Synthesis, antiviral activity and resistance of a novel small molecule HIV-1 entry inhibitor

Article abstract of DOI:10.1016/j.bmc.2015.11.006

One of the most critical requirements of the infection of the human immunodeficiency virus type 1 (HIV-1) is the interaction of its surface envelope glycoprotein gp120 with the cellular receptor CD4, which initiates virus entry to cells. Therefore, envelo

Full text of DOI:10.1016/j.bmc.2015.11.006

Bioorganic & Medicinal Chemistry 23 (2015) 7618–7628
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, antiviral activity and resistance of a novel small molecule
HIV-1 entry inhibitor
a
b
b
b
b
Francesca Curreli a, , Kashfia Haque , Lihua Xie , Qian Qiu , Jinfeng Xu , Weizhong Yong ,
⇑
Xiaohe Tong b, Asim K. Debnath a,
⇑
a Laboratory of Molecular Modeling and Drug Design, Lindsey F. Kimball Research Institute, New York Blood Center, New York, NY 10065, USA
b CPC Scientific, Inc., 1245 Reamwood Ave., Sunnyvale, CA 94089, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
One of the most critical requirements of the infection of the human immunodeficiency virus type 1 (HIV-
1) is the interaction of its surface envelope glycoprotein gp120 with the cellular receptor CD4, which ini-
tiates virus entry to cells. Therefore, envelope glycoprotein gp120 has been validated as a potential target
to develop HIV-1 entry inhibitors. Here we report the evaluation of a novel non-natural amino acid, ter-
med 882376, reported earlier as a precursor of a CD4-mimetic miniprotein, as HIV-1 entry inhibitor.
882376 showed HIV-1 inhibitory activity against a large panel of primary isolates of different subtype.
Moreover, genotyping of 882376 resistant HIV-1 virus revealed three amino acid substitutions in the
gp120 including one in the CD4 binding site suggesting that this molecule may bind to gp120 and prevent
its binding to CD4. Additional neutralization experiments indicate that 882376 is not active against
mutant pseudoviruses carrying the amino acid substitutions S375H and S375Y located in the ‘Phe43 cav-
ity’ which is the major site of CD4 binding, suggesting that this compound may interfere with the inter-
action between gp120 and CD4. The unnatural amino acid, 882376, is expected to serve as a lead for
further optimization to more potent HIV-1 entry inhibitors.
Received 10 September 2015
Revised 28 October 2015
Accepted 5 November 2015
Available online 10 November 2015
Keywords:
Miniprotein
Unnatural amino acid
HIV-1
Anti-HIV-1
Resistance
HIV entry
Envelope glycoprotein gp120
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
combination with 1 non-nucleoside reverse transcriptase inhibitor
(NNRTI), protease inhibitor (PI), integrase inhibitor (INI) or entry
HIV-1 is the etiological agent of the Acquired Immunodeficiency
Syndrome (AIDS). According to the World Health Organization
(WHO) this disease ranked 6th among the 10 leading causes of death
in the world in 2012 (http://www.who.int/mediacentre/factsheets/
fs310/en/; accessed on August 26, 2015). Based on the 2014 Global
Statistics from UNAIDS about 36.9 million people are living with
HIV worldwide, about 2 million people were newly infected and
1.2 million people died from AIDS-related diseases world-wide
(http://www.unaids.org/sites/default/files/media_asset/20150714
_FS_MDG6_Report_en.pdf; accessed on 26th August, 2015). HIV-1 is
a highly variable virus which mutates rapidly. The currently used
treatment is termed the Highly Active Anti-Retroviral Therapy
(HAART), which uses combination of drugs targeting different steps
of the retrovirus life-cycle. There are five classes of drugs currently
approved by the US FDA, which are usually used in combination,
typically 2 nucleoside reverse transcriptase inhibitors (NRTIs) in
inhibitor.1,2 To date only two entry inhibitors are available, Maravi-
roc which targets the co-receptor CCR53,4 and Enfuvirtide, a peptide
drug which interacts with the N-terminal heptad repeat of gp41 of
HIV to form an inactive hetero six-helix bundle preventing infection
of host cells.5,6 Despite the success achieved with the HAART the
emergence of resistant viruses and the side effects of these therapies
highlight the need of novel anti-retroviral agents to overcome these
problems.
HIV-1 entry into the target cell is initiated by binding of the
viral surface glycoprotein gp120 to the cellular receptor CD4,
which induces the conformational change in gp120 that enable
its binding to the cellular coreceptors CCR5 or CXCR4.7,8 This bind-
ing in turn activates envelope glycoprotein gp41 which induces
fusion of the viral and cellular membrane followed by the entry
of the virus into the cells to initiate infection. One of the most
important residues in the gp120/CD4 interaction is Phe43 of CD4,
which deeply penetrates the cavity of gp120, referred to as the
‘Phe43 cavity’ and accounts for 23% of the total contact with
gp120. The hydrophobic and highly conserved Phe43 cavity has
been recognized as an attractive target for developing small mole-
⇑
Corresponding authors.
E-mail addresses: fcurreli@nybloodcenter.org (F. Curreli), adebnath@nyblood-
center.org (A.K. Debnath).
http://dx.doi.org/10.1016/j.bmc.2015.11.006
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
7619
cule inhibitors9–12 or CD4 mimetic ‘miniproteins’13,14 to fill the
cavity and disrupt the gp120/CD4 interaction and prevent HIV-1
entry to cells. Recently, Morellato-Castillo et al. reported the struc-
ture-based design of CD4-mimetic miniproteins.13 They modified
the Phe43 of CD4 with 11 non-natural phenylalanine derivatives
and one of the analogs M48U12 showed binding affinity of 8 pM
with HIV-1 YU2 gp120 and showed low nM antiviral potency
against several lab-adapted and primary HIV-1 isolates. The intro-
duction of the unnatural phenylalanine group containing cyclo-
replace Boc and assess their structure activity relationship (SAR).
We however, kept the Fmoc group in our set of compounds tested.
Fmoc-containing unnatural amino acids have been reported to be
good lead compounds against HIV-1 protease.15
2.2. Evaluation of antiviral activity of a series of unnatural
amino acids against HIV-1 pseudotyped with lab adapted HIV-
1HXB2 ENV
hexylmethyl amine moiety rendered M48U12
a
pico molar
We first evaluated the HIV-1 inhibitory activity of a series of 15
unnatural amino acid analogs (Table 1) against HIV-1 pseudotyped
with the HIV-1HXB-2 Env by using a single cycle infectivity assay. To
this end, we infected TZM-bl cells with the above pseudovirus in
the presence of increasing concentrations of the compounds. In
parallel, we evaluated the cytotoxicity of the compounds. Our
results indicated that 882376 displayed the best activity against
binder to gp120. Furthermore, M48U12 showed remarkable antivi-
ral activity against HIV-1 laboratory strains, primary isolates and
also transmitted/founder viruses. The X-ray structure of this pep-
tidomimetics bound to gp120 revealed that the cyclohexylmethy-
lamino phenyl moiety of the unnatural phenyl alanine residue
occupies about 80% of the volume of the Phe43 cavity making it
one of the best binders of gp120. Therefore, we are interested to
find out the contribution of this unnatural amino acid in
M48U12 on the antiviral activity and explore the possibility of
using the unnatural amino acid and its analogs as lead for further
optimization.
HIV-1 with a calculated IC50 of 4.5 0.4 lM and a CC50 > 50 lM
(with 30% toxicity at this dose) while the respective unprotected
form, 882896 (without BOC and Fmoc) showed no antiviral activ-
ity. Moreover, compounds 883597, 883598 and 883600 also dis-
played good anti-HIV-1 activity in the range 8.8–9.7 lM,
In this study we selected the unnatural amino acid, 882376,
from the most potent inhibitor, M48U12, prepared its Boc and
Fmoc substituted and other analogs and tested as HIV-1 entry inhi-
bitors. To our surprise when we removed these two protecting
groups we observed complete loss of antiviral activity. Since
882376 showed the best antiviral activity, we evaluated 882376
against a panel of primary HIV-1 Env-pseudotyped viruses from
different subtypes. Moreover, we isolated 882376 HIV-1 resistant
variants from HIV-1NL4-3 cloned virus obtained in the presence of
escalating doses of 882376 to identify three amino acid substitu-
tions in gp120 and two in the gp41 region.
however, they also showed higher toxicity than what we detected
for 882376. When we replaced the Boc protecting group with an
acetyl group (883594), the antiviral activity was also lost. Introduc-
tion of chloro acetyl group recovered some of the lost activity indi-
cating that a bulkier hydrophobic group is favored in that position.
We used chloro acetyl group just to understand the relative impor-
tance of bulkier groups despite the fact this moiety is known to be
reactive group. Importance of bulky group is also evident in com-
pounds 883597 and 883598 where a cyclohexylmethyl and benzyl
groups, respectively, were used. Activity dropped substantially
when a pent-4-enoyl group was used. This group has a more
extended and flexible structure. The importance of bulky
hydrophobic group was further evidenced in compound 883600
where a benzyl formate group was used. However, in Scheme 2
although different groups were used to generate SAR, the bulkier
hydrophobic group did not make any difference in antiviral activity
but notably the toxicity values were in general better.
