1,2,3-triazoles as amide bioisosteres: Discovery of a new class of potent HIV-1 Vif antagonists

Article abstract of DOI:10.1021/acs.jmedchem.6b00247

RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC₅₀ = 6 μM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, Id (IC₅₀ = 1.2 μM). We identified several potent HIV-1 inhibitors from a Id based library including 5ax (IC₅₀ = 0.01 μM), 5bx (0.2 μM), 2ey (0.4 μM), 5ey (0.6 μM), and 6bx (0.2 μ M).

Full text of DOI:10.1021/acs.jmedchem.6b00247

Brief Article
pubs.acs.org/jmc
1,2,3-Triazoles as Amide Bioisosteres: Discovery of a New Class of
Potent HIV‑1 Vif Antagonists
Idrees Mohammed,† Indrasena Reddy Kummetha,‡ Gatikrushna Singh,† Natalia Sharova,§
Gianluigi Lichinchi,†,‡ Jason Dang,‡ Mario Stevenson,§ and Tariq M. Rana*,†,‡
†Program for RNA Biology, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037, United States
‡Department of Pediatrics, University of California San Diego School of Medicine, 9500 Gilman Drive, MC 0762, La Jolla, California
92093 United States
§Division of Infectious Diseases, Miller School of Medicine, University of Miami, Miami, Florida 33136, United States
S
* Supporting Information
ABSTRACT: RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G)
expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 μM) by
isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 μM). We identified several potent HIV-1
inhibitors from a 1d based library including 5ax (IC50 = 0.01 μM), 5bx (0.2 μM), 2ey (0.4 μM), 5ey (0.6 μM), and 6bx (0.2
μM).
Therefore, new antiviral regimens are needed to eliminate these
viral reservoirs.
INTRODUCTION
■
Since the start of the AIDS epidemic in 1981, this disease has
led to the death of >30 million people globally. Although the
overall growth of the epidemic appears to be slowing, there
were nearly three million new infections and an estimated 1.8
million AIDS-related deaths in 2010. Over the past two
decades, more than 25 anti-HIV drugs have been developed
targeting several different stages of the virus life cycle.1 Among
these agents, HIV-1 reverse transcriptase and protease
inhibitors, when used in combinations in the highly active
antiretroviral therapy (cART), have proven to be highly
effective in reducing AIDS-related mortality throughout the
world.2 However, the development of drug resistance and toxic
side effects associated with cART have created a need for more
potent and less toxic therapies against other viral targets and
host−virus interactions.3 In patients on effective cART, plasma
viremia can be suppressed to below detectable levels for
extended intervals and the ability of cART to sustain this
aviremic state has promoted the view that cART is fully
suppressive and effectively stops all ongoing viral replication.
However, there is rapid recrudescence of plasma viremia upon
treatment interruption, regardless of the prior interval of viral
suppression, indicating the presence of long-lived viral
reservoirs that maintain viral persistence in the face of cART.
The HIV-1 accessory protein viral infectivity factor, Vif, is
essential for in vivo viral replication.4,5 HIV-1 Vif protein
targets an innate antiviral human DNA-editing enzyme,
APOBEC3G (A3G),6 which inhibits replication of retro-
viruses.7 A3G catalyzes critical hypermutations in the viral
DNA and acts as an innate weapon against retroviruses.5 Cells
that express A3G are “non-permissive” in that viral replication is
absolutely dependent on a functional Vif. In contrast, HIV-1
replication is Vif-independent in host cells that do not express
A3G (permissive cells). Because HIV-1 Vif has no known
cellular homologues, this protein represents an extremely
attractive, yet unrealized, target for antiviral intervention.
The RN-18 based class of small molecule Vif antagonists
reduce viral infectivity by enhancing A3G-dependent Vif
degradation, increasing A3G incorporation into virions, and
enhancing cytidine deamination of the viral genome.8−10 RN-
18 (1a) exhibits IC50 values of 4.5 and 6 μM in CEM cells and
H9 cells (nonpermissive cells), respectively. RN-18 does not
inhibit viral infectivity in MT4 cell line (permissive cells) even
at 100 μM, demonstrating that these inhibitors are Vif-specific.
Received: February 17, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
a
These findings provided the proof of concept that the HIV-1
Vif-A3G axis is a valid target for developing small molecule
based new therapies for AIDS or for enhancing innate
immunity against viruses.
Scheme 1. Synthesis of Isosteric Analogues of RN-18
We faced two major challenges for further development of
RN-18 based Vif antagonists as clinical candidates: (a) potency
and (b) metabolic stability. To address these questions, we
planned to explore isosteric replacement of the amide
functionality in RN-18. We reasoned to test a series of
conformationally restricted, biocompatible and metabolically
stable isosteric heterocyclic systems. Next, on the basis of the
activity, we would select and develop a suitable bioisosteric11
series to improve the both activity and pharmacological profiles.
