Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors incorporating aminothiochromane and aminotetrahydronaphthalene carboxamide derivatives as the P2 ligands

Article abstract of DOI:10.1016/j.ejmech.2018.09.046

We describe the design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors with carboxamide derivatives as the P2 ligands. We have specifically designed aminothiochromane and aminotetrahydronaphthalene-based carboxamide ligands to promote hydrogen bonding and van der Waals interactions in the active site of HIV-1 protease. Inhibitors 4e and 4j have shown potent enzyme inhibitory and antiviral activity. High resolution X-ray crystal structures of 4d- and 4k-bound HIV-1 protease revealed molecular insights into the ligand-binding site interactions.

Full text of DOI:10.1016/j.ejmech.2018.09.046

Accepted Manuscript
Design, synthesis, and X-ray studies of potent HIV-1 protease inhibitors incorporating
aminothiochromane and aminotetrahydronaphthalene carboxamide derivatives as the
P2 ligands
Arun K. Ghosh, Ravindra D. Jadhav, Hannah Simpson, Satish Kovela, Heather
Osswald, Johnson Agniswamy, Yuan-Fang Wang, Shin-ichiro Hattori, Irene T. Weber,
Hiroaki Mitsuya
PII:
S0223-5234(18)30818-3
10.1016/j.ejmech.2018.09.046
EJMECH 10757
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2018
Revised Date: 13 September 2018
Accepted Date: 14 September 2018
Please cite this article as: A.K. Ghosh, R.D. Jadhav, H. Simpson, S. Kovela, H. Osswald, J. Agniswamy,
Y.-F. Wang, S.-i. Hattori, I.T. Weber, H. Mitsuya, Design, synthesis, and X-ray studies of potent
HIV-1 protease inhibitors incorporating aminothiochromane and aminotetrahydronaphthalene
carboxamide derivatives as the P2 ligands, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.09.046.
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Graphical Abstract
Leave this area blank for abstract info.
Design, Synthesis, and X-Ray Studies of Potent
HIV-1 Protease Inhibitors incorporating amino-
thiochromane and aminotetrahydronaphthalene carboxamide derivatives as the P2
ligands. Arun K. Ghosh, Ravindra D. Jadhav, Hannah Simpson, Satish Kovela, Heather Osswald, Johnson
Agniswamy, Yuan-Fang Wang, Shin-ichiro Hattori, Irene T. Weber, and Hiroaki Mitsuya.
O
O
S
OMe
OH
H
N
N
S
O
O
NH
O
Ph
Boc
4e, Ki = 8 pM (HIVP)
IC50 = 47 nM (antiviral)

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Design, Synthesis, and X-Ray Studies of Potent HIV-1 Protease Inhibitors
incorporating aminothiochromane and aminotetrahydronaphthalene carboxamide
derivatives as the P2 ligands.
, a
Arun K. Ghosh , Ravindra D. Jadhav a, Hannah Simpsona, Satish Kovelaa, Heather Osswald,a Johnson
Agniswamyb, Yuan-Fang Wang b, Shin-ichiro Hattori c, Irene T. Weber b, and Hiroaki Mitsuya c,d,e
a Department of Chemistry and Department of Medicinal Chemistry, Purdue University, West Lafayette, IN 47907, United States
b Department of Biology, Molecular Basis of Disease, Georgia State University, Atlanta, Georgia 30303, United States
c Department of Refractory Viral Infection, National Center for Global Health & Medicine Research Institute, Tokyo 162-8655, Japan
d Departments of Hematology and Infectious Diseases, Kumamoto University Graduate School of Medical and Pharmaceutical Sciences, Kumamoto 860-8556, Japan
e Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, United
States
ARTICLE INFO
ABSTRACT
We describe the design, synthesis, and biological evaluation of a series of novel HIV-1 protease
inhibitors with carboxamide derivatives as the P2 ligands. We have specifically designed
aminothiochromane and aminotetrahydronaphthalene-based carboxamide ligands to promote
hydrogen bonding and van der Waals interactions in the active site of HIV-1 protease. Inhibitors
4e and 4j have shown potent enzyme inhibitory and antiviral activity. High resolution X-ray
crystal structures of 4d- and 4k-bound HIV-1 protease revealed molecular insights into the
ligand-binding site interactions.
