A Parallel Synthesis Approach to the Identification of Novel Diheteroarylamide-Based Compounds Blocking HIV Replication: Potential Inhibitors of HIV-1 Pre-mRNA Alternative Splicing

Article abstract of DOI:10.1021/acs.jmedchem.5b01357

A 256-compound library was evaluated in an anti-HIV screen to identify structural "mimics"of the fused tetracyclic indole compound 1 (IDC16) that conserve its anti-HIV activity without associated cytotoxicity. Four diheteroarylamide-type compounds, containing a common 5-nitroisobenzothiazole motif, were identified as active. In subsequent screens, the most potent compound 9 (1C8) was active against wild-type HIV-1_IIIB (subtype B, X4-tropic) and HIV-1 97USSN54 (subtype A, R5-tropic) with EC₅₀'s of 0.6 and 0.9 μM, respectively. Compound 9 also inhibited HIV strains resistant to drugs targeting HIV reverse transcriptase, protease, integrase, and coreceptor CCR5 with EC₅₀'s ranging from 0.9 to 1.5 μM. The CC₅₀ value obtained in a cytotoxicity assay for compound 9 was >100 μM, corresponding to a therapeutic index (CC₅₀/EC₅₀) of approximately 100. Further comparison studies revealed that, whereas the anti-HIV activity for compound 9 and the parent molecule 1 are similar, the cytotoxic effect for compound 9 was, as planned, markedly suppressed.

Full text of DOI:10.1021/acs.jmedchem.5b01357

Article
pubs.acs.org/jmc
A Parallel Synthesis Approach to the Identification of Novel
Diheteroarylamide-Based Compounds Blocking HIV Replication:
Potential Inhibitors of HIV‑1 Pre-mRNA Alternative Splicing
‡
†
†
†
†
Peter K. Cheung, David Horhant, Laura E. Bandy, Maryam Zamiri, Safwat M. Rabea,
Stoyan K. Karagiosov, Mitra Matloobi, Steven McArthur, P. Richard Harrigan, Benoit Chabot,⊥
and David S. Grierson*
†
†
§
‡,∥
,†
†
§
∥
Faculty of Pharmaceutical Sciences, Department of Microbiology and Immunology, and Department of Medicine, The University
of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
‡
British Columbia Centre for Excellence in HIV/AIDS, 608-1081 Burrard Street, Vancouver, British Columbia V6Z 1Y6, Canada
⊥
Dep
́
artement de microbiologie et d’infectiologie, Faculte
́ ́ ́ ́
de medecine et des sciences de la sante, Universite de Sherbrooke, 3201,
rue Jean-Mignault, Sherbrooke, Queb
́
ec J1E 4K8 Canada
*
S Supporting Information
ABSTRACT: A 256-compound library was evaluated in an anti-HIV screen to identify structural “mimics” of the fused
tetracyclic indole compound 1 (IDC16) that conserve its anti-HIV activity without associated cytotoxicity. Four
diheteroarylamide-type compounds, containing a common 5-nitroisobenzothiazole motif, were identified as active. In
subsequent screens, the most potent compound 9 (1C8) was active against wild-type HIV-1IIIB (subtype B, X4-tropic) and HIV-1
9
7USSN54 (subtype A, R5-tropic) with EC ’s of 0.6 and 0.9 μM, respectively. Compound 9 also inhibited HIV strains resistant
50
to drugs targeting HIV reverse transcriptase, protease, integrase, and coreceptor CCR5 with EC ’s ranging from 0.9 to 1.5 μM.
50
The CC50 value obtained in a cytotoxicity assay for compound 9 was >100 μM, corresponding to a therapeutic index (CC50/
EC ) of approximately 100. Further comparison studies revealed that, whereas the anti-HIV activity for compound 9 and the
50
parent molecule 1 are similar, the cytotoxic effect for compound 9 was, as planned, markedly suppressed.
INTRODUCTION
that act through unexplored mechanisms of action, which
■
bypass resistance, display minimal toxicity, and address the
Anti-retroviral therapy (ART) has changed the prognosis of
HIV infection to the point where, in developed countries, it is
considered a chronic infection. In light of this success in
9
problem of activating the latent viral pool. We are exploring
the potential of new anti-HIV agents that block HIV replication
through perturbation of HIV pre-mRNA alternative splicing.
Through random screening of compound libraries several
1
reducing morbidity, significant steps are now being taken to
bring ART to the developing world where the majority of HIV
infected people reside. The necessity of these initiatives is
underscored by the fact that, in spite of progress, an estimated 2
million new infections occur and approximately 1.2 million
people die from AIDS per year. In practice, the advantage of
ART therapy is that the use of a combination of drugs reduces
the chance of viral escape, which results from acquired drug
resistance to each individual drug. However, there are inherent
long-term limitations to ART, which include the necessity for
10−16
molecules
have been identified that either directly or
indirectly interfere with the events whereby HIV pre-mRNA is
1
7−20
alternatively spliced.
Pertinent to the present work,
21
2
,3
screening of the Institut Curie compound collection identified
the fused tetracyclic indole compound 1 (IDC16) (Figure 1) as
a molecule that blocks replication of HIV through inhibition of
the exonic splicing enhancer activity of SRSF1. This SR
protein plays a crucial role in human RNA splicing, and it has
been implicated in alternative splicing, mRNA stability, and
10
4
5
strict adherence to treatment schedules, drug toxicity, and the
emergence of drug-resistant HIV strains, which are no longer
sensitive to the drug cocktails employed. These issues take on
22
6
translation. In HIV-1 infected cells it specifically regulates
alternative splicing events that are vital to the production of
added significance as the scale and diversity of the populations
7
,8
receiving treatment increases.
There is need for the
Received: September 2, 2015
continued development of new drugs, and in particular those
©
XXXX American Chemical Society
A
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Figure 1. Strategy for the parallel synthesis of compound 1 mimics, showing the different linker motifs 2−7 that were investigated, the general
formula 8 for the series of 119 diheteroarylamides synthesized, and the structures of the compounds 9−12 identified as active in the preliminary anti-
HIV screen.
critical HIV proteins including Tat, Rev, and Nef. The RNA-
binding proteins Tat and Rev are key regulators for the
expression of HIV-1 viral genes, for the synthesis of full-length
genomic RNA and, ultimately, for the production of progeny
supplementary binding interactions not exploited by 1 could be
located, which improve potency and/or target selectivity.
As described in the present report, the screening of a small
molecule library of structural mimics of 1 led to the
identification of four active diheteroarylamide (DHA)-type
compounds (Figure 1). The most potent hit in this new class of
anti-HIV agents was carried forward to determine its anti-HIV
profile against HIV mutant strains resistant to the current drugs
used in ART.
1
7,23
virions.
Compound 1 and other pyridocarbazole alkaloid analogues
have been developed primarily as cytotoxic agents, acting,
among other mechanisms, as DNA intercalators and/or
24−28
topoisomerase II inhibitors.
Consequently, although 1
blocks HIV replication at concentrations lower than the
threshold of its cytotoxic effect (see ref 10 and results in
Figure 5), it is not a suitable molecule to carry down the drug
development process as an anti-HIV drug destined for long-
term use. Be this as it may, the significant anti-HIV activity
displayed by this molecule strongly suggests that it interacts
with its target SR protein in a way that it disrupts its contact
with other spliceosome components. It is possible, therefore,
that the information contained in the structure of 1 (geometry,
polarity, presence of aromatic rings), which is at the root of its
anti-HIV activity, could be translated into a different and more
flexible type of molecule that maintains this mechanism of
action, without displaying affinity for DNA. Further, it is
anticipated that such molecules would bind more strongly in
the putative site than the rigid and flat compound 1.