2. Results
2.1. Chemistry
The synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)
amino)-3-(4-((tert-butoxycarbonyl)(cyclohexylmethyl)amino)phe-
2.3. 882376 prevents cell-to-cell fusion
nyl)propanoic acid (882376) was performed by following
a
reported method.13 882896 was prepared from 882376 by remov-
ing Fmoc by 20% piperidine in DMF (Fig. 1).
HIV-mediated cell-to-cell fusion is a significant mode of HIV-1
spreading from infected cells to uninfected cells. To investigate
whether 882376 prevents HIV-1 spreading through cell-to-cell
fusion we co-cultured MAGI-CCR5 cells with HL 2/3 cells in the
presence of escalating concentrations of compounds for 24 h as
previously reported.16 We used BMS-378806 and NBD-556 which
have been reported to inhibit cell–cell fusion17,18 as controls.
BMS-378806 and NBD-556 inhibited cell-to-cell fusion with an
Analogs of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-
3-(4-((cyclohexylmethyl)amino)phenyl)propanoic acid (883594,
883596, 883597, 883598, 883599 and 883600) and ((S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-((cyclohexylmethyl)
amino)phenyl)propanoyl)-L-proline (902132, 902133, 902134,
902135, 902136 and 902137) were synthesized by using the syn-
thesis outline depicted in Schemes 1 and 2, respectively. All syn-
thesized compounds provided satisfactory analytical and
spectroscopic data to confirm their structures. Since Boc group is
located in the major binding site in the gp120 and is unstable in
acidic and basic conditions we wanted to explore other groups to
IC50 of 0.023 lM and 5.6 lM, respectively, while 882376 inhibited
this process with an IC50 of 10
lM displaying an important HIV-1
entry inhibitor trait (Fig. 2).
2.4. 882376 does not enhance HIV-1 entry into CD4-negative
cells
Previous studies have reported that the small molecule HIV-1
entry inhibitor NBD-556, which has been identified by our group
about a decade ago,18 can also act as CD4-agonist promoting
CCR5 binding and enhancing HIV-1 entry into CD4-negative cells
expressing CCR5.10,19 To assess whether 882376 performs as a
CD4-agonist or -antagonist, we infected CD4-negative Cf2Th-
CCR5 cells with recombinant CD4-dependent HIV-1ADA in the pres-
ence of escalating concentrations of compound. NBD-556 and
BMS-378806 (a highly potent small-molecule HIV-1 entry
inhibitor)20 were used as control. As reported previously, NBD-
Figure 1. The chemical structures of 882376 and 882896.

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F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
Scheme 1. Synthesis of compounds 883594–883600. Reagents and conditions: (a) pent-4-enoic anhydride/DIPEA in THF; (b) acetic anhydride/DIPEA in THF; (c) 2-
cyclohexylacetic anhydride/DIPEA in THF; (d) trifluoroacetic anhydride/DIPEA in THF; (e) Cbz-CI/DIPEA in THF; (f) 2-phenylacetyl chloride/DIPEA in THF; (g) Cl-CH2CONa/
IBCF/NMM in THF; (h) Pd/CH2 in MeOH.
Scheme 2. Synthesis of compounds 902132–902137.
556 enhanced HIV-1 entry into CD4-negative cells while BMS-
378806 as well as 882376 did not, suggesting that 882376 pos-
sesses CD4-antagonist properties (Fig. 3).
against a diverse set of HIV-1. We compared the antiviral activity
of 882376 with the activity of the small molecule NBD-556 (most
of this data was published recently16). Five representative dose-
dependent inhibition plots of the neutralization assay of selected
pseudoviruses [NIH # 11887 (clade A), # 11904 (clade A/D), #
11058 (clade B) #11909 (clade C) and # 11911 (clade D)] are
shown in Figure 4. As previously reported10,16 NBD-556 was active
against all the viral subtypes tested showing poor activity against
the viruses from subtype A and exhibiting an IC50 in the range of
2.5. Evaluation of antiviral activity of 882376 against a panel of
HIV-1 Env-pseudotyped reference viruses
The HIV-1 envelope glycoproteins are highly variable. There-
fore, we decided to test the inhibitory activity of 882376 against
a panel of HIV-1 Env-pseudotyped reference viruses of different
subtypes in a single-cycle infection assay to assess its efficacy
1.9–27.8
lM (Table 2). 882376 was equally active against all the
viruses tested displaying a better anti-HIV-1 activity profile than

F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
7621
Table 1
samples. DNAs were sequenced and analyzed. We detected one
amino acid substitution, K348E (frequency 1/7) in the sample
obtained from the culture grown in the presence of 25 lM of
Antiviral activity (IC50) and toxicity (CC50) of unnatural amino acids in TZM-bl cells
against HIV-1 pseudotyped with lab adapted HIV-1HXB2 ENV
Compound
IC50
(
lM)
CC50 (lM)
882376. By contrast, we identified 5 amino acids substitutions in
the sample obtained from the culture grown in the presence of
882376
882896
883594
883595
883596
883597
883598
883599
883600
902132
902133
902134
902135
902136
902137
4.5 0.4
>140
>50
ꢀ50
>140
>74
33
lM of 882376 (Table 3): K348R located in the C3 that was dif-
ferent (K348E) in the sample from the 25
lM culture; S411G
14.3 1.5
48
2
located in the V4; K432E located in the CD4 binding site; C604Y
located in the immunodominant region of gp41 and C764R also
located in the gp41 (Table 3). We detected the same amino acid
substitutions in the sample obtained from the culture grown in
22
1
>67
26
36
8.8 0.4
9.7 0.4
23.9 1.8
8.9 0.6
12.3 1.4
17.8 1.7
16.8 0.4
16.6 1.8
15.9 1.2
10.4 0.7
3
2
>69
35
1
the presence of to 42
(7/11).
lM of 882376 with a much higher frequency
ꢀ50
ꢀ60
>42
>57
>58
2.8. Impact of mutations on HIV-1 entry and resistance to
882376
24.5 1.5
As next step, we wanted to verify whether the above identified
amino acid substitutions were responsible for the resistance of the
HIV-1NL4-3 to 882376. We introduced single amino acid substitu-
tions into the pHXB2-Env expression vector. We infected U87-
CD4-CXCR4 cells with WT and mutant HIV-1HXB-2 luciferase-
expressing pseudoviruses in the absence or in the presence of
882376. We used NBD-556 and BMS-378806 as controls. As
reported in Table 4, we observed that the K348R substitution had
virtually no effect on antiviral activity as indicated by the low
IC50 detected that was similar to the IC50 obtained for the HIV-
NBD-556. The IC50 of this compound was in the range of 1.4–
12.5 M with an overall mean of 5.17 0.4 compared to the overall
mean of NBD-556 of 7.28 0.74 (p < 0.05) (Fig. 5). Moreover,
882376 as well as NBD-556 did not show activity against control
virus A-MLV, suggesting that the inhibitory activity of this com-
pound is also specific to HIV-1.
l
2.6. Selection of 882376 resistant viruses
1
HXB2 WT. The conservative substitution (K ? R) supports this find-
To further confirm the mechanism of action of the 882376 we
passaged the cloned HIV-1NL4-3 in Jurkat cells in the presence of
escalating doses of the compound. We collected cellular and viral
ing. Also, the substitution S411G only induced a 2-fold increase in
882376 IC50 and had no impact on NBD-556 and BMS-378806
IC50s. By contrast, we observed that the amino acid substitution
detected in the CD4 binding region K432E conferred resistance
not only to 882376 which exhibited an IC50 P 25, about 6-fold
the IC50 obtained for the HIV-1HXB2 WT but also to NBD-556 and
BMS-378806. Moreover, one of the two substitutions detected in
the gp41 region, C764R conferred about 3-fold increase of the
IC50 of 882376 while the C604Y substitution detected in the highly
conserved immunodominant region produced non-infectious virus
when expressed as a single mutation. The substitution of Cysteine
604 with Tyrosine (C604Y) in the unprocessed gp160 disrupts the
gp41 disulfide loop (C598/C604) which has been reported to be
critical to the cellular protease furin recognition site of gp16021
therefore, impedes the processing of gp160 resulting in non-infec-
tious viral particles. When substitutions S411G and K432E were
combined we noticed a slightly increase in viral infectivity suggest-
samples at 25
lM, 33
l
M and 42
lM of 882376. The dose in cul-
ture could not be escalated beyond 42
lM due to toxicity. HIV-
1NL4-3 virus obtained from 882376 cultures and virus obtained
from untreated cultures were re-passaged in fresh Jurkat cells
and tittered by infecting TZM-bl cells. The results suggested that
the viruses released in the culture supernatant had infectivity rate
very similar compared to the untreated control Wild Type (WT)
virus (data not shown).
2.7. Identification and characterization of 882376-resistant
mutants
To identify the amino acid mutations responsible for the HIV-1
resistance to 882376, we isolated genomic DNA from cellular
Figure 3. CD4-independent infectivity assay. CD4-negative Cf2Th-CCR5 cells were
infected with CD4-dependent HIV-1ADA in the presence of increasing concentrations
of 882376, NBD-556 and BMS-378806. Relative virus infectivity specifies the
amount of infection detected in the presence of the compounds relative to the
infection detected in the absence of the compounds. Three independent experi-
ments were performed in triplicate and the graph is representative of one
experiment; the values represent the mean standard deviation.
Figure 2. Cell-to-cell fusion inhibition assay. Inhibitory activity of 882376 and
NBD-556 on HIV-1 mediated fusion of MAGI-CCR5 cells and HL2/3 cells. Experi-
ments were performed in triplicate, and data is shown as mean standard
deviation.