Article history:
Received
Revised
Accepted
Available online
Keywords:
HIV-1 protease inhibitors
P2 ligand
2009 Elsevier Ltd. All rights reserved.
Drug resistance
Design and synthesis
X-ray crystal structure
———
Corresponding author. e-mail: akghosh@purdue.edu (AKG); Tel.: +1-765-494-5323; fax: +1-765-496-1612;

ACCEPTED MANUSCRIPT
approved inhibitors darunavir and nelfinavir-bound to HIV-1
1. Introduction
Design and development of HIV-1 protease inhibitors and their
protease.15,16,35 Our efforts led to very potent inhibitors with
picomolar enzyme inhibitory activity and low nanomolar antiviral
activity. The X-ray structural studies of a number of these
inhibitors provided structural insights into the ligand and HIV-1
protease interactions, particularly in the active site of the
enzyme.15,35 We have now further explored these molecular insights
and we report the design, synthesis, biological evaluation, and X-
ray structural studies of a new series of protease inhibitors
incorporating stereochemically defined aminothiochromane and
aminotetrahydronaphthalene carboxamide derivatives as the P2
ligands.
introduction in combination therapy represent a major innovation
for the treatment of HIV-1 infection and AIDS.1,2 These
combination antiretroviral therapies (ART) significantly improve
the life expectancy of HIV-1 infected patients.3,4 The mortality rates
for HIV/AIDS patients who are treated with ART, have become
close to those of the general population.5,6 However, the majority of
approved protease inhibitors in ART have limitations due to rapid
occurrence of resistant strains, high pill burdens and other side
effects.7,8 The last FDA approved protease inhibitor, darunavir (1,
Figure 1), has significantly improved properties.9,10 Darunavir has
been shown to maintain excellent potency against a broad-spectrum
of highly multidrug-resistant HIV-1 variants.11,12 Darunavir and
related derivative TMC126 (2) were specifically designed to
promote extensive hydrogen-bonding interactions with HIV-1
protease backbone atoms.13,14 Indeed, the X-ray structure of
darunavir-bound HIV-1 protease revealed these critical ligand-
binding site interactions which are now further utilized in our
molecular design.15,16 The design of protease inhibitors continue to
be an important area of research. There are recent reports of novel
PIs incorporating P2/P2' ligands.17-20 PIs were also designed with
new P1 and P2' ligands.21,22 Recently, a new class of piperazine
derived non-peptide inhibitors have been reported.23,24
2. Results and discussion
2.1 Chemistry
Based upon the X-ray structures of darunavir-bound HIV-1
protease and nelfinavir-bound HIV-1 protease, we designed 4-
aminothiochromane
and
4-aminotetrahydronaphthalene
carboxamide derivatives as the P2 ligand. The synthesis of 4-
aminothiochromane-6-carboxylic acid in optically active form is
shown in Scheme 1. Reaction of 4-mercaptobenzoic acid methyl
ester 5 with 3-bromopropionic acid in the presence of pyridine at
80 °C for 1 h provided the corresponding alkylated product. The
resulting acid was reacted with polyphosphoric acid (PPA) at 65 °C
for 6 h to afford 4-oxothiochromane derivative 6.36 Reaction of
ketone 6 with commercially available (R)-(+)-2-methyl-2-propane
sulfinamide in the presence of Ti(OEt)4 in THF at 70 °C furnished
sulfinyl imins 7.37,38 Imine derivative 7 was reduced with NaBH4 in
a mixture of THF and water at -50 °C to afford a mixture (3:1) of
diastereomeric sulfonamides 8 and 9 in 78% combined yield. The
diastereomers were separated by silica gel chromatography to
O
O
O
O
O
1. Br(CH2)2CO2H
Pyridine, 80 °C
MeO
MeO
EtO
EtO
2. PPA, 65 °C
(52%)
S
SH
6
5
(R)-(+)-t-butyl-
sulfinamide
Ti(OEt)4, THF
(76%)
O
S
O
S
O
N
HN
H
NaBH4
EtO
THF-H2O
S
S
(78%)
(dr = 3:1)
7
8
Figure 1. Structure of protease inhibitors 1-3, 4e.
