Benzamide-based thiolcarbamates: A new class of HIV-1 NCp7 inhibitors

Article abstract of DOI:10.1016/S0960-894X(02)00007-0

The HIV-1 nucleocapsid protein NCp7, which contains two highly conserved zinc fingers, is being used as a novel target for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of NC

Full text of DOI:10.1016/S0960-894X(02)00007-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 767–770
Benzamide-Based Thiolcarbamates: A New Class of
HIV-1 NCp7 Inhibitors
Atul Goel,a Sharlyn J. Mazur,a Rasem J. Fattah,b Tracy L. Hartman,c Jim A. Turpin,c
b,
Mingjun Huang,d William G. Rice,d Ettore Appellaa andJohn K. Inman *
aLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
bLaboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
cInfectious Disease Research Department, Southern Research Institute, 431 Aviation Way, Frederick, MD 21702, USA
dAchillion Pharmaceuticals, Inc., 300 George Street, New Haven, CT O6511, USA
Received26 October 2001; accepted17 December 2001
Abstract—The HIV-1 nucleocapsidprotein NCp7, which contains two highly conservedzinc fingers, is being usedas a novel target
for AIDS therapy due to its pivotal role in viral replication and its mutationally intolerant nature. Herein we report a new class of
NCp7 inhibitors that possess goodantiviral activity with low cellular toxicity. # 2002 Elsevier Science Ltd. All rights reserved.
The nucleocapsidp7 protein (NCp7) zinc finger
domains of human immunodeficiency virus type 1
(HIV-1) have been chosen andexploitedas targets in
the pursuit of alternative, novel anti-retroviral drugs.1
NCp7, a basic 55 amino acidprotein, results from spe-
cific post-translational processing of Pr55gag and
Pr160gag-pol precursor polyproteins2 andplays a pivotal
role in both early (reverse transcription3 andintegra-
showedthat these compounsd exhibitedsignificant
antiviral activity against laboratory andclinical isolates
of HIV in several infectedcell lines. Recently, we iden-
tifieda novel chemotype, pyridinioalkanoyl thiolesters
(PATEs),16 that inhibits viral replication in both acutely
andlatently HIV-1 infectedcells. PATEs possessed
NCp7 zinc finger reactivity andviruciadl activity.
However, efforts to generate compounds with sig-
nificantly improvedantiviral potencies were not suc-
cessful. The present study was focused on identifying a
new class of NCp7 inhibitors, thiolcarbamates (TICAs),
that possess improvedanti-retroviral potency.
5
6
tion4) andlate (protease processing andpackaging of
viral genomic RNA) stages of the viral replication cycle.
The Cys-(Xaa)2-Cys-(Xaa)4-His-(Xaa)4-Cys (CCHC)
motifs7 are present in both zinc fingers of NCp7 andare
8
highly conservedandmutationally intolerant.
Muta-
tional studies on virus infectivity indicated that mod-
ification in either zinc chelating, non-chelating and
adjacent sequences of the zinc finger domains results in
virions with defective RNA encapsidation and/or NCp7
function that renders them noninfectious.6,9
A variety of electrophilic agents have been identified
that cause HIV-1 inhibition by chemical modification of
Cys-sulfurs of the NCp7 zinc finger motifs. Studies on
NCp7 inhibitors, such as 3-nitrosobenzamide (NOBA),10
2,20-dithiobisbenzamides (DIBA)1a,11 andtheir benz-
isothiazolone derivatives (BITA),12 cyclic 2,20-dithio-
bisbenzamides (SRR-SB3),13 1,2-dithiane-4,5-diol-1,1-
dioxide (dithiane),14 and azodicarbonamide (ADA)15
Scheme 1. Reagents andconditions: (i) N-Hydroxysuccinimide/DIC/
THF-PrOH-2/25 ꢀC; (ii) H2N(CHR)mCONH2.HCl/Et3N/DMF/25 ꢀC;
(iii) TCEP.HCl/Et3N/DMF–H2O (9:1)/25 ꢀC; (iv) R0NCO/DMF/
25 ꢀC; (v) R0=CH2CH2Br, pyridine/under N2/25 ꢀC.
*Corresponding author. Fax: +1-301-496-0222; e-mail: jinman@
niaid.nih.gov
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00007-0