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F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
To further understand the possible implications of the amino
acid mutations on the envelope glycoproteins by this unnatural
amino acid we isolated and genotyped HIV-1 clones resistant to
882376. We identified three amino acid substitutions in gp120
and two in the gp41 region. The substitutions K432E located near
the gp120/CD4 binding region conferred the highest resistance
indicated by 6-fold increase in IC50 with respect to the WT (Table 4)
suggesting that the gp120/CD4 binding region may be the target of
Table 2
Neutralization activity (IC50) of 882376 against a panel of HIV-1 Env pseudoviruses
Subtype NIH #
IC50
(
l
M) SDa
NBD-556
9.5 0.3
20.2
8.1 1.5
ENVs
882376
Figure 4. Representative dose-dependent inhibition curves of 882376 against ENV
pseudotyped HIV-1. Experiments were performed in triplicate, and data is shown as
mean standard deviation.
A
11887
11888
11889
11890
11891
11892
11899
Q259env.w6
4.8 0.5
5.5 0.5
12.5 0.8
QB726.70M.ENV.C4c
QB726.70M.ENV.B3
QF495.23M.ENV.A1c
QF495.23M.ENV.A3c
QF495.23M.ENV.B2c
6
13.6 2.3 1.9 0.2
27.8 9.3 2.7 0.2
ing that these amino acid substitutions may be rescuing the virus
(data not shown). To better understand the binding site of
882376 we also investigated its inhibitory activity against pseu-
doviruses carrying the S375H or S375Y mutation located in the
Phe43 cavity. These mutations reduced the ability of 882376 to
inhibit HIV-1 as evidenced by the 4-fold increase in IC50. Further-
more, we tested 882376 with a set of mutated pseudoviruses based
on BMS-378806 resistance study.20 We selected the mutations that
conferred the highest resistance of HIV-1LAI to BMS-378806, the
two single mutations M475I and M434I. As expected, our results
indicated that these mutations induced resistance to 200 nM
BMS-378806 and only the M475I induced resistance to NBD-556
but both mutants showed marginal resistance to 882376 (about
3-fold). Taken together these results suggest that 882376 may
interfere with the gp120/CD4 interaction preventing HIV-1
infection.
20.4
7
2.8 0.3
12 1.3
6.7 2.3
3.5 0.4
1.QH359.21M.ENV.D1 3.6 0.6
BG505-T332Nc
KNH1144c
11.5
6
20 3.2
A/D
A2/D
A/E
11901
11903
11904
QA790.204I.ENV.A4c
QA790.204I.ENV.C8
QA790.204I.ENV.E2
12.6 2.7 5.2 0.9
9.7 1.8
15 1.7
5.5 0.5
3.2 0.06
11905
11906
11907
QG393.60M.ENV.A1
QG393.60M.ENV.B7c
QG393.60M.ENV.B8
CRF01_AE clone 269c
6.9 0.6
3.5 0.8
11.2 2.5
10.3 0.6
5.8 1.6
4.1
1
11603
(potential)
10.7 1.5 4.4 0.5
AA058
CM244
2.1 0.3
4.2 0.6
3
0.7
9.6 0.4
A/G
B
11597
11601
11602
11605
CRF02_AG clone 253
CRF02_AG clone 263
CRF02_AG clone 266c
CRF02_AG clone 278c
6
1.7
3.9 0.2
6.1 0.3
2.2 0.3
7.3 1.7
4.6 1.6
6.5 1.1
3
0.3
2.9. Mutant viruses do not infect CD4-negative cells
B41c
4.5
2
1
9.4 0.5
2.4 0.1
11563
11578
11018
11022
11023
11033
11035
11036
11037
11038
11058
p1058_11.B11.1550b,c 1.9
pWEAUd15.410.5017b 4.3 0.6
3
0.3
To verify whether the mutated viruses acquired a CD4-indepen-
dent phenotype that does not require interaction with CD4 for
infection, we infected, in parallel, CD4-negative ACTOne-CXCR4
cells and CD4-positive U87-CD4-CXCR4 cells with the same
amounts of mutant pseudovirus carrying a single amino acid sub-
stitution or with WT HIV-1HXB2 as a control. None of the mutant
pseudoviruses acquired the ability to efficiently infect the CD4-
negative ACTOne-CXCR4 cells (Fig. 6).
QH0692, clone 42c
PVO, clone 4c
2
0.6
1.4 0.2
3.2 0.2
3.2 0.1
6.3 0.3
2.8 0.2
4.1 0.6
4.2 0.7
5.3 0.5
5.7 0.2
3.5 0.7
4.1 0.6
TRO, clone 11c
pWITO4160 clone 33c 4.8 0.8
pREJO4541 clone 67c
pRHPA4259 clone 7c
3.4 0.2
4.3 0.5
pTHRO4156 clone 18c 8.4 2.4
pCAAN5342 clone A2
SC422661.8
4.1 0.3
4.8 0.7
C
11306
11307
11308
11309
11310
11311
11312
11313
11314
11315
11316
11317
11908
11909
Du156, clone 12c
Du172, clone 17c
Du422, clone 1c
ZM197M.PB7c
5
0.9
3.5 0.3
3.3 0.2
3.1 0.1
4.8 0.5
3.2 0.2
6.9 0.2
7.3 0.2
3.2 0.2
7.2 0.5
7.7 1.1
9.8 1.5
3.6 0.1
2.5 0.2
2.7 0.2
3. Discussion
4.8 0.2
6.1 0.5
3.8 0.3
4.1 0.7
6.6 0.8
6.1 0.3
Currently, after over 20 years of research, there is no effective
HIV vaccine available. The HAART is the only available treatment
which stabilizes the viremia but does not eradicate HIV and more
importantly does not prevent the spread of HIV. Moreover, the
emergence of recombinant and resistant viruses emphasizes the
need of novel anti-retroviral agents. In this study, we investigated
the effectiveness of 882376, a novel unnatural amino acid selected
from the most potent inhibitor M48U12,13 and its analogs as HIV-1
entry inhibitors that target and disrupt the gp120/CD4 binding.
Single cycle neutralization experiments indicated that 882376
inhibits HIV-1 infection regardless of the co-receptor usage as
demonstrated by its low micromolar antiviral activity against
CXCR4-tropic (lab-adapted HIV-1HXB2 and HIV-1NL4-3) and CCR5-
tropic (primary isolates, Table 2) HIV-1 viruses. Additionally,
882376 showed broad spectrum inhibition against a diverse panel
of HIV-1 Env-pseudotyped viruses exhibiting better activity
against subtypes A, A/G and B viruses.
ZM214M.PL15c
ZM233M.PB6c
ZM249M.PL1c
ZM53M.PB12c
14.6
1
ZM109F.PB4c
6.2 0.3
2.2 0.6
6.2 2.4
5.3 0.6
5.7 0.9
3.6 0.9
ZM135M.PL10ac
CAP45.2.00.G3
CAP210.2.00.E8c
QB099.391M.ENV.B1c
QB099.391M.ENV.C8c
D
11911
11912
11916
11917
11918
Control
QA013.70I.ENV.H1c
QA013.70I.ENV.M12c
QD435.100M.ENV.B5c
QD435.100M.ENV.A4
QD435.100M.ENV.E1
A-MLV
9.4 2.6
5.4 0.3
2.4 0.2
3.3 0.1
4.4
4.4 0.7
0.4
>50
1
4
0.2
8.4 0.9
10.2 0.6
>23
4
a
b
c
The reported IC50 are means standard deviations (n = 3).
R5X4-tropic viruses; all the rest were R5-tropic viruses.
NBD-556 data previously published (Ref. 16).

F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
7623
Figure 6. CD4-dependent (U87-CD4-CXCR4 cells) and CD4-independent (CD4-
negative ACTOne-CXCR4 cells) infectivity assay of mutant and WT HIV-1HXB2
pseudoviruses. Infectivity of the pseudoviruses detected in the ACTOne-CXCR4 cells
is reported as percentage of infectivity with respect to the infectivity detected in the
U87-CD4-CXCR4 cells. Values represent the mean standard deviation of five
independent experiments.
Figure 5. Comparison of IC50 obtained for NBD-556 and 882376 against a large
panel of HIV-1 pseudoviruses from different clades. The significance is referred as
the mean standard error.
The S375Y substitution was shown to confer the highest resistance
to entry inhibitors NBD-556, NBD-09027 and NBD-11008 as well
as BMS-378806.10 In this study we tested 882376 against two
mutant pseudoviruses carrying single substitution S375H and
S375Y. Also in these neutralization experiments we detected that
the IC50 of 882376 against both mutants increased by about 4-fold
with respect to the WT, again supporting the hypothesis that
882376 binds in the gp120/CD4 binding region. Another unique
feature of this compound is that induces an amino acid substitu-
tion in a highly conserved region of the gp41.21 This substitution
C604Y by abolishing the disulfide loop of gp41 (C598/C604) inhi-
bits the cellular protease furin to recognize its gp160 site and as
consequence, induces the production of non-infectious viruses
which carry an uncleaved gp160 incapable of binding the CD4 cel-
lular receptor.
