Synthesis and structure-activity analysis of diphenylpyrazolodiazene inhibitors of the HIV-1 Nef virulence factor

Article abstract of DOI:10.1016/j.bmcl.2014.02.045

HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as 'B9', binds directly to Nef and inhibits its dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo.

Full text of DOI:10.1016/j.bmcl.2014.02.045

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1702–1706
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity analysis of diphenylpyrazolodiazene
inhibitors of the HIV-1 Nef virulence factor
Prema C. Iyer a,b, Jielu Zhao a,b, , Lori A. Emert-Sedlak c, Kerry K. Moore d, Thomas E. Smithgall b,c,
,
⇑
Billy W. Day a,b,d
a Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA 15261, USA
b University of Pittsburgh Drug Discovery Institute, Pittsburgh, PA 15261, USA
c Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Bridgeside Point II, Suite 523, 450 Technology Drive, Pittsburgh, PA 15219, USA
d Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery.
A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase acti-
vation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 repli-
cation assays against a broad range of primary Nef variants. This compound, known as ‘B9’, binds directly
to Nef and inhibits its dimerization in cells as a possible mechanism of action. Here were synthesized a
diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical
modifications to each of the three rings present in the parent compound were identified that did not com-
promise antiviral action. These analogs will guide the development of next-generation compounds with
appropriate pharmacological profiles for assessment of antiretroviral activity in vivo.
Received 2 January 2014
Revised 12 February 2014
Accepted 18 February 2014
Available online 26 February 2014
Keywords:
HIV-1
HIV Nef
Nef inhibitors
Antiretroviral drug discovery
Ó 2014 Elsevier Ltd. All rights reserved.
HIV/AIDS remains as a persistent global public health problem.
According to UNAIDS, more than 2.5 million people have died of
HIV-related causes worldwide since the pandemic began in 1981
and 34 million are currently living with the virus. The advent of po-
tent combination antiretroviral therapy in the mid-1990s changed
the course of the HIV epidemic from a life-threatening illness to a
chronic condition.1 These drugs target HIV-1 enzymes critical to
the viral life cycle as well as fusion of the virus to the host cell.
Although antiretroviral drug cocktails have increased the life
expectancy of infected individuals, they do not clear the virus
and require lifelong administration. However, the rise of multi-
drug-resistant strains of HIV-1,2 together with uncertain vaccine
prospects,3 underscore the urgent need for new antiretroviral
drugs with mechanisms of action complementary to existing
agents.1
as well as the gp41 envelope glycoprotein essential for host cell fu-
sion with the virus.1 HIV-1 also encodes four unique accessory pro-
teins (Nef, Vif, Vpu, and Vpr) which combat host cell restriction
factors, promote viral growth and represent alternative targets
for antiretroviral drug discovery.4 Of these, HIV-1 Nef is a particu-
larly attractive drug target because of its roles in HIV-1 infectivity,
viral replication and immune escape of HIV-infected cells.5 Early
studies established that Nef is required for high-titer replication
of SIV and disease progression in non-human primates.6 Remark-
ably, targeted expression of Nef alone in the CD4+ cell compart-
ment of transgenic mice is sufficient to cause a severe AIDS-like
N
N
Cl
HO
The HIV-1 genome shares three genes with other retroviruses
(Gag, Pol, and Env) and also encodes proteins essential for regula-
tion of viral transcription (Tat) and RNA splicing (Rev). Currently
approved antiretroviral drugs target products of the HIV-1 Pol
and Env genes, including reverse transcriptase, integrase, protease
Site 1
H2N
S
N
N
Site 3
Site 2
NO2
1
⇑
Corresponding author. Tel.: +1 (412) 648 8106; fax: +1 (412) 624 8997.
Figure 1. Structure of the diphenylpyrazolodiazene Nef antagonist B9 [1] with
analog modification sites indicated. Colors of each modification site match the bars
in the antiviral data set shown in Figure 2.
E-mail address: tsmithga@pitt.edu (T.E. Smithgall).
Present address: Proctor and Gamble, Inc. 8700 Mason Montgomery Rd, Mason,
OH 45040, USA.
http://dx.doi.org/10.1016/j.bmcl.2014.02.045
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

