Design and Development of Highly Potent HIV-1 Protease Inhibitors with a Crown-Like Oxotricyclic Core as the P2-Ligand To Combat Multidrug-Resistant HIV Variants

Article abstract of DOI:10.1021/acs.jmedchem.7b00172

Design, synthesis, and evaluation of a new class of exceptionally potent HIV-1 protease inhibitors are reported. Inhibitor 5 displayed superior antiviral activity and drug-resistance profiles. In fact, this inhibitor showed several orders of magnitude improved antiviral activity over the FDA approved drug darunavir. This inhibitor incorporates an unprecedented 6-5-5 ring-fused crown-like tetrahydropyranofuran as the P2 ligand and an aminobenzothiazole as the P2′ ligand with the (R)-hydroxyethylsulfonamide isostere. The crown-like P2 ligand for this inhibitor has been synthesized efficiently in an optically active form using a chiral Diels-Alder catalyst providing a key intermediate in high enantiomeric purity. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.

Full text of DOI:10.1021/acs.jmedchem.7b00172

Article
pubs.acs.org/jmc
Design and Development of Highly Potent HIV‑1 Protease Inhibitors
with a Crown-Like Oxotricyclic Core as the P2-Ligand To Combat
Multidrug-Resistant HIV Variants
Arun K. Ghosh,*,† Kalapala Venkateswara Rao,† Prasanth R. Nyalapatla,† Heather L. Osswald,†
Cuthbert D. Martyr,† Manabu Aoki,§,∥,# Hironori Hayashi,§,⊥ Johnson Agniswamy,‡ Yuan-Fang Wang,‡
Haydar Bulut,# Debananda Das,# Irene T. Weber,‡ and Hiroaki Mitsuya§,⊥,#
†Department of Chemistry and Department of Medicinal Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana
47907, United States
‡Department of Biology, Molecular Basis of Disease, Georgia State University, Atlanta, Georgia 30303, United States
§Departments of Infectious Diseases and Hematology, Kumamoto University Graduate School of Biomedical Sciences, Kumamoto
860-8556, Japan
∥Department of Medical Technology, Kumamoto Health Science University, Kumamoto 861-5598, Japan
⊥Department of Refractory Viral Infection, National Center for Global Health and Medicine Research Institute, Tokyo 162-8655,
Japan
#Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, United States
S
* Supporting Information
ABSTRACT: Design, synthesis, and evaluation of a new class
of exceptionally potent HIV-1 protease inhibitors are reported.
Inhibitor 5 displayed superior antiviral activity and drug-
resistance profiles. In fact, this inhibitor showed several orders
of magnitude improved antiviral activity over the FDA
approved drug darunavir. This inhibitor incorporates an
unprecedented 6−5−5 ring-fused crown-like tetrahydro-
pyranofuran as the P2 ligand and an aminobenzothiazole as
the P2′ ligand with the (R)-hydroxyethylsulfonamide isostere. The crown-like P2 ligand for this inhibitor has been synthesized
efficiently in an optically active form using a chiral Diels−Alder catalyst providing a key intermediate in high enantiomeric purity.
Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms
of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.
active site interactions of numerous HIV-1 PIs bound to both
wild-type and mutant proteases. These studies revealed that the
backbone conformations of the active sites of various mutant
proteases are only minimally distorted compared to wild-type
HIV-1 protease.12,13 It is fundamentally very striking that a
protease cannot significantly alter its overall active site
backbone conformation without compromising its vital catalytic
fitness for viral replication.9,14 Therefore, a promising inhibitor
design strategy would be to maximize active site interactions
and particularly to promote a robust network of hydrogen-
bonding interactions with backbone atoms of wild-type HIV-1
protease. Such inhibitors will likely maintain those active site
interactions with other protease mutants.9,11 In essence, the
backbone binding strategies may lead to inhibitors that would
slow development of drug-resistant HIV-1 variants due to
possible reduction of catalytic fitness. HIV-1 protease in its
INTRODUCTION
■
The introduction of protease inhibitors (PIs) in combination
with antiretroviral therapies (ART) dramatically improved the
treatment of patients with HIV-1 infection and AIDS.1,2 The
ART regimens have demonstrated prolonged suppression of
HIV-1 replication in treated patients. This led to marked
improvements in HIV-related morbidity and mortality in
developed countries.3,4 Despite this progress, one major issue
of current PIs is that in many patients, HIV RNA levels have
relapsed due to the emergence of drug-resistant HIV-1 variants,
rendering a majority of current PIs ineffective within months.
This has raised a significant concern for long-term treatment
prognosis.5,6 Therefore, the design and development of new PIs
with broad-spectrum antiviral activity for treatment-experienced
patients remain an urgent priority.7,8
Our protein-X-ray structure-based design strategies are based
upon promoting extensive interactions in the active site of HIV-
1 protease, particularly with the backbone atoms.9−11 We have
examined and compared X-ray structural variations of critical
Received: February 2, 2017
© XXXX American Chemical Society
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active form is a homodimer containing catalytic aspartic acid
residues in the active site. As depicted in Figure 1, our inhibitor
Figure 1. Proposed model for the design of PIs to combat drug
resistance. The model suggests the formation of robust hydrogen
bonds by the P2 and P2′ ligands simultaneously in both the S2 and S2′
subsites. The transition-state hydroxyl group binds to catalytic
aspartates, and the P1 and P1′ ligands fill in the S1 and S1′ subsites.
Figure 2. Structures of HIV-1 protease inhibitors.
design model to combat drug resistance involves the design of
P2 and P2′ ligands that would form robust hydrogen bonds
with polar groups in S2 and S2′ regions.15 Furthermore, we
plan to fill in the hydrophobic S1 and S1′ subsites with
hydrophobic P1 and P1′ ligands. Among many possible classes
of transition-state binders, we plan to incorporate an (R)-
hydroxyethylamine sulfonamide isostere, as it can be
synthesized readily with varying P1 and P1′ substituents to
provide the nonpeptide drug-like scaffold.
RESULTS AND DISCUSSION
■
Our examination of the X-ray crystal structure of DRV-bound
HIV-1 protease and subsequent modeling suggested that the
bis-THF ring binding properties can be further optimized. In
particular, we planned to optimize the structural template of the
bis-THF ligand in DRV that could enhance the backbone
binding as well as improve hydrophobic interactions with the
protease active site. The bis-THF ligand in DRV and its
methoxy derivative 2 (TMC-126)23 show a distance of about
3.0−3.2 Å between the cyclic ether oxygens and the Asp30
backbone amide NH and a shorter and stronger hydrogen bond
with the Asp29 amide NH in the X-ray structure. Upon the
basis of this ligand-binding site interactions, we subsequently
designed a stereochemically defined hexahydrofuropyranol-
derived urethane as the P2-ligand in inhibitor 3. We speculated
that the bicyclic acetal on a 6−5 system would have favorable
alignment with backbone residues in the S2-subsite. Also, we
presumed that the extra methylene group on the tetrahy-
dropyran would increase van der Waals interaction in the active
site. Indeed, inhibitor 3 displayed excellent enzyme inhibitory
and antiviral activity (Ki = 2.7 pM and EC50 = 0.5 nM). Also,
this inhibitor maintained excellent potency against multidrug-
resistant HIV-1 variants similar to DRV and inhibitor 2.27 To
promote stronger hydrogen bonds with the top oxygen of bis-
THF, we further hypothesized that a larger seven-membered
ring could increase the dihedral angle of the bicyclic acetal
template and thereby put the top oxygen closer to the Asp30
backbone amide NH. Our preliminary model showed a more
optimal alignment of Asp30 NH with the top cyclic oxygen.