+
1. 6M HCl, 2-propanol
2. Boc2O, Et3N, CH2Cl2
O
S
(99%)
In our continuing studies towards the design and synthesis of
new class of PIs, we have structure-based designed a range of
exceptionally potent PIs with intriguing molecular features
including, GRL-6579, GRL-02031, GRL-0519, and more recently
GRL-0142.14,25-28 These inhibitors exhibit broad-spectrum antiviral
activity against highly multidrug-resistant HIV-1 mutant strains and
also show high genetic barrier to resistance.27,29 In these inhibitors,
we incorporated a variety of cyclic ether-derived templates as the
P2-ligand attached to the hydroxyethylamine sulfonamide isostere
with a urethane functionality.30 The major design concept behind
these PIs is to promote extensive hydrogen bonding with the HIV-1
protease active site backbone atoms like a molecular crab.13,14
Recently, we and others reported a variety of protease inhibitors
with benzoic acid amide derivatives as the P2-ligands.31-34 These
inhibitors were designed based upon the X-ray structures of FDA
HN
O
NHBoc
H
H
RO
S
S
10
11
(84%)
R = Et
R = H
9
1M aq LiOH
O
S
O
NHBoc
H
O
N
S
HO
EtO
S
11
ent-
7
ent-
Scheme 1. 4-Aminothiochromane-6-carboxylic acid
.

ACCEPTED MANUSCRIPT
provide R-sulfinyl inamide 8 as the major product. Sulfinamide 8
separated by silica gel chromatography using 30% ethyl acetate in
hexanes as the eluent. Reduction of 13 with L-selectride at 0 °C to
23 °C for 3 h afforded only disatereomer 15 in 96% yield.
was treated with HCl (6 M solution in isopropanol) in methanol at
23 °C for 1 h to provide the corresponding amine hydrochloride
salt. Reaction of this amine salt in CH2Cl2 with Boc2O in the
presence of triethylamine afforded Boc- protected amine derivative
10. Saponification of the ethyl ester with aqueous LiOH provided
carboxylic acid 11. For the synthesis of the enantiomeric amine
Treatment of sulfinamide 15 with HCl (6M solution in
isopropanol) in methanol at 23 °C for 2.5 h afforded the
corresponding amine which was reacted with Boc2O in the presence
of Et3N in CH2Cl2 at 0 °C to 23 °C for 12 h to provide optically
active Boc-derivative 16 in 85% yield over two-steps. For
introduction of the 5-hydroxyl group, Boc-amine derivative 16 was
oxidized using KMnO4 in the presence of MgSO4 in acetone at 0 °C
to 23 °C for 8 h to furnish the corresponding bromoketone
derivative in 61% yield. Treatment of the resulting bromoketone in
methanol in the presence of catalytic amount Pd(OAc)2, (2 mol%)
xantphos (4 mol%) and excess of Et3N under a CO-filled balloon at
70 °C for 3.5 h provided methyl ester derivative 17 in 80% yield.39
Ketoester 17 was converted to the corresponding Boc-aminoalcohol
derivative by a catalytic transfer hydrogenation reaction using the
Noyori catalyst RuCl(Mes)[R,R-ts-dpen] in DMF in the presence of
formic acid and Et3N at 60 °C for 12 h to afford the corresponding
alcohol as a single diastereomer in 94% yield.40,41 Saponification of
the resulting methyl ester with 1N aqueous LiOH in THF in the
presence of a few drops of MeOH at 23 °C for 12 h afforded
carboxylic acid 18 in 89% yield. Furthermore, reduction of
ketoester 17 with RuCl(p-Cym)[S,S-ts-dpen] under the same
reaction conditions mentioned above, provided the diastereomeric
alcohol as a single diastereomer in 93% yield. Basic ester
hydrolysis furnished ligand acid 19. The synthesis of enantiomeric
ligand carboxylic acid ent-18 was carried out from commercially
O
S
O
N
(R)-(+)-t-butyl-
sulfinamide
Br
Br
Ti(OEt)4, THF
(87%)
13
12
L-selectide
THF
(96%)
NaBH4
THF-H2O
O
S
O
(94%)
S
NH
NH
H
H
Br
Br
+
(dr = 2:1)
14
15
1. 6M HCl, 2-propanol
2. Boc2O, Et3N, CH2Cl2
(85%)
Boc
Boc
NH
HN
H
O
1. KMnO4
H
Br
MgSO4
OMe
2. Pd(OAc)2,
Xanthphos,
CO, Et3N
17
16
O
(49%)
available
methyl
8-oxo-5,6,7,8-tetrahydronaphthalene-2-
1. RuCl(Mes)[ts-dpen]-
(R,R), HCO2H, Et3N
2. aq LiOH, THF
1. RuCl(p-cym)[ts-dpen
(S,S), HCO2H, Et3N
2. aq LiOH, THF
(84%)
(93%)
OMe
OH
S
Boc
H
Boc
NH
O
NH
O
H2N
Ph
N
OH
+
H
S
H
OH
OH
O
O
NHBoc
O
20
11
18
19
EDC HCl, HOBt
DIPEA, THF
(63%)
OH
O
OH
S
OMe
Boc
H
OH
NH
OH
O
O
H
N
N
S
OH
OH
O
O
NH
O
4a
Boc
Ph
m-CPBA
CH2Cl2
ent-18
(92%)
O
O
S
Scheme 2. Synthesis of 8-amino-5-hydroxy-
tetrahydronaphthalene-2-carboxylic acid.