768
A. Goel et al. / Bioorg. Med. Chem. Lett. 12 (2002) 767–770
The existing methodologies17 for thiolcarbamate for-
mation are limitedby the needfor specializedreagents
such as transition metal complexes, andthe use of
strong bases, high temperatures and/or cumbersome
reagents such as phosgene. However, our synthetic
approach to preparing benzamide-based thiolcarb-
amates through the use of commercially available
reagents is straightforward(Scheme 1). The synthesis of
the 2,20-dithiobis(benzamides) (3) was accomplishedvia
coupling of N,N0-disuccinimidyl-2,20-dithiosalicylate (2,
and 6a–c) of this set showedsimilar EC values (0.16–
50
1.19 mM), but exhibiteda wide range of cellular toxicity
(IC50) values lying between 4 and200 mM (highest con-
centration tested). Changing the hydrophobicity by
preparing linear alkyl homologues (5a–d) did not
impact on their antiviral potencies. On the other hand,
increasing the steric bulk on the carbamoyl nitrogen
atom by substitution with isopropyl (5e,f) or tertiary
butyl (5g,h) groups reduced the cellular toxicity, result-
ing in good therapeutic indices. Compounds comprised
of a saturatedring ( 5i,j) at the carbamoyl nitrogen,
showedhigh cellular toxicities andcorresponidngly
poor therapeutic indices, while substitution with aro-
matic groups (5k–m) gave rise to less toxic compounds,
except for 5l. The effect of the electron withdrawing
bromo group at the terminus of the N-ethylcarb-
amoylthio group (5n,o) resultedin increasedcellular
toxicity. The replacement of the bromo group by a pyr-
idinio moiety (6a–c), yielding pyridinium salts, reduced
cellular toxicity while maintaining submicromolar
EC50s andtherapeutic indices >140. Thus, the combi-
nation of the carbamoylthio moiety andcertain sub-
stitutents like tertiary butyl andpyridinium moiety at
the carbamoylthio nitrogen with the carboxamide term-
inal yields antiviral agents with submicromolar EC50s
andreducedcytotoxicity. These branchedor cationic
structures may confer lower toxicity by slowing the
reverse of reaction iv (Scheme 1), which results in the
release of isocyanate.
0
preparedfrom the 2,2 -dithiosalicylic acid (1), N-hydro-
xysuccinimide and 1,3-diisopropylcarbodiimide) with
the desired amino acid amides. These disulfide benza-
mides (3) were reduced using tris(2-carboxyethyl)pho-
sphine hydrochloride (TCEP.HCl) in 90% DMF in
water under nitrogen atmosphere. The desired thiolcar-
bamates (5) were preparedby condensation of 4 with
the corresponding isocyanates at room temperature. All
bromo derivatives (5n,o) were convertedto their
respective pyridinium salts (6) by stirring in pyridine at
room temperature under nitrogen. All the synthesized
compounds were characterized by spectroscopic analyses,
andthe composition analysis data for one of the repre-
sentative compounds is given in the reference section.18
Compounds 5a–o and 6a–c were evaluatedfor their
antiviral activity (Table 1) with CEM-SS cells andHIV-
1RF virus using the HIV-1 cytoprotection assay.10b,14
Our initial efforts were focusedon the structure–activity
relationships (SARs) associatedwith substitution on the
nitrogen atom of the carbamoylthio moiety of the
TICAs as shown in Table 1. All the compounds (5a–o
We next determined if the observed advantages in anti-
viral activity were associatedwith Zn Finger reactivity.
Table 1. Antiviral activity of 5a–o and 6a–c was measuredby the HIV-1 cytoprotection assay a
S.N.
m
n
R
Z
Antiviral activity EC50(mM)
Cellular toxicity IC50(mM)
TI
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
6a
6b
6c
2
2
1
2
1
2
1
2
2
2
1
1
2
2
1
1
2
1
4
5
7
7
0
0
0
0
0
0
0
0
0
2
2
2
2
2
H
H
d-CH3
H
l-CH3
H
d-CH3
H
H
H
d-CH3
d-CH3
H
CH3
CH3
CH3
CH3
0.50
0.43
0.23
0.16
0.77
0.33
0.36
0.37
0.81
0.55
0.47
1.19
0.45
0.48
0.92
0.50
0.95
0.97
41.2
39.4
38.3
40.2
76
55.1
125
142
17.3
11.9
130
23.1
128
4.9
82
92
167
251
99
167
347b
384b
21
22
277
19
284
10
9
CH(CH3)2
CH(CH3)2
C(CH3)3
C(CH3)3
Cyc-Pentyl
Cyc-Hexyl
4-FC6H4
4-NO2C6H4
4-CF3C6H4
Br
H
d-CH3
H
H
d-CH3
Br
8.0
70
153
200
Py+Brꢁ
Py+Brꢁ
Py+Brꢁ
140
161
206
aAll determinations were performed in duplicate with triplicate determinations for each compound concentration. The variation of individual tri-
plicates was 10% or less.
bZinc finger reactivity was observedat 12 mM using previously described methodologies.19 EC50: effective concentration resulting in 50% cytopro-
tection; IC50: cellular growth inhibitory concentration causing 50% cytotoxicity; TI: therapeutic index (IC50/ EC50).