Since the conformation of a seven-membered ring is usually
labile, we sought to incorporate a bridged methylene group on
the seven-membered ring to constrain the conformational
flexibility. In essence, our new design will incorporate three
extra methylene groups over the bis-THF ligand. The positions
of these methylene groups may provide more favorable van der
Waals interactions in the hydrophobic space surrounding Ile47,
Val32, Leu76, Ile84, and Ile50′ residues. We have now designed
a crown-like-tetrahydropyranofuran (crn-THF) derivative as
the novel P2-ligand shown in inhibitor 4. A larger ring as the P2
Over the years, our structure-based design of a variety of P2-
ligands has been based upon cyclic-ether templates where the
ether oxygens are positioned to effectively mimic the carbonyl
oxygens of peptide bonds. These structural templates were
designed with a defined stereoconfiguration and structural
complementarity to effectively fill in the hydrophobic pockets
in the active site. Our laboratories have designed and
synthesized a range of exceptionally potent non-peptide HIV-
1 PIs showing good drug-like properties.16,17 One of these PIs
is the FDA approved drug darunavir (DRV, 1, Figure 2), which
was designed to promote extensive active site interactions with
the backbone atoms of the HIV-1 protease active site.18,19 DRV
has emerged as the first-line therapy for rescue treatment in
current U.S. Department of Health and Human Service
(DHHS) guidelines. DRV’s superb resistance profile is likely
due to its extensive interactions, particularly the network of
hydrogen bonding interactions in the active site as evidenced by
X-ray structural studies.9,11,20,21 The bis-THF ligand in DRV is
an intriguing pharmacophore. Both oxygens of the bis-THF
form very strong hydrogen bonds with Asp29 and Asp30
backbone amide NHs. Furthermore, the bicyclic ring forms nice
van der Waals interactions with residues in the S2 subsite.22−26
To further optimize the bis-THF structural template, we have
investigated structural templates that would enhance the
backbone-binding interactions, as well as further improve van
der Waals interactions within the S2 subsite of the HIV-1
protease active site. Herein, we report the design, synthesis, and
X-ray structural studies of a new class of PIs incorporating an
unprecedented 6−5−5 ring-fused crown-like tetrahydro-
pyranofuran as the P2 ligand with the (R)-hydroxyethyl-
sulfonamide isostere.
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ligand with some degree of flexibility may show better
adaptability to protease mutation. Furthermore, we speculated
that with new interactions in the S2-subsite, it may be necessary
to optimize the P2′-sulfonamide ligand as well. To promote
further hydrogen bonding interactions as well as to improve
hydrophobic contacts in the S2′-subsite, we have planned to
investigate inhibitors with a crn-THF P2-ligand in combination
with a benzothiazole derivative with a small alkylamine as
represented in inhibitor 5 to interact with the Asp30′ residue in
the S2′-subsite. In general, aminobenzothiazoles are structural
features of numerous medicinally important compounds and
these templates may further improve the inhibitors’ drug-like
properties.28,29
KHCO3 to provide alcohol 8 in 80% yield over two steps in
gram scale. Optical purity of alcohol 8 was determined to be
98% ee by chiral HPLC analysis [compound 8, [α]D −141.5 (c
1.04, CHCl3)]. Alcohol 8 was treated with TBSOTf in the
presence of 2,6-lutidine in CH2Cl2 at 0−23 °C for 45 min to
afford TBS ether 9 in 95% yield. Ester 9 was converted to
bicyclic acetal 10 in a three-step sequence involving (1)
reduction of the ester with LAH in THF at 0−23 °C for 1.5 h,
(2) one-pot oxidative cleavage of the olefin using Nicolaou’s
protocol,33 and (3) reduction of the resulting aldehyde with
DIBAL-H in CH2Cl2 at 0 °C for 4 h. Bicyclic acetal 10 (2:1
mixture) was obtained in 62% yield over three steps. The lactol
mixture was treated with trifluoroacetic acid (TFA) in CH2Cl2
at 0−23 °C for 15 h to afford bridged tricyclic derivative 11.
Removal of the TBS group with tetrabutylammonium fluoride
(TBAF) in THF at 0−23 °C for 2 h provided alcohol 12 in
68% yield over two steps. This exo-alcohol was converted to
endo-alcohol 13 by Dess−Martin oxidation at 0−23 °C for 3 h
followed by reduction of the resulting ketone with NaBH4 in
MeOH at 0 °C for 45 min. The desired bridged tricyclic ligand,
(3S,3aS,5R,7aS,8S)-hexahydro-4H-3,5-methanofuro[2,3-b]-
pyran-8-ol (13) was obtained in 76% yield over two steps. The
overall route is quite efficient and provided convenient access
to both endo- and exo-ligand alcohols in optically active form
(>99% ee). Both exo- and endo-alcohols 12 and 13, respectively,
were converted to the corresponding mixed activated carbonate
derivatives.34,35 Treatment of these alcohols with 4-nitrophenyl
chloroformate and pyridine in CH2Cl2 at 0−23 °C for 12 h
furnished carbonates 14 and 15 in excellent yields.
Our synthesis of the crn-THF P2 ligand is shown in Scheme
1. For the enantioselective synthesis of this unprecedented
a
Scheme 1. Synthesis of crn-THF Ligand 13
The synthesis of (R)-hydroxysulfonamide isostere 21 is
shown in Scheme 2. Azidooxirane 17 was prepared from
commercially available phenyl-2-buten-1-ol (16) using Sharp-
less epoxidation followed by epoxide ring opening and
conversion of the diol to epoxide 17.36−38 Reaction of this
oxirane with isobutylamine followed by treatment of the
a
Scheme 2. Synthesis of Sulfonamide Isostere 21
a
Reagents and conditions: (a) 6 (20 mol %), CH2Cl2, −78 °C; (b)
KHCO3, H2O2, 0 °C (80% for 2 steps); (c) TBSOTf, 2,6-lutidine, 0−
23 °C, CH2Cl2 (95%); (d) LAH, THF, 0−23 °C; (e) OsO4, NMO, 23
°C, acetone/H2O (10:1); then PhI(OAc)2, 23 °C; (f) DIBAL-H,
CH2Cl2, 0 °C, (62% for 3 steps); (g) TFA, CH2Cl2, 0 °C, (74%); (h)
TBAF, THF, 0−23 °C; (i) Dess−Martin periodinane, Na2HPO4,
CH2Cl2, 0−23 °C; (j) NaBH4, MeOH, 0 °C, (70% for 3 steps); (k) 4-
NO2-PhOCOCl, pyridine, CH2Cl2, 0−23 °C (83−95%).
ligand, we planned to utilize a chiral oxazaborolidinium cation
catalyzed Diels−Alder reaction developed by Corey and
Mukherjee.30,31 The requisite oxazaborolidinium cation 6 was
generated in situ from the corresponding oxazoborolidine (25
mol %) and triflimide (20 mol %) as reported.30 The Diels−
Alder reaction of readily available ethyl vinyl boronate 732 and
cyclopentadiene at −78 °C for 7 h provided the corresponding
cycloadduct which was oxidized with H2O2 in the presence of
a
Reagents and conditions: (a) i-BuNH2, i-PrOH, 65 °C; (b) 18,
CH2Cl2, Et3N, 23 °C, (90% over 2 steps); (c) Ph3P, THF−H2O (3:1),
23 °C; (d) Boc2O, THF−H2O (1:1), NaHCO3, 23 °C, (85% over 2
steps); (e) m-CPBA, CH2Cl2, 23 °C; (f) cyclopropylamine, THF, 65
°C, (91% over 2 steps); (g) TFA, CH2Cl2, 0 °C (99%).
C
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resulting amino alcohol with sulfonyl chloride 18 in CH2Cl2 in
the presence of triethylamine at 23 °C for 12 h provided azido
alcohol 19 in excellent yields (90%) over two steps. Staudinger
reduction39 of azide 19 with Ph3P in aqueous THF at 23 °C for
24 h followed by reaction of the resulting amine with Boc2O
afforded Boc-derivative 20 in excellent yield (85%) over two
steps. This methyl sulfide was converted to the
cyclopropylaminobenzothiazole derivative 21 in a three-step
sequence involving (1) mCPBA oxidation of sulfide to sulfone
at 23 °C for 12 h, (2) treatment of the resulting sulfone with
cyclopropylamine in THF at 65 °C for 12 h, and (3) treatment
of the resulting Boc-derivative with TFA in CH2Cl2 at 0 °C for
1 h. Amine 21 was obtained in excellent yield.
bis-THF carbonate 26 was reacted with benzothiazole
derivative 21 in CH3CN at 23 °C for 36 h to provide inhibitor
27 in good yields. Similarly, reaction of activated carbonate of
endo-crn-THF 14 with benzothiazole derivative 21 provided
inhibitor 5 in very good yield.
Our examination of the preliminary model of endo-crn-THF
containing inhibitor 25 and exo-crn-THF-derived inhibitor 24
indicated that the acetal oxygens in the endo-derivative are
suitably positioned to form hydrogen bonds with Asp30 and
Asp29 amide NHs. Also, the tricyclic scaffold of inhibitor 25
appeared to fill the hydrophobic pocket in the S2 site more
effectively than inhibitor 24. As can be seen in Table 1,
The synthesis of various inhibitors containing crn-THF as
the P2 ligand is shown in Scheme 3. For the synthesis of
Table 1. Enzymatic Inhibitory and Antiviral Activity of
Inhibitors
a
Scheme 3. Synthesis of PIs
inhibitor 25 containing endo-crn-THF ligand and a 4-
methoxybenzenesulfonamide as P2′-ligand exhibited an enzyme
inhibitory Ki of 14 pM compared to inhibitor 24 containing
exo-crn-THF ligand (Ki of 10.8 nM). We utilized the assay
protocol developed by Toth and Marshall.40 The correspond-
ing inhibitor 4, with a 4-aminobenzenesulfonamide as P2′-
ligand, displayed a comparable Ki value of 13 pM. We have
a
Reagents and conditions: (a) carbonate 14 or 15, DIPEA, CH3CN,
23 °C; (b) amine 21 and carbonate 26 or 14, DIPEA, CH3CN, 23 °C
(50−60%).
determined antiviral activity of these inhibitors in MT-2 human
41
T-lymphoid cells exposed to HIVLAI
.
