Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly

Article abstract of DOI:10.1016/j.bmcl.2010.10.131

The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit established structure-activity relationships. Replacement of the enamine functionality of the hit series with either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.

Full text of DOI:10.1016/j.bmcl.2010.10.131

Bioorganic & Medicinal Chemistry Letters 21 (2011) 398–404
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of
inhibitors of HIV-1 capsid assembly
⇑
Lee D. Fader , Richard Bethell, Pierre Bonneau, Michael Bös, Yves Bousquet, Michael G. Cordingley,
René Coulombe, Patrick Deroy, Anne-Marie Faucher, Alexandre Gagnon, Nathalie Goudreau,
Chantal Grand-Maître, Ingrid Guse, Oliver Hucke, Stephen H. Kawai, Jean-Eric Lacoste, Serge Landry,
Christopher T. Lemke, Eric Malenfant, Stephen Mason, Sébastien Morin, Jeff O’Meara, Bruno Simoneau,
Steve Titolo, Christiane Yoakim
Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Que., Canada H7S 2G5
a r t i c l e i n f o
a b s t r a c t
Article history:
The discovery of a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione series of inhibitors of HIV-1 capsid
assembly is described. Synthesis of analogs of the 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione hit estab-
lished structure–activity relationships. Replacement of the enamine functionality of the hit series with
either an imidazole or a pyrazole ring led to compounds that inhibited both capsid assembly and reverse
transcriptase. Optimization of the bicyclic benzodiazepine scaffold to include a 3-phenyl substituent led
to lead compound 48, a pure capsid assembly inhibitor with improved antiviral activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 September 2010
Revised 25 October 2010
Accepted 26 October 2010
Available online 31 October 2010
Keywords:
HIV
Capsid assembly inhibitor
Reverse transcriptase inhibitor
Benzodiazepine
The status of the HIV/AIDS epidemic has evolved with time and
by many estimates the global adult prevalence of HIV has stabi-
lized at slightly less than one percent of the world population.1
Highly active antiretroviral therapy (HAART) has improved the
prognosis and quality of life for individuals living with HIV such
that the disease is now thought of as a manageable, chronic disor-
der with life expectancy estimated as great as twenty years from
the start of HAART. Currently, there are twenty five approved
direct-acting antiretrovirals for the treatment of HIV and the
majority of them fall into either the nucleoside reverse transcrip-
tase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibi-
tor (NNRTI) or protease inhibitor (PI) mechanistic class. In spite
of this impressive arsenal of treatment options for HIV infected
individuals, the development of resistance, patient compliance
and drug tolerability are major factors that challenge the manage-
ment of HIV. In response to emerging cross-resistance within
mechanistic classes and multi-drug resistance in general, discovery
efforts have gradually shifted away from these major drug classes,
giving rise to marketed drugs with new mechanisms of action, such
as the CCR5 antagonist maraviroc2 and the integrase (IN) inhibitor
raltegravir.3
As part of our own interest in this area, we recently disclosed
the identification of two structurally distinct chemical series that
inhibit the assembly of the viral capsid protein p24 (CA).4 The
assembly of CA is an essential step in HIV life cycle and thus
presents an opportunity for small molecules to disrupt viral repli-
cation.5 After budding of immature HIV virions, proteolytic cleav-
age of the Gag polyprotein yields matrix (MA), CA, nucleocapsid
(NC) and p6, which allows for assembly of CA into a conical core
that encapsulates the viral RNA complexed with NC, RT and IN.
Assembly of CA is driven by three distinct intermolecular pro-
tein–protein interactions: (a) interaction between CA C-terminal
domain (CTD) and CA N-terminal domain (NTD); (b) CTD dimeriza-
tion; and (c) interaction between NTD of adjacent CA molecules.6
The finding that CA assembly can be recapitulated in vitro has
led to the development of a high throughput capsid assembly assay
(CAA) that, based on the prior work of Sundquist et al.,7 employed a
recombinant CA-NC protein construct and oligonucleotide tem-
plate, as previously described.4,8 Using this assay, we screened a
cluster collection of ꢀ13,000 compounds and identified a 1,5-dihy-
drobenzo[b][1,4]diazepine-2,4-dione hit series exemplified by
compound 1. Herein, we describe our efforts toward optimization
of this hit and the discovery of potent inhibitors of capsid assembly
and viral replication.