A. Goel et al. / Bioorg. Med. Chem. Lett. 12 (2002) 767–770
Table 2. Antiviral range of action studies of select TICAs
769
a
CompdPBMCs
Monocyteb
(EC50 mM)
TNF-a Induced U1 cellsc
(EC50 mM)
Chronically infected
cellsd (EC50 mM)
Virus inactivatione
(EC50 mM)
(EC50 mM)
RT
p24
5g
5h
6a
6b
1.1
0.8
2.7
1.9
13.9
28.7
28.4
27.1
ND
ND
5.1
2.26
ND
ND
2.5
0.84
ND
ND
20
0.9
1
ND
0.9
10.8
Data shown are representative of triplicate assays in at least two separate experiments. ND means not determined.
aExpression of supernatant RT activity was determined 6 days post-infection of human PBMCs with the pediatric clinical isolate HIV-1RoJo.
bHuman peripheral blood monocytes were cultured in vitro for 6 days and infected with HIV-1 Ba-L, and virus replication detected by commercial
p24 ELISA.
cU1 cells were induced with 10 ng/mL TNF-a andthe expression of supernatant RT activity andp24 protein determinedat day 3 post-treatment.
dCEM-SS cells chronically infectedwith the SK-1 strain of HIV-1 (CEM/SK-1) were assessedfor supernatant RT activity following 6 days of
treatment.
eCell-free HIV-1 IIIB was incubatedfor 4 h incubation with TICAs, removedby centrifugation (18,000 g, 90 min, 4 ꢀC) andresidual infectivity
determined on HeLa CD4 LTR b-gal cells by chemiluminescence. EC50: concentration resulting in 50% suppression of virus replication.
Presumably, transacylation of the carbamoyl group to
Zn finger cysteine sulfur atoms occurs in the manner
shown for thiolesters.19 This mechanism is currently
being investigated. The lead compounds (5g and 5h; TI
>300) were evaluatedfor their ability to eject zinc from
purifiedrecombinant NCp7 in vitro in a continuous
fluorescence-based assay as previously described.19 The
test compoundat 10 or 12 mM concentration resultedin
nearly quantitative release of zinc from 1 mM NCp7,
while addition of 6 mM releasedapproximately 25% of
the zinc (data not shown). The release of zinc occurs at
lower concentrations of TICAs than was foundfor the
PATEs.19 These results demonstrate that TICAs retain
NCp7 reactivity andare more active comparedto our
earlier described PATEs.16
of this family of potent NCp7 inhibitors holds promise
for the development of broad spectrum antiretrovirals.
Discovery of TICAs that inhibit virus replication in
chronically andlatently infectedcells shouldallow for
the development of novel antiviral therapies that can
address some of the limitations of current therapies.
Acknowledgements
This research was supportedby the Intramural AIDS
TargetedAntiviral Program of the office of the Director
of the National Institutes of Health (A.G. andE.A.).
We wish to thank Noel F. Whittaker for mass spectral
analyses of compounds, and Ms. Karen Watson, Lu
Yang andMr. Mark C. Osterling for their excellent
technical assistance.
The discovery of the susceptibility of the NCp7 protein
to electrophilic agents has resultedin the identification
of a suite of secondary cell-based assays as NCp7 rele-
vant inhibitor models. The methodologies for and use of
cell-basedassays to assess potential Zn finger inhibitors
has been extensively reported.1a,11,14ꢁ16 Table 2 shows
that compounds 5g, 5h, 6a, and 6b can inactivate cell-
free virus andinhibit virus replication in acute (PBMCs
andmonocytes), chronic andin a post-integrative
(latent) model of HIV-1 replication (TNF-a induced U1
cells). The TICAs are shown to be low micromolar
inhibitors of virus replication andprevent both new
infection andthe expression of virus from infectedcells.
We have preliminary data (not shown) indicating that
cross-linking of gag precursors occurredin U1 cells
treatedwith TICAs; visualization was done by native
andreudcing electrophoresis followedby Western
staining for NCp7 andp24.
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(brs, 1H, NH2), 7.30–7.60 (m, 5H, Ar-H, NH2 ), 8.06 (s, 1H,
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