As shown, inhibitor 24
with an exo-crn-THF P2-ligand showed no appreciable antiviral
activity (IC50 > 1 μM). However, inhibitor 25 with an endo-crn-
THF displayed an antiviral IC50 value of 2.7 nM. Inhibitor 4,
with a DRV isostere, showed an antiviral IC50 value of 2.8 nM.
Antiviral activity of inhibitor 27, with a bis-THF as the P2-
ligand and a benzothiazole as the P2′-ligand, showed a Ki value
of 10 pM and an improved antiviral IC50 value of 1.9 nM
compared to DRV. Inhibitor 5 with an endo-crn P2-ligand
showed Ki value of 40 pM and significant improvement in
antiviral potency with an IC50 value of 0.26 nM. In comparison,
inhibitors with 4-methoxybenzenesulfonamide as the P2′-
ligand, activated carbonates of the endo- and exo-crn-THF 14
and 15 respectively were reacted with known34,35 4-
methoxybenzenesulfonamide isostere 22 in the presence of
diisopropylethylamine (DIPEA) at 23 °C for 72 h to furnish
inhibitors 24 and 25 in good yields. Similarly, reaction of
activated carbonate 14 with 4-aminobenzenesulfonamide
isostere 23 provided inhibitor 4. For the synthesis of inhibitors
with benzothiazolesulfonamide as the P2′-ligand, known34,35
D
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Table 2. Antiviral Activity of Novel Three Compounds against Highly DRV-Resistant HIV-1 Variants
a
mean IC50 SD in nM (fold-change)
LPV
4
DRV
3.8 0.7
4
27
5
HIV-1NL4‑3
20
3.3 1.2
2.0 0.8
0.39 0.06
R
HIV-1DRV
>1000 (>50)
>1000 (>50)
>1000 (>50)
34 19 (9)
30 10 (9)
340 18 (103)
7.7 2 (4)
30 5 (15)
0.17 0.01 (0.4)
1.8 0.5 (5)
P20
P30
P51
R
HIV-1DRV
360 10 (95)
3200 300 (842)
R
HIV-1DRV
>1000 (>303)
480 83 (240)
35 12 (90)
a
R
R
R
MT-4 cells (1 × 104) were exposed to 50 TCID50 of wild-type HIV-1NL4‑3, HIV-1DRV P20, HIV-1DRV P30, or HIV-1DRV
and cultured in the
P51
presence of various concentrations of each PI, and the IC50 values were determined using the p24 assay. The amino acid substitutions identified in
protease of HIV-1DRV P20, HIV-1DRV P30, and HIV-1DRV
R
R
R
P51
compared to HIV-1NL4‑3 include L10I/I15V/K20R/L24I/V32I/M36I/M46L/L63P/
V82A/L89M, L10I/I15V/K20R/L24I/V32I/M36I/M46L/L63P/K70R/V82A/I84V/L89M, and L10I/I15V/K20R/L24I/V32I/L33F/M36I/
M46L/I54M/L63P/K70Q/V82I/I84V/L89M, respectively. All assays were conducted in triplicate, and the data shown represent mean values
(
standard deviation) derived from the results of three independent experiments.
Table 3. Antiviral Activity of Inhibitor 5 against Highly PI-Resistant HIV-1 Variants
mean IC50 SD in nM (fold-change)
virus species
wild-type
LPV
4
ATV
DRV
5
HIV-1NL4‑3
20
4.2 0.7
3.8 0.7
0.39 0.06
a
R
ivHIVPI
HIV-1SQV‑5μM
HIV-1APV‑5μM
HIV-1LPV‑5μM
HIV-1IDV‑5μM
HIV-1NFV‑5μM
HIV-1ATV‑5μM
HIV-1TPV‑15μM
>1000 (>50)
310 18 (16)
>1000 (>50)
170 20 (9)
42 7 (2)
510 14 (121)
3.8 2.5 (0.9)
45 19 (11)
62 10 (15)
17 3 (4)
45 2 (12)
25 21 (7)
430 40 (113)
30 17 (8)
8.2 3.8 (2)
36 3 (9)
0.03 0.01 (0.08)
0.27 0.1 (0.7)
0.0026 0.0006 (0.007)
0.012 0.013 (0.03)
0.028 0.02 (0.07)
0.19 0.06 (0.5)
330 30 (17)
>1000 (>50)
>1000 (>238)
>1000 (>238)
39 5 (10)
0.057 0.03 (0.15)
a
In vitro PI-selected HIV-1 variants. The amino acid substitutions identified in protease of HIV-1SQV‑5μM, HIV-1APV‑5μM, HIV-1LPV‑5 μM, HIV-1IDV‑5μM
,
HIV-1NFV‑5μM, HIV-1ATV‑5μM, and HIV-1TPV‑15μM compared to the wild-type HIV-1NL4‑3 include L10I/N37D/G48V/I54V/L63P/G73C/I84V/L90M,
L10F/V32I/L33F/M46L/I54M/A71V, L10F/V32I/M46I/I47A/A71V/I84V, L10F/L24I/M46I/I54V/L63P/A71V/G73S/V82T, L10F/K20T/
D30N/K45I/A71V/V77I, L23I/E34Q/K43I/M46I/I50L/G51A/L63P/A71V/V82A/T91A, and L10I/L33I/M36I/M46I/I54V/K55R/I62V/
L63P/A71V/G73S/V82T/L90M/I93L, respectively. Numbers in parentheses represent fold-changes in IC50 values for each isolate compared to
the IC50 values for wild-type HIV-1NL4‑3. All assays were conducted in triplicate, and the data shown represent mean values ( standard deviation)
derived from the results of three independent experiments.
darunavir and saquinavir showed IC50 values of 3.2 nM and 21
nM, respectively.
wild-type HIV-1NLR4‑3 were similar to DRV. These DRV-
resistant HIV-1DRV variants are highly resistant to all current
clinically used PIs including DRV and nucleoside/nucleotide
reverse transcriptase inhibitors (NRTIs) such as tenofovir.
Inhibitor 27, containing a cyclopropylaminobenzothiazole as
the P2′ ligand on DRV, showed an improved antiviral activity
While current antiretroviral therapy and treatment guidelines
are updated regularly with the availability of new drugs or drug-
effect information, PIs continue to be a critical element of
current ART regimens. PIs are widely used for the treatment of
naive and experienced HIV/AIDS patients. However, heavily-
ART regimen-experienced patients tend to have drug failure
with many of the currently available PIs including daruna-
vir.42,43 Therefore, design and discovery of more potent PIs
showing a high genetic barrier are very important to effective
long-term treatment options. Since our development of DRV,
our design objectives include the design of highly potent PIs
that maintain potency against a variety of existing multi-PI-
resistant HIV-1 variants with better selectivity index and safety
profiles. Also, it is important that the new PIs do not permit, or
substantially delay, the emergence of HIV-1 variants resistant to
these PIs. We therefore examined both potent crn-THF
containing PIs (4, 5) and the bis-THF-derived inhibitor 27
against DRV-resistant HIV-1 variants. In these assays, MT-4
cells (1 × 104) were exposed to wild-type HIV-1 and three
compared to inhibitor 4 and DRV. As can be seen, the fold-
R
differences in the IC50 values of 27 against HIV-1DRV
and
P20
R
HIV-1DRV
compared to wild-type HIV-1NL4‑3 were 4- and
P30
15-fold, respectively, while the fold-differences for DRV were 9-
and 95-fold, respectively. These results indicated that the
benzothiazole P2′-ligand exerts a more favorable interaction in
the S2′ site compared to 4-aminobenzenesulfonamide of DRV.
Inhibitor 5 containing crn-THF as the P2 ligand and
aminobenzothiazole as the P2′-ligand potently blocked the
replication of wild-type HIV-1NL4‑3 by a factor of 10 compared
to DRV. Furthermore, this inhibitor suppressed the replication
of all three DRV-resistant variants. Of particular note, the fold-
R
R
differences of 5 against HIV-1DRV
and HIV-1DRV
P20
P30
compared to HIV-1NL4‑3 were 0.4- and 5-fold, respectively.