The benzodiazepine hit 1 had an IC50 value in the CAA of 1.4
but was inactive in a counter screen involving assembly of Rous
lM,
⇑
Corresponding author. Tel.: +1 450 682 4640; fax: +1 450 6824189.
E-mail address: lee.fader@boehringer-ingelheim.com (L.D. Fader).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.131

L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
399
Table 1
Effect of modification at the 3- and 5-positions of the benzodiazepine core
IC50 (CAA) 1.4 µM
O
O
N
Cl
IC50 (RSV) >100 µM
EC50 17 µM
O
N
N
Cl
N
H
N
H
X
1
O
R
Figure 1. Profile of hit compound 1 identified by screening compound collection
using the capsid assembly assay.
R
X
IC50
>46
(lM)
H
H
6a
7
H
sarcoma virus capsid, consistent with selective inhibition of
assembly of only the HIV-1 CA (Fig. 1). Furthermore, compound 1
displayed modest antiviral activity in a viral replication assay,
H
H
1.4
N
H
OMe
OH
having an EC50 value of 17 l
M.9 With these promising results in
hand, we prepared analogs of compound 1 in hopes of establishing
structure–activity relationships. In doing so, we were particularly
concerned with the inherent electrophilicity of the enamine
moiety, which could lead to problems of chemical instability and
even to conjugation to biomolecules in vivo.
8
9
0.85
N
H
N
H
H
0.67
The synthesis of analogs of benzodiazepine 1 is outlined in
Scheme 1. The route began with SNAr of appropriately substituted
2-chloro- or 2-fluoronitrobenzene derivatives 2 with a primary
amine, which gave nitroanilines 3. The nitro group of compounds
3 was then reduced to give substituted phenylenediamines 4,
which then participate in a cyclization reaction to give benzodiaze-
pines 5. If R1 was a hydrogen (i.e., compounds 6a–9), then aniline
was used in the SNAr reaction to give compounds 3 (i.e., R = Ph)
and compounds 5 were directly converted to the final inhibitors
(i.e., compounds 7–9). If R1 was a small alkyl group, the N1 phenyl
group was installed using copper mediated arylation of the anilide
as originally described by Barton,10 where the phenyl group was
transferred from triphenylbismuth. Finally, the enamine substitu-
ent at the 3-position was introduced via a three-component con-
densation between compounds 6, Bredereck’s reagent and a
primary amine to give inhibitors 10–17.
N
10
11
12
Me
Et
0.59
0.47
>9
N
H
OMe
OMe
OMe
N
H
N
H
Me
observed flexibility in the size and polarity of the substituent on
the nitrogen atom of the enamine. For example, the polarity of
the group had little effect on inhibition of capsid assembly, as is
illustrated by the fact that the butyl side chain of compound 1
could be replaced with the more polar 2-methoxyethyl or
2-hydroxyethyl chains without any loss in potency (cf. compound
We began developing SAR in this series with modification of the
enamine substituent at the 3-position of the benzodiazepine core
(Table 1). It was noteworthy that the enamine functionality was
important for potency since the C3 unsubstituted analog 6a
suffered a complete loss of potency in the CAA. In general, we
NH2
a
b or c
NO2
NO2
Y
Y
Y
NH
R
NH
R
X
4
3
2
d
H
N
O
O
O
O
O
N
N
N
f
e
Y
Y
Y
N R2
H
N
R
N
R1
O
R
5
6
7-17
6a Y = 7-Cl, R = H
f
Scheme 1. Synthesis of analogs 7–20. Reagents and conditions: (a) RNH2, TEA, THF; (b) Fe/AcOH, 100 °C; (c) Sn/aq HCl; (d) simultaneous co-addition of 4/THF and
CH2(COCl)2/THF into THF; (e) Ph3Bi, Cu(OAc)2, TEA or pyr, DCM; (f) Bredereck’s reagent, 140 °C then R2NH2, rt.

400
L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
Table 3
1 to compounds 7 or 8, respectively). We also found that this posi-
tion tolerated large or small substituents very well, as was illus-
trated by comparison of compound 7 or 8 to compound 9. For
this study, we also varied the N5 group and the general finding
was that small alkyl groups were well tolerated, even leading to
moderate improvements in IC50 value of 2- to 3-fold (cf. compound
7 to compounds 10 and 11). However, aromatic groups, such as the
p-tolyl, led to a dramatic loss in potency (cf. compounds 10 and
12).