RESULTS AND DISCUSSION
■
In this communication, we describe the successful identification
of potent bioisosteric analogues of RN-18. Initially, we designed
and synthesized four test molecules by substituting the amide
functionality in the lead molecule with isosteric heterocyclic
systems such as 1,3,4-oxadiazole12 1b, 1,2,4-oxadiazole13 1c,
1,4-disubstituted-1,2,3-triazole14 1d, and 1,5-disubstituted-
1,2,3-triazole15 1e (Figure 1).
a
Reagents and conditions: (a) SOCl2, cat. DMF, benzene, 80 °C, 2 h;
(b) CH3OH, TEA, 0 °C−rt, 2 h; (c) NH2NH2·H2O, 80 °C, 3 h; (d) o-
anisic acid, POCl3, 110 °C, 8 h; (e) 4-nitrothiophenol, K2CO3, 5 mol
%, CuI, DMF, 110 °C, 8 h; (f) 2-iodobenzoic acid, DCC, DMF, rt to
100 °C, 8 h; (g) trimethylsilyl acetylene, 1 mol % PdCl2(PPh3)2, 1 mol
% CuI, NEt3, rt, 12 h; (h) NaOH (aq), ethanol/THF (1:1), rt, 1 h; (i)
NaN3, 10 mol % CuSO4·5H2O, CH3OH, rt, 8 h; (j) 1k, 5 mol %
CuSO4·5H2O, 10 mol % Na ascorbate, t-BuOH/H2O (1:1), rt,
overnight; (k) NaNO2, 5 N HCl, −10 to −5 °C, 2 h; (l) KI, −10 to
−5 °C, 8 h; (m) 1k, 1 mol % Cp*RuCl(PPh3)2, benzene, 80 °C, 3 h.
Figure 1. Amide bioisosteres of 1a, RN-18.
1,3,4-Oxadiazole 1b was synthesized with the coupling of
hydrazine and 2-iodobenzoic acid (Scheme 1A). The one pot
coupling involves the formation of in situ methyl ester of 2-
iodobenzoic acid, which was later refluxed in the presence of
hydrazine hydrate to obtain the benzohydrazide derivative 1f
quantitatively. Benzohydrazide 1f was later reacted with o-anisic
acid in refluxing phosphoryl chloride, leading to the formation
of iodo intermediate 1,3,4-oxadiazole 1g. Intermediate 1g was
reacted with 4-nitrothiophenol under copper(I) catalyzed S-
arylation conditions,16 leading to the formation of 1b. Synthesis
of 1,2,4-oxadiazole 1c was started (Scheme 1B) with the
coupling between the commercially available N′-hydroxy-2-
methoxybenzimidamide and 2-iodobenzoic acid using dicyclo-
hexyldicarbodiimide,17 leading to the formation of the iodo
intermediate 1,2,4-oxadiazole 1h. S-Arylation of 1h with 4-
nitrothiophenol under copper(I) catalytic conditions led to the
formation of 3,5-disubstituted-1,2,4-oxadiazole, 1c.
Synthesis of 1,4-disubstituted-1,2,3-triazole analogue 1d
required two synthons, 2-ethynylaniline 1j and 1-azido-2-
methoxybenzene 1k (Scheme 1C). 2-Iodoaniline was reacted
with trimethylsilylacetylene under Sonogashira reaction con-
ditions catalyzed by bis(triphenylphosphine)palladium chloride
in the presence of triethylamine base and copper iodide as
cocatalyst,18 leading to the formation of TMS protected
ethynylaniline 1i, which was deprotected using sodium
hydroxide, affording the required synthon 2-ethynylaniline 1j.
Azide 1k was synthesized by following a Cham-Lam type of
coupling between 2-methoxyphenylboronic acid and sodium
azide catalyzed by copper sulfate at room temperature in
methanol.19 Copper-catalyzed click reaction20 between alkyne
1j and azide 1k generated triazole amine 1l quantitatively in t-
butanol/water (Scheme 1D). Triazole amine 1l was diazotized
using sodium nitrite in 5 N HCl around −10 °C and
concomitantly converted to iodotriazole 1m by reacting with
potassium iodide. Copper(I) catalyzed S-arylation of iodo-
triazole 1m using 4-nitrothiophenol in DMF solvent and
potassium carbonate led to the synthesis of 1d, IMA-53. 1,5-
Disubstituted-1,2,3-triazole 1e analogue was synthesized
initially by reacting alkyne 1j and azide 1k under ruthenium
catalyzed click chemistry conditions using Cp*RuCl(PPh3)2
catalyst in benzene at 80 °C,21 leading to the formation of
amine 1n (Scheme 1E). Diazotization, iodination (1o), and S-
arylation reaction sequences afforded 1,5-disubstituted-1,2,3-
triazole 1e.