Our approach to identify molecules with these features has
been to use parallel synthesis to construct a library of ring-
opened, and more conformationally mobile “mimics” of 1 and
to evaluate the library components for anti-HIV activity. In the
absence of high-resolution structural data for the SR proteins
and only a limited vision of their interactions at the molecular
level, the library design strategy adopted was to construct
molecules that retain a structural resemblance to the outline of
RESULTS
■
Chemistry and Preliminary Screen. The tetracyclic
indole compound 1 contains three nitrogen atoms in an
otherwise highly lipophilic polyaromatic structure. Taking as a
supposition that these polar heteroatoms play an important role
in interactions with the SR protein, the principle objective in
the synthesis of mimics of this molecule was to generate
structures that position nitrogen and/or oxygen functionality
into the same space occupied by its indole and A/D-ring
nitrogen atoms. Further, as the B- and C-rings in 1 may be
contributing little to binding, the idea was to replace one or
both of these rings by an appropriate spacer element that
connects the different motifs used as alternatives to the A- and
D-rings (Figure 1). Spacer elements explored, which replace
rings B and C, included a 1,2,4-oxadiazole motif 2, an extended
1
,2,4-oxadiazole-3-carboxamide system 3, and a simple amide
function 4. Fused bicyclic A/B-type ring systems, where the B-
ring is part of the spacer element and the C-ring is eliminated
included imidazopyridine 5, pyrazolopyridine 6, and pyridoi-
midazolone 7 systems. Using a parallel synthesis approach, a
diverse library of 256 compound 1 mimics was prepared by
combining A-and A/B-ring components with different D-ring
motifs (see Table 1 in the Supporting Information for a
complete listing of all compounds in the library).
1, when projected onto a flat (two-dimensional) surface. Of
importance was to also include molecules in the library that
incorporate functionality and/or structural motifs that project
beyond the outline of 1. It was expected that, in this way,
Focusing in on the subset of IDC16 mimics containing an
amide linker, a total of 119 compounds, represented by general
formula 8, were prepared by reaction of the acid chloride
B
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Figure 2. Structures of the different A- and D-ring subunits employed in the synthesis of the 119 dihereroarylamides 8 present in the compound
library.
derivative of the selected A-ring carboxylic acid components
with a diverse set of functionalized aromatic and heteroaromatic
amines (Figure 2). Looking at the set of D-ring components
explored, one see that, in some cases, substituents were situated
at different positions of the aromatic and heteroaromatic ring
that are similar in size to the chlorine atom in 1. In other cases,
more extended heteroatom-containing side chains were ex-
plored, and in still other cases the D-ring alternatives
corresponded to fused and nonfused bicylic motifs.
(3C2), 11 (2D3) and 12 (1E5) (microplate number and
compound location) (Figure 1), were identified as active,
inhibiting HIV-1 replication in the low micromolar range (see
Figure 1a−d in the Supporting Information for the screening
results). The immediately apparent feature of these molecules
was that, whereas the A-ring motif could be varied among
different pyridine/pyridinone structures, they all had in
common the presence of a 5-nitro substituted benzo[d]-
isothiazole motif as the D-ring subunit.
Preliminary evaluation of these and the other library
molecules was carried out in a cell-based anti-HIV screen, in
which reporter CEM-GXR cells were infected by coculture with
The four step preparation of compound 9 involved (Scheme
1a): (i) the conversion of 4-chloropyridine 13 to 4-
chloronicotinic acid 14 through reaction with LDA followed
30
31
1
% HIV-1NL4‑3 infected (GFP positive) cells at the start of the
by CO gas; (ii) refluxing this intermediate in H O at reflux;
2
2
32
assay (day 0). CEM-GXR cells are an immortalized CD4+ T-
lymphocyte line that expresses GFP due to the activation of the
(iii) reaction of the derived 4-pyridinone 15 with excess
33
methyl iodide to give 16 (using KHCO rather than NaOH as
3
29
Tat-dependent promoter upon HIV-1 infection. A different
library molecule in DMSO was added to the culture in each
microplate well immediately after the inoculation by infected
cells. The final library compound concentration was fixed at 2
μM, and the final DMSO concentration was <0.1%. The
percent of infected cells in the culture was determined by flow
cytometry on day 3. Four DHA-type compounds, 9 (1C8), 10
the base; 90% yield); and (iv) the reaction of the acid chloride
derivative of N-methyl-4-pyridinone-3-carboxylic acid 16 with
5-nitrobenzo[d]isothiazol-3-amine.
Compound 10 was prepared (Scheme 1a) by reaction of 4-
chloronicotinic acid 14 with the anion of 2-methoxyethanol to
give 17, followed by subsequent conversion of 17 to the acid
chloride and its reaction with 5-nitrobenzo[d]isothiazol-3-
C
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a
Scheme 1
was selected for further antiviral activity testing and therapeutic
index evaluation.
Given that the antiviral activity of anti-HIV drugs can vary
significantly due to the genetic variation of HIV-1 subtypes and
its coreceptor usage, 9 was evaluated in the above-described
anti-HIV screen assay of viral spread using different HIV-1
subtypes and tropisms. In the experiment, the culture contained
serial dilutions of molecule 9 in final concentrations between
1
00 nM and 10 μM, and the half maximal effective
concentration (EC ) values were calculated by nonlinear
50
regression with GraphPad Prism software. Compound 9
exhibited activity against wild-type HIV-1IIIB (subtype B, X4-
tropic), and HIV-1 97USSN54 (subtype A, R5-tropic) with
EC50 values of 0.6 and 0.9 μM, respectively (Figure 3A,B). To
validate these EC50 values, the anti-HIV drug, emtricitabine
(
(
FTC) was included in the anti-HIV assay as a positive control
Figure 3A). Serial dilutions of FTC in final concentrations
between 1 nM and 10 μM were tested, and an EC value of
5
0
35
0
.039 μM was determined.
To assess its potential against drug-resistant strains, the
antiretroviral activity of the 9 was tested against key HIV-1
strains resistant to at least one of the current four major drug
target categories: (non)-nucleoside reverse transcriptase
inhibitor (N)NRTI, protease inhibitor (PI), integrase inhibitor
(
INI), and coreceptor inhibitor (CRI). A summary of
mutations in HIV-1 proteins associated with resistance to
antiviral drugs can be found in Table 1.
a
Reagents and conditions: (i) (ref 30) LDA, THF, −78 °C, then CO ;
2
Compound 9 was evaluated against the isolate (E00443),
which greatly reduces susceptibility to both NNRTI’s and
NRTI’s. Indeed, this isolate harbors a variety of mutations,
including the widely recognized NNRTI-resistance mutation
K103N that confer high level resistance to efavirenz (EFV) and
nevirapine (NVP). This virus also carries the NRTI-resistance
mutations D67N, K70R, Y115F, Q151M, M184V, and K219Q,
which confer high-level resistance to lamivudine (3TC),
abacavir (ABC), zidovudine (AZT), and emtricitabine
(
ii) (ref 31) H O reflux 2 h; (iii) KHCO , DMF-H O, then MeI, 100
2
3
2
°C, 1 h; (iv) SOCl , RT or 90 °C, then 5-nitrobenzo[d]isothiazol-3-
2
amine, DMF or THF, Et N or K CO , rt, 10−24 h; (v) 2-
3
2
3
methoxyethan-1-ol, Na, 90 °C, then 4-chloronicotinic acid, 90 °C, 4
h; (vi) 2-bromoethyl methyl ether, Cs CO , DMF, 100 °C, 4 h; (vii)
2
3
LiOH, THF-H O, rt, 4 h.
2
amine. In a similar way (Scheme 1b), the commercially
available 2-methoxypyridine-4-carboxylic acid 18 was treated
with SOCl , and the derived acid chloride intermediate was
36
(FTC). In the assay compound 9 remained active, with an
EC50 of 1.3 μM (Figure 3C).
2
reacted with 5-nitrobenzo[d]isothiazol-3-amine to give 11.