Tolerated modifications of the N1 phenyl group
R
O
N
CF3
N
N
H
OMe
O
Me
R
IC50
4.0
(lM)
We then conducted several positional scans around the benzene
ring of the benzodiazepine core. The case of a chlorine atom was
illustrative of the general trends we observed (cf. compounds 10,
13–15, Table 2). It was generally found that lipophilic substituents
were well tolerated at all positions of the phenyl ring, but the 7-po-
sition was convincingly preferred. Having this information, we also
conducted a broad screen at the 7-position and found that cyclo-
propyl and CF3 might be tolerated as chlorine atom replace-
ments.11 However, over the course of subsequent investigations,
it was found that only the 7-CF3 and 7-Cl substituents were
broadly tolerated and so development of new SAR was narrowed
to include just these modifications.
Cl
18
Cl
19
20
7.8
S
0.50
Extensive effort was made to modify the N1–Ph group. Almost
all substitutions that were attempted at this position led to a
dramatic loss in potency. The only substitutions tolerated on the
phenyl ring were the m- and p-Cl groups, although at a significant
loss in potency (Table 3, compounds 18 and 19, respectively).
However, it was found that substitution of the phenyl group with
a 2-thiophenyl group led to an equipotent analog (cf. compound
20 to 10).
Concurrent with our early hit to lead activity, we conducted de-
tailed NMR and X-ray crystallographic experiments to elucidate
the binding mode of this series on the CA-NC protein. These studies
revealed that the 1,5-dihydro-benzo[b][1,4]diazepine-2,4-diones
bind to the NTD of CA.12 A full account of these studies will be dis-
closed in due course but two key findings were used to guide the
next phase of our lead optimization. First, both of the carbonyl oxy-
gen atoms on the benzodiazepine scaffold participated in hydrogen
bonding interactions within the binding pocket. Second, the enam-
ine side chain protruded from the binding pocket, which accounted
for broad flexibility encountered during our SAR study. Based on
these observations and due to our concern about the enamine
functionality (vide supra), we began to search for a modification
capable of replacing this functional group. Our solution involved
incorporation of a heterocycle that mimicked both the hydrogen
bonding interaction and sp2-character of the C3 atom. A key design
consideration was the knowledge that all the enamine inhibitors
described above were prepared and tested as mixtures of enamine
stereoisomers. This fact prompted us to evaluate the pyrazole iso-
mer exemplified by compound 21 as a N/Ph trans-isomer mimic
and the imidazole analog 22 as a N/Ph cis-isomer mimic (Fig. 2).
The general synthetic strategy used to prepare analogs of both
the imidazole and pyrazole scaffold is illustrated in Scheme 2.
From heterocyclic amino ester 23,13,14 we introduced the N5-alkyl
group in the three-step acylation, alkylation and deprotection
Table 2
Effect of 5-, 6-, 7- and 8-substitution on the benzodiazepine core
O
N1
Y
N5
Me
N
H
OMe
O
N1
IC50
0.59
0.93
(lM)
Y
N5
N1
Cl
10
13
N5
Cl
N1
N5
N1
sequence leading to compound 26.
A nucleophilic aromatic
14
2.6
N5
substitution then allowed for the installation of the appropriately
substituted benzene ring destined to be part of the benzodiazepine
core. The seven-membered ring was then formed through a nitro
reduction and concomitant cyclization sequence to provide com-
pound 28. For the imidazole and pyrazole series, we relied on Bar-
ton’s Cu/Bi-based chemistry for introduction of the N1-phenyl
group. At this stage, the deprotection of the PMB group was done
under acidic conditions to give building block 30 and the point of
diversity was introduced by mild, regioselective alkylation to give
compound 31.
Cl
N1
N5
15
16
1.6
Cl
CF3
N1
0.89
N5
N1
We first examined structure–activity relationships in the pyra-
zole series in an effort to mimic the N/Ph trans-isomer of the enam-
ine series. The unsubstituted compound 30a had an IC50 value of
17
1.4
N5
0.30 lM in the capsid assembly assay and an EC50 of 7.8 lM in

L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
401
Table 4
Effect of pyrazole substitution
O
O
O
N
N
N
Cl
Cl
NHR
O
N
N
N
Cl
N
N
R
H
O
Me
Me
N
R
N / Ph cis isomer
N / Ph trans isomer
Me
R
IC50
(lM)
EC50
7.8
(lM)
30a
0.30
H
32
0.7
2.2
1.5
7.0
—
Me
31a
33
16
OMe
N
O
O
N
34
35
6.8
1.0
16
N
N
N
N
Cl
Cl
R
N
N
0.90
N
O
R
N
Me
Me
CO2H
36
37
0.60
0.60
2.0
4.7
21
trans isomer mimic
22
cis isomer mimic
Figure 2. Design of enamine stereoisomer mimics.
