
bifunctional chimeric NRTI/NNRTI and examining the
interaction with HIV-1 RT at the biochemical level.
Among the key issues in the design of a chimeric NRTI/
NNRTI joined by a flexible tether are (1) selection of optimal
NRTI and NNRTIs, (2) linkage points for the NRTI and
NNRTI, and (3) linker length and composition. We have
designed chimeric NRTI-linker-NNRTI bifunctional inhibitors
aided by computer modeling and the available structural
information.26,27
In the design of the bifunctional NRTI-linker-NNRTI, 2′,3′-
didehydrothymidine (d4T, Figure 1) was chosen as the NRTI,
on the basis of our mechanistic studies showing that d4T
triphosphate (d4TTP) binds to HIV-1 RT with comparable
binding affinity to the natural substrate.28 More importantly,
B
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
includes an ether linkage from the central pyrimidine ring to
wing II and a cyano group at the para position as shown in
Figure 1. With the aid of computer modeling, we have used the
available three-dimensional structures of the catalytic RT/
DNA/dNTP complex along with the RT/TMC inhibitor
complexes to guide our selection of the most feasible NRTI and
NNRTI linker positions as well as to estimate the optimal
linker length between the two binding sites (Figure 2).27
Selection of the NNRTI attachment point to the linker and the
route from the NNRTI-binding pocket (NBP) to the
polymerization site poses a challenge, since the opening of
the NBP points away from the nucleotide-binding site.
Modeling and structural information of RT/TMC inhibitor
complexes have demonstrated the importance of the two aryl
substituents on the pyrimidine ring, often referred to as wings I
and II, and their contact with key hydrophobic residues in the
NBP.26,27,35 Especially, the cyanodimethylphenoxy ring (wing
I) of TMC125 interacts with Y181, Y188, and the conserved
W229 residues. The recently approved TMC278 demonstrates
that the long cyanovinyl functionality at the para position of
wing I maintains low nanomolar potency and even improves
the antiviral activity against mutant strains.36 Therefore, a
plausible possibility for NNRTI linker attachment would
involve a linker attached to the para position of the
dimethylphenoxy ring of TMC125 leading out of the pocket
via alkynyl attachment in the region of the W229 and F227
interactions. The flexibility of the residues lining the NBP is an
important feature in regard to linker attachment. Upon NNRTI
binding, the side chains of Y181 and Y188 flip from being
pointed toward the entrance of the putative binding pocket
nearly 180° from their initial position, to point toward the
polymerization active site, forming the NBP.37 The pocket itself
is formed upon NNRTI binding, and the pocket is not seen in
structures of RT without NNRTI bound. We predicted, on the
basis of our molecular model and aforementioned literature,
that this flexibility of Y181, Y188, and W229 could
accommodate a long linker and allow an NRTI-linker-
NNRTI bifunctional inhibitor to span the NBP and the active
site (Figure 3A,B). The feasibility of this position on the TMC
scaffold was further validated by the crystal structure of the
HIV-1 RT/TMC278 complex, which demonstrates that the p-
cyanovinyl arm of wing I of TMC278 protrudes into a
hydrophobic tunnel in the pocket that opens to the active site
(Figure 3C).35 This study focuses on the development of novel
bifunctional compounds utilizing a TMC scaffold as the
NNRTI portion, which is coupled to a d4T moiety with a
PEG linker (Figure 2). We have utilized an integrated approach
combining organic synthesis, molecular modeling, and in vitro
enzyme inhibition assays in order to realize our goal of
identifying a bifunctional inhibitor that demonstrates binding at
both sites simultaneously with a concomitant increase in
potency.
RESULTS AND DISCUSSION
■
A PEG linker was chosen for the bifunctional inhibitor design,
in view of optimal water solubility while retaining significant
organic lipophilicity and flexibility. Molecular modeling using
the X-ray structures of RT/TMC12027 and the ternary
structure of RT38 together suggested a four-PEG spacer was
optimal to bridge both binding sites and allow proper base
pairing at the active site (Figure 3B). In addition, we recognize
the importance of linker addition and the attachment point to
the NNRTI entity from our previous study with the TIBO
bifunctional series with decreased antiviral activities compare to
8-Cl-TIBO.33 Alignment of the RT/8-Cl-TIBO structure39 with
the RT/TMC278 structure35 shows that the chlorine atom on
the benzene ring of 8-Cl-TIBO projects into the base of the
hydrophobic tunnel to some extent, where the cyanovinyl
substituent of wing I of TMC278 binds in a subpocket
generated by residues F227 and W229. Although the
functionalization at the 8-Cl position could point toward the
active site of the enzyme, our results demonstrated that placing
a PEG tether at this position severely impairs potency by 2000-
fold.33 This result implies that either the 8-Cl-TIBO lacks
flexibility or the neighboring residues in this region are perhaps
less flexible than the residues lining the hydrophobic tunnel
accepting the cyanovinyl substituent of TMC278. Therefore,
the present study focused on utilizing d4T-4PEG-TMC as our
bifunctional prototype. Previous studies on C-5-substituted
d4T derivatives yielded mixed results but mostly resulted in
inactive analogues.24,25,40−43 Moreover, earlier attempts to
show efficient HIV replication inhibition of bifunctional
compounds all failed.44,45 We hypothesized that one of the
possible reasons for these unsuccessful attempts could be
impaired phosphorylation of the nucleoside end of bifunctional
analogues catalyzed by cellular kinases. Consequently, the
synthesis of a metabolically active triphosphate form of our
d4T-4PEG-TMC bifunctional nucleoside was conducted to
directly address the phosphorylation requirement for in vitro
incorporation assays catalyzed by HIV-1 RT.
The synthesis of 5′-benzoyl-d4T-4PEG-TMC (1a, Figure 2)
began with targeting quantities of the known pyrimidine
derivative 2,46 which underwent nucleophilic substitution in
high yield to 3 with 4-iodo-2,6-dimethylphenol by use of
cesium carbonate as base (Scheme 1). Subsequent Sonogashira
coupling with the monopropargyl ether of 4-PEG-diol47 gave
tethered TMC-derivative 4, followed by hydroxyl group
propargylation to give 5, which was subjected to a further
Sonogashira coupling with 5′-benzoyl-5-iodo-d4U48 to afford
bifunctional benzoate (1a) in around 60% isolated yield.
Deprotection of 1a with sodium methoxide, quenching with
acetic acid, and direct silica-gel flash chromatography without a
workup afforded bifunctional nucleoside, d4T-4PEG-TMC
(1b). Transformation to its triphosphate derivative (1c) proved
Figure 3. Molecular modeling predicts binding of d4T-4PEG-TMC.
(A) Molecular modeling suggests a 4PEG linker would be long enough
to span the NNRTI binding pocket and the active site. (B) The PEG
linker is predicted to extend from the NNRTI binding pocket toward
the active site through a hydrophobic tunnel, similar to the tunnel
identified in the TMC278 bound structure (pdb ID 2ZD1).35 This
would allow proper base-pairing of the NRTI portion with the
templating A. (C) TMC278 bound structure.