To obtain compound 12 (Scheme 1c), the 4-hydroxyl group
Compound 9 similarly retained its potency (EC50 of 1.40
μM; Figure 3D) against the PI-resistant isolate (2948), which
contains substitutions G48V and L90M. This combination
reduces the susceptibility to atazanavir (ATV) and lopinavir
(LPV), currently the two of the three most frequently
3
4
in pyridinone 19 was O-alkylated to give 20 through reaction
with 2-methoxy-1-bromoethane, and the ester function in 20
was subsequently hydrolyzed to give the acid intermediate 21 in
high overall yield. Conversion of 21 to 12 was again achieved
through the acid chloride route. With the exception of
compound 12, the isolated yields for the diheteroarylamide
products in the library were generally low (2−30%), due to the
formation of multiple side products and consequent difficulties
encountered during column chromatography. However, all
diheteroarylamide compounds were obtained in >95% purity,
37
prescribed HIV protease inhibitors.
To determine whether 9 targets HIV-1 integrase, it was
tested against a clinical isolate (11845), which harbors G140S
and Q148H substitutions that reduce susceptibility to
raltegravir (RAL) and elvitegravir (EVG) more than 100-
38
fold and reduce sensitivity toward dolutegravir (DTG) up to
39
10-fold. Compound 9 showed activity against this isolate with
an EC50 of 1.36 μM (Figure 3E).
1
1
as determined by H NMR. In the H NMR spectra of the four
active compounds, the three peaks for the benzisothiazole motif
Lastly, 9 retained activity (EC50 of 0.9 μM; Figure 3F)
against an HIV-1 strain that is resistant to the entry inhibitor-
based drug maraviroc (MVC). This maraviroc-resistant variant
derived from a R5 strain of HIV-1BaL contains five mutations
within the envelope V3 loop: A19T, L20F, T22A, E25D, and
(
singlet between δ 8.85 and 9.40 for H-4′, and doublets for the
adjacent H-6′/7′ protons between δ 8.13 and 8.17 and δ 7.75
and 7.77) were readily distinguished from the absorptions for
the pyridine/pyridinone protons of the A-ring components.
Anti-HIV Activity of 9 against Wild-Type and Drug-
Resistant HIV Strains. Assessment of the four initial hits by
dose−response relations indicated that compounds 10 and 12
did not respond to the increasing doses. This correlates with
40
I26V.
The Cytotoxicity and Therapeutic Index of 9. To
establish that the observed antiviral activities of 9 were not the
result of cytotoxicity after exposure, therapeutic indexes were
determined. The therapeutic index (TI = CC /EC ) provided
their poor DMSO-H O solubility. In contrast, compounds 9
2
50
50
and 11 displayed good response. As the antiviral potency of 9
was approximately 3-fold higher than that for 11, compound 9
the relative effectiveness of compound 9 in antiviral activities
compared to inducing cell death. In other words, a high
D
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Figure 3. Anti-HIV-1 activity of 9 against wild-type subtypes A and B and drug-resistant viruses. (A) HIV-1IIIB (subtype B), (B) 97USSN54 (subtype
A), (C) (N)NRTI-resistant isolate, (D) PI-resistant isolate, (E) INI-resistant isolate, (F) Maraviroc-resistant R5 strain. The anti-HIV activity for
emtricitabine (FTC) as a positive control is also shown in (A). In the presence of increasing 9 concentrations, HIV-1 infectivity on CEM-GXR cells
was assessed by measuring GFP positive cells 3 days after infection. The inhibition curves were then fitted by nonlinear regression with GraphPad
Prism software, allowing EC50 calculation. Results represent the average of three independent determinations.
Table 1. Mutations in Drug-Resistant HIV-1 Strains
Associated with Resistance to ARV Drugs
therapeutic index signifies desirable antiviral activity with
minimal cell toxicity. The potential adverse effect of 9 on
CEM-GXR cells viability was determined by the Guava
ViaCount assay. This assay differentially stains viable and
nonviable cells based on their permeability to the DNA-binding
dyes and uses the forward scatter (FSC) properties to
distinguish free nuclei and cellular debris from intact cells to
quantify cell count. CEM-GXR cells were incubated with 9 at
concentrations ranging between 0.5 and 100 μM for 24 h. At
the highest concentration tested (100 μM of 9), a decrease in
cell viability from 98.0 to 68.8% (±6.8) (Figure 4) was
observed. The cytotoxic concentration resulting in the 50%
death of the host cells (CC ) was, thus, >100 μM. As the
a
drug-
resistant
HIV-1
strain
resistance to
ARV drugs
viral protein
primary mutations
E00443
reverse
transcriptase
K103N, D67N, K70R,
Y115F, Q151M, M184V,
K219Q
EFV, NVP, 3TC,
ABC, AZT,
FTC
2
1
948
protease
integrase
G48V, L90M
ATV, LPV
1845
G140S, Q148H
RAL, EVG,
DTG
MVC-res
envelop-V3
A19T, L20F, T22A, E25D, MVC
I26V
a
50
Abbreviations: efavirenz (EFV), nevirapine (NVP), lamivudine
3TC), abacavir (ABC), zidovudine (AZT), emtricitabine (FTC),
atazanavir (ATV), lopinavir (LPV), raltegravir (RAL), elvitegravir
EVG), dolutegravir (DTG), and maraviroc (MVC).
EC s in all the tested viruses ranged between 0.6 and 1.5 μM,
(
50
the calculated therapeutic indexes are >66−100 (Table 2).
Comparative Study of 1 and 9 on Cell Viability and
Anti-HIV Activity. To study further cell viability versus anti-
(
E
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four initial hit molecules. Although, at this stage, the screening
results provide only a primitive guide to future SAR studies on
this subunit, it is interesting to note the absence of activity for
the 4-pyridinone-based compounds 22 (1B8) and 23 (1D8)
and the 2-pyridinone molecules 24 (4B2) and 25 (4F3), which
contain a single five-membered heterocycle as the D-ring
subunit (Figure 6). It is further noteworthy that the isomeric
benzothiazole compounds 26 (4C2) and 27 (4A4), where the
benzo-ring has an “extended” spatial orientation versus the
“
bent orientation found in 9 and 12, were also inactive.
However, replacement of the benzisothiazole subunit by a
related isoquinoline motif, as in the 12-type compound 28
Figure 4. Evaluation of 9 on CEM-GXR cell viability in the Guava
ViaCount assay. Cells were analyzed after 24 h incubation with
compound 9 in concentrations ranging between 0.5 and 100 μM.
Results are expressed as the percentage (%) of viable cells ± SEM of
three independent experiments.
(
4C3), also resulted in a loss of activity.
Important to future SAR studies on the active mimics of 1
was the observation that considerable latitude exists to replace
the A-ring of 1 by alternate substructures. Indeed, the position
of the pyridine nitrogen atom relative to the carboxamide
function can be changed, a 2-pyridinone system is tolerated,
and the indole nitrogen in 1 can be replaced by the 4-
pyridinone carbonyl oxygen atom, as in 9, or a 2-
methoxyethanol side chain, as in 10 and 12. Interestingly,
compound 29 (2C3), lacking this oxygen substituted side
chain, is inactive.
Table 2. Antiviral Potencies of Compound 9 in Cells
Infected with Wild-Type or Drug-Resistant Virus Strains
a
WT and drug
resistant virus
resistance to drug
category
CC50
EC50
(μM)
therapeutic
b
c
d
(μM)
index
HIV-1IIIB
none
none
RTI
PI
>100
>100
>100
>100
>100
>100
0.6
0.9
1.3
1.4
1.5
0.9
>166
>111
>77
97USSN54
E00443
Importantly, the activity versus viability data in Figure 5
revealed a distinct advantage of 9 over 1 as an anti-HIV agent.