OH
the viral replication assay (Table 4).15 Addition of a methyl group
to give analog 32 led to a 2-fold loss in potency in the CAA, but
had no effect on the antiviral activity. However, installation of
the slightly larger ethyl group led to a 3-fold increase in the IC50
value compared to compound 32. Similar results were obtained
with polar groups that were reminiscent of those used in our inves-
tigation of the enamine hit series. For example, incorporation of an
ether or dimethylamino group (compounds 33 and 34) both lead to
5- to 10-fold loss in the CAA, accompanied by similar losses in anti-
viral potency. However, potency was restored with the larger, aro-
matic benzyl groups, such as those found in compounds 35–37.
Not only were IC50 values in the sub-micromolar range for these
compounds, but antiviral potency reached the sub-micromolar le-
vel for the first time in the case of the basic analog 35. Although
these results were encouraging, it was difficult to account for the
EtO2C
b
X
N
EtO2C
a
EtO2C
X
X
N
Y
Y
Y
OMe
H2N
OMe
N
HN
O
OMe
N
CF3
CF3
O
24
25
23
c
EtO2C
N
H
N
O
N
X
Cl
e
Y
d
EtO2C
X
X
N
OMe
Y
Y
N
N
OMe
O2N
N
H
28
27
26
OMe
Cl
f
O
N
O
g
h
N
N
N
N
Cl
Cl
O
N
Cl
X
X
Y
H
X
Y
N
Y
N
N
31a X = CH, Y = N
31b Y = CH, X = N
29
30a X = CH, Y = N
30b Y = CH, X = N
OMe
Scheme 2. General synthetic pathway for heterocyclic analogs in Tables 4 and 5. Reagents and conditions: imidazole series X = N, Y = CH; pyrazole series X = CH, Y = N; (a)
TFAA, TEA, DCM; (b) MeI, NaHMDS, DMF; (c) NaOEt/EtOH; (d) NaHMDS, 2-fluoro-5-chloronitrobenzene, THF; (e) Fe/AcOH; (f) Ph3Bi, Cu(OAc)2, Pyr, DCM; (g) TFMSA/TFA; (h)
EtI, K2CO3, DMF.

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L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
relatively steep SAR observed in the pyrazole series without recall-
ing that the N/Ph cis-isomer was preferred in our X-ray studies
involving the enamine series. With this in mind, we turned our
attention to the imidazole series designed to mimic the N/Ph cis-
enamine isomer.
H
N
O
N
N
N
We began our investigation of the imidazole series by making
modifications common to the pyrazole series in order to allow a di-
rect comparison. The unsubstituted analog 30b was among the
most potent analogs tested, having an IC50 value similar to that
of unsubstituted pyrazole 30a, but an EC50 value about 2-fold low-
er (Table 5). The SAR began to diverge upon substitution of the
imidazole nitrogen, where small alkyl groups were well tolerated,
giving lower IC50 values compared to similar substitutions ex-
plored in the pyrazole series (cf. compounds 38 and 31b to com-
pound 30b). Polar substituents were also well tolerated in this
series and benzyl groups also improved potency in both the CA
assembly and antiviral assays. As with the pyrazole series, the ben-
zylic amine 43 led to analogs that reached sub-micromolar activity
in the antiviral assay. Taken together, the results supported the
hypothesis that the cis-enamine stereoisomer was the more potent
of the two.
Over the course of our investigation of the pyrazole and imidaz-
ole series, trends in antiviral potency often did not correlate to SAR
observed in the CAA. Cognizant of the structural similarity of the
pyrazole and imidazole series to that of the NNRTI nevirapine
(44, Fig. 3), several analogs were profiled in an antiviral assay that
used engineered HIV virus having the clinically relevant K103N/
Y181C double mutation in reverse transcriptase associated with
resistance to NNRTIs. If compounds showed no shift in this assay
relative to the one using wild type virus, that would help us rule
out a mixed mechanism of action composed of both NNRTI and
capsid assembly inhibition. However, if a shift was observed, sug-
gesting some level of inhibition of RT, this might account for the
unusual SAR that was emerging in these series. A number of ana-
logs tested did show a shift between the two antiviral assays,
and so a full evaluation of the collection of capsid assembly inhib-
itors was undertaken. The results are presented in Figure 4 as a plot
of EC50 for the wild type (WT) virus versus EC50 for the double mu-
tant (DM) virus. The ideal situation for a series of capsid assembly
44
Figure 3. Structure of the NNRTI nevirapine.