This PI also maintained IC50 values of 35 nM against HIV-
R
R
R
DRV-resistant variants HIV-1DRV P20, HIV-1DRV P30, and HIV-
1DRV P51, the most multi-PI/NRTI-resistant HIV-1 variants.
R
1DRV
and subjected to various concentrations of each PI.
We then examined whether inhibitor 5 was active against a
variety of HIV-1 variants that had been selected in vitro with
each of seven FDA-approved PIs, SQV, APV, LPV, IDV, NFV,
ATV, and TPV. Each of these HIV-1 variants were selected in
vitro by propagating HIV-1NL4‑3 in the presence of increasing
concentrations of each PI (up to 5 μM) in MT-4 cells.41,44
P51
IC50 values were determined using p24 assay.41,44 The results
are shown in Table 2. PI 4 containing the crn-THF as the P2
ligand on the DRV isostere displayed comparable antiviral
activity to DRV. The fold-differences in the IC50 value of 4
against all three DRV-resistant HIV-1 variants compared to
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They were shown to have acquired multiple amino acid
substitutions in the protease of the virus that are associated
with viral resistance to each PI drug. Each variant was highly
resistant to the PI with which the variant was selected.41,44 The
results are shown in Table 3. As shown, two current clinically
used PIs, LPV and ATV, lost significant activity against the
seven HIV-1 variants. DRV showed relatively better results;
however, it too failed to block replication of each variant very
effectively. DRV displayed an IC50 value fold-change ranging
from 2- to 113-fold. On the contrary, inhibitor 5 maintained
superior activity against all seven HIV-1 variants showing
significantly more potent antiviral activity compared to wild-
type HIVNL4‑3. Inhibitor 5 exerted very potent antiviral activity
with IC50 values ranging from 0.0026 to 0.27 nM. Our detailed
X-ray crystallographic studies of inhibitors 5 and 25-bound
HIV-1 protease provided molecular insight into the binding
properties responsible for the superior bioactivity of inhibitor 5.
The X-ray crystal structure of the wild type HIV-1 protease
cocrystallized with inhibitor 25 was refined to an R factor of
17.9% at the high resolution of 1.53 Å (PDB code 5ULT).45
The crystal structure contains the protease dimer and the
inhibitor bound to HIV-1 protease in two orientations related
by a 180° rotation with 55/45% relative occupancies. The
overall structure is very similar to the structure with HIV-1
protease and DRV21 with root-mean-square difference of 0.25
Å for Cα atoms. The largest difference between corresponding
Cα atoms is 0.8 Å. The inhibitor is bound in the active site
cavity by forming a series of hydrogen bonding interactions and
numerous weaker CH···O interactions with the main chain
atoms of HIV-1 protease. As shown in Figure 3, the major
novel crn-THF like tricyclic P2-ligand in inhibitor 25, which
forms a crown-like shape. The tricyclic crown scaffold is
suitably substituted with two acetal oxygens to interact with
backbone residues and the side chain Asp30 carboxylate group
in S2 subsite. Indeed, both oxygens form hydrogen bonds with
the amide groups of protease residues Asp29 and Asp30,
comparable to the interactions of darunavir. The new P2-ligand
is conformationally rigid and larger than the P2 bis-THF ligand
in DRV. Our structural analysis revealed that the crn-THF
ligand displaces residues 45′−48′ by up to 0.8 Å, resulting in an
enlarged inhibitor-binding cavity. The new ligand forms very
strong hydrogen bonds with backbone atoms in the S2-site as
well as with the side chain Asp29 carboxylate group.
Furthermore, it also makes significantly enhanced van der
Waals interactions in the S2-site compared to DRV’s bis-THF
ligand (distances of Ile47, Val32, and Leu76 are significantly
shorter).
We also determined the X-ray crystal structure of inhibitor 5-
bound wild-type HIV-1 protease at a 1.7 Å resolution (PDB
code 5TYR).44 In brief, a wild-type protease derived from wild-
type HIV-1NL4‑3 carrying no amino acid substitutions (PRNL4‑3
)
was complexed with inhibitor 5. The inhibitor occupied the
protease active site in two distinct conformations. The key
inhibitor−protease interactions are shown in Figure 5. The
major conformation shows that the inhibitor is bound in the
active site through a network of strong hydrogen bonds with
backbone atoms as well as with catalytic aspartates. In
particular, both oxygens of the crn-THF ligand formed strong
hydrogen bonds with backbone amide NHs of Asp29 and
Asp30. Furthermore, the crn-scaffold was involved in significant
nonbonded van der Waals interactions with the HIV-1
protease. The crn-THF makes significantly better hydrophobic
contacts than the bis-THF ligand in DRV. The P2′ ligand’s
thiazole amine nitrogen also makes strong hydrogen bonds with
the backbone NH of Asp30′, and the cyclopropylamine
nitrogen forms additional polar interactions with the side
chain carboxylate of Asp30′. This P2′ ligand also makes more
hydrophobic contacts with the protease than the 4-
aminobenzenesulfonamide ligand of DRV. These extensive
molecular interactions must be responsible for the high affinity
for HIV-1 protease as well as its robust antiviral activity against
multidrug-resistant HIV-1 variants. We have also compared the
binding properties of the crn-THF ligand in the X-ray
structures for inhibitors 5 and 25 to see the effect of the
cyclopropylaminobenzothiazole in inhibitor 5. As shown in
Figure 6, the hydrogen bonding distances for inhibitors are
nearly identical. Leu76 appears to be 0.4 Å closer to the crn-
THF ligand in inhibitor 5; however, Val32 shifted 0.5 Å further
compared to inhibitor 25. Overall, the binding properties of the
crn-THF ligand in both inhibitors are very similar.
Figure 3. Inhibitor 25-bound X-ray structure of HIV-1 protease (PDB
code 5ULT). The major orientation of the inhibitor is shown. The
inhibitor carbon atoms are shown in cyan, water molecules are red
spheres, and the hydrogen bonds are indicated by dotted lines.
CONCLUSIONS
■
In summary, we investigated a new class of HIV-1 protease
inhibitors by promoting ligand−backbone interactions in the
active site. We designed an unprecedented crown-like
hexahydrofuropyranylurethane as the P2-ligand and a
cyclopropylaminobenzothiazole as the P2′-ligand with a (R)-
hydroxysulfonamide isostere. This combination of ligands
provided inhibitor 5 which displayed remarkable enzyme
inhibitory and antiviral activity. Of particular importance,
inhibitor 5 remained highly potent against DRV-resistant
HIV-1 variants, many of which are multi-PI- and multi-NRTI-
resistant HIV-1 variants. Inhibitor 5 also showed highly potent
conformation of the inhibitor forms hydrogen bonding
interactions of its urethane NH with the carbonyl oxygen of
Gly27. The inhibitor also forms tetracoordinated water-
mediated interactions connecting the inhibitor carbonyl oxygen
and sulfonamide oxygen with the amides of Ile50 and Ile50′ in
the flaps. Furthermore, the p-methoxy group of the P2′-
sulfonamide forms a hydrogen bond with the amide NH of
Asp30′ as well as with its side chain carboxyl group.
As highlighted in Figure 4, the majority of differences of
inhibitor 25 and DRV are confined to the interactions of the
F
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Article
Figure 4. Side by side comparison of binding properties of P2 bis-THF moiety in darunavir-bound HIV-1 protease (left, cyan), X-ray structure (PDB
code 2IEN) with the P2 crn-THF moiety of inhibitor 25-bound HIV-1 protease (right, purple), X-ray structure (PDB code 5ULT) inside the S2
subpocket. Both groups are located close to the periphery of the protease active site and form three hydrogen bonds in a similar fashion (black
dashes). The crn-THF is bulkier with extended triangle-shaped surface pointing toward the hydrophobic region consisting of Ile47, Val32, and
Leu76. The crn-THF forms closer van der Waals interactions with Ile47 compared to the bis-THF.
examination of inhibitors containing bis-THF and aminobenzo-
thiazole as the P2 and P2′-ligands led to very potent compound
27. However, inhibitor 5 showed superior antiviral activity and
drug-resistance profiles several orders of magnitude improved
over DRV. Furthermore, inhibitor 5 showed improved
lipophilicity (clogP = 5.3) over darunavir (clogP = 2.9). Our
X-ray structural studies of 25-bound and 5-bound HIV-1
protease revealed molecular insight into the ligand-binding site
interactions responsible for its exceptional drug-resistance
profiles. It appears that the crn-THF ligand with defined
configuration formed robust hydrogen bonding interactions
with the backbone amide NHs of Asp29 and Asp30.