C
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 1. Synthesis of d4TTP-4PEG-TMC Bifunctional Derivative
a
Reagents and conditions: (a) 4-Iodo-2,6-dimethylphenol, Cs2CO3, DMF, Δ, 85%. (b) Monopropargyl ether of 4-PEG diol (or n-PEG diol),
Pd(PPh3)4 (10 mol %), CuI (50 mol %), THF, DMF, 85%. (c) (i) TsCl, NEt3, CH2Cl2; (ii) propargyl alcohol, NaH, THF, Δ, 92%. (d) 5′-Benzoyl-
5-iodo-d4U, Pd(PPh3)4 (10 mol %), CuI (50 mol %), THF, DMF, 59%. (e) NaOMe, MeOH, 94%. (f) (i) 2-Chloro-1,3,2-benzodioxaphosphorin-4-
−
one, pyr, DMF, 0 °C to rt; (ii) bis(tributylammonium) pyrophosphate; (iii) I2; (iv) 1 M Et3NH+ HCO2 , 37%.
to be problematic in that direct phosphorylation by Yoshikawa’s
procedure49 with POCl3 and trimethyl phosphate failed to give
the desired product. Convergent cross-coupling of the 5′-
triphosphate of 5-iodo-d4U as a tetra(triethylammonium) salt
with alkyne 5 via a homogeneous50 Sonogashira reaction in
N,N-dimethylformamids (DMF) appeared to produce the
product by thin-layer chromatography (TLC), but separation
from the copper reagent proved to be problematic. Finally, the
bifunctional nucleoside (1b) was successfully converted into
d4TTP-4PEG-TMC (1c) via the procedure of Ludwig and
Eckstein.51 The product was purified by Sephadex (dieth-
ylaminoethyl, DEAE) ion-exchange chromatography with
aqueous triethylammonium bicarbonate (TEAB, 1 M)/H2O
as the mobile phase to afford 1c as a film. All final compounds
for in vitro testingTMC-derivative (Figure 1), the propargyl-
4PEG-TMC-derivative (5), and d4TTP-4PEG-TMC (1c)
were obtained in moderate yields, and conformation of
structure and purity were provided by NMR spectroscopy
(1H, 13C, and 31P) as well as HPLC and high-resolution mass
spectrometry (HRMS), in preparation for the inhibition assays.
Initially, we examined the ability of d4TTP-4PEG-TMC to
serve as a substrate for incorporation by HIV-1 RT.
Incorporation assays under single-turnover conditions demon-
strated 100% conversion of the D23 substrate to an elongated
primer chain terminated with the bifunctional monophosphate,
indicated as a D23-d4TMP-4PEG-TMC product band that
migrates significantly higher than the D23 primer substrate or a
D24-mer elongated by one dNTP in Figure 4A. Control
experiments lacking magnesium chloride were also performed,
demonstrating that formation of the elongated product
required divalent metal ions. We next sought to address
whether the linker location at the C-5 position on d4TTP
would affect base paring. In other words, was incorporation of
d4TTP-4PEG-TMC base-specific? For this purpose, another
primer/template (P/T) for G incorporation (D24/D36) was
utilized, which failed to produce an elongated primer opposite a
template dCMP as shown in lane 3 (Figure 4B). The negative
control experiment lacking magnesium chloride was repeated
for D24/D36 P/T (lane 2), and no elongation was observed as
well. These findings illustrate the requirement of Mg2+ as a
divalent metal ion and, more importantly, the base-specific
incorporation of the bifunctional molecule possessing a
thymine nucleobase when utilizing D23/D36 but not D24/
D36 primer/template containing a guanine nucleobase. More-
over, as mentioned above, the altered mobility of the D23-
d4TMP-4PEG-TMC band that is higher in distance compared
to a standard mobility distance of a single nucleotide elongated
product band strongly supports the incorporation of the large
bifunctional compound.
After illustrating that d4TTP-4PEG-TMC bound to the
active site and served as a substrate for incorporation by HIV-1
RT, we evaluated the inhibition activity of the bifunctional
inhibitor at the biochemical level via a steady-state competition
assay to determine its potency (Figure 4C). The IC50 values
were determined in triplicate and averaged for in vitro
inhibition of RT for TMC-derivative, TMC-4PEG-propargyl,
and triphosphate of the bifunctional nucleoside. The parent
TMC-derivative had an IC50 value of 13 3 nM. Elaboration of
the TMC-derivative with a 4-PEG linker, to give the NNRTI
tether propargyl-4PEG-TMC, results in a 10-fold decrease in
D
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Incorporation and potent HIV-1 RT inhibition of d4TTP-4PEG-TMC is base-specific. (A) Sequence of D23/D36 P/T for correct base
pairing used in biochemical assays. d4TTP-4PEG-TMC serves as a substrate for HIV-1 RT-catalyzed polymerization when it is used as a primer for
dTTP incorporation. Lane 1 represents the 0 time point, and lane 2 serves as a negative control lacking Mg2+. Lane 3 represents the incorporated
bifunctional triphosphate (indicated as *D23-d4TMP-4PEG-TMC) product band by HIV-1 RT. (B) Sequence of D24/D36 P/T for incorrect base
pairing used in biochemical assays. d4TTP-4PEG-TMC does not serve as a substrate for HIV-1 RT when it is used as a primer for dGTP
incorporation. Lane 1 represents the 0 time point, and lane 2 serves as a negative control lacking Mg2+. Lane 3 shows the lack of an incorporated
bifunctional triphosphate product band. (C) Inhibition of dTTP incorporation is shown in bar graphs for d4TTP (13 000 nM), TMC-derivative (13
nM), propargyl-4PEG-TMC (122 nM), and d4TTP-4PEG-TMC (3 nM). Additionally, the calculated IC50 values for 1:1 mixtures of d4TTP/TMC-
derivative and d4TTP/propargyl-4PEG-TMC are 9 nM and 114 nM, respectively. IC50 values were determined in triplicate and averaged. (D)
Inhibition of dTTP incorporation (correct base pairing) is shown in blue; inhibition of dGTP incorporation (incorrect base pairing) is shown in red.
Whereas d4TTP-4PEG-TMC is very potent with proper base pairing (3 nM), its ability to inhibit dGTP incorporation is only slightly more potent
(42 nM) than the propargyl-4PEG-TMC (64 nM). Results for TMC-derivative and propargyl-4PEG-TMC are similar for inhibition of dTTP
incorporation or dGTP incorporation. Potent RT inhibition of d4TTP-4PEG-TMC is therefore base-specific.
potency (122 14 nM). Analysis of the metabolically active
d4TTP-4PEG-TMC exhibited a low nanomolar inhibition (3
1 nM). This enhancement is approximately 4.3-fold improve-
ment relative to the TMC-derivative, a 40-fold improvement
over propargyl-4PEG-TMC, and a 4300-fold improvement
relative to d4TTP (13 000 2000 nM). True binding affinity
may in fact be higher, as this value may represent stoichiometric
binding to the enzyme (as our experimental setup utilized 6 nM
active sites of HIV-1 RT). Additional experiments demon-
strated that 1:1 mixtures of d4TTP/TMC-derivative (9 1.5
since the bifunctional nucleoside triphosphate was not
incorporated opposite a templating C, it would be less potent
in inhibiting dGTP incorporation due to the diminished
binding interactions at the active site. Under these conditions,
the IC50 values of the TMC-derivative (9
3 nM) and
propargyl-4PEG-TMC (64 7 nM) were similar to results for
inhibition of dTTP incorporation, as expected since these
inhibitors are not predicted to interact with the active site
(Figure 4D). However, when the bifunctional triphosphate
would not be properly base-paired for incorporation at the
active site, the polymerase inhibition potency of d4TTP-4PEG-
TMC was significantly reduced (42 10 nM), indicating that
the potent inhibition seen during dTTP incorporation is linked
to the proper base pairing of the NRTI moiety. Our findings
suggest that simultaneous occupation of the NNRTI binding
pocket and the active site is occurring to yield the high level of
potency with d4TTP-4PEG-TMC bifunctional inhibitor.
nM) and d4TTP/propargyl-4PEG-TMC (114
30 nM)
showed no significant change in inhibition compared to the
NNRTI or NNRTI tether component alone (Figure 4C),
implying that having both components physically attached as
one molecule is essential for the increase in potency.
After demonstrating that proper base pairing is essential for
incorporation of the NRTI component of our bifunctional
triphosphate, we hypothesized that the increased potency of
d4TTP-4PEG-TMC was linked to proper base pairing at the
active site. Therefore, we performed a similar set of biochemical
experiments to explore this by simply changing the nucleotide
and primer/template used in the assay. We would predict that
CONCLUSIONS AND FUTURE DIRECTIONS
■
Although we have shown that the nucleoside triphosphate
moiety of the bifunctional can bind at the active site and be
incorporated, further work is required to prove simultaneous
E
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
on RediSep amine-functionalized plates. All synthesized final
compounds were determined to be ≥95% pure by LC-MS. Each
binding at both sites. One potential avenue to establish the
binding of the inhibitor will be through obtaining an X-ray
crystal structure of the bifunctional triphosphate bound to HIV-
1 RT. Crystallization trials are currently underway in an attempt
to determine structures of d4TTP-4PEG-TMC bound to HIV-
1 RT in the presence and absence of primer/template. Further
evidence suggesting interaction at both the active site and the
NNRTI binding pocket could be obtained by determining the
effect of NRTI resistance mutations (Q151M) or NNRTI
resistance mutations (Y181C) on the activity of d4TTP-4PEG-
TMC in a steady-state inhibition assay.