DM / WT = 5
DM / WT = 2
DM / WT = 1
25000
20000
15000
10000
5000
0
imidazoles
enamines
pyrazoles
0
5000
10000
EC50 (WT) nM
15000
20000
25000
Figure 4. Plot of EC50 (K103N/Y181C) versus EC50 (WT) for different subseries of
capsid assembly inhibitors.
inhibitors is to trend along the orange line in Figure 4 where the
shift between the assays is essentially 1. For the pyrazole series
(blue circles), the shift was as high as 15-fold between the two as-
says and Figure 4 shows that this was generally found across the
whole series. In the case of the imidazole series (yellow circles),
the shift was not as dramatic as with the pyrazole series, but for
the most potent compounds, a shift was apparent and could be
as high as fivefold. It should be noted that when the results shown
in Figure 4 indicated that the NNRTI mechanism may be contribut-
ing to antiviral potency, we performed enzymatic assays with WT
RT and DM RT and were able to show similar, often more dramatic
shifts between the two assays (data not shown). In these cases, this
confirmed some contribution of the NNRTI mechanism to antiviral
potency and explained the unusual SAR we had observed.
The primary objective of our research program was to discover
a new antiretroviral belonging to a novel mechanistic class that
could be administered to HIV-infected individuals with no limita-
tion with respect to past treatment history and potential resistance
profile. Therefore, we concentrated our effort on the search for a
pure capsid assembly inhibitor devoid of inhibition of reverse
transcriptase. To move forward, we took a step back from the imid-
azole and pyrazole series in order to implement a different strategy
toward structural replacement of the enamine group. Examination
of the data presented in Figure 4 revealed that the original enamine
series remained clustered along the orange dotted line, indicating
little or no contribution from inhibition of RT to the antiviral activ-
ity of these compounds. Furthermore, a remaining avenue that we
had not yet investigated was substitution of the C3 atom of the
benzodiazepine core that was sp3-hybridized. In hopes that main-
Table 5
Effect of substitution of the imidazole ring
O
N
N
Cl
R
N
N
Me
R
IC50
(l
M)
EC50
3.8
(lM)
30b
0.21
H
38
0.3
2.9
5.2
5.8
Me
31b
39
0.22
0.36
OMe
NH2
40
0.37
4.8
41
0.56
7.3
OH
CO2H
42
43
0.43
0.17
6.1
OMe
N
0.90
H

L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
403
Table 6
ture–activity relationships at several positions on the molecular
framework. Furthermore, rationally designed replacements of the
potentially reactive enamine functional group led to the potent
imidazole and pyrazole series of capsid assembly inhibitors. Unfor-
tunately, these two new series displayed a mixed mechanism of ac-
tion in viral replication assays that involved inhibition of HIV
reverse transcriptase.18 Finally, reinvestigation of the bicyclic ben-
zodiazepine scaffold identified the 3-phenylbenzodiazepine lead
candidate showing similar potency to the original hit compound
in the capsid assembly assay, a >5-fold improvement in antiviral
potency and no inhibition of reverse transcriptase. Importantly,
this series is devoid of the enamine functional group, a potential
liability from the perspective of in vivo toxicity due to its electro-
philic nature. Efforts to optimize this new lead with the objective
of identifying a direct-acting antiretroviral that operates through
a novel mechanism of action and with a unique resistance profile
will be reported in due course.
Effect of substituted sp3-C3 on potency
O
O
N
N
CF3
R
H
R
IC50
16
(l
M)
EC50 WT/DM
M)
CC50
M)
(
l
(
l
45
46
À/À
À/À
—
—
10% inhibition @ 1.4
40% inhibition @ 3.8
1.2
47
48
>20/À
—
3.0/2.7
>20
Acknowledgments
The authors wish to acknowledge Jean-François Mercier and
Elizabeth Wardrop for IC50 and EC50 value determinations, respec-
tively. Jean-François Mercier is also acknowledged for conducting
the screen that identified hit compound 1. The authors would also
like to thank Professor Wesley Sundquist for insightful discussions.