Furthermore, the crown-scaffold of this new ligand is larger
than the bis-THF of DRV and displaces residues 45′−48′ in the
active site by 0.8 Å. This hydrophobic space was nicely filled by
the crown-scaffold resulting in an enhanced van der Waals
Figure 5. Inhibitor 5-bound HIV-1 protease X-ray structure is shown
interaction in the S2 site. The aminobenzothiazole P2′-ligand
(PDB code 5TYR). The inhibitor carbon atoms are shown in magenta,
also formed robust hydrogen bonds in the S2′-site, leading to
and hydrogen bonds are shown by dotted lines.
an enhancement of ligand-binding site interactions with these
combination of ligands. The crn-THF ligand has been
antiviral activity against a variety of HIV-1 variants selected in
vitro with other approved PIs. These values are significantly
better than other approved PIs including darunavir. Our
synthesized efficiently in an optically active form using Corey’s
chiral Diels−Alder catalyst providing a key intermediate in high
enantiomeric purity. The basic design of novel ligands and the
Figure 6. Comparison of the binding properties of the P2 crn-THF moiety of inhibitor 25-bound HIV-1 protease (panel A, purple), X-ray structure
(PDB code 5ULT) and inhibitor 5-bound HIV-1 protease (panel B, green), X-ray structure (PDB code 5TYR) inside the S2 subpocket. The
hydrogen bonds with the backbone atoms are shown in black dashes. The crn-THF in both inhibitors 5 and 25 form closer van der Waals
interactions.
G
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Article
solution of Tf2NH (0.281 g in 2 mL of abs CH2Cl2, 1 mmol) was
added, and the resulting solution was stirred at −25 °C for 25 min.
Solvent was then removed carefully under reduced pressure at −25 °C.
A solution of ethyl vinyl boronate 7 (1.13 g in 3 mL of CH2Cl2, 5
mmol) was added to the resulting residue, and the mixture was cooled
to −78 °C. Cyclopentadiene (2.1 mL) was then added over 2 h by
using a syringe pump and stirred at −78 °C for another 5 h. After this
period, the reaction mixture was diluted with Et2O, warmed to 23 °C,
and filtered through a Celite pad. The filtrate was concentrated under
reduced pressure and the crude product was purified by silica gel
column chromatography (10% EtOAc in hexane) to afford Diels−
Alder adduct.
examination of ligand combination for maximizing interactions
in the enzyme active site are powerful approaches for the next
generation of protease inhibitors with broad spectrum activity
against multidrug resistant HIV-1 variants.
EXPERIMENTAL SECTION
■
All moisture-sensitive reactions were carried out in oven-dried
glassware under an argon atmosphere unless otherwise stated.
Anhydrous solvents were obtained as follows: diethyl ether and
tetrahydrofuran were distilled from sodium metal/benzophenone
under argon. Toluene and dichloromethane were distilled from
calcium hydride under argon. All other solvents were reagent grade.
Column chromatography was performed using Silicycle SiliaFlash F60
230−400 mesh silica gel. Thin-layer chromatography was carried out
To a stirred solution of the above Diels−Alder adduct and KHCO3
(5 mL, 10 mmol, 2 M aqueous solution) in THF/EtOH (20 mL, 3:1)
was added H2O2 (2 mL, 30% wt solution in H2O, 20 mmol) slowly at
0 °C. The resulting mixture was stirred for 4.5 h at 0 °C. Upon
completion, the reaction mixture was quenched by the addition of
saturated aqueous Na2S2O3 and extracted with EtOAc. The extracts
were washed with H2O, saturated aqueous NaCl, dried (Na2SO4), and
concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography (30% EtOAc in hexane) to afford
using EMD Millipore TLC silica gel 60 F254 plates. H NMR and 13C
1
NMR spectra were recorded on a Varian INOVA300, Bruker ARX400,
Bruker DRX500, or Bruker AV-III-500-HD. Low-resolution mass
spectra were collected on a Waters 600 LCMS instrument or by the
Purdue University Campus-Wide Mass Spectrometry Center. High-
resolution mass spectra were collected by the Purdue University
Campus-Wide Mass Spectrometry Center. HPLC analysis and
purification were done an on Agilent 1100 series instrument using a
YMC Pack ODS-A column of 4.6 mm i.d. for analysis and either 10
mm i.d. or 20 mm i.d. for purification. The purity of all test
compounds was determined by HPLC analysis to be ≥95% pure.
(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl ((2S,3R)-4-((4-Amino-N-isobutylphenyl)sulfonamido)-3-hy-
droxy-1-phenylbutan-2-yl)carbamate (4). Compound 14 (18.3
mg, 0.057 mmol) was treated with isostere 23 (25 mg, 0.063 mmol) by
following the procedure outlined for inhibitor 24 to give inhibitor 4
20
1
8 (730 mg, 80% over two steps). [α]D −141.5 (c 1.04, CHCl3); H
NMR (400 MHz, CDCl3) δ 6.13 (dd, J = 5.7, 2.7 Hz, 1H), 6.06 (dd, J
= 5.6, 3.3 Hz, 1H), 4.13−4.04 (m, 3H), 3.07 (s, 1H), 2.77−2.74 (m,
1H), 2.66 (s, 1H), 2.61 (t, J = 3.0 Hz 1H), 1.88 (d, J = 8.7 Hz, 1H),
1.63 (dd, J = 8.7, 1.6 Hz, 1H), 1.22 (t, J = 7.1 Hz, 3H); 13C NMR (100
MHz, CDCl3) δ 173.8, 137.3, 134.7, 75.7, 60.6, 55.2, 50.6, 46.4, 44.2,
14.4.
Ethyl (1S,2R,3R,4R)-3-((tert-Butyldimethylsilyl)oxy)bicyclo-
[2.2.1]hept-5-ene-2-carboxylate (9). To a stirred solution of 8
(730 mg, 4 mmol) in dichloromethane (15 mL) were added 2,6-
lutidine (1.39 mL, 12 mmol) and TBSOTf (1.38 mL, 6 mmol) at 0 °C
under argon atmosphere. The reaction mixture was warmed to 23 °C
and stirred for 1 h. Upon completion, the reaction mixture was
quenched by the addition of saturated aqueous NaHCO3 and extracted
with dichloromethane. The extracts were washed with saturated
aqueous NaCl, dried (Na2SO4), and concentrated under reduced
pressure. The crude product was purified by silica gel column
1
(26 mg, 80%). H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 8.5 Hz,
2H), 7.31−7.27(m, 2H), 7.25−7.20 (m, 3H), 6.68 (d, J = 8.3 Hz, 2H),
5.42 (d, J = 6.6 Hz, 1H), 5.08 (d, J = 8.5 Hz, 1H), 4.77 (dd, J = 8.8, 5.8
Hz, 1H), 3.91−3.83 (m, 3H), 3.74 (d, J = 9.4 Hz, 1H), 3.59 (dd, J =
11.2, 8.0 Hz, 1H), 3.54 (dd, J = 9.2, 6.5 Hz, 1H), 3.13 (dd, J = 15.0, 8.4
Hz, 1H), 3.04 (dd, J = 14.0, 3.5 Hz, 1H), 2.99−2.91 (m, 2H), 2.83
(dd, J = 13.9, 9.1 Hz, 1H), 2.76 (dd, J = 13.3, 6.6 Hz, 1H), 2.72−2.67
(m, 1H), 2.66−2.62 (m, 1H), 2.37−2.27 (m, 1H), 1.81 (d, J = 11.4
Hz, 2H), 1.43 (dt, J = 11.8, 3.7 Hz, 1H), 0.92 (d, J = 6.6 Hz, 3H),
0.88−0.86 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 155.7, 150.8,
137.8, 129.6, 129.5, 128.7, 126.7, 126.3, 114.3, 104.5, 75.1, 72.8, 68.6,
60.0, 59.0, 55.2, 53.9, 45.0, 42.1, 37.5, 35.6, 31.7, 27.5, 23.6, 22.8, 20.3,
20.1, 14.3. LRMS-ESI (m/z): 596.4 [M + Na]+. HRMS-ESI (m/z):
[M + Na]+ calcd for C29H39N3O7SNa, 596.2407; found 596.2402.