Table 3
Amino acid substitutions detected by sequencing HIV-1NL4-3 resistant to different
doses of 882376
Mutation
Frequency
33
Location
25
lM
l
M
42 lM
K348R
S411G
K432E
C604Y
C764R
(K/E) 1/7
0/7
0/7
0/7
0/7
1/6
1/6
1/6
1/6
1/6
7/11
7/11
7/11
7/11
7/11
C3
V4
CD4 binding site
gp41 (immunodominant region)
gp41
The numbering was based on HIV-1HXB2 in the Los Alamos HIV database.
4. Conclusion
Table 4
Sensitivity of mutated-pseudoviruses to 882376
We can conclude that this unnatural amino acid 882376
showed a wide spectrum of inhibition against a diverse panel of
HIV-1 viruses and although an X-ray structure of 882376 bound
to gp120 core is not available our resistance studies and the exper-
iments performed with the mutant viruses indicate that 882376
may target the CD4 binding site in gp120 and disrupts the
gp120/CD4 interaction preventing HIV-1 infection. Therefore,
882376 has the potential as a lead compound for further optimiza-
tion to more potent HIV-1 gp120 targeted entry inhibitors.
882376 (
>50
lM)
NBD-556 (
lM)
BMS-378806 (nM)
Toxicity CC50
HXB2 WT IC50
>100
7.4 0.8
—
ꢀ1
4
0.9
882376 mutants
K348R
S411G
K432E
C764R
5.6
1
7.3 0.2
7.4 2.6
>30
7.8 1.2
22.3 1.2
ꢀ1
8.4 1.6
P25
11.6 0.2
12.2 0.9
ꢀ1
ꢀ25
ꢀ1
S411G/K432E
ꢀ25
Phe43 cavity mutants
S375H
S375Y
>21
>21
>20
>30
>200
>200
5. Experimental
BMS-378806 mutants
5.1. Cells and plasmids
M475I
M434I
>15
15.6 2.6
>20
1.2 0.8
>200
ꢀ25
TZM-bl cells (a HeLa cell line that expresses CD4, CXCR4, and
CCR5 and also luciferase under the control of the HIV-1 pro-
moter),23,24 HL 2/3 cells (from Drs. B.K. Felber and G.N. Pavlakis),25
MAGI-CCR5 cells (from Dr. J. Overbaugh),26 Jurkat cells27 and U87-
CD4-CXCR4 cells (from H. Deng and D. R. Littman)28 were obtained
through the NIH ARP. The CD4 negative Cf2Th-CCR5 cells were
kindly provided by Dr. J.G. Sodroski.29 The HEK 293T cells were
purchased from ATCC. The ACTOne-CXCR4 cells, a modified HEK
293T cell line, expressing coreceptor CXCR4 without CD4 were pur-
chased from Codex BioSolutions, Inc., Gaithersburg, MD.
this compound. This finding was also supported by the poor antivi-
ral activity of 882376 against two mutant pseudoviruses, M475I
and M434I carrying amino acid substitutions located at or near
the CD4 binding pocket, as shown by the 3-fold increase in IC50
with respect to the WT virus (Table 4). CD4 binds the HIV gp120
between the inner and outer domain creating a conserved cavity,
known as the Phe43 cavity. Amino acid substitutions in this region
especially the residue 375 (S/Y, S/H or S/W) may partially fill the
cavity conferring resistance to neutralization by HIV-1 entry inhi-
bitors that target the gp120/CD4 binding pocket. In fact, the substi-
tution S375H was suggested to be responsible for the resistance of
the CRF01_AE HIV-1 to a potent entry inhibitor, BMS-599793.22
The full-length, replication and infection-competent chimeric
DNA pNL4-3 (from M. Martin),30 the Env-deleted proviral back-
bone plasmids pNL4-3.Luc.R-.E-DNA (from N. Landau)31,32 and
the pSG3 DNA (from J.C. Kappes and X. Wu)24,33 were obtained

7624
F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
through the NIH ARP. The Env-deleted proviral backbone plasmid
pNL4-3KFS DNA was kindly provided by Dr. E. Freed from NIH/NCI.
The HIV-1 Env molecular clone expression vector pHXB2 (X4)
DNA was obtained through the ARP from K. Page and D. Littman.34
The Env expression vector pSVIIIenv-ADA DNA was kindly pro-
vided by Dr. J.G. Sodroski.29 The HIV-1 Env molecular clones of
gp160 genes for HIV-1 Env pseudovirus production were obtained
as follows: the clones representing the standard panels A, A/D, A2/
D and D and panel C (QB099.391M.ENV.C8, QB099.391M.ENV.B1)
were obtained through the NIH ARP from J. Overbaugh.35,36 The
HIV-1 Env molecular clones of subtype A/G and the CRF01_AE
clone 269 were obtained through the NIH ARP from D. Ellenberger,
B. Li, M. Callahan, and S. Butera.37 The AE clones AA058 and CM244
were kindly provided by Drs. R.J. McLinden and A.L. Chenine from
US Military HIV Program, Henry M. Jackson Foundation (Silver
Spring, MD). The HIV-1 Env molecular clones of standard reference
subtype B SC422661.8, QH0692 clone 42, PVO clone 4 and TRO
clone 11 were obtained through the NIH ARP from D. Montefiori,
F. Gao, and M. Li. B.H. Hahn and J.F. Salazar-Gonzalez provided
pWITO4160 clone 33, pREJO4541 clone 67 and pRHPA4259 clone
7. B.H. Hahn and D.L. Kothe provided pTHRO4156 clone 18 and
pCAAN5342 clone A2.24,33,38 The subtype B pWEAUd15.410.5017
and p1058_11.B11.1550 were obtained through the NIH ARP from
Drs. B.H. Hahn, B.F. Keele and G.M. Shaw.39 The subtype C HIV-1
reference panel of Env clones were also obtained through the
NIH ARP from Drs. D. Montefiori, F. Gao, S.A. Karim and G. Ramjee
(Du156 clone 12 and Du172 clone 17); from Drs. D. Montefiori, F.
Gao, C. Williamson and S.A. Karim (Du422 clone 1), from Drs. B.
H. Hahn, Y. Li and J.F. Salazar-Gonzalez (ZM197M.PB7, ZM233M.
PB6, ZM249M.PL1 and ZM214M.PL15); from Drs. E. Hunter and C.
Derdeyn (ZM53M.PB12, ZM135M.PL10a and ZM109F.PB4); from
Drs. L. Morris, K. Mlisana and D. Montefiori, (CAP45.2.00.G3 and
CAP210.2.00.E8).
5.2.3. General procedure for the synthesis of (S)-2-((((9H-
fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-
((cyclohexylmethyl)amino)phenyl)propanoic acid derivative
(883594, 883596, 883597, 883598, 883599, 883600)
To a solution of 2 (100 mg, 0.18 mmol) and DIPEA (65
ll,
0.37 mmol) in dry THF (4 mL) a solution of an acid anhydride or
acid chloride (0.28 mmol) in dry methylene chloride (1 mL) was
added dropwise with stirring at 0 °C in about 20 min. The reaction
mixture was then allowed to warm up to room temperature and
stirred for 2–3 h. It was then diluted with methylene chloride
(50 mL) and washed with water (30 mL), saturated sodium bicar-
bonate (30 mL), brine (30 mL) and dried over anhydrous Na2SO4.
The solvent was removed in vacuo to give crude (S)-2-((((9H-fluo-
ren-9-yl)methoxy)carbonyl)amino)-3-(4-((cyclohexylmethyl)
amino)phenyl)propanoic acid derivative, which was purified by
HPLC to obtain pure derivatives 883594 (33 mg, 96% purity),
883596 (54 mg, 98.8% purity), 883597 (73 mg, 99% purity),
883598 (54 mg, 99% purity), 883599 (25 mg, 98.9% purity),
883600 (53 mg, 97.9% purity).
883594: Yield: 33%; 1H NMR (400 MHz, DMSO-d6) d: 7.896 (d,
J = 7.2 Hz, 2H), 7.820 (d, J = 8.8 Hz, 1H), 7.656 (m, 2H), 7.675–
7.629 (m, 2H), 7.357–7.316 (m, 4H), 7.138 (d, J = 8.0 Hz, 1H),
4.242–4.114 (m, 4H), 3.476–3.424 (m, 2H), 3.329–3.279 (m, 1H),
3.159–3.117 (m, 1H), 2.913–2.851 (m, 1H), 1.669–1.434 (m, 7H),
1.248 (m, 2H), 0.989 (s, 3H), 0.785–0.726 (m, 2H). ESI-MS: 540.7
(C33H36N2O5, [M+H]+).
883596: Yield: 42.6%; 1H NMR (400 MHz, DMSO-d6) d: 7.820 (d,
J = 7.2 Hz, 2H), 7.752 (d, J = 8.8 Hz, 1H), 7.598–7.548 (m, 2H),
7.359–7.305 (m, 4H), 7.247–7.241 (m, 2H), 7.158 (d, J = 7.6 Hz,
1H), 4.180 (m, 1H), 4.098–4.045 (m, 3H), 3.469–3.417 (m, 1H),
3.107–3.063 (m, 1H), 2.860–2.798 (m, 1H), 1.480–1.331 (m, 5H),
1.238–1.166 (m, 2H), 0.932 (m, 3H), 0.781–0.706 (m, 2H). ESI-
MS: 594.6, (C33H33F3N2O5, [M+H]+).
The ENV pseudotyped genes of BG505.T332N, KNH1144 and
B41 were kindly provided by Dr. J.P. Moore of the Weil Cornell
Medical College, NY.