We believe this study offers a major advance toward
establishing proof-of-concept and the feasibility of designing
bifunctional RT inhibitors with a binding mode that confirms
that the NRTI (d4T) moiety is binding in the dNTP pocket in
a base-specific manner and with improved potency relative to
the parent NRTI and NNRTI moieties. Building on these
results, further optimization studies are in progress to more
fully exploit the potential additive binding energies of the NRTI
and NNRTI. Additional work is in progress to prepare various
lengths of PEG linker for the bifunctional compounds in
nucleoside form, as well as their prodrug versions to obtain
optimal potency at the cellular level utilizing HIV-1 infected
human T-cells. The triphosphates of these derivatives will also
be synthesized and evaluated via similar biochemical experi-
ments. Since intracellular phosphorylation may be a potential
barrier for these bifunctional RT inhibitors, we are currently
exploring an alternative approach for delivery involving the
encapsulation of the bifunctional inhibitor triphosphates in
biodegradable nanogels. These nanogel formulations have been
successfully used in cancer chemotherapy as drug delivery
systems for cytotoxic nucleoside analogues.52,53 Another avenue
that we are exploring is using nucleotide-competing RT
inhibitors (NcRTIs)54 instead of NRTIs in our bifunctional
inhibitor designs, since NcRTIs are distinct from the chain-
terminator NRTIs and do not require any phosphorylation by
cellular kinases.
1
compound identity was verified by H NMR, 13C NMR, and HRMS,
1
and additionally by 31P NMR for 1c. H and 13C NMR spectra were
recorded on a Bruker Avance DPX (at 500 and 400 MHz,
respectively) in solvents CDCl3 and MeOH-d4, with the resonances
for CDCl3 taken as δ 7.24 ppm for 1H and δ 77.0 ppm for 13C, and for
1
MeOH-d4 as δ 3.30 ppm for H and δ 49.0 ppm for 13C to serve as
internal references (Cambridge Isotope Laboratories, Inc.). The data
for 1H NMR spectra are reported as follows: chemical shift in parts per
million, ppm (multiplicity, coupling constant in hertz, integration).
Those for 13C NMR spectra are reported in terms of chemical shifts to
one significant figure relative to the solvent references as described
above. High-resolution mass spectra (ESI/MS) were obtained from
the Keck Biotechnology Resource Laboratory, Yale University.
4-[4-(4-Iodo-2,6-dimethylphenoxy)pyrimidin-2-ylamino]-
benzonitrile (3). A mixture of 4-(4-chloropyrimidin-2-ylamino)-
benzonitrile46 (2) (600.0 mg, 2.60 mmol), 4-iodo-2,6-dimethylphe-
nol57 (838.0 mg, 3.38 mmol), and cesium carbonate (2.20 g, 6.76
mmol) in dry DMF (12 mL) was heated to 90 °C.59 After 3 h, TLC
(EtOAc/hexane = 50:50) revealed a more polar product and
consumption of compound 2. The mixture was poured into water
(100 mL) and the product was extracted into EtOAc (3 × 50 mL).
The combined extracts were washed with brine (50 mL), followed by
water (2 × 50 mL), and then dried over Na2SO4. The solvent was
evaporated in vacuo to furnish a residue that was subjected to silica-gel
column chromatography with EtOAc/hexane (50:50) as eluent to
1
produce 3 as a white solid (982.0 mg, 2.22 mmol, 85%). H NMR
(CDCl3, 500 MHz) δ (ppm) 8.31 (d, J = 6.0 Hz, 1H), 7.48 (s, 2H),
7.42 (d, J = 8.5 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.31 (s, 1H, NH),
6.47 (d, J = 6.0 Hz, 1H), 2.06 (s, 6H). 13C NMR (CDCl3, 500 MHz) δ
(ppm) 168.9, 159.7, 159.1, 149.9, 143.3, 137.5, 133.4, 133.1, 119.3,
118.1, 104.7, 99.5, 89.9, 16.0. HRMS (EI+) m/z calcd for C19H15IN4O
[M + H]+ 443.0363, found 443.0357.
4-(4-{4-[3-(2-{2-[2-(2-Hydroxyethoxy)ethoxy]ethoxy}ethoxy)prop-
1-ynyl]-2,6-dimethylphenoxy}pyrimidin-2-ylamino)benzonitrile (4).
To a deoxygenated solution of iodopyrimidine (3) (494 mg, 1.12
mmol) and 2-(2-{2-[2-(prop-2-ynyloxy)ethoxy]ethoxy}ethoxy)-
ethanol47 (780 mg, 3.36 mmol) in dry DMF (1.5 mL) and dry
THF (3.0 mL) were added successively dry triethylamine (562.0 mg,
5.55 mmol, 0.80 mL), copper(I) iodide (105.7 mg, 0.56 mmol), and
tetrakis(triphenylphosphine)palladium(0) (128.3 mg, 0.11 mmol).
The reaction mixture was stirred at 25 °C under a nitrogen
atmosphere for 3 h, after which TLC (DCM/MeOH = 3:97) revealed
a more polar product and consumption of starting material 3. The
mixture was poured into ethylenediaminetetraacetic acid (EDTA)
solution (20 mL, 5%) and the organic product was extracted into
EtOAc (3 × 50 mL). The combined organic extracts were washed with
brine (20 mL) and dried over Na2SO4. The solvent was evaporated in
vacuo to furnish a residue that was subjected to silica-gel column
chromatography with DCM/MeOH (95:5) as eluent to produce 4 as a
light-brown oil (522.0 mg, 0.956 mmol, 85%). 1H NMR (CD3OD, 400
MHz) δ (ppm) 8.37 (d, J = 5.6 Hz, 1H), 7.44 (d, J = 9.2 Hz, 2H), 7.35
(d, J = 9.2 Hz, 2H), 7.27 (s, 2H), 6.60 (d, J = 5.6 Hz, 1H), 4.46 (s,
2H), 3.71−3.79 (m, 4H), 3.53−3.66 (m, 12H), 2.08 (s, 6H). 13C
NMR (CDCl3, 500 MHz) δ (ppm) 169.1, 159.6, 159.3, 150.0, 143.4,
133.0, 132.1, 131.2, 120.3, 119.3, 118.1, 104.6, 99.5, 85.6, 85.1, 72.5,
70.7, 70.6, 70.6, 70.5, 70.4, 69.2, 61.8, 59.2, 16.3. HRMS (EI+) m/z
calcd for C30H34N4O6 [M + H]+ 547.2551, found 547.2528.
4-[4-(4-{3-[2-(2-{2-[2-(Prop-2-ynyloxy)ethoxy]ethoxy}ethoxy)-
ethoxy]prop-1-ynyl}-2,6-dimethylphenoxy)pyrimidin-2-ylamino]-
benzonitrile (5). Dry triethylamine (278 mg, 2.75 mmol, 0.4 mL)
followed by p-toluenesulfonyl chloride (349.0 mg, 1.83 mmol) and 4-
dimethylaminopyridine (11.2 mg, 0.009 mmol) were added to alcohol
4 (500.0 mg, 0.92 mmol) dissolved in dry dichloromethane (5 mL).