OMe
OMe
OH
H
N
N
O
S
derivative ent-11 (enantiomer of compound 11), 4-oxochromane
derivative 6 was reacted with commercially available (S)-(-)-2-
methyl-2-propane sulfinamide to obtain the corresponding imine
which was reduced with NaBH4 to provide mixture of
diastereomers. Separation of diastereomers followed by reaction of
the major isomers with 6 M HCl, and protection of amine as a Boc-
derivative, and aqueous LiOH hydrolysis as described above
resulted in enantiomeric acid ent-11.
O
NH
O
Boc
4b
Ph
TFA, CH2Cl2
(83%)
O
O
S
OH
H
N
N
S
O
O
NH2
O
O
4c
Ph
The
synthesis
of
optically
active
amino-
NHBoc
H
hydroxytetrahydronaphthalene carboxylic acids is shown in
Scheme 2. Commercially available bromotetralone 12 was reacted
with (R)-(+)-2-methyl-2-propane sulfinamide in the presence of
Ti(OEt)4 in THF at 66 °C for 10 h to provide sulfinyl imine 13 in
87% yield. Reduction of 13 with NaBH4 in a mixture of (98:2) THF
and water at -50 °C to 23 °C for 3 h provided 14 and 15 in 2:1 ratio
in 94% combined yield. The diastereomeric sulfinamides were
EtO
4d, 4e, 4f
S
ent-
11
Scheme 3. Synthesis of protease inhibitors 4a-f.

ACCEPTED MANUSCRIPT
carboxylate and (S)-t-butyl sulfinamide to provide imine which
was reduced with NaBH4 to obtain mixture (2:1) of
corresponding enantiomeric ligand ent-19 was then converted to
inhibitors 4k and 4l as described above. The full structures of these
a
diastereomers. The major diastereomer was was treated with 6 M
HCl to provide the corresponding amine. Protection of the
resulting amine as Boc-derivative. This was converted to
carboxylic acid ent-18 by following the same sequence of reactions
as described above.
inhibitors are shown in Table 2.
2.2 HIV-1 Protease inhibitory and antiviral activity
Our preliminary model of inhibitor 4a that we created in the
Nelfinavir-bound HIV-1 protease active site,35 indicated that the
thiochroman heterocycle with (S)-Boc-amine functionality can
interact with Asp29 and Asp30 backbone NHs in the S2 subsite,
while the thiochroman moiety would fill the hydrophobic pocket.
The results of HIV-1 protease inhibitory Ki and antiviral IC50
values are shown in Table 1. The assay protocol for HIV-1 protease
activity is similar to the report of Toth and Marshall.42 Antiviral
activity was determined in MT-4 human T-lymphoid cells exposed
to HIV-1NL4-3 (subtype B) as described by us previously.11 We
chose to utilize a hydroxyethylaminesulfonamide isostere with 4-
methoxybenzene sulfonamide as the P2' ligand as in inhibitor 2. As
can be seen, inhibitor 4a with 4-(R)-aminothiochroman
The synthesis of various inhibitors containing the (R)-
hydroxyethylaminesulfonamide isostere and various thiochromane
derivatives as the P2-ligand is shown in Scheme 3. Optically active
thiochromane carboxylic acid 11 with the known aminoalcohol
2025,26 was reacted with EDC and HOBt in the presence of
diisopropyl-ethylamine (DIPEA) in THF at 23 °C for 8 h to furnish
inhibitor 4a in 63% yield. Oxidation of thiochromane derivative 4a
with mCPBA in CH2Cl2 at 0 °C to 23 °C for 6 h afforded sulfone
derivative 4b in 92% yield. Treatment of 4b with trifluoroacetic
acid (TFA) in CH2Cl2 at 23 °C for 3 h furnished the amine
derivative 4c in 83% yield. Enantiomeric ligand acid ent-11 was
converted to inhibitors 4d-f as described above. The full structures
of these inhibitors are shown in Table 1.