Both compounds exhibited comparable EC50 values and had
little effect on cell viability at 1 μM concentration. However, at
higher concentrations, toxicity toward 1 was strongly
manifested, whereas, the mimic 9 remained minimally toxic
to the CEM-GXR cells. This result was highly encouraging, as it
confirmed that, unlike 1, compound 9 displays no significant
affinity for DNA. However, when taking into consideration
long-term anti-HIV therapy, an important issue in moving
forward with development of this molecule is the presence of
the 5-nitro substituent on the isobenzoisothiazole motif. A
priority will thus be given to establishing whether the nitro is
crucial to activity, and if so, whether it can be replaced by a
2
1
948
>71
1845
INI
>66
MVC-res
CRI
>111
a
Abbreviations: reverse transcriptase inhibitor (RTI), protease
inhibitor (PI), integrase inhibitor (INI), and coreceptor inhibitor
b
(
CRI). Cytotoxic concentration resulting in the death of 50% of the
c
host cells (CC ) measured by ViaCount assay (Millipore). Effective
50
concentration resulting in 50% reduction of viral infected cells
d
measured by GFP expression signal. Therapeutic index calculated as
the ratio of CC50 to EC50.
HIV activity, a comparison study was made between 1 and the
mimic 9 (Figure 5A,B). Serial dilutions of both molecules in
final concentrations between 62.5 nM and 16 μM were added
in the culture containing either CEM-GXR cells alone for the
viability study or in one percent of HIV-1IIIB infected CEM-
GXR cells for antiviral activity assessment. At 1 μM
concentration, both molecules showed comparable antiviral
activities with 18.4% infected cells detected in 9 and 17.7%
detected in 1. The half-maximal effective concentration (EC )
41
suitable bioisostere motif, which is less prone to bioactivation.
Finally, having discovered a new prototype anti-HIV agent,
which was inspired by the structure of 1, it remains to
determine whether, like the parent compound, 9 blocks HIV
replication through perturbation of HIV-1 pre-mRNA alter-
native splicing. In this context, it is noteworthy that the anti-
HIV agent 30 (ABX-464), whose development was also
inspired by the structure of 1, interfered with HIV alternative
50
values for both molecules were calculated using the Igor Pro
software. Compounds 1 and 9 exhibited equivalent activity
against HIV-1IIIB with EC50 values of 0.959 ± 0.026 and 0.902
1
6
splicing in a manner that does not implicate SRSF1.
Considering that 1 and 9 are, in several ways, structurally
dissimilar, one cannot, therefore, assume that they share the
same mechanism of action. This line of reasoning was
reinforced by the fact that compound 31 (1H7) (Figure 6),
which mostly closely follow the outline of 1, was inactive. This
suggested that the amide linker in the DHA compounds may
not correctly emulate the B- and C-rings of 1 (i.e., capture the
same interactions these rings are implicated in upon binding to
the SR protein) and/or that the extra phenyl ring in the
bennzoisothiazole system captured additional interactions,
which compensate for this possible deficiency. To establish
the mode of action of 9, very detailed experiments have been
carried out, which both eliminated SRSF1 as the target of 9 and
confirmed that it deregulates HIV-1 pre-mRNA alternative
splicing in a complex process involving other SR proteins. The
±
0.113 μM, respectively. Similar effects on cell viability were
observed for both molecules at 1 μM, as 89.6% viable cells were
detected in 9 and 85.5% were found in 1. At higher
concentrations, however, the cell viability dropped considerably
for 1 (33.2% at 4 μM, 19.9% at 8 μM, and 11.9% at 16 μM)
reflecting its cytotoxicity, while only minimal effect on viability
was observed for 9 (81.5% at 4 μM, 80.9% at 8 μM, and 78.7%
at 16 μM).
DISCUSSION AND CONCLUSIONS
■
Taken together, the anti-HIV assay results strongly suggest that
compound 9 does not inhibit HIV replication through a
mechanism involving inhibition of the four current HIV protein
drug targets. Looking at the structure of this molecule, the
importance of the 5-nitro isobenzoisothiazole system to activity
was evident from its appearance as the D-ring component in all
42
results of these ongoing studies serve to validate the approach
taken to design 9 as HIV alternative splicing inhibitor. They
F
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Journal of Medicinal Chemistry
Article
Figure 5. Evaluation of anti-HIV-1 activity (column) and GXR-CEM cell viability (line) for 1 (A) and 9 (B) at concentrations between 62.5 nM and
6 μM (0 provides the value when no compound was added). HIV-1 infection in CEM-GXR cells was assessed by measuring GFP positive cells 3
1
days after infection. CEM-GXR cell viability was measured after 24 h incubation within the same range of concentrations. Results were expressed
from three independent experiments for both the anti-HIV-1 activity and cell viability assays.
further provide the tools necessary for continued research to
identify the optimal 9 analog that blocks HIV replication
without detrimental effect on normal cellular splicing/
alternative splicing processes.
compound 11 contained trace amounts (approximately 5%) of an
unidentified impurity (see 1H NMR spectrum in the Supporting
Information). For the biological assays: nelfinavir, efavirenz and 3′-
azido-3′-deoxythymidine (AZT) were obtained from Sigma-Aldrich
(St. Louis, MO). Maraviroc, raltegravir, elvitegravir, dolutegraver, and
emtricitabine were obtained from Cedarlane Laboratories (Ontario,
Canada). Indicator cell line CEM-GXR was obtained from Dr. Mark
Brockman (Simon Fraser University). The (N)NRTI-resistant virus
EXPERIMENTAL SECTION
■
Experimental General. All chemicals were purchased from Sigma-
Aldrich or Oakwood Chemicals, and they were used without
purification unless mentioned. All solvents were dried and kept
(
E00443) was obtained from Dr. Zabrina Brumme (Simon Fraser
University). The HIV-1 CXCR4-tropic laboratory-adapted strain NL4-
, and IIIB, HIV-1 CCR5-tropic BaL, integrase inhibitor-resistant virus
11845), protease inhibitor-resistant virus (2948), and HIV-1 subtype
1
13
3
(
under N . H and C NMR spectra were recorded at 400 and 100
2
MHz, respectively, on a Bruker AC 400 Ultrashield 10 spectropho-
tometer. Chemical shifts are expressed in ppm, (δ scale). When peak
multiplicities are reported, the following abbreviations are used: s
A (4114) were obtained through the NIH AIDS Research and
Reference Reagent Program, Division of AIDS, NIAID, NIH).
1-Methyl-N-(5-nitrobenzo[d]isothiazol-3-yl)-4-oxo-1,4-dihydro-
pyridine-3-carboxamide (9). Thionyl chloride (0.23 mL, 3.13 mmol,
(
singlet), d (doublet), m (multiplet), dd (doublet of doublet).
Coupling constants are reported in Hz. Low- and high-resolution mass
spectra were recorded on a AB Sciex UHPLC/MS/MS System and a
Thermo Scientific Q Exactive Orbitrap High-Resolution Mass
Spectrometer, respectively. Flash column chromatography was
performed using silica gel (Silicycle, Siliaflash F60, 40−63 μm, 230−
1
.2 equiv) was added dropwise to an ice-bath cooled suspension of 1-
methyl-4-oxo-1,4-dihydropyridine-3-carboxylic acid 16 (400 mg, 2.6
mmol) in dry CH Cl (6 mL) under a flow of nitrogen. After the
2
2
addition, the reaction mixture was warmed to room temperature and
then stirred at room temperature for 2 h. The resulting solution was
then concentrated under reduced pressure, and the crude acyl chloride
was dried in vacuo for 1 h. The solid product was dissolved in DMF (3
4
00 mesh) or on a Biotage Isolera purification system, (PartnerTech
Atvidaberg AB) using prepacked silica gel columns (Biotage, part no.
FSKO-1107-0010, FSKO-1107-0025, or FSKO-1107-0050). Inter-
mediates 15−21 and the final compounds 9, 10, and 12 were
mL) containing Et N (4.5 mL) and cooled to 0 °C. Then a solution of
3
1
confirmed to be >95% pure by H NMR spectroscopy. Final
5-nitrobenzo[d]isothiazol-3-amine (612.5 mg, 3.13 mmol) in DMF (8
G
DOI: 10.1021/acs.jmedchem.5b01357
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2
-Methoxy-N-(5-nitrobenzo[d]isothiazol-3-yl)isonicotinamide
(
11). Thionyl chloride (10 mL) was added to acid 18 (500 mg, 3.27
mmol) (commercially available), and the mixture was heated at 90 °C
for 1h. The excess of thionyl chloride was removed under reduced
pressure, and the crude acyl chloride was suspended in THF (30 mL).