The antiviral activities of the four synthesized RN-18
analogues were measured against wild-type HIV-1 both in
B
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
nonpermissive H9 and permissive MT-4 cells (see details of
methods in Supporting Information (SI)). In all the antiviral
activity measurements, RN-18 (1a) was used as a positive
control and the cells cultured without any inhibitor served as a
negative control. The IC50 values of the bioisosteric analogues
of RN-18 are presented in Table 1. Both 1,3,4-oxadiazole 1b
These observations were well in-line with the structural
similarities in the 3D orientations except the 1,5-disubstituted-
1,2,3-triazole 1e, which has a twisted structure (see SI,
crystallographic data). 1,3,4-Oxadiazole and 1,2,4-oxadiazole
heterocyclic systems have both planarity and dipole moment
similar to the amide functionality. Similarly, 1,4-disubstituted
and 1,5-disubstituted 1,2,3-triazoles possess strong dipole
moment beside having better H-bond accepting (N(2) and
N(3)) and H-bond donating (triazole C(5)-H) capacity than
an amide functionality.22 However, in the current biochemical
context, 1,4-disubstituted-1,2,3-triazle 1d analogue showed
both improved antiviral activity (IC50 = 1.2 μM) and selectivity
(no activity in MT4 cells).
Having discovered 1d as a potent and specific inhibitor of
Vif-A3G axis, we decided to optimize the analogue to generate
a new class of anti-HIV drug candidates for clinical develop-
ment. We designed and synthesized an 84-membered library
using a parallel format exploring various substitution patterns in
ring-A, ring-C, and bridge A−B in the 1d structure (Table 2).
In this direction, the synthetic scheme for 1d (Scheme 1D) was
followed. Synthetic schemes (see SI, Schemes 1S−6S),
experimental procedures, and characterization data of all the
84 members of the library are given in the SI. Antiviral activities
of the library were determined against wild-type HIV-1 both in
nonpermissive H9 and permissive MT-4 cells. The IC50 values
for important compounds are presented in Table 2. Antiviral
activities of the complete library is given in the SI, Table 1S.
None of the 84 compounds exhibited antiviral activities at 50
μM in nonpermissive MT4 cells indicating the requirement of
Vif for their function, which is quite remarkable. Further
analysis of a selective set of potent compounds showed dose-
dependent inhibition of HIV-1 in H9 cells with no significant
toxicity at 50 μM as measured by MTS cell viability assays (SI,
Figures 1S,2S).
For a few selected compounds (2dx, 2ey, 2gy, 5ax, 5bx, 5gy,
and 5ey), we then determined whether the analogues could
upregulate A3G and downregulate Vif in a manner similar to
RN-18 and 1d. Immunoblots for A3G and Vif in the presence
of compounds are shown in Figure 3, which clearly showed that
the new inhibitors exert the anti-HIV activity via the same
mechanism as observed for RN-18 and 1d. Of the 84 members
library, about 30 compounds inhibited HIV-1 with IC5o values
in the range of 0.01−5 μM in the nonpermissive H9 cells.
Among them, the 5ax exhibited the most potent activity with
an IC50 of 10 nM, which is about 1000-fold more potent than
the original lead molecule, RN-18. Similarly, 2ey, 5bx, 5ey, and
6bx exhibited IC50 values in the range of 0.2−0.6 μM and 2ax,
2dx, 2ex, 2fx, 3ax, 3dx, 3fx, 3fy, 5ay, 6ex, 6fx, 6ey, and 6fy in
the range of 1−3 μM. Three water-soluble choline salts 2gy,
4gy, and 5gy exhibited IC50 values of 0.2, 0.7, and 0.5 μM,
respectively. Overall, the SAR of the library showed striking
sensitivity toward the three variables (Z-bridge, R1 and R2
substituents) tested in this study. Among various SAR findings,
few of the noteworthy ones are in general sulfide (−S−) as
bridge Z exhibited overall better activity compared with sulfone
(−SO2−) bridge (in the case of RN-18 sulfone derivative
showed better activity).9 However, sulfones (−SO2−) showed
better activities when the R2 substituent was an amino group.
This study has found replacements such as −COOCH3,
−COOH, −CF3, −NH2, and −choline carboxylate for the nitro
functionality in RN-18.
Table 1. IC50 Values of the Isosteric Analogues of RN-18
antiviral activity (IC50 μM)
compd
H9 cells
MT4 cells
a
1a, RN-18
6
NA
1b
1c
1d
1e
6.8
6.8
1.2
15
50
a
NA
a
NA
25
a
NA = no activity even at 50 μM conc.