The reaction was left stirring for 3 h at room temperature, after which
time TLC (DCM/MeOH = 5:95) revealed a less polar product and
consumption of starting material 4. The mixture was poured into
saturated aqueous sodium bicarbonate (20 mL) and extracted with
dichloromethane (3 × 50 mL). The combined organic extracts were
EXPERIMENTAL SECTION
■
Chemistry. Unless stated otherwise, reactions were performed in
oven-dried glassware under a nitrogen atmosphere (house nitrogen,
dried with Drierite and KOH) with dry, deoxygenated solvents
(distilled or passed over a column of activated alumina). 2-Thiouracil
and 4-aminobenzonitrile were purchased from Sigma−Aldrich
Chemical Co. and used as received. The key starting material, 4-(4-
chloropyrimidin-2-ylamino)benzonitrile (2), and the TMC-derivative,
4-[4-(4-iodo-2,6-dimethylphenoxy)pyrimidin-2-ylamino]benzonitrile,
that we used in our bifunctional compounds were synthesized
according to literature procedures.46,55,56 Sephadex ion-exchange
(DEAE) adsorbent was purchased from Sigma, while 4-iodo-2,6-
dimethylphenol,57 2-(2-{2-[2-(prop-2-ynyloxy)ethoxy]ethoxy}-
ethoxy)ethanol,47 and 5-iodo-5′-benzoyl-d4U48,58 were also prepared
according to literature procedures. All reactions were magnetically
stirred and monitored by thin-layer chromatography. TLC was
performed on E. Merck silica-gel 60 F254 precoated plates (0.25
mm) or RediSep amine-functionalized silica gel containing a 254 nm
fluorescent indicator for triphosphate synthesis and visualized by UV
fluorescence quenching or Hanessian staining. Except for triphosphate
synthesis, traditional flash chromatography and a CombiFlash
Companion system were used for all of the separations with normal-
phase silica gel utilizing repackable columns with 35−60 μm average
particle size (230−400 mesh). The triphosphate synthesis purification
was carried out on DEAE-Sephadex with increasing percentages of
aqueous TEAB (1 M, triethylammonium bicarbonate)/H2O mixtures
as the mobile phase. TLC for the latter utilized TEAB/MeOH = 6:4
F
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
washed with brine (20 mL) and then dried (Na2SO4). Evaporation of
the solvent in vacuo gave a light brown oil (658.0 mg), which was
taken to the next step directly by adding it to a solution of propargyl
alcohol (526 mg, 9.39 mmol, 0.54 mL) dissolved in dry THF (5 mL)
to which sodium hydride (375 mg, 60%, 9.39 mmol) had been added.
The reaction mixture was heated to 60 °C for 3 h, after which time
TLC (DCM/MeOH = 5:95) indicated the formation of a more polar
product and consumption of the intermediate. The mixture was
poured into saturated aqueous sodium bicarbonate (20 mL) and
extracted with EtOAc (3 × 50 mL), and the combined organic extracts
were washed with brine (20 mL) and then dried (Na2SO4).
Evaporation of the solvent in vacuo gave a residue that was subjected
to column chromatography on silica gel with DCM/MeOH mixtures
to furnish 5 as a light-yellow oil (493.0 mg, 0.84 mmol, 92% for two
d4U-Propynyl-4-PEG-propynyl-TMC-triphosphate (1c). (For sim-
plicity, we refer to this structure as d4TTP-4PEG-TMC in text.) The
bifunctional nucleoside 1b (95.0 mg, 0.120 mmol) was dissolved in a
1:1 mixture of DMF/pyridine (1.0 mL) and the flask was cooled to
−20 °C. 2-Chloro-1,3,2-benzodioxaphosphorin-4-one51 (30.0 mg,
0.148 mmol) dissolved in THF (0.5 mL) was added slowly, and the
reaction was left to warm to room temperature. After 60 min,
bis(tributylammonium) pyrophosphate (92.0 mg, 0.168 mmol) in
DMF (0.5 mL) was added, followed by triethylamine (0.5 mL), and
the solution was left stirring for 1 h. I2 (43.0 mg, 0.170 mmol)
dissolved in pyridine/water (2 mL, 98:2) was then added, and the
solution was left stirring for 15 min before being quenched by aqueous
Na2S2O3 (0.5 M, 0.5 mL). TEAB (1 M, 3.0 mL) was immediately
added and the solution was left stirring for 2 h at room temperature.
The solution was then evaporated to dryness before being chromato-
graphed on a Sephadex ion-exchange (DEAE) column (3.0 g) with
aqueous TEAB as the mobile phase. The bifunctional triphosphate
eluted at around 0.6 M. Fractions identified from TLC (on amine-
impregnated silica gel plates with TEAB/MeOH = 3.5/6.5) were
combined and the solvent was evaporated. Following several additions
of methanol with pumping, tris(tiethylammonium) triphosphate 1c
(60.0 mg, 0.045 mmol, 37%) was obtained as a colorless oil. 1H NMR
(CD3OD, 500 MHz) δ (ppm) 8.32 (m, 1H), 7.78 (s, 1H), 7.43 (m,
2H), 7.33 (m, 2H), 7.23 (s, 2H), 6.85 (m, 1H), 6.55 (m, 2H), 5.88 (m,
1H), 5.02 (m, 1H), 4.42 (s, 2H), 4.32 (s, 2H), 4.12 (m, 2H), 3.54−
3.74 (m, 16H), 3.13 (br s, 18H), 2.04 (s, 6H), 1.25 (br s, 27H). 13C
NMR (CD3OD, 75 MHz) δ (ppm) 170. 4, 164.1, 161.2, 160.8, 151.8,
151.6, 145.9, 145.4, 136.6, 133.7, 133.1, 132.8, 126.5, 121.5, 120.4,
119.6, 104.6, 100.5, 100.1, 91.8, 90.9, 87.5, 86.6, 86.0, 78.4, 71.5, 71.5,
71.5, 71.5, 71.5, 71.4, 70.2, 70.2, 68.4, 59.8, 59.7, 47.2, 16.4, 9.1. 31P
NMR (CD3OD, 202.4 MHz) δ (ppm) −8.9 (d, J = 20.2 Hz), −9.8 (d,
J = 20.2 Hz), −22.1 (t, J = 20.2 Hz). HRMS (EI+) m/z calcd for
tetraphosphoric acid C42H46N6O19P3 [M − H]+ 1031.2036, found
1031.2035.
High-Pressure Liquid Chromatography of d4TTP-4PEG-TMC (1c).
d4TTP-4PEG-TMC was further analyzed by HPLC on a DNA Pac
PA-100 analytical column (Dionex, Sunnyvale, CA) under the
following conditions: mobile phase A, 0.05 M triethylammonium
bicarbonate (TEAB), pH 8.0; mobile phase B, 0.5 M TEAB, pH 8.0.
Mobile phase flow rate was 1 mL/min with a gradient of 100% A to
50% A/50% B for 10 min and then to 100% B for 5 min, followed by
100% B for 10 min. One Gaussian peak was seen with a retention time
of 20 min (Figure S1, Supporting Information). Absorbance was
measured at 260 nm.
Biology. Expression and Purification of HIV-1 RT. C-Terminal
histidine-tagged heterodimeric p66/p51 wild-type HIV-1 RT was
expressed and purified as described previously60 from a clone
generously provided by Stephen Hughes, Paul Boyer, and Andrea
Ferris (Frederick Cancer Research and Development Center,
Frederick, MD).
Nucleoside Triphosphates. dTTP and dGTP were purchased from
GE Biosciences. d4TTP was purchased from Moravek Biochemicals
(Brea, CA). Concentrated stocks were diluted and concentrations
were verified spectrophotometrically by determining absorbance at
260 nM by use of known extinction coefficients for each nucleotide.