Table 1. Structures and potency of inhibitors 4a-f.a,b
Entry
Inhibitor Structure
Ki
IC50
(nM)
(nM)
1
26.7
0.10
18
>1000
476
2
3
4
>1000
>1000
0.38
5
0.008
15.8
47
6
>1000
Scheme 4. Synthesis of protease inhibitors 4g-f.
The synthesis of various aminotetrahydronapthalene derivatives
as the P2 ligands is shown in Scheme 4. Coupling of acid
containing Boc-aminoalcohol derivative 18 with amine 20 using
EDCI and HOBt in the presence of DIPEA in THF to provide
inhibitor 4g. Removal of Boc-group by exposure to TFA in CH2Cl2
at 23 °C for 3 h provided inhibitor 4h with aminoalcohol
functionalities. Coupling of diastereomeric ligand acid 19 with
amine 20 under similar coupling conditions afforded inhibitor 4i.
Removal of the Boc group with TFA provided inhibitor 4j. The
a
All antiviral assays were performed using MT-4 cells and HIVNL4-3
(subtype B). Values are the mean value of at least two experiments. bThe
IC50 values of amprenavir (APV), saquinavir (SQV), indinavir (IDV),
and darunavir (DRV) were 0.03, 0.015, 0.03, and 0.003 µM,
respectively.
carboxamide as the P2 ligand, showed a HIV-1 protease inhibitory
Ki of 26.7 nM, but did not show any appreciable antiviral activity

ACCEPTED MANUSCRIPT
bonding interactions, we oxidized the ring sulfur to its sulfone
Table 2. Structures and potency of inhibitors 4g-l.a,b
derivative. The resulting inhibitor 4b showed improvement of
potency, exhibiting enzyme Ki of 0.1 nM. It also exhibited
improvement of antiviral activity with an IC50 value of 476 nM
(entry 2). The removal of the Boc-group provided 4-amine
derivative 4c which displayed significant loss of activity (entry 3).
We then examined stereochemical effect and inhibitor 4d with a 4-
(S)-aminothiochroman carboxamide as the P2 ligand showed potent
enzyme inhibitory activity with a Ki of 0.38 nM. However,
inhibitor 4d did not exhibit appreciable antiviral activity (IC50 > 1
µM). We oxidized the ring sulfur to its sulfone derivative 4e. This
led to significant improvement of enzyme inhibitory activity with a
Ki of 8 pM (entry 5). Inhibitor 4e also showed very good antiviral
activity (IC50 = 47 nM). We presume that the improvement of
activity is due to formation of a hydrogen bond through one of the
sulfone oxygens of the P2 ligand. Removal of the Boc-group
Entry
Inhibitor Structure
Ki
IC50
(nM)
(nM)
1
0.3
>1000
>1000
254
2
3
4
5
17
0.14
0.06
1.63
Table 3. Selectivity Index for selected inhibitors.a
Inhibitor
CC50 (µM)
Selectivity Indexa
>210
232
4b
>100
4e
4i
>100
33.3
>2128
131
>1000
4j
35.1
151
6
1.10
>1000
a Each selectivity index denotes a ratio of CC50 to IC50.
a All antiviral assays were performed using MT-4 cells and HIVNL4-3
(subtype B). b The IC50 values of amprenavir (APV), saquinavir (SQV),
indinavir (IDV), and darunavir (DRV) were 0.03, 0.015, 0.03, and 0.003
µM, respectively.
provided inhibitor 4f, which showed substantial loss of enzyme
inhibitory and antiviral activity similar to inhibitor 4c (entries 3 and
6). In general, this series of inhibitors showed low cytotoxicity
(CC50) values in MT4 cells. The selectivity index of selected
inhibitors are shown in Table 3.