(IC50 = 6.8 μM) and 1,2,4-oxadiazole 1c (IC50 = 6.8 μM) based
analogues exhibited cell-based antiviral activity in the non-
permissive H9 cells similar to the lead molecule RN-18 (IC50
=
6 μM). Interestingly, 2,5-disubstituted-1,3,4-oxadiazole 1b
showed nonspecific antiviral activity with IC50 of 50 μM in
permissive MT4 cells, whereas the 1,4-disubstituted-1,2,3-
triazole based analogue 1d exhibited remarkably better anti-
HIV activity (IC50 = 1.2 μM in H9 cells) and specificity (no
activity in MT4 cells). On the contrary, 1,5-disubstituted-1,2,3-
triazole 1e analogue exhibited comparatively lesser potency
(IC50 = 15 μM in H9 cells) with nonspecific activity in the
permissive cells (IC50 = 25 μM in MT4 cells).
Next, to determine the mechanism of these bioisosteres of
RN-18, we analyzed Vif degradation and rescue of A3G levels
in the presence of these compounds and compared with RN-18.
293FT cells coexpressing hemagglutinin (HA)-tagged A3G and
green fluorescent protein (GFP)-tagged Vif or ΔVif were
treated with various compounds (50 μM) for 16 h (see SI,
methods details). The cell extracts were then analyzed by
immunoblotting with anti-HA-A3G, anti-GFP-Vif, and anti-
GAPDH antibodies (Figure 2). All the bioisostere analogues of
RN-18 resulted in restoring A3G levels in the presence of Vif
and down-regulated Vif expression, indicating that these
analogues (1b, 1c, 1d, and 1e) are capable of antagonizing
Vif function similar to RN-18. However, analogues 1b and 1e
also exhibited some nonspecific activity (Table 1).
Figure 2. Bioisosteric analogues of RN-18 enhance A3G levels and
reduce Vif expression. 293FT cells coexpressing HA-tagged A3G and
GFP-tagged Vif or ΔVif were incubated in the presence (50 μM) or in
the absence of the compounds for 16 h. See SI for the structure of
negative control (8s). Anti-HA-A3G, anti-GFP-Vif, and anti-GAPDH
antibodies were used for immunoblotting (see SI for details).
C
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
Table 2. IC50 Values of the Library
antiviral activity (IC50 μM)
compd
Z
R1
R2
NO2
H9 Cells
Figure 3. Triaozle-based Vif antagonist small molecules enhance A3G
levels and reduce Vif expression. 293FT cells coexpressing HA-tagged
A3G and GFP-tagged Vif or ΔVif were incubated in the presence (50
μM) or in the absence of the compounds for 16 h. Choline chloride
was used as a negative control.
2ax (1d)
2dx
2ex
2fx
S
S
S
S
H
H
H
H
H
H
H
H
H
H
1.2
2.6
2.5
1.0
13.8
4.3
4.8
0.4
8.2
0.2
1.1
8
CF3
NH2
COOH
NO2
2ay
2cy
2dy
2ey
2fy
SO2
SO2
SO2
SO2
SO2
SO2
S
OCH3
CF3
EXPERIMENTAL SECTION
■
Details of general procedures and materials are described in the SI.
Parallel synthesis was performed using Carousel 6 (Radleys Discovery
NH2
COOH
1
Technologies). H and 13C NMR spectra were recorded using a 400
a
2gy
3ax
3bx
3dx
3fx
CC
MHz Jeol JNM-ECS spectrometer (equipped with a 5 mm proton/
multifrequency autotune and an auto sample changer) with
trimethylsilane (TMS) as the internal reference. The spectra are
reported in ppm on the δ scale. ESI MS was performed on Waters
micromass model ZQ 4000 using methanol. HRMS was performed on
Agilent Technologies 6224A MS-TOF. Purity of the tested
compounds was determined using Waters 2695 module HPLC
equipped with Waters 996 photodiode detector at 254 nm. Purity of
the final compounds mentioned in Tables 1 and 2 was found to be
≥95% in HPLC. X-ray structural determination was performed at
UCSD facility using Bruker diffractometer with CCD detectors and
low-temperature cryostats.
2-Iodobenzohydrazide (1f). A suspension of 2-iodobenzoic acid
(2.48 g, 10 mmol) and SOCl2 (1.43 g, 12 mmol) in dry benzene 25
mL was refluxed for about 2 h at 80 °C in the presence of catalytic
DMF (2 drops). Benzene and excess SOCl2 were removed under
reduced pressure. The residue obtained was slowly treated with
methanol (25 mL) at 0 °C, and triethylamine (5 mL) was added
followed by stirring at room temperature for 2 h. To the above mixture
hydrazine hydrate (1.0 g, 20 mmol) was added dropwise, and the
mixture was refluxed at 80 °C for about 3 h. TLC showed the
completion of the reaction. The reaction mixture was dried under
reduced pressure and extracted with AcOEt (2 × 25 mL). The organic
extract was sequentially treated with saturated solution of NaHCO3,
brine, and anhydrous Na2SO4. Flash column chromatography using
AcOEt:hexane (1:1) afforded colorless amorphous solid 1f (1.99 g,
76% yield).