Potassium carbonate (902 mg, 6.54 mmol, 2 equiv) and 5-
nitrobenzo[d]isothiazol-3-amine (637 mg, 3.27 mmol) were then
added, and the resulting mixture was stirred at room temperature for
1
0 h. The mixture was then filtered, and the solid material was washed
with THF. The filtrate was concentrated in vacuo and flash column
chromatographed (silica-gel: 0−2% MeOH in CH Cl ), providing 11
2
2
1
as a yellow solid (20 mg, 2%). H NMR (DMSO-d ) δ 13.18 (br s,
6
1
1
3
1
H), 9.70 (s, 1H), 8.47 (d, J = 5.2 Hz, 1H), 8.16 (dd, J = 1.2, 9.6 Hz,
H), 7.77 (d, J = 9.6 Hz, 1H), 7.63 (d, J = 5.2 Hz, 1H), 7.55 (s, 1H),
.97 (s, 3H); 13C NMR (DMSO-d ) δ 164.04, 148.12, 141.74, 120.70,
6
20.03, 115.48, 109.86, 53.81 (6 peaks for quaternary carbons are
−
missing); HRMS (HESI) m/z: calcd for C H N O S [M − H]
14
9
4
4
3
29.03500, found 329.03531.
-(2-Methoxyethoxy)-1-methyl-N-(5-nitrobenzoisothiazol-3-yl)-
-oxo-1,6-dihydropyridine-3-carboxamide (12). 4-(2-methoxye-
4
6
thoxy)-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic acid 21 (100
mg, 0.40 mmol) was dissolved in an excess of thionyl chloride (1 mL),
and the reaction mixture was heated at 85 °C for 1 h. The residual
thionyl chloride was then removed in vacuo. 5-nitrobenzo[d]-
isothiazol-3-amine (66 mg, 0.34 mmol) in THF (3 mL) containing
triethylamine (0.077 mL, 0.548 mmol) was then added, and the
reaction mixture was stirred under nitrogen at room temperature
overnight. The precipitate formed was removed by vacuum filtration,
and the concentrated filtrate was washed with acetone. The acetone
washed concentrate was silica-gel flash column chromatographed
(
EtOAc/MeOH; 9:1), providing compound 12 as a yellow solid (80
1
mg, 58%). H NMR (DMSO-d ) δ 12.34 (s, 1H), 9.36 (s, 1H), 8.54
6
(
4
s, 1H), 8.16 (d, 1H, J = 10 Hz), 7.75 (d, 1H, J = 10 Hz), 6.05 (s, 1H),
.28 (s, 2H), 3.81 (s, 2H), 3.50 (s, 3H), 3.22 (s, 3H); C NMR
1
3
(
DMSO-d ) δ 163.43, 162.93, 162.71, 161.83, 157.79, 145.62, 141.62,
6
1
3
4
22.27, 122.03, 119.95, 119.38, 104.40, 96.15, 69.58, 68.18, 58.06,
−
6.50; HRMS (HESI) m/z: calcd for C H N O S [M − H]
17
15
4
6
Figure 6. Library components providing cursory SAR on compound 9
and the structure of 30 (ABX-464).
03.07178; found 403.06946.
4
-Oxo-1,4-dihydropyridine-3-carboxylic acid (15). Adapting the
31
procedure by Ross, 4-chloronicotinic acid 14 (1.29 g, 0.77 mmol)
was dissolved in 20 mL of water and refluxed for 2 h. After the solution
was cooled in an ice bath, a sufficient quantity of conc. HCl was added
mL) was added dropwise, and the reaction mixture was warmed to
room temperature and stirred for 24 h. The DMF was removed under
reduced pressure, and the solid mixture was washed with acetone (30
mL), filtered and concentrated. The solid obtained was purified twice
(if necessary) to ensure that the pH ≤ 4. The precipitate that formed
was collected by suction filtration and washed with water. The aqueous
filtrate was then concentrated and suspended in approximately 10 mL
of water. After cooling at 0 °C, the precipitated material was again
by flash column chromatography (DCM/MeOH 95:5) to afford 9 as a
1
red-brown solid (80 mg, 9%). H NMR (DMSO-d ) δ 15.58 (s, 1H),
6
8
.85 (s, 1H), 8.75 (d, J = 2.0 Hz, 1H), 8.16 (dd, J = 9.6, 2.3 Hz, 1H),
collected by suction filtration, washed with Et O, and dried in vacuo to
afford 15 as a colorless solid (800 mg, 75%). H NMR (DMSO-d ) δ
2
8.04 (d, J = 7.10 Hz, 1H), 7.77 (d, J = 9.6 Hz, 1H), 6.75 (d, J = 7.10
1
13
6
Hz), 1H), 3.92 (s, 3H); C NMR (DMSO-d ) δ 176.41, 157.91,
6
1
2.94 (bs, 1H), 8.60 (s 1H), 8.07 (m, 1H), 6.73 (d, J = 7.1 Hz, 1H);
1
47.09, 143.80, 141.60, 122.45, 122.15, 120.08, 119.69, 118.22 (3
13
C NMR (DMSO-d ) δ 179.43, 166.73, 143.38, 141.34, 118.13,
6
peaks for quaternary carbons are missing); HRMS (HESI) m/z: calcd
for C H N O S [M + H] 329.03500, found 329.03531
[M − H]−
−
115.77; HRMS (HESI) m/z: calcd for C
38.01967, found 138.01811.
-Methyl-4-oxo-1,4-dihydropyridine-3-carboxylic acid (16).
H NO
6 4 3
14
9
4
4
1
4
-(2-Methoxyethoxy)-N-(5-nitrobenzo[d]isothiazol-3-yl)-
nicotinamide (10). Thionyl chloride (1.8 mL) was added to acid 17
50 mg, 0.25 mmol), and the mixture was heated at 90 °C for 1 h. The
1
Adapting the procedure in ref 32, 4-oxo-1,4-dihydropyridine-3-
carboxylic acid 15 (792 mg, 5.7 mmol) was dissolved in DMF/water
(
excess of thionyl chloride was removed under reduced pressure, and
the crude acyl chloride was suspended in THF (3 mL). Potassium
carbonate (70 mg, 0.51 mmol) and 5-nitrobenzo[d]isothiazol-3-amine
(5/1) (12 mL), and KHCO (590 mg, 5.9 mmol) was added. The
3
mixture was heated at 40 °C for 1 h. After cooling to room
temperature, a white solid was collected by suction filtration and
washed with DMF (2 mL). To the filtrate was added MeI (1.42 mL,
(
50 mg, 0.26 mmol) were then added, and the resulting mixture was
stirred at room temperature for 10 h. The solvent was removed in
0
.023 mol, 4 equiv), and the mixture was heated at 100 °C for 1 h. It
vacuo, and the solid obtained was silica-gel flash column chromato-
was then concentrated in vacuo, and the residue was washed with
acetone, filtered, and rinsed with a 1/1 mixture of acetone/MeOH.
The solid material was triturated with hot MeOH, and the MeOH
washes were concentrated and dried in vacuo to afford 16 as a
colorless solid (0.745 g, 85%). H NMR (DMSO-d
graphed (EtOAc to EtOAc/MeOH 9:1). Compound 10 was isolated
1
pure as a yellow solid (5 mg, 5%). H NMR (DMSO-d ) δ 13.05 (br s,
6
1
1
=
H), 9.45 (d, J = 2.3 Hz, 1H), 8.79 (br s, 1H), 8.66 (d, J = 5.9 Hz,
H), 8.16 (dd, J = 9.7, 2.3 Hz, 1H), 7.77 (d, J = 9.7 Hz, 1H), 7.37 (d, J
5.9 Hz 1H,), 4.37 (m, 2H), 3.71 (m, 2H), 3.20 (s, 3H); 13C NMR
1
) δ 8.70 (d, J = 2.3
6
(
DMSO-d ) δ 163.56, 162.82, 162.41, 157.79, 154,30, 150.66, 141,77,
Hz, 1H), 8.10 (dd, J = 7.8, 2.3 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 3.89
(s, 3H); 13C NMR (DMSO-d
) δ 178.29, 165.54, 147. 31, 145.43,
118.56, 115.60, 34.88; HRMS (HESI) m/z: calcd for C H NO [M +
6
1
22.27, 122.03, 120.02, 119.64, 118.36, 108.69, 69.79, 68.24, 58.17;
6
−
HRMS (HESI) m/z: calcd for C H N O S [M − H] 373.06121,
16
13
4
5
7
8
3
+
found 373.06107.