Labeling and Annealing of Oligonucleotides. Primers and
templates used for incorporation and removal studies were synthesized
at the Keck Facility at Yale University and purified by 20%
polyacrylamide denaturing gel electrophoresis. The sequences of
primers and templates used in this study are as follows: D23 (5′-TCA
GGT CCC TGT TCG GGC GCC AC-3′), D24 (5′-TCA GGT CCC
TGT TCG GGC GCC ACT-3′), and D36 (5′-TCT CTA GCA GTG
GCG CCC GAA CAG GGA CCT GAA AGC-3′). The primers D23
and D24 were 5′-32P-labeled with T4 polynucleotide kinase (New
England Biolabs) as previously described.61 [γ-32P]ATP was purchased
from GE Healthcare. Bio-Spin columns for the removal of excess
[γ-32P]ATP were purchased from Bio-Rad. Annealing of the primers,
D23 and D24, and template D36 were carried out by mixing a 1:1.4
molar ratio of purified primer/template at 90 °C for 5 min, 50 °C for
10 min, and 0 °C for 10 min. The annealed primer and template were
1
steps). H NMR (CDCl3, 500 MHz) δ (ppm) 8.28 (d, J = 6.0 Hz,
1H), 8.05 (s, 1H, NH), 7.35 (m, 4H), 7.18 (s, 2H), 6.44 (d, J = 6.0 Hz,
1H), 4.38 (s, 2H), 4.11 (d, J = 2.5 Hz, 2H), 3.72−3.67 (m, 4H), 3.66−
3.58 (m, 12H), 2.37 (t, J = 2.5 Hz, 1H), 2.03 (s, 6H). 13C NMR
(CDCl3, 500 MHz) δ (ppm), 168.9, 159.4, 159.1, 149.9, 143.5, 132.8,
131.9, 131.1, 120.0, 119.2, 118.1, 104.2, 99.2, 85.5, 84.9, 79.5, 74.4,
70.5, 70.4, 70.4, 70.4, 70.3, 70.2, 69.1, 68.9, 59.0, 58.2, 16.1. HRMS
(EI+) m/z calcd for C33H36N4O6Na [M + Na]+ 607.2527, found
607.2500.
Benzoyl-d4U-propynyl-4-PEG-propynyl-TMC (1a). (For simplicity,
we refer to this structure as 5′-benzoyl-d4T-4PEG-TMC in text, while
formally it might best be considered as a uridine rather than thymidine
derivative.) A mixture of (tetrakis)triphenylphosphinepalladium(0)
(30.0 mg, 0.026 mmol) and copper(I) iodide (30.0 mg, 0.16 mmol)
was added rapidly to a solution of 5-iodo-5′-benzoyl-d4U58 (110 mg,
0.25 mmol) and alkyne 5 (99.0 mg, 0.17 mmol) in a degassed mixture
of THF and DMF (6 mL, 2:1) at room temperature under nitrogen.
TLC indicated reaction to a more polar product to be complete after 3
h with complete consumption of alkyne. Workup involved adding
EDTA solution (20 mL, 5%) and extracting the product into EtOAc
(3 × 50 mL). Drying (Na2SO4) and evaporation of solvent, followed
by column chromatography of the residue on silica gel with EtOAc/
hexane mixtures as eluent, furnished the bifunctional compound 1a
(91.0 mg, 59%). 1H NMR (CDCl3, 500 MHz) δ (ppm) 11.82 (1H, s),
8.95 (1H, s), 8.42 (1H, d, J = 5.7 Hz), 7.99 (2H, m), 7.67 (1H, s), 7.56
(1H, m), 7.43 (m, 2H), 7.32 (m, 4H), 7.22 (s, 2H), 6.94 (m, 1H), 6.47
(d, J = 5.7 Hz, 1H), 6.39 (m, 1H), 5.96 (m, 1H), 5.19 (m, 1H), 4.67
(dd, J = 4.5, 10.0 Hz, 1H), 4.49 (dd, J = 3.0, 10.0 Hz, 1H), 4.43 (s,
2H), 4.20 (s, 2H), 3.58−3.80 (m, 16H), 2.06 (s, 6H). 13C NMR
(CDCl3, 500 MHz) δ (ppm) 169.2, 166.2, 162.2, 159.0, 158.9, 150.3,
150.1, 143.6, 142.5, 133.7, 133.4, 132.8, 132.1, 131.2, 129.7, 129.1,
128.7, 126.8, 120.2, 119.5, 118.2, 104.2, 100.2, 99.0, 90.6, 90.1, 85.6,
85.2, 85.1, 77.0, 70.6, 70.5, 70.5, 70.5, 70.4, 70.3, 69.2, 69.2, 65.0, 59.2,
59.0, 16.2. HRMS (EI+) m/z calcd for C49H48N6O11 [M + H]+
897.3453, found 897.3464.
d4U-Propynyl-4-PEG-propynyl-TMC (1b). (For simplicity, we refer
to this structure as d4T-4PEG-TMC in text; see note above.) To a
solution of benzoate 1a (38.0 mg, 0.042 mmol) dissolved in methanol
(3 mL) at 0 °C was added a solution of sodium methoxide in
methanol (0.5 M, 0.1 mL). After 2 h, the solution was allowed to warm
to room temperature. Once TLC indicated the complete consumption
of starting material, acetic acid was added (3.0 mg, 0.05 mmol) and
methanol was removed on the rotoevaporator. The residue was
immediately chromatographed on silica gel with EtOAc/MeOH
mixtures to obtain nucleoside 1b (31.5 mg, 94%) as a colorless oil.
1H NMR (CDCl3, 500 MHz), δ (ppm) 11.54 (br s, 1H), 8.88 (br s,
1H), 8.38 (d, J = 5.2 Hz, 1H), 8.34 (s, 1H), 7.33 (m, 4H), 7.22 (s,
2H), 6.98 (m, 1H), 6.47 (d, J = 5.2 Hz, 1H), 6.34 (d, J = 5.8 Hz, 1H),
5.82 (d, J = 5.8 Hz, 1H), 4.94 (m, 1H), 4.44 (s, 2H), 4.32 (s, 2H), 3.94
(dd, J = 3.0, 11.2 Hz, 1H), 3.84 (dd, J = 2.0, 11.2 Hz, 1H), 3.60−3.80
(m, 16H), 3.48 (br s, 1H, OH), 2.06 (s, 6H). 13C NMR (CDCl3, 500
MHz) δ (ppm) 169.2, 162.4, 159.1, 158.9, 150.5, 150.2, 145.2, 143.6,
135.0, 132.8, 132.1, 131.3, 126.0, 120.1, 119.5, 118.3, 104.2, 99.4, 99.0,
90.1, 89.1, 87.7, 85.7, 85.0, 77.9, 70.5, 70.5, 70.5, 70.4, 70.4, 70.3, 69.2,
69.0, 62.7, 59.2, 59.2, 16.3. HRMS (EI+) m/z calcd for C42H44N6O10
[M + H]+ 793.3191, found 793.3204.
G
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
then analyzed by 15% nondenaturing polyacrylamide gel electro-
phoresis to ensure complete annealing. Concentrations of the
oligonucleotides were determined by UV absorbance at 260 nm by
use of calculated extinction coefficients.
Incorporation Assays. Incorporation experiments were performed
under single-turnover conditions: 50 μM d4TTP-4PEG-TMC and 10
mM MgCl2 were mixed with 250 nM RT (active sites) and 50 nM 5′-
labeled primer/template to initiate the reaction. Negative controls
were performed under identical conditions without MgCl2. The
reaction was allowed to proceed for 30 min at 37 °C, after which the
reaction was quenched with 0.3 M EDTA. Reaction products were
subjected to 20% denaturing polyacrylamide gel electrophoresis and
analyzed on a Bio-Rad Molecular Imager FX.
resulting structure was checked for consistency, and a second run of
300 steps was restarted form this point using the same degrees of
freedom, during which the energy decreased further to −187.2 kcal/
mol. The entire system was then allowed to relax for successive
optimizations of 100 and 300 steps, which reduced the total energy
from 8490.5 to −3398.0 kcal/mol. The Cα rms deviation of the final
structure to the initial 1rtd is only 0.27 Å, which shows that there was
relatively little distortion created by the process.
ASSOCIATED CONTENT
* Supporting Information
■
S
One figure showing the HPLC trace of d4TTP-4PEG-TMC
(1c). This material is available free of charge via the Internet at
http://pubs.acs.org.
IC50 Determination. RT (6 nM active sites, based on pre-steady-
state active-site determination) was preincubated for at least 15 min
with 1 μM 5′-labeled primer/template prior to mixing with
appropriate concentrations of inhibitor and allowed to incubate for a
minimum of 15 additional minutes on ice. DMSO concentrations were
kept constant at less than 2%. DMSO alone was added as a no-
inhibitor control for each set of experiments. Reactions were initiated
with the addition of 5 μM dNTP and 10 mM MgCl2 and were
quenched after 15 min at 37 °C with 0.3 M EDTA. All concentrations
represent final concentrations after mixing. Reaction products were
subjected to 20% denaturing polyacrylamide gel electrophoresis and
quantitated on a Bio-Rad Molecular Imager FX. Product formation
was plotted as a function of inhibitor concentration and fitted to a
hyperbola to generate IC50 curves. IC50 values are defined as the
concentration of inhibitor that inhibits steady-state single nucleotide
incorporation by 50%.