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
3-OCH3
4-OCH3
4-OCH3
4-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
5-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-OCH3
6-F
NO2
S
COOCH3
CF3
S
1.9
2.8
4.3
4.7
12.4
1.4
7.1
12
S
COOH
a
3gx
3by
3ey
3fy
S
CC
SO2
SO2
SO2
S
COOCH3
NH2
COOH
COOH
CF3
4fx
4dy
4gy
5ax
5bx
5fx
SO2
SO2
S
a
CC
0.7
0.01
0.2
4.5
1.0
4.6
0.6
0.5
0.2
1.5
1.9
1.5
1.2
3.9
7.8
4.9
15
NO2
S
COOCH3
COOH
NO2
S
5ay
5by
5ey
5gy
6bx
6ex
6fx
SO2
SO2
SO2
SO2
S
COOCH3
NH2
a
CC
COOCH3
NH2
S
S
COOH
NH2
6ey
6fy
SO2
SO2
S
COOH
NO2
7ax
7bx
7fx
S
6-F
COOCH3
COOH
NH2
2-(2-Iodophenyl)-5-(2-methoxyphenyl)-1,3,4-oxadiazole
(1g). A 50 mL round-bottom flask was discharged with o-anisic acid
(0.30 g, 2 mmol) and benzohydrazide 1f (0.52 g, 2 mmol) followed by
the addition of POCl3 (8 mL). The suspension was refluxed at 110 °C
for 8 h until TLC showed depletion of the starting materials. The
reaction mixture was poured into cold saturated solution of K2CO3
followed by extraction with AcOEt (2 × 25 mL). The organic extract
was treated with saturated solution of NaHCO3 and dried over
anhydrous Na2SO4. TLC (AcOEt:hexane, 1:1) showed two new spots
with almost equal intensity. Flash chromatographic separation of the
upper spot using AcOEt:hexane (1:1) afforded colorless amorphous
solid 1g (0.41 g, 55% yield).
2-(2-Methoxyphenyl)-5-(2-((4-nitrophenyl)thio)phenyl)-
1,3,4-oxadiazole (1b). S-Aylation procedure described for the
synthesis of 1d (see below) was followed for the synthesis of 1b
using the intermediate 1g and 4-nitrothiophenol as starting materials.
Flash chromatography using AcOEt:hexane (1:3) afforded the
compound as a light-yellow amorphous solid (0.219 g, 82% yield),
which was crystallized using a mixture of DCM and methanol to afford
light-yellow crystalline 1b.
S
6-F
7ey
SO2
6-F
a
Choline carboxylate.
CONCLUSION
■
In summary, this study reports three major findings: (a) 1,4-
disubstituted-1,2,3-triazole system is a suitable bioisostere in the
RN-18 context, (b) discovery of a new class of potent Vif
antagonists as preclinical candidates for novel AIDS therapy,
and (c) generation of potent chemical modulators for
perturbing and understanding the Vif-A3G axis. Further
optimization of 1,4-disubstituted-1,2,4-oxadiazole, 1c, and
preclinical studies for the selected 1,4-disubstituted-1,2,3-
triazole based Vif antagonists are in progress.
D
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
5-(2-Iodophenyl)-3-(2-methoxyphenyl)-1,2,4-oxadiazole
(1h). A solution of 2-iodobenzoic acid (0.99 g, 4 mmol) in 15 mL of
dry DMF was cooled to 0 °C followed by the addition of
dicyclohexylcarbodiimide (1.24 g, 6.0 mmol) under N2 atmosphere,
and the reaction mixture was stirred for an hour at 0 °C. To the above
mixture was added commercially available N′-hydroxy-2-methoxyben-
zimidamide (0.664 g, 4 mmol) and stirred for 30 min at 0 °C. Then for
3 h stirring was continued at room temperature. Gradually, the
reaction mixture was heated up to 110 °C and kept for 8 h. The
reaction mixture was later poured into ice-cold water and diluted using
AcOEt (20 mL). Dicyclohexylurea crystals formed were separated by
filtration. Filtrate organic layer was treated with saturated solution of
K2CO3, brine, and anhydrous Na2SO4. Flash chromatography using
AcOEt:hexane (1:3) afforded the required 1h as a colorless amorphous
solid (1.2 g, 80% yield).
3-(2-Methoxyphenyl)-5-(2-((4-nitrophenyl)thio)phenyl)-
1,2,4-oxadiazole (1c). S-Aylation procedure described for the
synthesis of 1d was followed for the synthesis of 1c using the
intermediate 1h and 4-nitrothiophenol as starting materials. Flash
chromatography using AcOEt:hexane (1:3) afforded the compound as
a light-yellow amorphous solid (0.225 g, 84% yield), which was
crystallized using a mixture of DCM and methanol to afford light-
yellow crystalline 1c.
raphy using AcOEt:hexane (1:3) afforded the triazole amine 1l as a
light-brown amorphous solid (0.467 g, 88% yield).