P. C. Iyer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1702–1706
1703
O
O
To circumvent this problem, Emert-Sedlak et al. developed an indi-
rect assay linking Nef to the activation of Hck, a Src-family kinase
and host cell effector protein for Nef.12,13 This kinase-coupled Nef
assay was then automated, enabling HTS of a large and diverse
chemical library of more than 220,000 compounds.13 Four com-
pound classes emerged from this screen that potently inhibited
HIV-1 infectivity and replication in multiple cell lines. In particular,
a unique diphenylpyrazolodiazene scaffold (1) was identified that
blocked Nef-dependent enhancement of HIV-1 replication with
submicromolar potency. Furthermore, this compound was shown
to bind directly to recombinant purified Nef protein by surface
plasmon resonance, and to inhibit Nef dimerization in a cell-based
fluorescence complementation assay. Other work has shown that
dimerization is critical to many Nef functions in the context of
HIV infection,14,15 supporting the idea that Nef dimerization block-
ers may represent a new class of antiretroviral therapies. In this
Letter, we describe the synthesis and virological evaluation of ana-
logs that are focused on systematic modifications to Sites 1–3 in
this compound (Fig. 1).
The re-synthesis of the diphenylpyrazolodiazene hit compound
1 was accomplished in three steps following a literature protocol.16
In this procedure, 3-chloroaniline was diazotized and treated with
ethyl 4-nitrobenzoylacetate to give the oxopropanoate intermedi-
ate 2, which was then treated with thiosemicarbazide to give 1
(Scheme 1). For Site 1 SAR, substituted anilines 3–9 and pyridines
10–11 were treated with ethyl 4-nitrobenzoylacetate to give the
oxopropanoate intermediates 12, which were then treated with
thiosemicarbazide to give compounds 13–21 (Scheme 2).
NH2
N2
OEt
NaNO2, CH3COOH,
HCl
O2N
Cl
NaOAc, MeOH
Cl
S
OEt
N
N
Cl
Cl
N
N
HO
H2N
S
N
H
NH2
O
N
N
H2N
O
CH3COOH
120 oC, 5 h
NO2
NO2
2
1
Scheme 1. Re-synthesis of hit compound, B9 [compound 1].
syndrome, supporting the singular importance of this viral protein
in HIV-1 pathogenesis.7 Conversely, strains of HIV-1 with defective
Nef alleles have been isolated from a subset of patients with long-
term, non-progressive HIV infection, implicating Nef as a critical
virulence factor for human AIDS.8–10 Taken together, these findings
make a strong case for Nef as a valid target for antiretroviral drug
discovery.11
Drug discovery campaigns targeting Nef have been hampered
by the lack of a screening assay. Nef does not exhibit any known
enzymatic or biochemical activity amenable to HTS assay design.
N2
NH2
55
54
53
O
O
OEt
X
X
NaNO2, CH3COOH,
HCl
Site 3
O2N
52
or
(urea)
or
N
51
50
49
40
NaOAc, MeOH
Y
NH2
N2
N
Y
39
Site 3
38
37
28
27
26
21
20
19
18
17
N
X
= 2-Cl
4-Cl
3-F
3
X
4
OEt
O
N
N
Y
OEt
O
N
N
5
Site 2
2-F
6
or
O
O
4-F
2-OMe
3-OMe
7
8
9
Y = Cl
10
11
NO2
NO2
H
12
X
Site 1
16
N
N
HO
15
14
13
S
H2N
N
N
B9 parent
S
X = 2-Cl
4-Cl
3-F
13
14
15
16
17
1
H2N
NO2
N
H
NH2
or
0
25
50
75
2-F
CH3COOH
120 oC, 5 h
4-F
N
HIV Infectivity, %Control
2-OMe 18
3-OMe 19
= Cl
H
Y
Figure 2. Assessment of Nef inhibitor action on Nef-dependent enhancement of
antiviral activity in the TZM-bl HIV infectivity assay. Compounds (3 M) were
N
N
HO
Y
20
21
l
S
added to cultures of TZM-bl reporter cells followed by infection with wild-type HIV-
1 (NL4-3 strain) in 96-well plates. Viral infectivity was assessed as luciferase
activity 48 h later as described.13 Data were corrected for luciferase activity
observed with cells infected with Nef-defective HIV, and are expressed as mean
percent of control values from untreated cells SEM. Nef enhances HIV infectivity
by 2- to 3-fold in this assay.13 Activity for compound 1 (red bar) is highlighted as a
reference control (dotted line).
N
N
H2N
NO2
Scheme 2. Synthesis of B9 Site 1 precursors 3–11 and analogs 13–21.