SV-A-MLV-env was obtained through the NIH ARP from Drs. N.
Landau and D. Littman.
883597: Yield: 62.7%; 1H NMR (400 MHz, DMSO-d6) d: 7.891 (d,
J = 7.2 Hz, 2H), 7.842 (d, J = 8.8 Hz, 1H), 7.697 (d, J = 7.2 Hz, 1H),
7.627 (d, J = 7.2 Hz, 1H), 7.430–7.305 (m, 6H), 7.048 (d, J = 7.6 Hz,
1H), 4.272–4.232 (m, 1H), 4.110 (s, 3H), 3.417 (s, 1H), 3.325–
3.274 (m, 1H), 3.173–3.131 (m, 1H), 2.900–2.837 (t, J = 12.6 Hz,
1H), 1.737–1.328 (m, 13H), 1.242 (s, 1H), 1.010 (m, 5H), 0.807–
0.776 (m, 3H), 0.538–0.425 (m, 2H). ESI-MS: 622.8 (C39H46N2O5,
[M+H]+).
5.2. Synthesis
5.2.1. Synthesis of 882376 and 882896
883598: Yield: 46.8%; 1H NMR (400 MHz, DMSO-d6) d: 7.888 (d,
J = 7.2 Hz, 2H), 7.832 (d, J = 8.8 Hz, 1H), 7.658–7.606 (m, 2H),
7.405–7.300 (m, 6H), 7.158–7.084 (m, 4H), 6.880 (d, J = 6.8 Hz,
2H), 4.287–4.229 (m, 1H), 4.114 (s, 3H), 3.498–3.446 (m, 2H),
3.358–3.146 (m, 4H), 2.920–0.859 (t, J = 12.2 Hz, 1H), 1.562–
1.446 (m, 5H), 1.262–1.243 (m, 1H), 1.006–0.949 (m, 3H), 0.811–
0.753 (m, 2H). ESI-MS: 616.8 (C39H40N2O5, [M+H]+).
The synthesis of 1 (882376) was performed by following a
reported method.13 882896 was prepared from 882376 by remov-
ing protecting groups Boc and Fmoc by 4.0 N HCl in Dioxane and by
20% piperidine in DMF, respectively. The molecular mass of these
molecules and all molecules reported subsequently were deter-
mined by low resolution mass spectrometry (LR-MS).
882376: 1H NMR (400 MHz, CD3Cl) d: 7.766 (d, J = 6 Hz, 2H),
7.588–7.556 (m, 2H), 7.408–7.378 (m, 2H), 7.324–7.294 (m, 2H),
7.110 (s, 4H), 4.721–4.706 (m, 1H), 4.435–4.326 (m, 2H), 4.217–
4.189 (m, 1H), 3.457 (d, J = 5.6 Hz, 2H), 3.153 (s, 2H), 1.663 (s,
4H), 1.447 (s, 9H), 1.255 (s, 4H), 1.131–1.117 (m, 3H). ESI-MS:
598.7 (C36H42N2O6, [M+H]+).
883599: Yield: 21.5%; 1H NMR (400 MHz, DMSO-d6) d: 7.894 (d,
J = 7.2 Hz, 2H), 7.825 (d, J = 8.8 Hz, 1H), 7.682–7.616 (m, 2H),
7.434–7.396 (m, 2H), 7.367–7.305 (m, 4H), 7.114 (d, J = 8.0 Hz,
1H), 5.567–5.500 (m, 1H), 4.790–4.727 (m, 2H), 4.278–4.220 (m,
1H), 4.151–4.105 (m, 3H), 3.330–3.278 (m, 1H), 3.166–3.124 (m,
1H), 2.908–2.846 (m, 1H), 2.098–1.855 (m, 4H), 1.550–1.432 (m,
5H), 1.243 (s, 1H), 0.999 (m, 3H), 0.842–0.708 (m, 2H). ESI-MS:
580.7. ESI-MS: 580.7 (C36H40N2O5, [M+H]+).
882896: 1H NMR (400 MHz, CD3Cl) d: 7.406 (s, 4H), 4.074–4.028
(m, 1H), 3.243–3.229 (m, 4H), 1.727–1.578 (m, 6H), 1.202–0.997
(m, 5H). ESI-MS: 276.4 (C16H24N2O2, [M+H]+).
883600: Yield: 42%; 1H NMR (400 MHz, DMSO-d6) d: 7.892 (d,
J = 7.2 Hz, 2H), 7.809 (d, J = 8.4 Hz, 1H), 7.673 (t, J = 6.8 Hz, 2H),
7.424–7.388 (m, 2H), 7.337–7.276 (m, 7H), 7.234–7.220 (m, 1H),
7.145–7.125 (d, J = 8 Hz, 2H), 5.073–5.000 (m, 2H), 4.210–4.136
(m, 4H), 3.383–3.330 (m, 2H), 3.123–3.080 (m, 1H), 2.891–2.829
(t, J = 12.4 Hz, 1H), 1.546–1.464 (m, 5H), 1.283–1.243 (m, 1H),
0.980–0.961 (m, 3H), 0.773–0.709 (m, 2H). ESI-MS: 632.4
(C39H40N2O6, [M+H]+).
5.2.2. Synthesis of 2
A solution of 1 (1.5 g, 2.5 mmol) in 20 mL of 4.0 N HCl/Dioxane
was stirred at room temperature for 2 h, the reaction mixture was
monitored by TLC. The 1 was reacted completely then the reaction
mixture was concentrated under vacuum to give 2, which was used
to next step without further workup.

F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
7625
5.2.4. Synthesis of 3
To a solution of 1 (1 g, 1.67 mmol) and Na2CO3 (213 mg,
2.0 mmol) in DMF (10 mL), Benzyl bromide (240 l, 2.0 mmol)
was added dropwise with stirring at room temperature and stirred
overnight at room temperature. Then the reaction mixture was
diluted with DCM (100 mL) and washed with 5% H2PO3
(100 mL ꢁ 3), water (100 mL), brine (100 mL), dried over anhy-
dride Na2SO4, the solvent was removed in vacuo to give 1.5 g of
3, which was used to the next step without further purification.
5.3.1. Synthesis of 20
To
a
solution of
1
(580 mg, 0.818 mmol) in HCl/dioxane
l
(3.3 mol/L), the solution was stirred at room temperature for 2 h.
The reaction completion was confirmed by TLC (DCM/MeOH/
AcOH = 40:1:0.5). The solution was concentrated in vacuo to obtain
20 (500 mg, yield = 100%).
5.3.2. Synthesis of 902132
(a) (18 mg, 0.252 mmol) was added in the mixture of a solution
of 2 (75 mg, 0.126 mmol) and Et3N (33 mg, 0.315 mmol) in dry THF
(1.0 mL). The mixture was stirred at room temperature for 0.5 h.
The completion of reaction was confirmed by TLC (DCM/MeOH/
AcOH = 40:1:0.5). The solution was poured into water and
extracted by ether. The organic phase was collected and washed
by brine and dried over Na2SO4. Then it was concentrated and
the compound was Prep-HPLC to get 902132 (32 mg) as a white
solid.
5.2.5. Synthesis of 4
A solution of 3 (1.5 g, 1.67 mmol) in 20 mL of 4.0 N HCl/Dioxane
was stirred at room temperature for 2 h, the reaction mixture was
monitored by TLC. After the reaction was complete, the reaction
mixture was concentrated under vacuum to give 4, which was used
to the next step without further purification.
5.2.6. Synthesis of 5
Yield: 39%; 1H NMR (400 MHz, DMSO-d6) d: 7.819 (d, J = 8 Hz,
3H), 7.643–7.570 (m, 2H), 7.356–7.322 (m, 3H), 7.264–7.227 (m,
2H), 6.966–6.882 (m, 2H), 5.993–5.951 (d, 1H), 5.816–5.749 (m,
1H), 5.295 (d, J = 10.4 Hz, 1H), 4.439–4.328 (m, 1H), 4.223–4.192
(m, 1H), 4.028 (s, 2H), 3.588–3.419 (m, 3H), 2.921–2.757 (m, 2H),
2.107–2.061 (m, 1H), 1.920–1.689 (m, 4H), 1.482–1.309 (m, 5H),
1.169 (s, 2H), 0.906–0.860 (m, 3H), 0.730–0.657 (m, 2H). ESI-MS:
649.8 (C39H43N3O6, [M+H]+).
To a solution ClCH2CO2Na (932 mg, 8 mmol) in anhydrous THF
(8 mL) isobutyl chloroformate (1.27 mL, 9.6 mmol) was added
dropwise with stirring at 0 °C for 15 min, filtered and the filtrate
was added to a solution of 4 (500 mg, 0.8 mmol) in anhydrous
THF (4 mL), the reaction mixture was stirred at room temperature
for 1 h, the solvent was removed in vacuo and the residue was dis-
solved in DCM (100 mL), washed with 5% H3PO4 (100 mL), brine
(100 mL), dried over anhydride Na2SO4, the solvent was removed
in vacuo to give 510 mg of 5, which was used to next step without
further purification.
5.3.3. Synthesis of 902133
A
solution of 2 (80 mg, 0.13 mmol) and Et3N (33 mg,
0.315 mmol) in dry THF (1.0 mL), (b) (21 mg, 0.2 mmol) was added
in the mixture. The mixture was stirred at room temperature for
0.5 h. The reaction completion was confirmed by TLC (DCM/
MeOH/AcOH = 40:1:0.5). The solution was pour into water and
extracted by ether. Collecting the organic phase was washed
by brine and dried over Na2SO4. Then it was concentrated and
the compound was Prep-HPLC to get 902133 (30 mg) as a white
solid.