4-(2-Iodophenyl)-1-(2-methoxyphenyl)-1H-1,2,3-triazole
(1m). In a 50 mL round-bottom flask triazole amine 1l (0.266 g, 1
mmol) was dissolved in 10 mL of 5 N HCl at 0 °C and stirred for 30
min. Sodium nitrite (82.8 mg, 1.2 mmol) dissolved in a minimum
amount of water was added dropwise to the above mixture at −10 °C.
Stirring was continued at −10 °C for a period of 2 h to get diazozium
salt in situ. Urea (∼50 mg) was added to the reaction mixture to
remove any excess nitrous acid generated in situ. In a separate beaker,
KI (0.249 g, 1.5 mmol) was dissolved in 5 mL of deionized water and
kept at −5 °C. To this solution of KI was added the diazonium
hydrochloride solution drop-by-drop using dropping funnel. After
addition, stirring was continued for a period of 8 h at room
temperature. The reaction mixture was later diluted with 20 mL of
AcOEt and 10 mL of deionized water. Small amount of iodine
liberated in the reaction was quenched by the addition of sodium
dithionite. Organic layer was separated and was sequentially treated
with saturated NaHCO3, saturated brine, and anhydrous Na2SO4
followed by adsorption on silica gel. Flash chromatography using
AcOEt:hexane (1:3) afforded 1m as a colorless amorphous solid
(0.293 g, 78% yield).
1-(2-Methoxyphenyl)-4-(2-((4-nitrophenyl)thio)phenyl)-1H-
1,2,3-triazole (1d). In a dry 25 mL two-neck round-bottom flask,
iodo triazole 1m (0.25 g, 0.66 mmol, 1 equiv) was dissolved in 5 mL of
dry DMF followed by the addition of anhydrous K2CO3 (0.110 g, 0.79
mmol, 1.2 equiv) and catalyst copper iodide (6.31 mg, 0.033 mmol, 5
mol %). The resulting mixture was stirred for 10 min under N2
pressure. 4-Nitrothiophenol (0.123 g, 0.79 mmol, 1.2 equiv) dissolved
in 2 mL of anhydrous DMF was added to the above reaction mixture
and stirred at 110 °C for 8 h. The reaction mixture was poured then
into ice-cold water followed by extraction with AcOEt (2 × 10 mL).
Organic extractions were treated sequentially with saturated K2CO3
solution and anhydrous Na2SO4. The dried organic extract was
adsorbed on silica gel and flash chromatography using AcOEt:hexane
(1:3) afforded 1d as a light-yellow amorphous solid (0.219 g, 82%
yield). The amorphous solid was crystallized using a mixture of DCM
and methanol to afford light-yellow crystalline 1d.
2-(1-(2-Methoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline (1n). 2-
Ethynylaniline 1j (0.234 g, 2 mmol) and 1-azido-2-methoxybenzene
1k (0.298 g, 2 mmol) were dissolved in 10 mL of anhydrous benzene.
To the above stirred solution, Cp*RuCl(PPh3)2 (15.90 mg, 0.02
mmol, 1 mol %) catalyst was added and the reaction mixture was
refluxed at 80 °C under N2 pressure for 3 h until TLC showed the
completion of the reaction. Benzene was removed under reduced
pressure, and the viscous residue was extracted with DCM (2 × 10
mL). The combined extractions were dried over Na2SO4 followed by
adsorption on neutral alumina. Flash chromatography using
AcOEt:hexane (2:3) afforded 1n as a brown amorphous solid (0.488
g, 92% yield).
2-((Trimethylsilyl)ethynyl)aniline (1i). In a dry 500 mL two-
necked round-bottom flask, 2-iodoaniline (25.0 g, 0.114 mol) was
dissolved in 250 mL of deoxygenated triethylamine. To this solution,
PdCl2(PPh3)2 catalyst (0.8 g, 1.14 mmol, 1 mol %) and copper(I)
iodide cocatalyst (0.217 g, 1.14 mmol, 1 mol %) were added. The
mixture was stirred for 15 min at room temperature under N2 pressure.
To this mixture, trimethylacetylene (11.21 g, 0.114 mol) was added
and stirred for 12 h at room temperature. Triethylamine was removed
under reduced pressure to get a crude viscous residue. The residue was
dissolved in AcOEt (250 mL) treated with saturated brine and Na2SO4
and adsorbed on neutral alumina. Flash column chromatography using
AcOEt:hexane (1:9) afforded pale-yellow liquid 1i (18.37 g, 85%
yield).