Molecular Modeling of [d4T-4PEG-TMC] Bound to HIV-1 RT/
Template/Primer. Creation of the molecular model of the bifunctional
nucleoside bound to the binary complex of RT/template/primer was
based on a composite of structural data. Since there is no crystal
structure of RT complexed with both a NRTI and a NNRTI, a model
was created by starting from the crystal structures of the RT template/
primer complex from Huang et al.38 (PDB entry 1rtd) and the Das et
al.27 complex of TMC120−R147681 NNRTI (PDB entry 1s6q). First,
both of these files were read into the UCSF Chimera program62 and
superimposed by use of the Matchmaker utility. A composite protein
was then created by combining residues 1a−92a, 108a−178a, 241a−
554a, and 3b−249b, template, primer, and four Mg2+ ions from 1rtd
and residues 93a−107a and 179a−240a and the ligand from 1s6q.
The initial 3D structure of the bifunctional ligand bf4 was created by
drawing it in ChemDraw (version 6.0, CambridgeSoft Corp.,
Cambridge, MA, 2000), followed by energy minimization using the
MM2 force field in Chem3D (version 5.0, CambridgeSoft Corp.,
Cambridge, MA, 2000) and writing as a PDB file. After reading this
structure in Chimera, the nucleotide end of this molecule was
superimposed on the dNTP of the composite protein created above,
and a few of the dihedrals on the poly(ethylene glycol) linker were
manually changed to g+ or g− to superimpose as best as possible the
NNRTI end on the TMC120 crystallographic ligand. The side-chain
torsions χ1 and χ2 of Y188a were adjusted by −30° and +30°,
respectively, to avoid a severe steric clash. Finally, the crystallographic
dNTP and TMC120 were then removed and the resulting complex
was exported as a PDB file.
AUTHOR INFORMATION
Corresponding Author
*Telephone 203-785-4526; fax 203-785-7670; e-mail karen.
anderson@yale.edu.
■
Author Contributions
∥C.M.B., T.J.S., and P.I. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH Grant GM49551 to K.S.A.,
NIH Grants AI44616 and GM32136 to W.L.J., and a Fulbright
Fellowship to R.H..
ABBREVIATIONS USED
■
HIV-1, human immunodeficiency virus type 1; RT, reverse
transcriptase; NRTIs, nucleoside reverse transcriptase inhib-
itors; NNRTIs, non-nucleoside reverse transcriptase inhibitors;
HAART, highly active antiretroviral therapy; dNTP, deoxy-
nucleoside triphosphate; d4T, β-D-(+)-2′,3′-didehydro-3′-deox-
ythymidine; 8-Cl-TIBO, 8-chlorotetrahydroimidazobenzodiaze-
pinone; WT, wild type; TEAB, triethylammonium bicarbonate;
DMSO, dimethyl sulfoxide; PEG, poly(ethylene glycol);
TBDMS, tert-butyldimethylsilyl; MW, molecular weight; Tris,
tris(hydroxymethyl)aminomethane
REFERENCES
■
(1) World Health Organization. Progress Report 2011: Global HIV/
AIDS response. http://www.who.int/hiv/pub/progress_report2011/
en/index.html (accessed January 14, 2013).
(2) World Health Organization. Antiretroviral therapy for HIV
infection in adults and adolescents: Recommendations for a public
health approach, 2010 revision. http://www.who.int/hiv/pub/arv/
adult2010/en/index.html (accessed January 14, 2013).
(3) Menendez-Arias, L. Molecular basis of human immunodeficiency
virus drug resistance: an update. Antiviral Res. 2010, 85, 210−231.
(4) Martinez-Cajas, J. L.; Petrella, M.; Wainberg, M. A. Clinical
significance and biological basis of HIV drug resistance. In
Antimicrobial Resistance and Implications for the Twenty-First Century;
Springer: New York, 2008; pp 231−261.
(5) Mansky, L. M. Retrovirus mutation rates and their role in genetic
variation. J. Gen. Virol. 1998, 79 (Pt. 6), 1337−1345.
(6) Coffin, J. M. HIV population dynamics in vivo: implications for
genetic variation, pathogenesis, and therapy. Science 1995, 267, 483−
489.
(7) Perelson, A. S.; Neumann, A. U.; Markowitz, M.; Leonard, J. M.;
Ho, D. D. HIV-1 dynamics in vivo: virion clearance rate, infected cell
life-span, and viral generation time. Science 1996, 271, 1582−1586.
(8) Rhodes, T.; Wargo, H.; Hu, W. S. High rates of human
immunodeficiency virus type 1 recombination: near-random segrega-
The creation of the model was completed by reading the PDB files
created by Chimera in Schrodinger’s Maestro 7.5 (version 7.5,
̈
Schrodinger, LLC, New York, NY, 2006) and adding the hydrogen
̈
atoms needed at protonation states appropriate to pH = 7. The
following set of energy minimizations were sequentially run with the
Impact program (version 4.0, Schrodinger LLC, New York, 2006)
̈
using the OPLS_2001 force field with a distance-dependent dielectric
ε = 4r, a 12 Å cutoff for nonbonded interactions, and the steepest-
descent algorithm. First, the newly created ligand and the segments of
the protein chains originally from the 1s6q structure (93a−107a and
179a−240a) as well as all the residues within 4 Å of those were
allowed to move, while the rest were kept fixed at their original
positions during a 100-step optimization during which the total energy
of the system decreased from 6.1 × 1014 to 927.0 kcal/mol. The
H
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
tion of markers one kilobase apart in one round of viral replication. J.
Virol. 2003, 77, 11193−11200.
(26) Udier-Blagovic, M.; Tirado-Rives, J.; Jorgensen, W. L. Validation
of a model for the complex of HIV-1 reverse transcriptase with
nonnucleoside inhibitor TMC125. J. Am. Chem. Soc. 2003, 125, 6016−
6017.
(27) Das, K.; Clark, A. D., Jr.; Lewi, P. J.; Heeres, J.; De Jonge, M. R.;
Koymans, L. M.; Vinkers, H. M.; Daeyaert, F.; Ludovici, D. W.; Kukla,
M. J.; De Corte, B.; Kavash, R. W.; Ho, C. Y.; Ye, H.; Lichtenstein, M.
A.; Andries, K.; Pauwels, R.; De Bethune, M. P.; Boyer, P. L.; Clark, P.;
Hughes, S. H.; Janssen, P. A.; Arnold, E. Roles of conformational and
positional adaptability in structure-based design of TMC125-R165335
(etravirine) and related non-nucleoside reverse transcriptase inhibitors
that are highly potent and effective against wild-type and drug-resistant
HIV-1 variants. J. Med. Chem. 2004, 47, 2550−2560.
(9) Levy, D. N.; Aldrovandi, G. M.; Kutsch, O.; Shaw, G. M.
Dynamics of HIV-1 recombination in its natural target cells. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 4204−4209.
(10) Goody, R. S.; Muller, B.; Restle, T. Factors contributing to the
inhibition of HIV reverse transcriptase by chain-terminating
nucleotides in vitro and in vivo. FEBS Lett. 1991, 291, 1−5.
(11) Bienstock, R. J.; Copeland, W. C. Molecular insights into NRTI
inhibition and mitochondrial toxicity revealed from a structural model
of the human mitochondrial DNA polymerase. Mitochondrion 2004, 4,
203−213.
(12) Bailey, C. M.; Anderson, K. S. A mechanistic view of human
mitochondrial DNA polymerase gamma: providing insight into drug
toxicity and mitochondrial disease. Biochim. Biophys. Acta 2010, 1804,
1213−1222.