H] 154.04987, found 154.04977.
H
DOI: 10.1021/acs.jmedchem.5b01357
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4
-(2-Methoxyethoxy)nicotinic acid (17). A suspension of sodium
HIV-1 Inhibition Assay for 9. Compound 9 was further evaluated
in the human T-cell reporter assay based on CEM-GXR cells with the
following modifications. The inhibition assay was performed in a 96-
well plate and was inoculated by cells that were infected with different
viral strains of interest (Table 2). These strains of viruses were chosen
based on their subtypes (A and B), coreceptor usage (CCR5, and
CXCR4), and resistance to four major drug targets (reverse
transcriptase, protease, integrase, and coreceptor). The culture
contains serial dilutions of molecule 9 in the final concentrations
between 100 nM and 10 μM. The inhibition curves were then fitted by
nonlinear regression with GraphPad Prism software, allowing EC50
calculation. All data are reported as the mean ± s.e.m. from three
independent determinations.
(
9
1.15 g, 50 mmol) in 2-methoxyethan-1-ol (14 mL) was heated at
0°−100 °C under nitrogen until the sodium was consumed. 4-
Chloronicotinic acid 14 (1.07 g, 6.79 mmol) was then added in one
portion, and the reaction mixture was heated at 90 °C for 4 h. After
cooling to room temperature, the reaction mixture was poured into ice
water (75 mL) and extracted with CH Cl (3 × 30 mL). The aqueous
phase was concentrated under reduced pressure to about 1/5 of the
initial volume and acidified with 6 N HCl at 0 °C until pH 3.5−4.0 was
attained. The precipitate formed was filtered, washed with ice water
and ether, and dried in vacuo. Subsequent crystallization from
ethanol/ether gave 17 (545 mg, 41%) as a white solid. H NMR
DMSO-d ) δ 12.96 (br s, 1H), 8.67 (s, 1H), 8.53 (d, J = 5.7 Hz, 1H),
3
3
2
2
1
(
6
7
.18 (d, J = 5.9 Hz, 1H), 4.26 (m, 2H), 3.68 (m, 2H), 3.31 (s, 3H);
ViaCount Viability Assay. The ViaCount assay was performed
with the guava easyCyte 8HT flow cytometer according to the
manufacturer’s manual (Guava Technologies, Inc., Hayward, CA,
1
3
C NMR (MeOH-d ) δ 171.96, 163.91, 146.39, 145.92, 121.17,
4
1
13.20, 72.58, 71,21, 59.53; HRMS (HESI) m/z: calcd for C H NO
9
12
4
4
+
USA). In brief, CEM-GXR cells were seeded in 96-well plates 8 × 10
[
M + H] 198.07608, found 198.07617.
Ethyl 4-(2-Methoxyethoxy)-1-methyl-6-oxo-1,6-dihydropyridine-
cells/well in the absence or presence of compound 9 in concentrations
ranging between 0.5 and 100 μM. Twenty-four h later, 25 μL of cell
suspension was mixed with 225 μL of Guava ViaCount reagent, and
the mixture was incubated for 5 min at room temperature. Sample
acquisition and data analysis were performed with the selection of
EasyFit analysis feature using the ViaCount software module. All data
are reported as the mean ± s.e.m. from three independent
determinations.
34
3
-carboxylate (20). To a solution of 19 (4 g, 20.30 mmol) in DMF
(
120 mL) were added Cs CO (8.4 g, 25.78 mmol) and 2-bromoethyl
2 3
methyl ether (2.4 mL, 25.54 mmol). The yellow colored mixture was
stirred for 4 h at 100 °C and then concentrated under reduced
pressure. The resulting orange solid was taken-up in water and CH Cl2
and extracted with CH Cl (4 × 50 mL). The combined organic layers
were dried over Na SO and concentrated. The crude product mixture
2
2
2
2
4
was silica-gel flash chromatographed (0−2% of MeOH in EtOAc),
Antiviral Activity and Cell Viability for 1 and 9. The same
experimental protocols were followed as stated in the HIV-1 Inhibition
Assay for 9 and in the ViaCount Viability Assay with the following
exception: The concentration range for both 1 and 9 was chosen
between 62.5 nM and 16 μM. The half-maximal effective
1
providing compound 20 as a colorless solid (4.45 g, 86%). H NMR
(
CDCl ) δ 8.07 (s, 1H), 5.83 (s, 1H), 4.22 (dd, 2H, J = 14.3 Hz, J =
3
7
3
1
3
2
.1 Hz), 4.04 (t, 2H, J = 4.5 Hz), 3.75 (t, 2H, J = 4.2 Hz), 3.49 (s, 3H),
.41 (s, 3H), 1.30 (t, 3H, J = 7.1 Hz); 13C NMR (CDCl ) δ 165.74,
3
concentration (EC ) values were calculated in Igor Pro software in
63.92, 163.36, 145.14, 104.62, 97.09, 70.23, 68.35, 60.88, 59.37,
50
+
which 1 antiviral data were fit to a sigmoidal curve f(x) = a + [b/(1 +
7.29, 14.24; HRMS (HESI) m/z: [M + H] : calcd for C H NO
12
18
5
e[
=
̂
(c − x)/d])], while 9 antiviral data were fit to a hyperbolic curve f(x)
a + [bx/(1 + cx)].
56.11795; found 256.11765.
4
-(2-Methoxyethoxy)-1-methyl-6-oxo-1,6-dihydropyridine-3-car-
boxylic acid (21). A mixture of 20 (7.8 g, 30.6 mmol) and LiOH (2.2
g, 52.8 mmol) in THF/H O (2−1) (470 mL) was stirred for 4 h at
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01357.
2
■
room temperature. The mixture was then acidified to pH 1 using
S
concentrated HCl. The product was extracted with CH Cl , and the
2
2
aqueous layer was washed with CH Cl (4 × 25 mL). The combined
2
2
organic layers were dried over Na SO , filtrated, and concentrated to
2
4
1
afford 21 as a colorless solid (6.18 g, 89%). H NMR (CDCl ) δ 8.34
3
(
3
1
(
s, 1H), 5.99 (s, 1H), 4.26 (t, 2H, J = 9 Hz), 3.80 (t, 2H, J = 9 Hz),
Composition of the 256-membered library of compound
1 “mimics” (Table 1), plus bar graph presentation of the
preliminary anti-HIV screening results (Figure 1a−d),
and NMR ( H and C) and high-resolution mass
spectral data for the active compounds 9−12 and
synthetic intermediates 15−21 described in the Exper-
imental Section (PDF)
.57 (s, 3H), 3.45 (s, 3H); 13C NMR (CDCl ) δ 164.18, 163.85,
3
63.85, 147.03, 102.77, 97.58, 69.54, 68.96, 59.38, 37.73; HRMS
+
HESI) m/z: [M − H] : calcd for C H NO 228.08665 ; found
1
13
10
14
5
2
28.08621.
Preliminary Library Screen for Anti-HIV-1 Activity. The anti-
HIV-1 activity of the library molecules was measured using a human
29
T-cell reporter assay based on CEM-GXR cells. These cells naturally
express the HIV-1 receptor CD4 and coreceptor CXCR4 and have
been engineered to expresses the other HIV-1 coreceptor, CCR5.