2-Ethynylaniline (1j). A 1 M aqueous solution of NaOH (2.64 g,
65.95 mmol, 1.2 equiv) was added to a solution of 2-ethynylaniline 1i
(18.0 g, 54.96 mmol, 1 equiv) dissolved in 200 mL of ethanol/THF
(1:1). Stirring was continued at room temperature for about 1 h until
TLC showed complete disappearance of the starting material. Organic
solvents were evaporated under reduced pressure, and the residue was
diluted by adding 50 mL of deionized water and extracted with DCM
(2 × 100 mL). Organic extractions were dried over brine and Na2SO4
and adsorbed on neutral alumina. Flash column chromatography using
AcOEt:hexane (1:4) afforded colorless pale-yellow liquid 1j (6.18 g,
96% yield).
1-Azido-2-methoxybenzene (1k). To a solution of 2-methox-
yphenylboronic acid (1.52 g, 10 mmol) in 20 mL of methanol, sodium
azide (0.78 g, 12.0 mmol) was added and stirred. To this mixture
CuSO4·5H2O (0.249 g, 1 mmol, 10 mol %) was added and stirred at
room temperature for about 8 h until TLC showed completion of the
reaction. Methanol was removed under reduced pressure, and the
residue was treated with saturated solution of sodium bicarbonate
followed by extraction with DCM (2 × 20 mL). Organic extractions
were dried over anhydrous Na2SO4 and adsorbed on silica gel. Flash
column chromatography using AcOEt:hexane (1:9) afforded colorless
dark-brown liquid 1k (1.34 g, 90% yield).
2-(1-(2-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)aniline (1l). 2-
Ethynylaniline 1j (0.234 g, 2 mmol) and 1-azido-2-methoxybenzene
1k (0.298 g, 2 mmol) were dissolved in 10 mL of a mixture of tert-
butanol and deionized water (1:1) in a 50 mL round-bottom flask. To
the stirred solution sodium ascorbate (39.62 mg, 0.2 mmol, 10 mol %)
and CuSO4·5H2O (24.97 mg, 0.1 mmol, 5 mol %) were added. Stirring
was continued overnight until TLC showed the completion of the
reaction. t-Butanol was removed under reduced pressure, and the
viscous residue was extracted with DCM (2 × 10 mL). The combined
organic extractions were treated with saturated brine and anhydrous
Na2SO4 followed by adsorption on neutral alumina. Flash chromatog-
5-(2-Iodophenyl)-1-(2-methoxyphenyl)-1H-1,2,3-triazole
(1o). Procedure described for the synthesis of intermediate 1m was
followed using 1n as starting material to afford colorless amorphous
solid 1o (0.282 g, 75% yield).
1-(2-Methoxyphenyl)-5-(2-((4-nitrophenyl)thio)phenyl)-1H-
1,2,3-triazole (1e). S-Arylation procedure described for the synthesis
of 1d was followed for the synthesis of 1e using the intermediate 1o
and 4-nitrothiophenol as starting materials. Flash chromatography
using AcOEt:hexane (1:3) afforded the compound as a light-yellow
amorphous solid (0.227 g, 85% yield), which was crystallized using a
mixture of DCM and methanol to afford light-yellow crystalline 1e.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00247.
E
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
(12) Ko, E.; Liu, J.; Perez, L. M.; Lu, G.; Schaefer, A.; Burgess, K.
Universal peptidomimetics. J. Am. Chem. Soc. 2011, 133, 462−477.
(13) Borg, S.; Vollinga, R. C.; Labarre, M.; Payza, K.; Terenius, L.;
Luthman, K. Design, synthesis, and evaluation of Phe-Gly mimetics:
heterocyclic building blocks of pseudopeptides. J. Med. Chem. 1999,
42, 4331−4342.
(14) Valverde, I. E.; Bauman, A.; Kluba, C. A.; Vomstein, S.; Walter,
M. A.; Mindt, T. L. 1,2,3-Triazoles as amide bond mimics: triazole scan
yields protease-resistant peptidomimetics for tumor targeting. Angew.
Chem., Int. Ed. 2013, 52, 8957−8960.
(15) Tam, A.; Arnold, U.; Soellner, M. B.; Raines, R. T. Protein
prosthesis: 1,5-disubstituted[1,2,3]triazoles as cis-peptide bond
surrogates. J. Am. Chem. Soc. 2007, 129, 12670−12671.
(16) Sperotto, E.; van Klink, G. P. M.; de Vries, J. G.; van Koten, G.
Ligand-free copper-catalyzed C-S coupling of aryl iodides and thiols. J.
Org. Chem. 2008, 73, 5625−5628.
(17) (a) Liang, G.-B.; Feng, D. D. An improved oxadiazole synthesis
using peptide coupling reagents. Tetrahedron Lett. 1996, 37, 6627−
6630. (b) Kumar, D.; Patel, G.; Johnson, E. O.; Shah, K. Synthesis and
anticancer activities of novel 3,5-disubstituted-1,2,4-oxadiazoles. Bioorg.
Med. Chem. Lett. 2009, 19, 2739−2741.
(18) Sonogashira, K.; Tohda, Y.; Hagihara, N. A convenient synthesis
of acetylenes: catalytic substitutions of acetylenic hydrogen with
bromoalkenes, iodoarenes and bromopyridines. Tetrahedron Lett.