(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl ((2S,3R)-4-((2-(Cyclopropylamino)-N-isobutylbenzo[d]-
thiazole)-6-sulfonamido)-3-hydroxy-1-phenylbutan-2-yl)-
carbamate (5). Compound 14 (14 mg, 0.044 mmol) was treated with
isostere amine 21 (26 mg, 0.052 mmol) by following the procedure
1
chromatography (5% Et2O in hexane) to afford 9 (1.13 g, 95%). H
NMR (400 MHz, CDCl3) δ 6.13 (dd, J = 5.7, 2.7 Hz, 1H), 6.04 (dd, J
= 5.6, 3.2 Hz, 1H), 4.13−4.04 (m, 2H), 4.03 (q, J = 2.4, 1.5 Hz, 1H),
3.03 (s, 1H), 2.68−2.62 (m, 1H), 2.57 (dd, J = 3.5, 2.3 Hz, 1H), 1.90
(d, J = 8.5 Hz, 1H), 1.60 (dq, J = 8.4, 1.6 Hz, 1H), 1.23 (t, J = 7.1 Hz,
3H), 0.88 (s, 9H), 0.07 (d, J = 3.8 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 173.9, 137.5, 134.6, 76.0, 60.3, 55.5, 51.4, 46.7, 44.2, 25.9,
18.1, 14.4, −4.7. LRMS-ESI (m/z): 319.1 [M + Na]+.
tert-Butyl-(((3S,7aS,8R)-hexahydro-4H-3,5-methanofuro[2,3-
b]pyran-8-yl)oxy)dimethylsilane (11). To a stirred solution of
ester 9 (1.13 g, 3.8 mmol) in THF (15 mL) was added LAH (360 mg,
9.5 mmol) at 0 °C under argon atmosphere. The reaction mixture was
warmed to 23 °C and stirred for 1 h. Upon completion, the reaction
mixture was quenched slowly by the addition of 3 N NaOH solution,
forming a white suspension. The suspension was filtered and washed
with EtOAc. The solvent was removed under reduced pressure, and
the sample was used in the next step without further purification.
To the above crude in acetone/water (10:1, 35.2 mL) were added
2,6-lutidine (0.88 mL, 7.6 mmol), NMO (670 mg, 5.7 mmol), and
OsO4 (0.48 mL, 4% in H2O, 0.076 mmol) at 23 °C. The reaction
mixture was stirred for 24 h and monitored by TLC, and then
PhI(OAc)2 (1.84 g, 5.7 mmol) was added. After stirring for 15 h, the
reaction mixture was quenched by the addition of saturated aqueous
Na2S2O3 and extracted with EtOAc. The extracts were washed with
saturated aqueous CuSO4, H2O, saturated aqueous NaCl, dried
(Na2SO4), and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography (25%
EtOAc in hexane) to afford the mixture of aldehydes (980 mg).
To a stirred solution of the above aldehydes (980 mg, 3.42 mmol)
in dichloromethane (15 mL) at 0 °C was added DIBAL-H (7.2 mL,
7.2 mmol) under argon atmosphere, and the mixture was stirred at the
same temperature for 4 h. After this period, the reaction mixture was
1
outlined for inhibitor 24 to give inhibitor 5 (28 mg, 97%). H NMR
(500 MHz, CDCl3) δ 8.07 (s, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.54 (d, J
= 8.5 Hz, 2H), 7.29 (t, J = 7.4 Hz, 2H), 7.22 (dd, J = 13.6, 7.0 Hz,
3H), 5.41 (d, J = 6.7 Hz, 1H), 5.33−5.27 (m, 1H), 4.78 (dd, J = 8.7,
5.8 Hz, 1H), 3.96−3.84 (m, 4H), 3.73 (d, J = 9.3 Hz, 1H), 3.61−3.50
(m, 2H), 3.19 (dd, J = 14.9, 8.3 Hz, 1H), 3.11−2.96 (m, 3H), 2.84 (dt,
J = 13.4, 7.3 Hz, 2H), 2.75 (tt, J = 6.7, 3.5 Hz, 1H), 2.69 (q, J = 7.3 Hz,
1H), 2.66−2.60 (m, 1H), 2.35−2.28 (m, 1H), 1.85 (dt, J = 14.4, 7.0
Hz, 1H), 1.80 (d, J = 12.1 Hz, 1H), 1.46−1.39 (m, 1H), 0.95−0.91
(m, 4H), 0.89−0.86 (m, 4H), 0.81−0.77 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 173.3, 155.8, 137.8, 131.3, 130.4, 129.5, 128.7, 126.7,
125.5, 121.0, 118.6, 104.4, 75.1, 72.9, 68.5, 60.0, 58.9, 55.4, 53.8, 45.0,
42.1, 37.5, 35.5, 31.7, 27.4, 26.8, 23.63, 22.7, 20.3, 20.0, 14.2, 8.0.
LRMS-ESI (m/z): 693.3 [M + Na]+. HRMS-ESI (m/z): [M + Na]+
calcd for C33H42N4O7S2Na, 693.2393; found 693.2389.
Ethyl (1S,2R,3R,4R)-3-Hydroxybicyclo[2.2.1]hept-5-ene-2-
carboxylate (8). To an oven and flame-dried two-neck round-
bottom flask was added a solution of oxazaborolidine (5 mL, 1.25
mmol, 0.25 M solution in toluene), and the solvent was removed
under reduced pressure. The resulting residue was dissolved in 2 mL
of abs CH2Cl2, and the clear solution was cooled to −25 °C. A
H
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Article
quenched by the addition of saturated aqueous solution of sodium
potassium tartrate and stirred vigorously at 23 °C for 2 h until two
layers become clear. Organic layer was separated, and aqueous layer
was extracted with dichloromethane. Combined extracts were washed
with saturated aqueous NaCl, dried (Na2SO4), and concentrated under
reduced pressure. The crude product was purified by silica gel column
chromatography (60% EtOAc in hexane) to afford 10 in 2:1 ratio (670
mg, 62% for 3 steps).
To a stirred solution of 10 (670 mg, 2.32 mmol) in dichloro-
methane (60 mL) was added TFA (2.3 mL) at 0 °C under argon
atmosphere. The reaction mixture was warmed to 23 °C and stirred
for 15 h. Upon completion, the reaction mixture was quenched by the
addition of saturated aqueous NaHCO3 and extracted with dichloro-
methane. The extracts were washed with saturated aqueous NaCl,
dried (Na2SO4), and concentrated under reduced pressure. The crude
product was purified by silica gel column chromatography (5% EtOAc
in hexane) to afford 11 (465 mg, 74%). 1H NMR (400 MHz, CDCl3)
δ 5.36 (d, J = 6.8 Hz, 1H), 4.11 (dd, J = 9.0, 1.2 Hz, 1H), 3.97 (dd, J =
8.9, 7.0 Hz, 1H), 3.87−3.79 (m, 2H), 3.51 (d, J = 11.5 Hz, 1H), 2.70
(q, J = 5.9 Hz, 1H), 2.42 (t, J = 7.1 Hz, 1H), 2.06−2.01 (m, 1H), 1.97
(dt, J = 11.1, 4.4 Hz, 1H), 1.83 (d, J = 11.3 Hz, 1H), 0.86 (s, 9H), 0.04
(d, J = 3.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 104.2, 86.3, 73.3,
64.9, 52.7, 45.5, 44.6, 25.9, 25.6, 18.1, −4.6. LRMS-ESI (m/z): 293.1
[M + Na]+.
(3S,7aS,8R)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
ol (12). To a stirred solution of 11 (460 mg, 1.7 mmol) in THF (10
mL) was added TBAF (2.6 mL, 2.6 mmol) at 0 °C under argon
atmosphere. The reaction mixture was warmed to 23 °C and stirred
for 2 h. Upon completion, solvent was removed under reduced
pressure. The crude product was purified by silica gel column
chromatography (85% EtOAc in hexane) to afford 12 (248 mg, 92%).
[α]D20 −24.48 (c 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ 5.39 (d,
J = 6.8 Hz, 1H), 4.17 (dd, J = 9.0, 1.1 Hz, 1H), 3.99 (dd, J = 9.0, 6.9
Hz, 1H), 3.95 (s, 1H), 3.86 (dd, J = 11.5, 8.5 Hz, 1H), 3.56 (d, J = 11.5
Hz, 1H), 2.75 (q, J = 5.8 Hz, 1H), 2.47 (t, J = 7.0 Hz, 1H), 2.15−2.09
(m, 1H), 1.99 (dt, J = 11.6, 4.3 Hz, 1H), 1.91 (d, J = 11.7 Hz, 1H),
1.72 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 104.1, 85.7, 73.3, 64.7,
51.8, 45.5, 44.5, 25.5. LRMS-ESI (m/z): 179.0 [M + Na]+.
HRMS-ESI (m/z): [M + Na]+ calcd for C8H12O3Na, 179.0684; found
179.0680.