(IC50 > 1 µM). Since sulfone oxygens are known43,44 to form strong
Figure 2. Stereoview of the X-ray structure of inhibitor 4d (turquoise)-bound HIV-1 protease (PDB code: 6DV0). All strong active site
hydrogen bonding interactions of inhibitor 4d with HIV-1 protease are shown as dotted lines.

ACCEPTED MANUSCRIPT
Figure 3. Stereoview of the X-ray structure of inhibitor 4k (green)-bound HIV-1 protease (PDB code: 6DV4). All strong active site
hydrogen bonding interactions of inhibitor 4k with HIV-1 protease are shown as dotted lines.
Since both sulfone derivatives 4b and 4e are significantly more
potent than the corresponding sulfides 4a and 4d, we speculated
that one of the sulfone oxygens may have formed hydrogen
bonding interactions with a residue in the active site. We, therefore,
designed tetrahydronapthalene carboxamide derivatives with
aminoalcohol substitution on the ring to mimic the interactions of
Boc-amine and sulfone functionalities of inhibitor 4e. The results
are shown in Table 2. Inhibitor 4g with a (R)-hydroxy derivative as
the P2 ligand showed good enzyme activity, but antiviral activity
in Figure 2. Inhibitor 4k, on the other hand, contains a
tetrahydronaphthalene carboxamide with (R)-Boc-amine and (S)-
hydroxyl functionalities as the P2 ligand and a stereoview of the
active site interactions is shown in Figure 3.
In inhibitor 4d-bound HIV-1 protease structure, the bulky
sulfur atom in the thiochroman group provides hydrophobic
interactions with the side chains of Asp29, Asp30 and Ile47. The
amide of the carbamate group forms a hydrogen bond of 2.6 Å
length with the carbonyl oxygen atom of Gly48 in the flap, and
carbonyl oxygen forms a hydrogen bond of 3.4 Å with NH2 moiety
of guanidinium side chain of Arg8’. As can be seen from the X-ray
structure of 4d, the significant improvement of enzyme inhibitory
and antiviral activity of the corresponding sulfone derivative 4e
could be due to formation of hydrogen bonding interactions of the
sulfone oxygens with the backbone NH’s of Asp29 and Asp30
located in the S2 subsite. The t-butyl group forms van der Waals
interactions with the hydrophobic side chains of Pro81, Val82 and
Phe53. In the inhibitor 4k-HIV-1 protease complex, the hydroxyl
oxygen on the cyclohexane ring is in an equivalent location to the
methoxy oxygen of the P2′ ligand and forms similar hydrogen
bonds with the main chain amides of Asp29 and Asp30 with bond
lengths of 3.5 Å and 3.1 Å, respectively, and a 2.8 Å-long hydrogen
bond with the carboxylate oxygen of the Asp30 side chain. The
different chiral orientation relative to inhibitor 4d shifts the
carbamate away from the flap residue Gly48. The carbamate amide
and carbonyl oxygen can only form hydrogen bonds via one or two
water intermediates to the amide atom of Asp29 and main chain
oxygen and amide of Gly49, respectively. The terminal t-butyl
group embeds between the side chains of Arg8’, Pro81 and Val82,
forming a C-H...π interaction with the guanidinium group of Arg8’
and van der Waals interactions with Pro81 and Val82. These
differences in the P2 group may contribute to the inhibitor potency
against HIV-1 protease.
was
> 1 µM. The corresponding amine derivative 4h is
significantly less potent (entry 2). The 4(S)-hydroxy derivative 4i
showed improvement of both enzyme inhibitory and antiviral
activity with an IC50 value of 254 nM. The corresponding
aminoalcohol derivative 4j exhibited 1-fold improvement in
enzyme activity, but showed comparable antiviral activity to its
Boc-derivative 4i. We also examined the stereochemical effect of
the Boc derivative 4k and the corresponding amine derivative 4l.
Both compounds were less potent.