(13) Brown, J. A.; Pack, L. R.; Fowler, J. D.; Suo, Z. Pre-steady-state
kinetic analysis of the incorporation of anti-HIV nucleotide analogs
catalyzed by human X- and Y-family DNA polymerases. Antimicrob.
Agents Chemother. 2011, 55, 276−283.
(14) Koczor, C. A.; Lewis, W. Nucleoside reverse transcriptase
inhibitor toxicity and mitochondrial DNA. Expert Opin. Drug Metab.
Toxicol. 2010, 6, 1493−1504.
(15) Desai, V. G.; Lee, T.; Delongchamp, R. R.; Leakey, J. E.; Lewis,
S. M.; Lee, F.; Moland, C. L.; Branham, W. S.; Fuscoe, J. C.
Nucleoside reverse transcriptase inhibitors (NRTIs)-induced expres-
sion profile of mitochondria-related genes in the mouse liver.
Mitochondrion 2008, 8, 181−195.
(16) Sarafianos, S. G.; Marchand, B.; Das, K.; Himmel, D. M.;
Parniak, M. A.; Hughes, S. H.; Arnold, E. Structure and function of
HIV-1 reverse transcriptase: molecular mechanisms of polymerization
and inhibition. J. Mol. Biol. 2009, 385, 693−713.
(17) Smerdon, S. J.; Jager, J.; Wang, J.; Kohlstaedt, L. A.; Chirino, A.
J.; Friedman, J. M.; Rice, P. A.; Steitz, T. A. Structure of the binding
site for nonnucleoside inhibitors of the reverse transcriptase of human
immunodeficiency virus type 1. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
3911−3915.
(18) Spence, R. A.; Kati, W. M.; Anderson, K. S.; Johnson, K. A.
Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleo-
side inhibitors. Science 1995, 267, 988−993.
(28) Vaccaro, J. A.; Parnell, K. M.; Terezakis, S. A.; Anderson, K. S.
Mechanism of inhibition of the human immunodeficiency virus type 1
reverse transcriptase by d4TTP: an equivalent incorporation efficiency
relative to the natural substrate dTTP. Antimicrob. Agents Chemother.
2000, 44, 217−221.
(29) World Health Organization. Antiretroviral therapy for HIV
infection in adults and adolescents, 2010. http://www.who.int/hiv/
pub/arv/adult2010/en/index.html (accessed January 14, 2013).
(30) Ruth, J. L.; Cheng, Y. Selective antiviral agents. The metabolism
of 5-propyl-2′-deoxyuridine and effects on DNA synthesis in herpes
simplex virus type 1 infections. J. Biol. Chem. 1982, 257, 10261−10266.
(31) Rong, F.-G.; Soloway, A. H. Synthesis of 5-tethered carborane-
containing pyrimidine nucleosides as potential agents for DNA
incorporation. Nucleosides Nucleotides 1994, 13, 2021−2034.
(32) Rong, F.-G.; Soloway, A. H.; Ikeda, S.; Ives, D. H. Synthesis and
biochemical activity of 5-tethered carborane-containing pyrimidine
nucleosides as potential agents for DNA incorporation. Nucleosides
Nucleotides 1995, 14, 1873−1887.
(33) Piao, D.; Basavapathruni, A.; Iyidogan, P.; Dai, G.; Hinz, W.;
Ray, A. S.; Murakami, E.; Feng, J. Y.; You, F.; Dutschman, G. E.;
Austin, D. J.; Parker, K. A.; Anderson, K. S. Bifunctional inhibition of
HIV-1 reverse transcriptase: a first step in designing a bifunctional
triphosphate. Bioorg. Med. Chem. Lett. 2013, 23, 1511−1518.
(34) Li, D.; Zhan, P.; De Clercq, E.; Liu, X. Strategies for the design
of HIV-1 non-nucleoside reverse transcriptase inhibitors: Lessons from
the development of seven representative paradigms. J. Med. Chem.
2012, 55, 3595−3613.
(35) Das, K.; Bauman, J. D.; Clark, A. D., Jr.; Frenkel, Y. V.; Lewi, P.
J.; Shatkin, A. J.; Hughes, S. H.; Arnold, E. High-resolution structures
of HIV-1 reverse transcriptase/TMC278 complexes: strategic
flexibility explains potency against resistance mutations. Proc. Natl.
Acad. Sci. U.S.A. 2008, 105, 1466−1471.
(36) Janssen, P. A.; Lewi, P. J.; Arnold, E.; Daeyaert, F.; de Jonge, M.;
Heeres, J.; Koymans, L.; Vinkers, M.; Guillemont, J.; Pasquier, E.;
Kukla, M.; Ludovici, D.; Andries, K.; de Bethune, M. P.; Pauwels, R.;
Das, K.; Clark, A. D., Jr.; Frenkel, Y. V.; Hughes, S. H.; Medaer, B.; De
Knaep, F.; Bohets, H.; De Clerck, F.; Lampo, A.; Williams, P.; Stoffels,
P. In search of a novel anti-HIV drug: multidisciplinary coordination in
the discovery of 4-[[4-[[4-[(1E)-2-cyanoethenyl]-2,6-
dimethylphenyl]amino]-2- pyrimidinyl]amino]benzonitrile (R278474,
rilpivirine). J. Med. Chem. 2005, 48, 1901−1909.
(19) Rittinger, K.; Divita, G.; Goody, R. S. Human immunodeficiency
virus reverse transcriptase substrate-induced conformational changes
and the mechanism of inhibition by nonnucleoside inhibitors. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 8046−8049.
(20) Iyidogan, P.; Anderson, K. S. Understanding the molecular
mechanism of sequence dependent tenofovir removal by HIV-1
reverse transcriptase: differences in primer binding site versus
polypurine tract. Antiviral Res. 2012, 95, 93−103.
(21) Basavapathruni, A.; Bailey, C. M.; Anderson, K. S. Defining a
molecular mechanism of synergy between nucleoside and nonnucleo-
side AIDS drugs. J. Biol. Chem. 2004, 279, 6221−6224.
(22) Basavapathruni, A.; Vingerhoets, J.; de Bethune, M. P.; Chung,
R.; Bailey, C. M.; Kim, J.; Anderson, K. S. Modulation of human
immunodeficiency virus type 1 synergistic inhibition by reverse
transcriptase mutations. Biochemistry 2006, 45, 7334−7340.
(23) Velazquez, S.; Alvarez, R.; San-Felix, A.; Jimeno, M. L.; De
Clercq, E.; Balzarini, J.; Camarasa, M. J. Synthesis and anti-HIV activity
of [AZT]-[TSAO-T] and [AZT]-[HEPT] dimers as potential
multifunctional inhibitors of HIV-1 reverse transcriptase. J. Med.
Chem. 1995, 38, 1641−1649.
(37) Hsiou, Y.; Ding, J.; Das, K.; Clark, A. D., Jr.; Hughes, S. H.;
Arnold, E. Structure of unliganded HIV-1 reverse transcriptase at 2.7 A
resolution: implications of conformational changes for polymerization
and inhibition mechanisms. Structure 1996, 4, 853−860.
(38) Huang, H.; Chopra, R.; Verdine, G. L.; Harrison, S. C. Structure
of a covalently trapped catalytic complex of HIV-1 reverse tran-
scriptase: implications for drug resistance. Science 1998, 282, 1669−
1675.
(39) Ding, J.; Das, K.; Moereels, H.; Koymans, L.; Andries, K.;
Janssen, P. A.; Hughes, S. H.; Arnold, E. Structure of HIV-1 RT/TIBO
R 86183 complex reveals similarity in the binding of diverse
nonnucleoside inhibitors. Nat. Struct. Biol. 1995, 2, 407−415.
(40) Younis, Y.; Hunter, R.; Muhanji, C. I.; Hale, I.; Singh, R.; Bailey,
C. M.; Sullivan, T. J.; Anderson, K. S. [d4U]-Spacer-[HI-236] double-
(24) Renoud-Grappin, M.; Fossey, C.; Fontaine, G.; Laduree, D.;
Aubertin, A. M.; Kirn, A. Imidazo[1,5-b]pyridazine-d4T conjugates:
synthesis and anti-human immunodeficiency virus evaluation. Antiviral
Chem. Chemother. 1998, 9, 205−223.