(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl (4-Nitrophenyl)carbonate (14). To a stirred solution of 13 (75.6
mg, 0.484 mmol) in dichloromethane (4 mL) were added pyridine
(0.18 mL, 2.2 mmol) and 4-nitrophenyl chloroformate (292 mg, 1.45
mmol) at 0 °C under argon atmosphere. The reaction mixture was
warmed to 23 °C and stirred for 12 h. Upon completion, solvent was
removed under reduced pressure. The crude product was purified by
silica gel column chromatography (35% EtOAc in hexane) to afford 14
1
(147 mg, 94%). H NMR (400 MHz, CDCl3) δ 8.32−8.26 (m, 2H),
7.41−7.36 (m, 2H), 5.52 (d, J = 6.8 Hz, 1H), 5.02 (dd, J = 9.2, 5.7 Hz,
1H), 4.28 (d, J = 9.5 Hz, 1H), 4.05 (d, J = 11.6 Hz, 1H), 3.86 (dd, J =
9.5, 6.6 Hz, 1H), 3.74 (dd, J = 11.6, 7.9 Hz, 1H), 2.96 (q, J = 7.1 Hz,
1H), 2.79 (q, J = 6.8 Hz, 1H), 2.59 (q, J = 5.5 Hz, 1H), 1.95 (d, J =
12.2 Hz, 1H), 1.56 (dt, J = 12.2, 4.2 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 155.5, 152.1, 145.6, 125.5, 121.8, 104.5, 79.9, 68.5, 59.7,
45.1, 42.0, 37.5, 23.7. LRMS-ESI (m/z): 343.9 [M + Na]+.
(3S,7aS,8R)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl (4-Nitrocyclohexa-1,5-dien-1-yl)carbonate (15). Alcohol 12
(9.5 mg, 0.061 mmol) was treated with 4-nitrophenyl chloroformate
(24.6 mg, 0.122 mmol) by following the procedure above to afford 15
1
(17.5 mg, 90%). H NMR (400 MHz, CDCl3) δ 8.31−8.26 (m, 2H),
7.41−7.35 (m, 2H), 5.45 (d, J = 6.7 Hz, 1H), 4.78 (s, 1H), 4.28 (d, J =
9.3 Hz, 1H), 4.03 (dd, J = 9.3, 6.6 Hz, 1H), 3.95 (dd, J = 11.7, 8.6 Hz,
1H), 3.66 (d, J = 11.7 Hz, 1H), 2.88−2.81 (m, 1H), 2.73 (t, J = 6.8 Hz,
1H), 2.49−2.43 (m, 1H), 2.05−1.95 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 155.5, 152.2, 145.6, 125.5, 121.8, 104.0, 92.4, 72.8, 64.0,
49.7, 45.3, 41.4, 26.1.
N-((2R,3S)-3-Amino-2-hydroxy-4-phenylbutyl)-2-(cyclopro-
pylamino)-N-isobutylbenzo[d]thiazole-6-sulfonamide (21). To
a stirred solution of 20 (360 mg, 0.62 mmol) in dichloromethane (5
mL) was added mCPBA (321 mg, 1.86 mmol) at 0 °C under argon
atmosphere, and the mixture was stirred at 23 °C for 12 h. After this
period, the reaction mixture was quenched by the addition of saturated
aqueous Na2S2O3 (1 mL) and extracted with dichloromethane. The
extracts were washed with saturated aqueous NaHCO3, dried
(Na2SO4), and concentrated under reduced pressure. To the crude
product in dry THF (3 mL) at 23 °C under argon atmosphere was
added cyclopropylamine (0.13 mL, 1.86 mmol), and the mixture was
stirred at 65 °C for 12 h. After this period, solvent was removed under
reduced pressure and the crude product was purified by silica gel
column chromatography (35% EtOAc in hexane) to give Boc
derivative (334 mg, 91% over two steps).
To a stirred solution of the above Boc derivative (334 mg, 0.57
mmol) in dichloromethane (5 mL) was added TFA (1 mL) at 0 °C
under argon atmosphere, and the mixture was stirred at 23 °C for 1 h.
After this period, solvent was removed under reduced pressure. The
crude was washed with saturated aqueous NaHCO3, dried (Na2SO4),
and concentrated under reduced pressure. The crude product was
purified by silica gel column chromatography (5% MeOH in CH2Cl2)
to afford 21 (275 mg, 99% yield). 1H NMR (500 MHz, CDCl3) δ 8.11
(d, J = 1.5 Hz, 1H), 7.71 (dd, J = 8.5, 1.6 Hz, 1H), 7.49 (d, J = 8.5 Hz,
1H), 7.31 (t, J = 7.4 Hz, 2H), 7.22 (dd, J = 12.5, 7.1 Hz, 3H), 3.85−
3.78 (m, 1H), 3.31 (d, J = 7.4 Hz, 2H), 3.19−3.11 (m, 1H), 3.07 (dd, J
= 13.3, 8.3 Hz, 1H), 3.01−2.87 (m, 2H), 2.73 (tt, J = 6.7, 3.5 Hz, 1H),
2.56−2.46 (m, 1H), 1.96−1.85 (m, 1H), 0.95−0.92 (m, 4H), 0.90−
0.88 (m, 4H), 0.79−0.76 (m, 2H); 13C NMR (125 MHz, CDCl3) δ
173.1, 155.6, 138.9, 131.2, 131.0, 129.4, 128.8, 126.6, 125.5, 121.0,
118.6, 73.1, 58.7, 55.8, 52.7, 39.1, 31.7, 27.3, 26.8, 22.8, 20.4, 20.1,
14.3, 8.0. LRMS-ESI (m/z): 489.2 [M]+, 490.2 [M + H]+. HRMS-ESI
(m/z): [M + H]+ calcd for C24H33N4O3S2, 489.1994; found 489.1986.
(3S,7aS,8R)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl ((2S,3R)-3-hHydroxy-4-((N-isobutyl-4-methoxyphenyl)-
sulfonamido)-1-phenylbutan-2-yl)carbamate (24). To a stirred
solution of activated alcohol 15 (17.3 mg, 0.054 mmol) and isostere
22 (24 mg, 0.059 mmol) in acetonitrile (2 mL) was added DIPEA (42
μL, 0.24 mmol) at 23 °C under argon atmosphere. The reaction
mixture was stirred at 23 °C until completion. Upon completion,
solvents were removed under reduced pressure and crude product was
(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
ol (13). To a stirred solution of 12 (100 mg, 0.64 mmol) in
dichloromethane (4 mL) were added Na2HPO4 (50 mg, 0.35 mmol)
and DMP (352 mg, 0.83 mmol) at 0 °C under argon atmosphere. The
reaction mixture was warmed to 23 °C and stirred for 3 h. Upon
completion, the reaction mixture was quenched by the addition of
saturated aqueous Na2S2O3 and extracted with dichloromethane. The
extracts were washed with saturated aqueous NaHCO3, saturated
aqueous NaCl, dried (Na2SO4), and concentrated under reduced
pressure. The crude product was purified by silica gel column
chromatography (40% EtOAc in hexane) to afford ketone (88 mg,
1
89%). H NMR (400 MHz, CDCl3) δ 5.57 (d, J = 7.0 Hz, 1H), 4.21
(d, J = 8.7 Hz, 1H), 3.83−3.76 (m, 2H), 3.68 (d, J = 11.2 Hz, 1H),
3.04−2.96 (m, 1H), 2.56 (t, J = 6.8 Hz, 1H), 2.39−2.32 (m, 1H), 2.09
(d, J = 12.0 Hz, 1H), 1.96−1.87 (m, 1H); 13C NMR (100 MHz,
CDCl3) δ 220.8, 104.1, 71.8, 63.3, 49.6, 46.6, 42.8, 23.3. LRMS-ESI
(m/z): 177.1 [M + Na]+.
To a stirred solution of above ketone (87.9 mg, 0.57 mmol) in
MeOH (4 mL) was added NaBH4 (108 mg, 2.85 mmol) at 0 °C, and
the mixture was stirred at the same temperature for 1 h. Upon
completion, the reaction mixture was quenched by the addition of
saturated aqueous NH4Cl and extracted with EtOAc. The extracts
were washed with saturated aqueous NaCl, dried (Na2SO4), and
concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography (65% EtOAc in hexane) to afford
13 (76 mg, 85%). [α]D20 −9.27 (c 1.03, CHCl3); 1H NMR (400 MHz,
CDCl3) δ 5.43 (d, J = 6.4 Hz, 1H), 4.42 (d, J = 9.4 Hz, 1H), 4.23 (dd,
J = 8.8, 5.7 Hz, 1H), 4.06 (d, J = 11.4 Hz, 1H), 3.74 (dd, J = 9.0, 6.2
Hz, 1H), 3.64 (dd, J = 11.4, 7.8 Hz, 1H), 2.70−2.58 (m, 2H), 2.23 (q,
J = 5.5 Hz, 1H), 1.98 (s, 1H), 1.80 (d, J = 12.0 Hz, 1H), 1.44 (dt, J =
12.0, 3.9 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 104.4, 72.5, 68.2,
59.4, 45.3, 43.0, 39.4, 23.9. LRMS-ESI (m/z): 179.0 [M + Na]+.