Upon HIV-1 infection, CEM-GXR cells express GFP due to the
activation of an exogenous Tat-driven LTR-GFP expression cassette,
and the level of infection was monitored using flow cytometric analysis
SMILES string for all compounds prepared (XLS)
AUTHOR INFORMATION
Corresponding Author
*Phone +1 604 8273353. E-mail: dgrierso@mail.ubc.ca.
■
(
guavaSoft 2.2 software, Guava HT8, Millipore). Cells were grown in
RPMI-1640 media (Invitrogen) supplemented with 10% fetal calf
serum (Invitrogen) and 100 U per ml penicillin-streptomycin. During
propagation, CEM-GXR cells were selected with 0.2 mg/mL G418
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
(
Invitrogen) and 0.1 mg/mL hygromycin B (Calbiochem). Antiviral
activity was evaluated in the assay that measures inhibition of HIV-1
spread in a coculture of CEM-GXR cells containing 1% of HIV-1NL4−3
infected (GFP positive) cells. Infection was performed in T25 flasks or
Notes
The authors declare no competing financial interest.
6
4
in 96-well plates containing 1 × 10 or 8 × 10 CEM-GXR cells,
respectively. The library molecules at 2 μM concentration and
commercially available anti-HIV-1 reagents zidovudine 2 μM and
nelfinavir 100 nM were added to the culture immediately after the
inoculation by infected cells. All compounds were prepared in DMSO
at the final concentration in culture that were <0.1% DMSO. The
percent of infected cells in the culture was determined by flow
cytometry on day 3.
ACKNOWLEDGMENTS
■
This work was generously supported by grants from the
Canadian Institutes of Health Research (CIHR) (and
HOP90183 and HOP120238). Special thanks goes to Dr.
Florence Mahuteau-Betzer (Institut Curie, Fr) for providing a
sample of compound 1 (IDC16).
I
DOI: 10.1021/acs.jmedchem.5b01357
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Wainberg, M. A.; Speck, R. F.; Scherrr, D.; Tazi, J. Long lasting control
of viral rebound with a new drug ABX464 targeting rev-mediated viral
RNA biogenesis. Retrovirology 2015, 12, 30.
ABBREVIATIONS USED
■
CCR5, C−C chemokine receptor type 5; CXCR4, C−X−C
chemokine receptor type 4; SRSF1, serine-/arginine-rich
splicing factor 1; Tat, trans-activator of transcription; Rev,
regulator of viral protein expression; nef, negative regulatory
factor; GFP, green fluorescent protein; LTR-GFP, long
terminal repeat-green fluorescent protein
(17) Stoltzfus, C. M. Regulation of HIV-1 Alternative RNA splicing
and its role in virus replication. Adv. Virus Res. 2009, 74, 1−40.
(18) Karn, J.; Stoltzfus, C. M. Transcriptional and posttranscriptional
regulation of HIV gene expression. Cold Spring Harbor Perspect. Med.
2012, 4, a006916.
(19) Tazi, J.; Bakkour, N.; Marchand, V.; Ayadi, L.; Aboufirassi, A.;
Branlant, C. Alternative splicing: regulation of HIV-1 multiplication as
REFERENCES
■
a target for therapeutic action. FEBS J. 2010, 277, 867−876.
(
1) Logan, R. M.; Sterne, J. A.; Hernandez-Diaz, S.; Robins, J. M.;
(20) Hryckiewicz, K.; Bura, M.; Kowala-Piaskowska, A.; Bolewska, B.;
Sabin, C.; Bansi, L.; van Sighem, A.; de Wolf, F.; Costagliota, D.;
Lanoy, E.; Bucher, H. C.; von Wyl, V.; Casbona, J.; del Amo, J.;
Moreno, S.; Justice, A.; Goulet, J.; Lodi, S.; Philips, A.; Seng, R.;
Meyer, L.; Perez-Hoyos, S.; Garcia de Olalla, P.; Herman, M. A. The
effect of combined antiretroviral therapy on the overall mortality of
HIV-infected individuals. AIDS 2010, 24, 123−137.
2) Statistics: Worldwide. amfAR, The Foundation for AIDS
Research; http://www.amfar.org/About-HIV-and-AIDS/Facts-and-
Stats/Statistics--Worldwide/ (Accessed September 2, 2014).
3) Hankins, C. Overview of the current state of the epidemic.
Current HIV/AIDS Reports 2013, 10, 113−123.
4) Chesney, M. Adherence to HAART regimens. AIDS Patient Care
STDs 2003, 17, 169−177.
5) Deeks, S. G.; Lewin, S. R.; Havlir, D. V. The end of AIDS: HIV
infection as a chronic disease. Lancet 2013, 382, 1525−1533.
6) Paredes, R.; Clotet, B. Clinical management of HIV-1 resistance.
Antiviral Res. 2010, 85, 245−265.
7) Gupta, R. K.; Jordan, M. R.; Sultan, B. J.; Hill, A.; Davis, D. H. J.;
Gregson, J.; Sawyer, A. W.; Hamers, R. L.; Ndembi, N.; Pillay, D.;
Bertagnolio, S. Global trends in antiretroviral resistance in treatment-
naive individuals with HIV after rollout of antiretroviral treatment in
resource-limited settings: a global collaborative study and meta-
regression analysis. Lancet 2012, 380, 1250−1258.
8) Hamers, R. L.; Kityo, C.; Lange, J. M.; de Wit, T. F.; Mugyenyi, P.
Global threat from drug resistant HIV in sub-Saharan Africa. Br. Med.
J. 2012, 344, e4159.
Mozer-Lisewska, I. HIV RNA splicing. HIV & AIDS Review 2011, 10,
1−64.
21) Florent, J.-C. Chimiotheque de petites molecules, outil de
recherche pour la biologie chimique. Biol. Aujourd'hui 2013, 207, 39−
4.
22) Zhong, X. Y.; Wang, P.; Rosenfeld, M. G.; Fu, X. D. SR proteins
6
(
5
(
(
in vertical integration of gene expression from transcription to RNA
processing to translation. Mol. Cell 2009, 35, 1−10.
(23) Stoltzfus, C. M.; Madsen, J. M. Role of viral splicing elements
(
and cellular RNA binding proteins in regulation of HIV-1 alternative
splicing. Curr. HIV Res. 2006, 4, 43−45.
(
(24) Rivalle, C.; Wendling, F.; Tambourin, P.; Hloste, J. M.; Bisagni,
E.; Cherman, J. C. Antitumor amino-substituted pyrido[3′,4′:4,5]-
pyrrolo[2,3-g]isoquinolines and pyrido[4,3-b]carbazole derivatives:
synthesis and evaluation of compounds resulting from new sidechain
and heterocycle modifications. J. Med. Chem. 1983, 26, 181−185.
(
(
(25) Miller, C. M.; McCarthy, F. O. Isolation, biological activity and
(
synthesis of the natural product ellipticine and related pyridocarba-
zoles. RSC Adv. 2012, 2, 8883−8918.
(26) Poljakova, J.; Eckschlager, T.; Hrabeta, J.; Hrebakova, J.;
Smutny, S.; Frei, E.; Martinek, V.; Kizek, R.; Stiborova, M. The
mechanism of cytotoxicity and DNA adduct formation by the
anticancer drug ellipticine in human neuroblastoma cells. Biochem.
Pharmacol. 2009, 77, 1466−1479.
(
(27) Stiborova, M.; Poljakova, J.; Martinkova, E.; Borek-Dohalska, L.;
Eckschlager, T.; Kizek, R.; Frei, E. Ellipticine cytotoxicity to cancer cell
(
9) (a) Chun, T. W.; Fauci, A. S. HIV reservoirs: pathogenesis and
obstacles to viral eradication and cure. AIDS 2012, 26, 1261−1268.
b) Taube, R.; Peterlin, B. M. Lost in transcription: molecular
mechanisms that control HIV latency. Viruses 2013, 5, 902−927.