1975, 16, 4467−4470.
(19) Tao, C.-Z.; Cui, X.; Li, J.; Liu, A.-X.; Liu, L.; Guo, Q.-X. Copper-
catalyzed synthesis of aryl azides and 1-aryl-1,2,3-triazoles from
boronic acids. Tetrahedron Lett. 2007, 48, 3525−3529.
(20) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed regiose-
lective “ligation” of azides and terminal alkynes. Angew. Chem., Int. Ed.
2002, 41, 2596−2599.
(21) Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.;
Sharpless, K. B.; Fokin, V. V.; Jia, G. Ruthenium-catalyzed cyclo-
addition of alkynes and organic azides. J. Am. Chem. Soc. 2005, 127,
15998−15999.
Details of general procedures, synthetic schemes,
characterization data, antiviral activities, and immuno-
blotting experiments (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 858-246-1100. Fax: 858-246-1600. E-mail: trana@
ucsd.edu.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported in part by grants from the NIH MH
100942 and DA 039562.
■
ABBREVIATIONS USED
■
APOBEC3G (A3G), apolipoprotein B mRNA editing enzyme
catalytic polypeptide like 3G; CC, choline carboxylate; DCM,
dichloromethane; DMF, dimethylformamide; TMS, trimethyl-
silyl; TLC, thin-layer chromatography; THF, tetrahydrofuran;
Vif, viral infectivity factor
REFERENCES
■
(1) Mehellou, Y.; Clercq, E. D. Twenty-six years of anti-HIV drug
discovery: where do we stand and where do we go? J. Med. Chem.
2010, 53, 521−538.
(2) Thompson, M. A.; Aberg, J. A.; Cahn, P.; Montaner, J. S. G.;
(22) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba,
G.; Genazzani, A. A. Click chemistry reactions in medicinal chemistry:
applications of the 1,3-dipolar cycloaddition between azides and
alkynes. Med. Res. Rev. 2008, 28, 278−308.
Rizzardini, G.; Telenti, A.; Gatell, J. M.; Gunthard, H. F.; Hammer, S.
̈
M.; Hirsch, M. S.; Jacobsen, D. M.; Reiss, P.; Richman, D. D.;
Volberding, P. A.; Yeni, P.; Schooley, R. T. Antiretroviral treatment of
adult HIV infection: 2010 recommendations of the International AIDS
Society-USA panel. JAMA 2010, 304, 321−333.
(3) Volderbing, P. A.; Deeks, S. G. Antiretroviral therapy and
management of HIV injection. Lancet 2010, 376, 49−62.
(4) Gabuzda, D. H.; Lawrence, K.; Langhoff, E.; Terwillliger, E.;
Dorfman, T.; Haseltine, W. A.; Sodroski, J. Role of vif in replication of
human immunodeficiency virus type 1 in CD4+ T lymphocytes. J.
Virol. 1992, 66, 6489−6495.
(5) Strebel, K.; Daugherty, D.; Clouse, K.; Cohen, D.; Folks, T.;
Martin, M. A. The HIV A (sor) gene product is essential for virus
infectivity. Nature 1987, 328, 728−730.
(6) Sheehy, A. M.; Gaddis, N. C.; Choi, J. D.; Malim, M. H. Isolation
of a human gene that inhibits HIV-1 infection and is suppressed by the
viral Vif protein. Nature 2002, 418, 646−650.
(7) Malim, M. H.; Bieniasz, P. D. HIV restriction factors and
mechanisms of evasion. Cold Spring Harbor Perspect. Med. 2012, 2,
a006940.
(8) Ali, A.; Wang, J.; Nathans, R. S.; Cao, H.; Sharova, N.; Stevenson,
M.; Rana, T. M. Synthesis and structure-activity relationship studies of
HIV-1 virion infectivity factor (Vif) inhibitors that block viral
replication. ChemMedChem 2012, 7, 1217−1229.
(9) Mohammed, I.; Parai, M. K.; Jiang, X.; Sharova, N.; Singh, G.;
Stevenson, M.; Rana, T. M. SAR and lead optimization of an HIV-1
Vif-APOBEC3G axis inhibitor. ACS Med. Chem. Lett. 2012, 3, 465−
469.
(10) Nathans, R.; Cao, H.; Sharova, N.; Ali, A.; Sharkey, M.; Stranska,
R.; Stevenson, M.; Rana, T. M. Small-molecule inhibition of HIV-1 Vif.
Nat. Biotechnol. 2008, 26, 1187−1192.
(11) Meanwell, N. A. Synopsis of some recent tactical application of
bioisosterism in drug design. J. Med. Chem. 2011, 54, 2529−2591.
F
DOI: 10.1021/acs.jmedchem.6b00247
J. Med. Chem. XXXX, XXX, XXX−XXX