We thank Claudio Sturino, François Bilodeau and Stephanie War-
ner for critical proof-reading during preparation of the manuscript.
taining a bicyclic scaffold was the key to minimizing the NNRTI
mechanism of action, we initiated prospecting efforts that revealed
that addition of both simple and more complex alkyl substitutions
did not improve potency (Table 6, cf. compounds 45–47 to com-
pound 6a). To our surprise, the directly attached phenyl analog
48 regained the potency lost in the CAA caused by excising the en-
amine functionality. Furthermore, compound 48 showed good po-
tency in the antiviral assay with an acceptable window versus
cytotoxicity and, importantly, showed no shift when the wild type
and double mutant assay results were compared.
The synthetic scheme we used to prepare analog 48 is shown in
Scheme 3.16,17 A salient feature of the route included the relatively
short sequence from commercially available ethyl ester 49. Impor-
tantly, there are a plethora of commercially available phenyl acetic
acid building blocks, which will allow for the rapid introduction of
diversity at the 3-position.
References and notes
1. UNAIDS/WHO ‘AIDS epidemic update 2009’, November, 2009, Geneva,
Available online at http://data.unaids.org/pub/Report/2009/JC1700_Epi_
Update_2009_en.pdf (Accessed July 16, 2010).
2. For a recent review of this target, including a discussion on maraviroc see:
Tilton, J. C.; Doms, R. W. Antiviral Res. 2010, 85, 91.
3. For a recent review of this target, including a discussion on raltegravir see:
McColl, D. J.; Chen, X. Antiviral Res. 2010, 85, 101.
4. Titolo, S., Mercier, J.-F.; Wardrop, E.; von Schwedler, U.; Goudreau, N.; Lemke,
C.; Faucher, A.-M.; Yoakim, C.; Simoneau, .B.; Sundquist, W. I.; Mason, S.
‘Discovery of potent HIV-1 capsid assembly inhibitors.’ 17th Conference on
Retroviruses and Opportunistic Infections 2010. Oral abstract 50.
5. For a comprehensive review of emerging HIV targets, including a detailed
discussion of the structure and function of CA, see: Adamson, C. S.; Freed, E. O.
Antiviral Res. 2010, 119.
6. (a) Ganser-Pornillos, B. K.; Yeager, M.; Sundquist, W. I. Curr. Opin. Struct. Biol.
2008, 18, 203; (b) Pornillos, O.; Ganser-Pornillos, B. K.; Kelly, B. N.; Hua, Y.;
Whitby, F. G.; Stout, C. D.; Sundquist, W. I.; Hill, C. P.; Yeager, M. Cell 2009, 137,
1282.
In conclusion, we have used our capsid assembly assay to
screen a focused collection of compounds and identified a hit series
based on a 1,5-dihydrobenzo[b][1,4]diazepine-2,4-dione scaffold.
Hit-to-lead activity based on hit compound 1 established struc-
7. Sundquist, W. I.; Wang, H.; Hill, C. P.; Stemmler, T. L.; Davis, D. R.; Alam, S. PCT
Int. Appl. WO 2003013425, 2003, p 127.
8. Our modification to the assay described by Sundquist et al. in Ref. 7, and its
adaptation to a plate format suitable for high-throughput screening is the
subject of a manuscript in preparation.
9. All EC50 determinations were made as previously described: see: Yoakim, C.;
Deroy, P.; Duplessis, M.; Gagnon, A.; Goulet, S.; Hucke, O.; Lemke, C.;
Surprenant, S. PCT Int. Appl. WO 2008067644, 2008, p 90.
10. Barton, D. H. R.; Finet, J. P.; Khamsi, J. Tetrahedron Lett. 1986, 2731, 3615.
11. Examples of substitutions that lead to dramatic loss in potency included: C-
linked amides, N-linked amides, ureas, N-linked sulfonamides, C-linked
acylsulfonamides, N-linked imides, amines, carboxylate acid and esters,
cyano, methoxy, hydroxyl, methanolyl,benzyl ethers, C2–C4 alkyl, sulfones,
heterocycles.