5.2.7. Synthesis of 883595
A reaction mixture of 5 (510 mg, 0.77 mmol) and Pd/C (100 mg)
in MeOH (10 mL) was stirred in an atmosphere of H2 overnight, the
reaction mixture was filtered to remove Pd/C, the solvent was
removed in vacuo to give crude of 883595 (310 mg, HPLC purity:
86%), and which was purified by HPLC to give pure of 883595
(115 mg, HPLC purity is 99%).
883595: Yield: 37%; 1H NMR (400 MHz, DMSO-d6) d: 7.895 (d,
J = 7.2 Hz, 2H), 7.821 (d, J = 8.8 Hz, 1H), 7.679–7.625 (m, 2H),
7.434–7.366 (m, 4H), 7.343–7.301 (m, 2H), 7.222 (d, J = 8.0 Hz,
1H), 4.251–4.114 (m, 4H), 3.899–3.849 (m, 2H), 3.499–3.447 (m,
1H), 3.364–3.312 (m, 1H), 3.171–3.128 (m, 1H), 2.928–2.866 (m,
1H), 1.570–1.486 (m, 5H), 1.243 (s, 1H), 0.998 (m, 3H), 0.841–
0.750 (m, 2H). ESI-MS: 575.1 (C33H35ClN2O5, [M+H]+).
Yield: 35%; 1H NMR (400 MHz, DMSO-d6) d: 7.899–7.851 (m,
2H), 7.729–7.655 (m, 2H), 7.440–7.404 (m, 2H), 7.346 (d,
J = 7.2 Hz, 4H), 7.015 (d, J = 8 Hz, 2H), 4.832 (s, 1H), 4.705 (s, 1H),
4.449–4.414 (m, 1H), 4.292–4.269 (m, 1H), 4.125–4.080 (m, 3H),
3.384–3.333 (m, 2H), 2.955–2.786 (m, 2H), 2.160–2.132 (m, 1H),
2.016–1.870 (m, 4H), 1.588–1.463 (m, 5H), 1.245 (s, 4H), 0.977–
0.718 (m, 6H). ESI-MS: 663.8 (C40H45N3O6, [M+H]+).
5.3. Synthesis of 10
5.3.4. Synthesis of 902134
(c) (40 mg, 0.25 mmol) was added in a solution of 2 (100 mg,
0.168 mmol) and Et3N (43 mg, 0.42 mmol) in dry THF (1.5 mL).
The mixture was stirred at room temperature for 0.5 h. The reac-
tion completion was confirmed by TLC (DCM/MeOH/
AcOH = 40:1:0.5). The solution was poured into water and
extracted by ether. Collecting the organic phase was washed by
brine and dried over Na2SO4. Then it was concentrated and
the compound was Prep-HPLC to get 902134 (65 mg) as a white
solid.
Fmoc-Pro-CTC resin (6.7 g, 2.0 mmol) was swelled in DMF
(100 mL) for 2 h. The suspension was filtered. 20% piperidine in
DMF (10 mL) was added into the resin. The suspension was kept
at room temperature for 0.5 h while a stream of nitrogen was bub-
bled through it. Then the suspension was filtered. The resin was
washed with DMF (6 ꢁ 100 mL). Fmoc-(S)-2-amino-3-(4-(tert-
butoxycarbonyl(cyclohexylmethyl)amino)phenyl)propanoic acid
(1.56 g, 2.6 mmol), HATU (0.936 g, 2.47 mmol), N-methylmorpho-
line (0.572 mL, 5.2 mmol) and DMF 20 mL were added into the
resin. The suspension was kept at room temperature for 2 h while
a stream of nitrogen was bubbled through it. When the ninhydrine
test indicated the completion of the coupling, the peptidyl resin
was washed with DMF (5 ꢁ 10 mL), MeOH (2 ꢁ 10 mL), DCM
(2 ꢁ 10 mL) and MeOH (2 ꢁ 10 mL), then it was dried under vac-
uum overnight to give the peptidyl resin (7.5 g).
Yield: 55%; 1H NMR (400 MHz, DMSO-d6) d: 7.897–7.831 (m,
3H), 7.714–7.656 (m, 2H), 7.439–7.402 (m, 2H), 7.343–7.304 (m,
3H), 7.230 (s, 1H), 7.121 (s, 1H), 6.999 (d, J = 4 Hz, 2H), 6.765 (d,
J = 4.8 Hz, 1H), 4.429–4.418 (m, 1H), 4.295–4.264 (m, 1H), 4.117
(m, 3H), 3.666–3.598 (m, 2H), 3.506–3.457 (m, 2H), 2.955–2.766
(m, 2H), 2.181–1.841 (m, 4H), 1.585–1.464 (m, 5H), 1.245 (s, 1H),
1.036–0.772 (m, 5H). ESI-MS: 705.9 (C41H43N3O6S, [M+H]+).
100 mL of AcOH/TFE/DCM (1:2:7) was added to the peptidyl
resin (7.5 g) in a glass vessel. The mixture was stirred for 1 h. The
suspension was filtered. The filtration was collected and the sol-
vent was removed under vacuum to give 10 as a white solid. 95%
of purity by UV220 (1.3 g, yield: 94.9%).
5.3.5. Synthesis of 902135
(d) (40 mg, 0.25 mmol) was added at ꢂ20 °C to a solution of 2
(100 mg, 0.168 mmol) and Et3N (43 mg, 0.42 mmol) in dry THF
(1.5 mL). The mixture was stirred at ꢂ20 °C-room temperature

7626
F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
for 0.5 h. The reaction completion was confirmed by TLC (DCM/
MeOH/AcOH = 40:1:0.5). The solution was poured into water and
extracted by ether. Collecting the organic phase was washed by
brine and dried over Na2SO4. Then it was concentrated and the
compound was Prep-HPLC to get 902135 (25.1 mg) as a white
solid.
5.5. Evaluation of antiviral activity
5.5.1. Single-cycle infection assay in TZM-bl cells
The antiviral activity of the synthesized compounds against
HIV-1 pseudotyped viruses expressing HXB-2 Env or a HIV-1 Env
from the panel of standard reference was measured as previously
described.9 Briefly, TZM-bl cells were plated in a 96-well tissue cul-
ture plate at 105 cells/mL and incubated overnight. Fifty microliters
of a test compound at graded concentrations was mixed with 50 ll
of the HIV-1 pseudovirus. After incubation at 37 °C for 30 min, the
Yield: 21%; 1H NMR (400 MHz, DMSO-d6) d: 7.890–7.843 (m,
3H), 7.692–7.656 (m, 2H), 7.415 (d, J = 8.0 Hz, 4H), 7.333–7.299
(m, 2H), 7.114–7.052 (m, 2H), 5.846 (d, J = 2.4 Hz, 1H), 5.411 (d,
J = 2.8 Hz, 1H), 4.500–4.398 (m, 1H), 4.312–4.280 (m, 1H), 4.111–
4.086 (m, 2H), 3.010–2.967 (m, 1H), 2.879–2.821 (m, 1H), 2.086
(s, 3H), 1.962–1.869 (m, 2H), 1.589–1.484 (m, 5H), 1.244 (s, 1H),
1.040–0.955 (m, 3H), 0.862–0.799 (m, 2H). ESI-MS: 703.8
(C42H45N3O7, [M+H]+).
mixture was added to the cells and incubated for 3 days. Cells were
washed and lysed with 50
lysate was transferred to a white 96-well plate and mixed with
100 l of the luciferase assay reagent. We immediately measured
ll of the cell lysis reagent; 20 ll of
l
the luciferase activity with a Tecan Infinite M1000 reader to calcu-
late the percent inhibition by the compounds and IC50s by using
GraphPad Software, Inc, California.
5.3.6. Synthesis of 902136
(e) (18 mg, 0.13 mmol) was added in a solution of 20 (62 mg,
0.10 mmol) and Et3N (25 mg, 0.25 mmol) in dry THF (1.0 mL).
The mixture was stirred at room temperature for 0.5 h. The
reaction completion was confirmed by TLC (DCM/MeOH/
AcOH = 40:1:0.5). The solution was poured into water and
extracted by ether. Collecting the organic phase was washed by
brine and dried over Na2SO4. Then it was concentrated and
the compound was Prep-HPLC to get 902136 (35 mg) as a white
solid.
5.5.2. Single-cycle infection assay in U87 cells
U87-CD4-CXCR4 cells were plated in 96-well plates at 104 cells/
well and incubated overnight as previously described.10 Briefly,
pseudoviruses were pre-incubated with escalating doses of com-
pounds for 30 min. The mixtures were then added to the respective
cells and incubated for 3 days. Cells were washed and lysed with
40
reagent and the luciferase activity was measured as described
above to calculate the IC50
ll of lysis reagent. Lysates were mixed with the luciferase assay
Yield = 51%; 1H NMR (400 MHz, DMSO-d6) d: 8.352 (s, 1H),
7.897–7.852 (m, 3H), 7.711–7.652 (m, 2H), 7.437–7.305 (m, 6H),
7.108 (d, J = 8 Hz, 2H), 5.774 (s, 1H), 4.457–4.423 (m, 1H), 4.292–
4.262 (m, 1H), 4.144–4.093 (m, 2H), 3.638–3.603 (m, 2H), 2.988–
2.942 (m, 1H), 2.861–2.804 (m, 1H), 2.180–1.997 (m, 2H), 1.922–
1.840 (m, 2H), 1.658–1.656 (m, 2H), 1.569–1.471 (m, 3H), 1.344
(m, 1H), 1.244 (m, 2H), 1.008–0.776 (m, 5H). ESI-MS: 690.8
(C40H42N4O7, [M+H]+).