2.3 X-Ray Crystal structure of inhibitor-bound HIV-1 protease
To obtain molecular insight into the ligand-binding site
interactions, we set up co-crystallization experiments with several
inhibitors and HIV-1 protease.45 The X-ray structures were
obtained for the wild-type HIV-1 protease co-crystallized
independently with inhibitors 4d (GRL-02815A) and 4k (GRL-
04315A) and were refined to a resolution of 1.20 Å and 1.14 Å,
respectively. The protease dimer structures were very similar to the
darunavir-bound HIV-1 protease complex15 with a RMSD of 0.15
Å for 198 equivalent Cα atoms, and the largest disparity of around
0.4-0.6 Å. In both structures, the active site of the protease dimer
was occupied by two alternate conformations of inhibitor related by
180º rotation with a relative occupancy of 0.60/0.40. The two
inhibitor conformations show similar interactions with the protease,
hence details are given for the major conformation. With the
exception of the P2 ligand, both inhibitors retain the hydrogen
bonds observed between darunavir and the main chain atoms of
protease. These inhibitors have distinctly different P2 ligands from
the bis-tetrahydrofuran in darunavir. Inhibitor 4d contains a
thiochroman heterocycle with (S)-Boc-amine functionality as the
P2 ligand and a stereoview of the active site interactions is shown
3. Conclusion
In summary, we have reported the structure-based design and
synthesis of a series of HIV-1 protease inhibitors incorporating
stereochemically
defined
amino-thiochroman
and
aminotetrahydronaphthalene carboxamide derivatives as the P2
ligands. We have investigated various stereoisomers in order to
promote effective hydrogen bonding interactions with backbone
atoms in the S2 subsite. These functionalized ligands were

ACCEPTED MANUSCRIPT
synthesized stereoselectively in optically active form by reduction
added and the resulting mixture was stirred at 65 °C for 6 h. After
this period, the reaction mixture was allowed to cool to room
temperature and quenched by the addition of cold water and
extracted with ethyl acetate (3×25 mL). The organic layer was
washed with saturated aq. NaHCO3 solution, water, saturated aq.
NaCl solution, and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure to give 6 (141 mg, 76 %) as a
yellow solid. Rf = 0.4 (30% EtOAc/hexanes). LRMS-ESI (m/z):
223 [M+H]+.
of chiral sulfinamide derivatives. Also, Noyori transfer
hydrogenation using chiral ruthenium catalyst provided selective
reduction of the 8-amino-5-tetralone derivatives to the
corresponding
5-hydroxy-naphthalene
derivatives.
Amide
derivatives of these ligands on a hydroxyethylamine sulfonamide
isostere provided potent inhibitors. Inhibitors 4e and 4j exhibited
very potent enzyme inhibitory activity in the picomolar range.
These inhibitors have also shown very good antiviral activity. To
obtain molecular insights into the ligand-binding site interactions,
we determined high resolution X-ray crystal structures of related
inhibitors 4d and 4k-bound HIV-1 protease. The structures show
Ethyl
carboxylate (7)
To a stirred solution of 6 (435 mg, 1.92 mmol) in THF (18
(S,E)-4-((tert-butylsulfinyl)imino)thiochromane-6-
key
interactions
of
amino-thiochroman
and
amino-
tetrahydronaphthalene ligands in the S2 subsite. Both amine
functionalities formed strong hydrogen bonds with the Asp30
backbone NH. This may explain the high enzyme inhibitory
activity of these inhibitors. Further design and ligand optimization
using X-ray structural insights are currently underway in our
laboratories.
mL) was added Ti(OEt)4 (1.0 mL, 4.805 mmol). The solution was
stirred at ambient temperature for 5 min before addition of (R)-(+)-
2-methyl-2-propanesulfinamide (291 mg, 2.4 mmol). Then reaction
mixture refluxed for 12 h. The reaction mixture was cooled to room
temperature and concentrated in vacuo, diluted with EtOAc (35
mL). Saturated NaHCO3 (17 mL) was added under vigourous
stirring and the slurry was filtered through a pad of celite. The
organic phase was separated, dried over Na2SO4 and concentrated
in vacuo. The crude residue was purified by silica gel column
chromatography (30% EtOAc/hexanes) to furnish 7 (500 mg, 76%).
Rf = 0.3 (30% EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ
8.81 (d, J = 2.0 Hz, 1H), 7.91 (dt, J = 8.3, 2.0 Hz, 1H), 7.28 (s, 1H),
4.36 (q, J = 7.1 Hz, 2H), 3.77 – 3.61 (m, 1H), 3.50 (ddd, J = 16.9,
6.7, 5.5 Hz, 1H), 3.14 (dd, J = 7.1, 5.6 Hz, 2H), 1.38 (t, J = 7.3 Hz,
3H), 1.35 (s, 9H); LRMS-ESI (m/z): 340 [M+H]+.