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P. C. Iyer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1702–1706
To assess the anti-HIV activity of the Site 1 analogs, we used the
electron-donating group (O-Me) at position 2 or 3 (18, 19) resulted
in a substantial loss of antiviral activity. Finally, pyridine substitu-
tion at Site 1 did not compromise activity, while addition of a
chloro group at position 5 of the pyridine ring reduced activity
somewhat (20, 21).
Modifications to Site 2 involved substituting the 4-nitro group
with electron-donating and withdrawing substituents (Scheme 3).
Replacement of the nitro group with a trifluoromethyl group (26)
maintained antiviral activity (Fig. 2). On the other hand, complete
removal of the nitro group (27) or replacement with an O-methyl
moiety (28) resulted in a significant loss of activity. These results
show that the nitro group can be effectively replaced by a triflu-
ormethyl substituent.
TZM-bl reporter cell line, in which the HIV-1 LTR is linked to the
transcription of luciferase.17 This system provides a convenient as-
say for initial events in the viral life cycle including attachment, en-
try, integration, and viral transcription, and is often used to assess
viral infectivity. Compound 1 has been previously shown to inhibit
Nef-dependent enhancement of HIV-1 infectivity in TZM-bl cells
with an IC50 value in the single-digit micromolar range.13
Compound 1 as well as analogs 13–21 were assayed for their
impact on Nef-dependent enhancement of HIV-1 infectivity, and
the results are shown in Figure 2. These results show that the posi-
tion of the chloro group is important for full activity, with the
4-chloro analog 14 equipotent to that of compound 1, while the
2-chloro analog 13 was less potent. Substitution of fluorine at
Site 1 positions 2, 3, or 4 (15–17) also compromised activity
relative to compound 1. Substitution of the halogen with an
To determine if the thiourea moiety is necessary for activity,
Site 3 modifications were made that removed the thiourea moiety
completely (37–40, Scheme 4) or substituted it with urea18 (49–55,
O
O
NH2
X
22
23
24
= CF3
= H
= OMe
N2
OEt
NaNO2, CH3COOH,
HCl
X
Cl
Cl
NaOAc, MeOH
N
S
Cl
H2N
HO
OEt
O
N
N
Cl
N
H
NH2
S
N
26
27
28
X
= CF3
= H
= OMe
O
N
H2N
CH3COOH
120 oC, 5 h
N
X
X
25
Scheme 3. Synthesis of Site 2 precursors 22–24 and analogs 26–28.
N2
NH2
O
O
OEt
NaNO2, CH3COOH
HCl
X
Z
X
or
or
NaOAc, MeOH
Y
NH2
N2
Z = F
= NO2
= H
33
34
35
N
N
Y
X = H
OMe
Y = H
Cl
29
30
31
32
X
X
N
N
OEt
N
N
HO
O
HN
N
O
37
38
X = H
OMe
Z = H
= NO2
Z
H2N NH2
Z
Z
or
or
CH3COOH
EtOH
N
N
Y
Z
Z = F
39
40
= Cl
H
= F
80 oC, 5 h
OEt
O
N
N
Y
N
N
Y
HO
HN
O
N
Z
Z
36
Scheme 4. Synthesis of Site 3 analogs, 37–40.

P. C. Iyer et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1702–1706
1705
NH2
or
N2
O
O
X
OEt
X
NaNO2, CH3COOH
HCl
Z
or
NaOAc, MeOH
Y
NH2
N2
Z
45
= NO2 46
= F
N
47
= H
N
X
41
= 3-Cl
Y
4-Cl 42
Y = H
5-Cl
43
44
X
X
OEt
O
N
N
N
N
HO
O
O
N
N
49
50
51
X
Z
= NO2
H2N
= 3-Cl
4-Cl Z = NO2
Z =
H2N
O
HN
Z
3-Cl
F
NH2
HCl
Z
or
or
Y = Cl Z = NO2
52
53
54
55
EtOH
Z
H
= NO2
Cl Z = F
80 oC, 5 h
N
N
Z
H
= F
Y
N
N
HO
OEt
N
N
Y
O
O
N
N
H2N
O
Z
Z
48
Scheme 5. Synthesis of Site 3 urea analogs, 49–55.
Scheme 5). Of the analogs lacking the thiourea, only compound 38
exhibited substantial antiviral effects, suggesting that substitution
at this position on the pyrazole ring is important for activity.
Replacement of the thiourea in the original structure with urea
(49) resulted in loss of activity, suggesting that the sulfur atom
plays a key role in antiretroviral activity. Previous docking studies
with compound 1 positioned the thiourea moiety between con-
served Nef residues Gln104, Gln107, and Asn 126 in the Nef dimer
interface. Modifications at this position, in the absence of other
changes, may impact this mode of interaction with the target pro-
tein. Of the remaining urea analogs, only compound 51 retained
appreciable antiviral activity, suggesting that substitution of a fluo-
rine atom for the nitro group in the original structure partially
compensates for the loss of the thiourea. None of the analogs
tested exhibited toxicity at the assay concentration in TZM-bl re-
porter cells, as determined by Cell Titer Blue cell viability assay
(data not shown).
in the design of analogs in which the diazene joining Sites 1 and 3
is replaced with carbon-based linkers.
Acknowledgments
This study was supported by National Institutes of Health
Grants AI57083 and AI102704 (to T.E.S.) and by the Common-
wealth of Pennsylvania (SAP 4100054875 to the University of Pitts-
burgh Drug Discovery Institute).
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.bmcl.
2014.02.045.
References and notes
In summary, we present the first structure–activity study of a
recently described pharmacological antagonist of HIV-1 Nef, a crit-
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chemical modifications can be made to all three rings in the origi-
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moieties are not essential for inhibition of Nef function. Taken to-
gether, these results will guide the development of next-genera-
tion compounds with appropriate pharmacological profiles for
assessment of Nef-directed antiretroviral activity in vivo. In partic-
ular, the SAR developed for Sites 1–3 as reported here will be useful
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