10) Bakkour, N.; Lin, Y.-L.; Marie, S.; Ayadi, L.; Mahuteau-Betzer,
F.; Nguyen, C. H.; Mettling, C.; Portales, P.; Grierson, D.; Chabot, B.;
Jeanteur, P.; Corbeau, P.; Tazi, J. Small-molecule inhibition of HIV
pre-mRNA splicing as a novel antiretroviral therapy to overcome drug
resistance. PLoS Pathog. 2007, 3, e159.
lines- a comparative study. Interdiscip. Toxicol. 2011, 4, 98−105.
(
28) Martinez, R.; Chacon-Garcia, L. The search of DNA-
(
intercalators as antitumoral drugs: what worked and what did not
work. Curr. Med. Chem. 2005, 12, 127−151.
(
(29) Brockman, M. A.; Tanzi, G. O.; Walker, B. D.; Allen, T. M. Use
of a novel GFP reporter cell line to examine replication capacity of
CXCR4- and CCR5-tropic HIV-1 by flow cytometry. J. Virol. Methods
2
(
006, 131, 134−142.
30) (a) Guillier, F.; Nivoliers, F.; Godard, A.; Marsais, F.;
(
11) Soret, J.; Bakkour, N.; Marie, S.; Durand, S.; Zekri, L.; Gabut,
Queguiner, G.; Siddiqui, M. A.; Sneikus, V. Combined metalation-
palladium-catalyzed cross coupling strategies. A formal synthesis of the
marine alkaloid amphimedine. J. Org. Chem. 1995, 60, 292−296.
M.; Fic, W.; Diva, G.; Rivalle, C.; Dauzonne, D.; Nguyen, C.-H.;
Jeanteur, P.; Tazi, J. Selective modification of alternative splicing by
indole derivatives that target serine-arginine-rich protein splicing
factors. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 8764−8769.
12) Wong, R. W.; Balachandran, A.; Ostrowski, M. A.; Cochrane, A.
Digoxin suppresses HIV-1 replication by altering viral RNA
(b) Marsais, F.; Trecourt, F.; Breant, P.; Queguiner, G. Directed
lithiation of 4-halopyridines: chemoselectivity, regioselectivity, and
application to synthesis. J. Heterocycl. Chem. 1988, 25, 81−87.
(31) Ross, W. C. J. The preparation of some 4-substituted nicotinic
acids and nicotinamides. J. Chem. Soc. C 1966, 1816−1821.
(32) Beak, P.; Covington, J. B.; Zeigler, J. M. Self-association and the
protomeric equilibria of 4-hydroxypyridine - 4-pyridone and related
compounds. J. Org. Chem. 1978, 43, 177−178.
(33) (a) Bernofsky, C. New synthesis of the 4- and 6-pyridones of 1-
methylnicotinamide and 1-methylnicotinic acid (trigonelline). Anal.
Biochem. 1979, 96, 189−200. (b) Di Marco, V. B.; Dean, A.; Ferlan, M.
G.; Yokel, R. A.; Li, H.; Venzo, A.; Bombi, G. G. Methyl-
hydroxypyridinecarboxylic acids as possible bidentate chelating agents
for aluminium(III): synthesis and metal-ligand solution chemistry. Eur.
J. Inorg. Chem. 2006, 2006, 1284−1293.
(
processing. PLoS Pathog. 2013, 9, e1003241.
13) Wong, R. W.; Balachandran, A.; Haaland, M.; Stoilov, P.;
(
Cochrane, A. Characterization of novel inhibitors of HIV-1 replication
that function via alteration of viral RNA processing and rev function.
Nucleic Acids Res. 2013, 41, 9471−9483.
14) Wong, R.; Balachandran, A.; Mao, A.; Dobson-Belaire, W.;
Grey-Owen, S.; Cochrane, A. Differential effect of Clk SR kinases on
(
HIV-1 expression: novel targets for therapy. Retrovirology 2011, 8, 47.
15) Fukuhara, T.; Hosoya, T.; Shimizu, S.; Sumi, K.; Oshiro, T.;
(
Yoshinaka, Y.; Suzuki, M.; Yamamoto, N.; Herzenberg, L. A.;
Herzenberg, L. A.; Hagiwara, M. Utilization of host SR protein
kinases and RNA-splicing machinery during viral replication. Proc.
Natl. Acad. Sci. U. S. A. 2006, 103, 11329−11333.
16) Campos, N.; Myburgh, R.; Garcel, A.; Vautrin, A.; Lapasset, L.;
Nadel, E. S.; Mahuteau-Betzer, F.; Najman, R.; Fornarelli, P.; Tantale,
(34) Wallace, E. M.; Lyssikatos, J.; Blake, J. F.; Seo, J.; Yang, H. W.;
Yeh, T. C.; Perrier, M.; Jarski, H.; Marsh, V.; Poch, G.; Livingston, M.
G.; Otten, J.; Hingorami, G.; Woessner, R.; Lee, P.; Winkler, J.; Koch,
K. Potent and selective mitogen-activated protein kinase kinase
(
K.; Basyuk, E.; Seveno, M.; Venables, J. P.; Pau, B.; Bertrand, E.;
J
DOI: 10.1021/acs.jmedchem.5b01357
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(
MEK) 1,2 inhibitors. 1. 4-(bromo-2-fluorophenylamino)-1-methyl-
pyridin-2(1H)-ones. J. Med. Chem. 2006, 49, 441−444.
35) Saag, M. S. Emtricitabine, a new antiretroviral agent with activity
against HIV and hepatitis B virus. Clin. Infect. Dis. 2006, 42, 126−131.
36) Wensing, A. M.; Calvez, V.; Gunthard, H. F.; Johnson, V. A.;
(
(
̈
Paredes, R.; Pillay, D.; Shafer, R. W.; Richman, D. D. Update of the
drug resistance mutations in HIV-1. Top. Antivir Med. 2014, 22, 642−
650.
(
37) Major HIV-1 drug resistance mutations updated March 9, 2015.
Stanford University; http://hivdb.stanford.edu/pages/download/
resistanceMutations_handout.pdf (accessed December 1, 2015).
(
38) Abram, M. E.; Hluhanich, R. M.; Goodman, D. D.; Andreatta, K.
N.; Margot, N. A.; Ye, L.; Niedziela-Majka, A.; Barnes, T. L.; Novikov,
N.; Chen, X.; Svarovskaia, E. S.; McColl, D. J.; White, K. L.; Miller, M.
D. Impact of primary elvitegravir resistance-associated mutations in
HIV-1 integrase on drug susceptibility and viral replication fitness.
Antimicrob. Agents Chemother. 2013, 57, 2654−63.
(
39) Underwood, M. R.; Johns, B. A.; Sato, A.; Martin, J. N.; Deeks,
S. G.; Fujiwara, T. The activity of the integrase inhibitor dolutegravir
against HIV-1 variants isolated from raltegravir-treated adults. JAIDS, J.
Acquired Immune Defic. Syndr. 2012, 61, 297−301.
(
40) Delobel, P.; Cazabat, M.; Saliou, A.; Loiseau, C.; Coassin, L.;
Raymond, S.; Requena, M.; Marchou, B.; Massip, P.; Izopet, J. Primary
resistance of CCR5-tropic HIV-1 to maraviroc cannot be predicted by
the V3 sequence. J. Antimicrob. Chemother. 2013, 68, 2506−2514.
(
41) Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.;
Callegari, E.; Henne, K. R.; Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.;
Nakai, Y.; O’Donnel, J. P.; Boer, J.; Harriman, S. P. A comprehensive
listing of bioactivation pathways of organic functional groups. Curr.
Drug Metab. 2005, 6, 161−225.
(
42) Shkreta, L.; Blanchette, M.; Toutant, J.; Wilhem, E.; Bell, B.;
Cochrane, A.; Harrigan, P. R.; Cheung, P. K.; Grierson, D. S.; Chabot,
B. submitted for publication.
K
DOI: 10.1021/acs.jmedchem.5b01357
J. Med. Chem. XXXX, XXX, XXX−XXX