O
O
a
EtO
EtO
NH2
NH
CF3
+
O
O
50
51
49
b
12. Other small molecules that bind to CANTD and disrupt capsid assembly have
been discovered. See the pioneering work of Michael Summers and coworkers:
(a) Tang, C.; Loeliger, E.; Kinde, I.; Kyere, S.; Mayo, K.; Barklis, E.; Sun, Y.; Huang,
M.; Summers, M. F. J. Mol. Biol. 2003, 327, 1013; (b) Kelly, B. N.; Kyere, S.; Kinde,
I.; Tang, C.; Howard, B. R.; Robinson, H.; Sundquist, W. I.; Summers, M. F.; Hill,
C. P. J. Mol. Biol. 2007, 373, 355; See also: (c) Li, J.; Tan, Z.; Tang, S.; Hewlett, I.;
Pang, R.; He, M.; He, S.; Tian, B.; Chen, K.; Yang, M. Bioorg. Med. Chem. 2009, 17,
3177.
13. (a) For the prior disclosures of the pyrazole derivative see: Bolea, C. PCT Int.
Appl. WO 2010079239 2010, p 107.; (b) Macor, J. E.; Bi, Y. U.S. Pat. Appl. Publ.
US 2002133008 2002, p 14.; (c) Bi, Y.; Stoy, P.; Adam, L.; He, B.; Krupinski, J.;
Normandin, D.; Pongrac, R.; Seliger, L.; Watson, A.; Macor, J. E. Bioorg. Med.
Chem. Lett. 2001, 11, 2461.
H
O
O
O
O
N
N
N
CF3
CF3
c
N
52
48
Scheme 3. Synthesis of 3-phenylbenzodiazepine analog 48. Reagents and condi-
tions: (a) NC–CO2Et, NaHMDS; (b) 50 + 51,
Cu(OAc)2, Pyr, DCM.
lW @ 160 °C, 25 min; (c) Ph3Bi,
14. For the prior disclosures of the imidazole derivative see: (a) Zavialov, I. A.;
Dahanukar, V. H.; Nguyen, H.; Orr, C.; Zhang, F.; Andrews, D. R. Org. Lett. 2004,

404
L. D. Fader et al. / Bioorg. Med. Chem. Lett. 21 (2011) 398–404
6, 3017; (b) Zavialov, I. A.; Dahanukar, V. H.; Nguyen, H.; Orr, C.; Andrews, D. R.
Org. Lett. 2004, 6, 2237.
15. The CC50/EC50 window (CI) was determined for selected capsid assembly
inhibitors presented in this study: CI was 5.2, 7.0, 8.3, 5.1 and >6.7 for
compounds 37, 30b, 41, 43 and 48, respectively.
1H, J = 8.3 Hz). UPLC-MS (column: HSST3, gradient of 5–100% MeCN/H2O with
0.06% TFA over 3.8 min, UV detection at 220 nm): rt = 1.54 (14.9%)/1.58 min
(85.1%), m/z (ES, +ve mode): 411.0 [M+H]+.
17. All evidence to date is consistent with the assigned tautomer that is sp3-
hybridized at C-3 for compound 48. For example, the resonance at 4.93, which
is assigned to the H-3 atom of compound 48, was unaffected by addition of
16. All inhibitors synthesized in this manuscript gave satisfactory 1H NMR, HPLC
and MS data, allowing us to confirm their identity and purity. Representative
data for compound 48 is included: Compound 48 exists as a mixture of
diastereomeric conformations that are separable by HPLC, but are not stable
enough to be isolated and characterized as individual atropisomers. The
following NMR data is given for the major set of resonances: 1H NMR
(400 MHz) d: 3.52, (s, 3H), 4.93 (s, 1H), 7.18 (d, 1H, J = 2.0 Hz), 7.27–7.32 (m,
5H), 7.36–7.40 (m, 1H), 7.44–7.50 (m, 5H), 7.77 (dd, 1H, J = 1.9, 8.6 Hz), 7.88 (d,
50 lL of D2O to a solution of 0.62 mg of compound 48 in 500 lL DMSO-d6, even
after prolonged incubation over several hours at room temperature.
18. A related pyrazole based tricyclic compound that lacks the N1-Ph substitution
has been reported to be inactive in HIV viral replication and recombinant
reverse transcriptase assays. See: Di Braccioa, M.; Grossia, G.; Romaa, G.;
Vargiub, L.; Murab, M.; Marongiu, M. E. Eur. J. Med. Chem. 2001, 36, 935.