.
5.5.3. Single-cycle infection assay in CD4-negative cells Cf2Th-
CCR5
Cf2Th-CCR5 were seeded at 6 ꢁ 103 cells/well in a 96 well tissue
culture plate and incubated at 37 °C overnight as previously
described.10,16 Briefly, 50
ll of a test compound at graded concen-
trations was mixed with an equal volume of the HIV-1ADA recom-
binant virus. After incubation at 37 °C for 30 min, the mixtures
were added to the cells and incubated for 48 h. Cells were washed
5.3.7. Synthesis of 902137
(f) (48 mg, 0.3 mmol) in dry THF (0.5 mL) was added dropwise
in a solution of 20 (150 mg, 0.25 mmol) and Et3N (51 mg, 0.5 mmol)
in dry THF (2.0 mL), at 0 °C. The mixture was stirred at 0 °C-room
temperature for 0.5 h. The completion of reaction was confirmed
by TLC (DCM/MeOH/AcOH = 40:1:0.5). The solution was poured
into water and extracted by ether. The organic phase was collected
and washed by brine and dried over Na2SO4. Then it was concen-
trated and the compound was prep-HPLC to get 902137 (56 mg)
as a white solid.
and lysed with 40 ll of cell culture lysis reagent. Lysates were
mixed with luciferase assay reagent and the luciferase activity
was immediately measured as described above.
5.6. Cell-to-cell fusion
To evaluate the ability of 882376 to block cell-to-cell fusion
mediated by HIV-1 we performed cell fusion assay as previously
described.16 MAGI-CCR5 cells (a HeLa cell clone expressing human
CD4, both co-receptors CXCR4 and CCR5 and HIV-LTR-b-gal)26,40
were used as target cells and HL 2/3 cells (a HeLa-derived cell line
which express HIV-1HXB2 Env on the surface and Tat and other viral
proteins in the cytoplasm and does not produce detectable
amounts of mature virions)25 were used as effector cells. Following
fusion of the two cell types, Tat induces the expression of b-gal
enzyme in the MAGI-CCR5 cells. Briefly, 1.5 ꢁ 104/well MAGI-
CCR5 cells were incubated for 1 h with escalating concentrations
of compounds then co-cultured with 7.5 ꢁ 103/well HL 2/3 cells
for 24 h. b-gal expression was quantified with the Beta-GloÒ Assay
System (Promega) following the manufacturer’s instructions. The
percent inhibition and the IC50 values were calculated using the
GraphPad Prism software.
Yield = 31%; 1H NMR (400 MHz, DMSO-d6) d: 7.893 (d,
J = 7.2 Hz, 3H), 7.763–7.725 (m, 1H), 7.660 (d, J = 7.6 Hz, 1H),
7.416–7.322 (m, 6H), 7.017–6.975 (m, 1H), 4.482 (m, 1H), 4.397–
4.387 (m, 1H), 4.304–4.014 (m, 2H), 3.508–3.163 (m, 3H),
2.992–2.819 (m, 2H), 2.338–2.143 (m, 2H), 2.017–0.830 (m, 3H),
1.555–1.447 (m, 8H), 1.346–1.244 (m, 5H), 1.015–1.005 (m, 6H),
0.862–0.679 (m, 6H). ESI-MS: 719.0 (C44H53N3O6, [M+H]+).
5.4. Pseudovirus preparation
The pseudoviruses capable of single-cycle infection were pre-
pared as previously described.9 Briefly, HEK293T cells were trans-
fected with a mixture of an Env-deleted proviral backbone plasmid
pNL4-3.Luc.R.E DNA or pSG3 DNA and an Env expression vector by
using FuGENE 6 (Promega). Amphotropic-MLV (A-MLV) pseu-
dovirus was prepared by transfecting 293T cells with a mixture
of the Env-deleted proviral backbone plasmid pNL4-3KFS DNA
and A-MLV Env expression vector by using Lipofectamin 2000
reagent (Life Technologies). Pseudovirus titers to identify the 50%
tissue culture infectious dose (TCID50) by infecting the different
cell types were determined as previously described.9
5.7. Cytotoxicity assay
The cytotoxicity of test compounds in TZM-bl and U87-CD4-
CXCR4 cells was measured by a colorimetric method using XTT
(PolySciences, Inc., Warrington, PA) as previously described.41
Briefly, 100
ll of the compound at graded concentrations was
added to an equal volume of cells (105/mL) and incubated for

F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
7627
3 days. Four hours after the addition of XTT, the soluble intracellu-
Acknowledgements
lar formazan was quantitated colorimetrically at 450 nm. The per-
cent cytotoxicity and the 50% cytotoxic concentration (CC50) values
were calculated.
This study was supported by funds from NIH – United States
Grant R01 AI104416 (A.K.D.) and from the intramural fund from
the New York Blood Center (A.K.D.).
5.8. Isolation and characterization of 882376 resistant viruses
Supplementary data
To select HIV-1 resistant variants, Jurkat cells were infected
with wild-type HIV-1NL4-3 in the presence of 25 and 33 lM of
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.11.006.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
882376. Viral replication was monitored by measuring p24
amounts in the culture medium by sandwich-ELISA. After 3 pas-
sages (10 days of culture) we detected small amounts of p24 in
the cell culture medium of the cells treated with 25
882376 and after 6 passages (20 days of culture) p24 was detected
in the culture medium of the cells treated with 33 M of 882376.
Thus, we escalated the concentration of the compound up to
42 M. The cell cultures were continuously renewed by adding
fresh cells during the procedure. We could not escalate the dose
beyond 42 M due to the toxicity of the compound. The cultures
lM of
References and notes
l
1. De, C. E. Int. J. Antimicrob. Agents 2009, 33, 307.
2. Fauci, A. S. Nat. Med. 2003, 9, 839.
l
3. Biswas, P.; Tambussi, G.; Lazzarin, A. Expert Opin. Pharmacother. 2007, 8, 923.
4. Lieberman-Blum, S. S.; Fung, H. B.; Bandres, J. C. Clin. Ther. 2008, 30, 1228.
5. Lalezari, J. P.; Eron, J. J.; Carlson, M.; Cohen, C.; Dejesus, E.; Arduino, R. C.;
Gallant, J. E.; Volberding, P.; Murphy, R. L.; Valentine, F.; Nelson, E. L.; Sista, P.
R.; Dusek, A.; Kilby, J. M. AIDS 2003, 17, 691.
6. Lalezari, J. P.; Luber, A. D. Drugs Today (Barc.) 2004, 40, 259.
7. Trkola, A.; Dragic, T.; Arthos, J.; Binley, J. M.; Olson, W. C.; Allaway, G. P.; Cheng-
Mayer, C.; Robinson, J.; Maddon, P. J.; Moore, J. P. Nature 1996, 384, 184.
8. Wu, L.; Gerard, N. P.; Wyatt, R.; Choe, H.; Parolin, C.; Ruffing, N.; Borsetti, A.;
Cardoso, A. A.; Desjardin, E.; Newman, W.; Gerard, C.; Sodroski, J. Nature 1996,
384, 179.
9. Curreli, F.; Choudhury, S.; Pyatkin, I.; Zagorodnikov, V. P.; Bulay, A. K.; Altieri,
A.; Kwon, Y. D.; Kwong, P. D.; Debnath, A. K. J. Med. Chem. 2012, 55, 4764.
10. Curreli, F.; Kwon, Y. D.; Zhang, H.; Yang, Y.; Scacalossi, D.; Kwong, P. D.;
Debnath, A. K. Antimicrob. Agents Chemother. 2014, 58, 5478.
11. Madani, N.; Schon, A.; Princiotto, A. M.; LaLonde, J. M.; Courter, J. R.; Soeta, T.;
Ng, D.; Wang, L.; Brower, E. T.; Xiang, S. H.; Kwon, Y. D.; Huang, C. C.; Wyatt, R.;
Kwong, P. D.; Freire, E.; Smith, A. B., III; Sodroski, J. Structure 2008, 16, 1689.
12. Narumi, T.; Arai, H.; Yoshimura, K.; Harada, S.; Hirota, Y.; Ohashi, N.;
Hashimoto, C.; Nomura, W.; Matsushita, S.; Tamamura, H. Bioorg. Med. Chem.
2013, 21, 2518.
13. Morellato-Castillo, L.; Acharya, P.; Combes, O.; Michiels, J.; Descours, A.;
Ramos, O. H.; Yang, Y.; Vanham, G.; Arien, K. K.; Kwong, P. D.; Martin, L.;
Kessler, P. J. Med. Chem. 2013, 56, 5033.
14. Vita, C.; Vizzavona, J.; Drakopoulou, E.; Zinn-Justin, S.; Gilquin, B.; Menez, A.
Biopolymers 1998, 47, 93.
15. Rutenber, E. E.; De Voss, J. J.; Hoffman, L.; Stroud, R. M.; Lee, K. H.; Alvarez, J.;
McPhee, F.; Craik, C.; Ortiz de Montellano, P. R. Bioorg. Med. Chem. 1997, 5,
1311.