(25) Pontikis, R.; Dolle, V.; Guillaumel, J.; Dechaux, E.; Note, R.;
Nguyen, C. H.; Legraverend, M.; Bisagni, E.; Aubertin, A. M.;
Grierson, D. S.; Monneret, C. Synthesis and evaluation of “AZT-
HEPT”, “AZT-pyridinone”, and “ddC-HEPT” conjugates as inhibitors
of HIV reverse transcriptase. J. Med. Chem. 2000, 43, 1927−1939.
I
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
drug inhibitors of HIV-1 reverse-transcriptase. Bioorg. Med. Chem.
2010, 18, 4661−4673.
(41) Delbederi, Z.; Fossey, C.; Fontaine, G.; Benzaria, S.; Gavriliu,
D.; Ciurea, A.; Lelong, B.; Laduree, D.; Aubertin, A. M.; Kirn, A.
Synthesis and antiviral activity of C-5 substituted beta-D- and beta-L-
D4T analogues. Nucleosides Nucleotides Nucleic Acids 2000, 19, 1441−
1461.
(58) Mansuri, M. M.; Starrett, J. E.; Wos, J. A.; Tortolani, D. R.;
Brodfuehrer, P. R.; Howell, H. G.; Martin, J. C. Preparation of 1-(2,3-
dideoxy-β-D-glyceropent-2-enofuranosyl)thymine (d4T) and 2′,3′-
dideoxyadenosine (ddA): General methods for the synthesis of 2′,3′-
olefinic and 2′,3′-dideoxynucleoside analogs active against HIV. J. Org.
Chem. 1989, 54, 4780−4785.
(59) Zhang, Y. M.; Razler, T.; Jackson, P. F. Synthesis of
pyrimido[4,5-b]indoles and benzo[4,5]furo[2,3-d]pyrimidines via
palladium-catalyzed intramolecular arylation. Tetrahedron Lett. 2002,
43, 8235−8239.
(60) Kerr, S. G.; Anderson, K. S. Pre-steady-state kinetic character-
ization of wild type and 3′-azido-3′-deoxythymidine (AZT) resistant
human immunodeficiency virus type 1 reverse transcriptase:
implication of RNA directed DNA polymerization in the mechanism
of AZT resistance. Biochemistry 1997, 36, 14064−14070.
(61) Murakami, E.; Feng, J. Y.; Lee, H.; Hanes, J.; Johnson, K. A.;
Anderson, K. S. Characterization of novel reverse transcriptase and
other RNA-associated catalytic activities by human DNA polymerase
gamma: importance in mitochondrial DNA replication. J. Biol. Chem.
2003, 278, 36403−36409.
(62) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera–a
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605−1612.
(42) Ciurea, A.; Fossey, C.; Benzaria, S.; Gavriliu, D.; Delbederi, Z.;
Lelong, B.; Laduree, D.; Aubertin, A. M.; Kirn, A. Synthesis of 5-
alkenylated D4T analogues via the Pd-catalyzed cross-coupling
reaction. Nucleosides Nucleotides Nucleic Acids 2001, 20, 1655−1670.
(43) Laduree, D.; Fossey, C.; Renoud-Grappin, M.; Fontaine, G.;
Camara, F.; Gavriliu, D.; Ciurea, A.; Aubertin, A. M.; Kirn, A. Synthesis
of novel C-5 substituted d4T analogues bearing linker arms as
potential anti-HIV agents. Nucleosides Nucleotides 1999, 18, 883−884.
(44) Velazquez, S.; Tunon, V.; Jimeno, M. L.; Chamorro, C.; De
Clercq, E.; Balzarini, J.; Camarasa, M. J. Potential multifunctional
inhibitors of HIV-1 reverse transcriptase. Novel [AZT]-[TSAO-T] and
[d4T]-[TSAO-T] heterodimers modified in the linker and in the
dideoxynucleoside region. J. Med. Chem. 1999, 42, 5188−5196.
(45) Gavriliu, D.; Fossey, C.; Ciurea, A.; Delbederi, Z.; Sugeac, E.;
Laduree, D.; Schmidt, S.; Laumond, G.; Aubertin, A. M. Synthesis and
anti-HIV activity of [d4U]-[Trovirdine analogue] and [d4T]-
[Trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse
transcriptase. Nucleosides Nucleotides Nucleic Acids 2002, 21, 505−533.
(46) Ludovici, D. W.; De Corte, B. L.; Kukla, M. J.; Ye, H.; Ho, C. Y.;
Lichtenstein, M. A.; Kavash, R. W.; Andries, K.; de Bethune, M. P.;
Azijn, H.; Pauwels, R.; Lewi, P. J.; Heeres, J.; Koymans, L. M.; de
Jonge, M. R.; Van Aken, K. J.; Daeyaert, F. F.; Das, K.; Arnold, E.;
Janssen, P. A. Evolution of anti-HIV drug candidates. Part 3:
Diarylpyrimidine (DAPY) analogues. Bioorg. Med. Chem. Lett. 2001,
11, 2235−2239.
(47) Hernandez, E.; Galan, A.; Rovira, C.; Veciana, J. Synthesis of a
series of symmetrically disubstituted diacetylenes with polychlor-
ophenyl rings as side groups and linear polyether chains as spacers.
Synthesis 1992, 1992, 1164−1169.
(48) Hunter, R.; Muhanji, C. I.; Hale, I.; Bailey, C. M.;
Basavapathruni, A.; Anderson, K. S. [d4U]-Butyne-[HI-236] as a
non-cleavable, bifunctional NRTI/NNRTI HIV-1 reverse-transcriptase
inhibitor. Bioorg. Med. Chem. Lett. 2007, 17, 2614−2617.
(49) Burgess, K.; Cook, D. Syntheses of nucleoside triphosphates.
Chem. Rev. 2000, 100, 2047−2060.
(50) Thoresen, L. H.; Jiao, G. S.; Haaland, W. C.; Metzker, M. L.;
Burgess, K. Rigid, conjugated, fluoresceinated thymidine triphos-
phates: syntheses and polymerase mediated incorporation into DNA
analogues. Chemistry 2003, 9, 4603−4610.
(51) Ludwig, J.; Eckstein, F. Rapid and efficient synthesis of
nucleoside 5′-O-(1-thiotriphosphates), 5′-triphosphates and 2′,3′-
cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphor-
in-4-one. J. Org. Chem. 1989, 54, 631−635.
(52) Vinogradov, S. V.; Zeman, A. D.; Batrakova, E. V.; Kabanov, A.
V. Polyplex nanogel formulations for drug delivery of cytotoxic
nucleoside analogs. J. Controlled Release 2005, 107, 143−157.
(53) Galmarini, C. M.; Warren, G.; Kohli, E.; Zeman, A.; Mitin, A.;
Vinogradov, S. V. Polymeric nanogels containing the triphosphate
form of cytotoxic nucleoside analogues show antitumor activity against
breast and colorectal cancer cell lines. Mol Cancer Ther 2008, 7, 3373−
3380.
(54) Maga, G.; Radi, M.; Gerard, M. A.; Botta, M.; Ennifar, E. HIV-1
RT inhibitors with a novel mechanism of action: NNRTIs that
compete with the nucleotide substrate. Viruses 2010, 2, 880−899.
(55) Spychala, J. A Facile preparation of N2-arylisocytosines. Synth.
Commun. 1997, 27, 1943−1949.
(56) Okada, M.; Yoden, T.; Kawaminami, E.; Shimada, Y.; Kudoh,
M.; Isomura, Y. Studies on aromatase inhibitors. III. Synthesis and
biological evaluation of [(4-bromobenzyl)(4-cyanophenyl)amino]-
azoles and their azine analogs. Chem. Pharm. Bull. 1997, 45, 482−486.
(57) Chabrier, P.; Seyden-Penne, J.; Fouage, A. M. C. R. Hebd.
Seances Acad. Sci. 1957, 245, 174−175.
J
dx.doi.org/10.1021/jm400160s | J. Med. Chem. XXXX, XXX, XXX−XXX