I
DOI: 10.1021/acs.jmedchem.7b00172
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
purified by silica gel column chromatography (45% EtOAc in hexane)
to give inhibitor 24 (26.4 mg, 83%). H NMR (400 MHz, CDCl3) δ
AUTHOR INFORMATION
Corresponding Author
*Phone: (765) 494-5323. Fax: (765) 496-1612. E-mail:
akghosh@purdue.edu.
■
1
7.69 (d, J = 8.7 Hz, 2H), 7.30−7.26 (m, 2H), 7.23−7.20 (m, 3H), 6.97
(d, J = 8.8 Hz, 2H), 5.38 (d, J = 6.6 Hz, 1H), 4.88 (d, J = 8.1 Hz, 1H),
4.50 (s, 1H), 4.19 (d, J = 9.0 Hz, 1H), 4.00−3.91 (m, 1H), 3.88−3.78
(m, 7H), 3.54 (d, J = 11.6 Hz, 1H), 3.12 (dd, J = 15.1, 8.2 Hz, 1H),
3.05−2.96 (m, 2H), 2.97−2.92 (m, 1H), 2.89−2.83 (m, 1H), 2.78 (dd,
J = 13.4, 6.7 Hz, 1H), 2.69 (s, 1H), 2.46 (t, J = 6.0 Hz, 1H), 2.02 (s,
1H), 1.86−1.80 (m, 2H), 1.75−1.70 (m, 1H), 0.91 (d, J = 6.6 Hz,
3H), 0.86 (d, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 163.2,
156.0, 137.8, 129.9, 129.6, 128.6, 126.6, 114.5, 104.0, 88.0, 73.0, 72.8,
64.4, 58.9, 55.8, 55.1, 53.8, 49.4, 45.2, 41.3, 35.6, 27.4, 25.9, 20.3, 20.0.
LRMS-ESI (m/z): 611.4 [M + Na]+. HRMS-ESI (m/z): [M + Na]+
calcd for C30H40N2O8SNa, 611.2404; found 611.2408.
ORCID
Arun K. Ghosh: 0000-0003-2472-1841
Irene T. Weber: 0000-0003-4876-7393
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health (Grant GM53386 to A.K.G. and Grant GM62920 to
I.T.W.). X-ray data were collected at the Southeast Regional
Collaborative Access Team (SER-CAT) beamline 22BM at the
Advanced Photon Source, Argonne National Laboratory. Use
of the Advanced Photon Source was supported by the U.S.
Department of Energy, Basic Energy Sciences, Office of
Science, under Contract W-31-109-Eng-38. This work was
also supported by the Intramural Research Program of the
Center for Cancer Research, National Cancer Institute,
National Institutes of Health, and in part by a Grant-in-Aid
for Scientific Research (Priority Areas) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan
(Monbu Kagakusho), a Grant for Promotion of AIDS Research
from the Ministry of Health, Welfare, and Labor of Japan, and
the Grant to the Cooperative Research Project on Clinical and
Epidemiological Studies of Emerging and Reemerging Infec-
tious Diseases (Renkei Jigyo) of Monbu-Kagakusho. The
authors thank the Purdue University Center for Cancer
Research, which supports the shared NMR and mass
spectrometry facilities. We thank Dr. Margherita Brindisi
(Purdue University) for helpful discussions.
(3S,7aS,8S)-Hexahydro-4H-3,5-methanofuro[2,3-b]pyran-8-
yl ((2S,3R)-3-Hydroxy-4-((N-isobutyl-4-methoxyphenyl)-
sulfonamido)-1-phenylbutan-2-yl)carbamate (25). Compound
14 (21.2 mg, 0.066 mmol) was treated with isoster 22 (29.7 mg, 0.073
mmol) by following the procedure above to afford inhibitor 25 (34.5
1
mg, 89%). H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 8.8 Hz, 2H),
7.32−7.26 (m, 2H), 7.25−7.20 (m, 3H), 6.97 (d, J = 8.8 Hz, 2H), 5.41
(d, J = 6.6 Hz, 1H), 5.14 (d, J = 8.5 Hz, 1H), 4.78 (dd, J = 8.6, 5.8 Hz,
1H), 3.92−3.80 (m, 7H), 3.74 (d, J = 9.1 Hz, 1H), 3.56 (ddd, J = 16.0,
10.2, 7.2 Hz, 2H), 3.14 (dd, J = 15.0, 8.2 Hz, 1H), 3.05 (dd, J = 14.2,
3.6 Hz, 1H), 3.02−2.92 (m, 2H), 2.81 (td, J = 14.9, 13.4, 7.7 Hz, 2H),
2.66 (td, J = 13.4, 10.7, 7.1 Hz, 2H), 2.35−2.27 (m, 1H), 1.83 (dd, J =
20.5, 9.2 Hz, 2H), 1.43 (dt, J = 11.9, 4.2 Hz, 1H), 0.91 (d, J = 6.6 Hz,
3H), 0.87 (d, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 163.2,
155.8, 137.8, 130.0, 129.6, 129.5, 128.7, 126.7, 114.5, 104.5, 75.1, 72.9,
68.6, 60.0, 58.9, 55.8, 55.3, 53.8, 45.0, 42.1, 37.5, 35.6, 31.7, 27.4, 23.6,
22.8, 20.3, 20.0. LRMS-ESI (m/z): 611.4 [M + Na]+. HRMS-ESI (m/
z): [M + Na]+ calcd for C30H40N2O8SNa, 611.2404; found 611.2401.
(3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl ((2S,3R)-4-((2-
(Cyclopropylamino)-N-isobutylbenzo[d]thiazole)-6-sulfonami-
do)-3-hydroxy-1-phenylbutan-2-yl)carbamate (27). Compound
26 (8 mg) was treated with isostere amine 21 by following the
procedure outlined for inhibitor 24 to give inhibitor 27 (15 mg, 79%).
1H NMR (500 MHz, CDCl3) δ 8.12−8.08 (m, 1H), 7.71 (dd, J = 8.5,
1.7 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.33−7.29 (m, 3H), 7.27−7.21
(m, 3H), 6.64 (s, 1H), 5.67 (d, J = 5.1 Hz, 1H), 5.04 (dt, J = 23.9, 7.5
Hz, 2H), 4.02−3.95 (m, 1H), 3.95−3.90 (m, 2H), 3.90−3.85 (m, 1H),
3.77−3.67 (m, 3H), 3.24 (dd, J = 14.8, 8.4 Hz, 1H), 3.16−3.09 (m,
1H), 3.05 (dt, J = 14.7, 7.4 Hz, 2H), 2.97−2.90 (m, 1H), 2.86 (dd, J =
13.3, 6.5 Hz, 2H), 2.80 (tt, J = 6.7, 3.5 Hz, 1H), 1.87 (dq, J = 13.3, 6.6
Hz, 1H), 1.71−1.64 (m, 1H), 1.52 (d, J = 9.8 Hz, 1H), 1.01−0.96 (m,
5H), 0.92 (d, J = 6.6 Hz, 3H), 0.85−0.81 (m, 2H); 13C NMR (125
MHz, CDCl3) δ 173.3, 155.8, 155.6, 137.8, 131.4, 130.4, 129.5, 128.7,
126.7, 125.5, 121.0, 118.7, 109.4, 73.6, 73.0, 70.9, 69.8, 59.1, 55.3, 53.9,
45.5, 35.8, 29.8, 27.5, 26.8, 25.9, 20.3, 20.0, 8.1. LRMS-ESI (m/z):
667.4 [M + Na]+. HRMS-ESI (m/z): [M + H]+ calcd for
C31H41N4O7S2, 645.2417; found 645.2423.
ABBREVIATIONS USED
■
THF, tetrahydrofuran; bis-THF, bis-tetrahydrofuran; PI,
protease inhibitor; crn-THF, crown-tetrahydrofuran; TFA,
trifluoroacetic acid; DRV, darunavir; APV, amprenavir; NRTI,
nucleotide reverse transcriptase inhibitor; LPV, lopinavir; ATV,
atazanavir
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge on the
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