16. Curreli, F.; Kwon, Y. D.; Zhang, H.; Scacalossi, D.; Belov, D. S.; Tikhonov, A. A.;
Andreev, I. A.; Altieri, A.; Kurkin, A. V.; Kwong, P. D.; Debnath, A. K. J. Med.
Chem. 2015.
l
were expanded to collect cellular and viral samples at different
doses. Viruses obtained from 882376 treated and untreated cul-
tures were re-passaged in fresh Jurkat cells. Twenty hours post-
infection the cells were washed with PBS and put back in culture.
Six days later, the supernatants were filtered to remove cell debris
and p24 was quantified by sandwich-ELISA. The viruses were nor-
malized by p24 amounts and tittered by infecting TZM-bl cells to
calculate TCID50
.
5.9. Genotyping of resistant and control viruses
To identify the amino acid substitutions in the gp160 responsi-
ble for conferring resistance to 882376, we extracted genomic DNA
from the cells producing control WT virus and form the cells pro-
ducing viruses resistant to 25 lM, 33 lM and 42 lM of 882376
by using a whole-blood DNA purification kit (Qiagen). The entire
Env coding region was amplified by PCR using the Expand High-
Fidelity DNA polymerase kit (Roche), cloned and sequenced. The
viral DNA sequences were analyzed with the Geneious R8 software
(Biomatters, New Zealand).
5.10. Drug sensitivity with ENV-mutated pseudovirus
17. Herschhorn, A.; Finzi, A.; Jones, D. M.; Courter, J. R.; Sugawara, A.; Smith, A. B.,
III; Sodroski, J. G. PLoS One 2011, 6, e26731.
18. Zhao, Q.; Ma, L.; Jiang, S.; Lu, H.; Liu, S.; He, Y.; Strick, N.; Neamati, N.; Debnath,
A. K. Virology 2005, 339, 213.
19. Schon, A.; Madani, N.; Klein, J. C.; Hubicki, A.; Ng, D.; Yang, X.; Smith, A. B., III;
Sodroski, J.; Freire, E. Biochemistry 2006, 45, 10973.
20. Lin, P. F.; Blair, W.; Wang, T.; Spicer, T.; Guo, Q.; Zhou, N.; Gong, Y. F.; Wang, H.
G.; Rose, R.; Yamanaka, G.; Robinson, B.; Li, C. B.; Fridell, R.; Deminie, C.;
Demers, G.; Yang, Z.; Zadjura, L.; Meanwell, N.; Colonno, R. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 11013.
21. Sen, J.; Jacobs, A.; Jiang, H.; Rong, L.; Caffrey, M. Protein Sci. 2007, 16, 1236.
22. Schader, S. M.; Colby-Germinario, S. P.; Quashie, P. K.; Oliveira, M.; Ibanescu, R.
I.; Moisi, D.; Mesplede, T.; Wainberg, M. A. Antimicrob. Agents Chemother. 2012,
56, 4257.
Amino acid substitutions were introduced into the pHXB2-Env
expression vector by site-directed mutagenesis (Stratagene) using
mutagenic oligonucleotides and verified by sequencing the entire
ENV gene of each construct. To measure the activity of the com-
pounds against the pseudoviruses expressing different mutations,
U87-CD4-CXCR4 cells were infected with the ENV-mutated pseu-
doviruses as described above.
5.11. Infectivity assay of CD4-negative cells
23. Platt, E. J.; Wehrly, K.; Kuhmann, S. E.; Chesebro, B.; Kabat, D. J. Virol. 1998, 72,
2855.
The infectivity assay of CD4-negative cells was performed as
previously described.10 Briefly, CD4-negative ACTOne-CXCR4 cells
were plated at 104 cells/well in a 96-well tissue culture plate.
The cells were infected with the wild-type (WT) HIV-1HXB2 and
with HIV-1HXB2-Env-mutant pseudoviruses expressing luciferase.
In parallel, as a control, we infected U87-CD4-CXCR4 cells at 104 -
cells/well with the same amounts of the respective pseudoviruses.
After 2 days of incubation, the cells were washed and lysed. The
luciferase activity was measured as described above. The infectiv-
ity of the pseudoviruses detected in the ACTOne-CXCR4 cells was
reported as the percentage of infection relative to the infectivity
detected in the U87-CD4-CXCR4 control cells.
24. Wei, X.; Decker, J. M.; Liu, H.; Zhang, Z.; Arani, R. B.; Kilby, J. M.; Saag, M. S.; Wu,
X.; Shaw, G. M.; Kappes, J. C. Antimicrob. Agents Chemother. 2002, 46, 1896.
25. Ciminale, V.; Felber, B. K.; Campbell, M.; Pavlakis, G. N. AIDS Res. Hum.
Retroviruses 1990, 6, 1281.
26. Chackerian, B.; Long, E. M.; Luciw, P. A.; Overbaugh, J. J. Virol. 1997, 71, 3932.
27. Weiss, A.; Wiskocil, R. L.; Stobo, J. D. J. Immunol. 1984, 133, 123.
28. Bjorndal, A.; Deng, H.; Jansson, M.; Fiore, J. R.; Colognesi, C.; Karlsson, A.;
Albert, J.; Scarlatti, G.; Littman, D. R.; Fenyo, E. M. J. Virol. 1997, 71, 7478.
29. Kolchinsky, P.; Mirzabekov, T.; Farzan, M.; Kiprilov, E.; Cayabyab, M.; Mooney,
L. J.; Choe, H.; Sodroski, J. J. Virol. 1999, 73, 8120.
30. Adachi, A.; Gendelman, H. E.; Koenig, S.; Folks, T.; Willey, R.; Rabson, A.; Martin,
M. A. J. Virol. 1986, 59, 284.
31. Connor, R. I.; Chen, B. K.; Choe, S.; Landau, N. R. Virology 1995, 206, 935.
32. He, J.; Choe, S.; Walker, R.; di, M. P.; Morgan, D. O.; Landau, N. R. J. Virol. 1995,
69, 6705.

7628
F. Curreli et al. / Bioorg. Med. Chem. 23 (2015) 7618–7628
33. Wei, X.; Decker, J. M.; Wang, S.; Hui, H.; Kappes, J. C.; Wu, X.; Salazar-Gonzalez,
J. F.; Salazar, M. G.; Kilby, J. M.; Saag, M. S.; Komarova, N. L.; Nowak, M. A.;
Hahn, B. H.; Kwong, P. D.; Shaw, G. M. Nature 2003, 422, 307.
34. Page, K. A.; Landau, N. R.; Littman, D. R. J. Virol. 1990, 64, 5270.
35. Blish, C. A.; Jalalian-Lechak, Z.; Rainwater, S.; Nguyen, M. A.; Dogan, O. C.;
Overbaugh, J. J. Virol. 2009, 83, 7783.
36. Long, E. M.; Rainwater, S. M.; Lavreys, L.; Mandaliya, K.; Overbaugh, J. AIDS RH
2002, 18, 567.
37. Kulkarni, S. S.; Lapedes, A.; Tang, H.; Gnanakaran, S.; Daniels, M. G.; Zhang, M.;
Bhattacharya, T.; Li, M.; Polonis, V. R.; McCutchan, F. E.; Morris, L.; Ellenberger,
D.; Butera, S. T.; Bollinger, R. C.; Korber, B. T.; Paranjape, R. S.; Montefiori, D. C.
Virology 2009, 385, 505.
Gonzalez, J. F.; Wei, X.; Decker, J. M.; Hahn, B. H.; Montefiori, D. C. J. Virol. 2005,
79, 10108.
39. Keele, B. F.; Giorgi, E. E.; Salazar-Gonzalez, J. F.; Decker, J. M.; Pham, K. T.;
Salazar, M. G.; Sun, C.; Grayson, T.; Wang, S.; Li, H.; Wei, X.; Jiang, C.; Kirchherr,
J. L.; Gao, F.; Anderson, J. A.; Ping, L. H.; Swanstrom, R.; Tomaras, G. D.; Blattner,
W. A.; Goepfert, P. A.; Kilby, J. M.; Saag, M. S.; Delwart, E. L.; Busch, M. P.;
Cohen, M. S.; Montefiori, D. C.; Haynes, B. F.; Gaschen, B.; Athreya, G. S.; Lee, H.
Y.; Wood, N.; Seoighe, C.; Perelson, A. S.; Bhattacharya, T.; Korber, B. T.; Hahn,
B. H.; Shaw, G. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 7552.
40. Yang, Q. E.; Stephen, A. G.; Adelsberger, J. W.; Roberts, P. E.; Zhu, W.; Currens,
M. J.; Feng, Y.; Crise, B. J.; Gorelick, R. J.; Rein, A. R.; Fisher, R. J.; Shoemaker, R.
H.; Sei, S. J. Virol. 2005, 79, 6122.
38. Li, M.; Gao, F.; Mascola, J. R.; Stamatatos, L.; Polonis, V. R.; Koutsoukos, M.;
Voss, G.; Goepfert, P.; Gilbert, P.; Greene, K. M.; Bilska, M.; Kothe, D. L.; Salazar-
41. Jiang, S.; Lu, H.; Liu, S.; Zhao, Q.; He, Y.; Debnath, A. K. Antimicrob. Agents
Chemother. 2004, 48, 4349.