4. Experimental Section
4.1. General experimental conditions
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 using EMD Millipore TLC silica gel 60 F254 plates.
1H NMR and 13C 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 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 was done an on Agilent 1100 series
instrument using a YMC Pack ODS-A column of 4.6 mm ID for
analysis and either 10 mm ID or 20 mm ID for purification. The
purity of all test compounds was determined by HPLC analysis to
be ≥95% pure.
Ethyl
carboxylate
(R)-4-(((R)-tert-butylsulfinyl)amino)thiochromane-6-
(8) and Ethyl (S)-4-(((R)-tert-
butylsulfinyl)amino)thiochromane-6-carboxylate (9)
To a srirred solution of 7 (400 mg, 1.22 mmol) in THF/H2O
o
(4 mL, 98:2) was added NaBH4 (139 mg, 3.68 mmol) at -50 C.
The resulting solution was warmed to room temperature over 3 h.
The solvent was then removed in vacuo and the resulting residue
was triturated with CH2Cl2. The solution was dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo to furnish a crude
product. The crude product was purified by flash column
chromatography over silica gel (30% ethyl acetate/hexanes) to
furnish 8 (250 mg, 60%) and 9 (76 mg, 18%).
Compound 8
23
Rf = 0.4 (50% EtOAc/hexanes); [α]D = -14.4 (c 0.83, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 8.05 (d, J = 1.9 Hz, 1H), 7.77 (dd, J =
8.3, 2.0 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 4.56 (dt, J = 10.2, 5.5
Hz, 1H), 4.34 (q, J = 7.1 Hz, 2H), 3.49 (d, J = 9.2 Hz, 1H), 3.21 –
3.06 (m, 2H), 2.43 (qd, J = 6.2, 5.4, 2.7 Hz, 2H), 1.37 (t, J = 7.1
Hz, 3H), 1.27 (s, 9H); LRMS-ESI (m/z): 342 [M+H]+.
4.2 Synthesis of inhibitors
Methyl 4-oxothiochromane-6-carboxylate (6)
A mixture of 3-bromopropionic acid (0.91 g, 5.95 mmol) and
p-(carbomethoxy)thio-phenol (1 g, 5.95 mmol) was placed in a
round-bottom flask. The flask was heated slowly with the aid of an
oil bath. When the mixture melted, giving a homogeneous solution,
pyridine (0.96 mL, 11.90 mmol) was added and the reaction was
Compound 9
23
1
Rf = 0.3 (50% EtOAc/hexanes); [α]D = +32.2 (c 0.75, CHCl3); H
NMR (400 MHz, CDCl3) δ 7.95 (d, J = 1.9 Hz, 1H), 7.81 (dd, J =
8.3, 1.9 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 4.69 (d, J = 4.7 Hz, 1H),
4.35 (q, J = 7.1 Hz, 2H), 3.34 (td, J = 12.7, 3.0 Hz, 1H), 3.17 (s,
1H), 2.85 (dt, J = 12.4, 4.0 Hz, 1H), 2.58 (dq, J = 14.5, 3.9 Hz, 1H),
2.07 – 1.93 (m, 2H), 1.38 (t, J = 7.1 Hz, 3H), 1.23 (s, 9H). LRMS-
ESI (m/z): 342 [M+H]+.
o
allowed to proceed under an atmosphere of nitrogen at 80 C for 1
h. After this period, the product was dissolved in ethyl acetate and
extracted repeatedly with aqueous bicarbonate. Acidification of the
bicarbonate
layer
afforded
3-((4-
(methoxycarbonyl)phenyl)thio)propanoic acid (982 mg, 69%) as an
amorphous crystals. LRMS-ESI (m/z): 241 [M+H]+.
Ethyl
carboxylate (10)
To a solution of 8 (350 mg, 1.02 mmol) in MeOH (10 mL) was
(R)-4-((tert-butoxycarbonyl)amino)thiochromane-6-
To the above 3-((4-(methoxycarbonyl)phenyl)-thio)propanoic
acid (200 mg, 0.832 mmol), 1.5 g of polyphosphoric acid was

ACCEPTED MANUSCRIPT
added 6 M HCl in Isopropanol (4mL) at 23 oC under argon
atmosphere. The reaction mixture was stirred at 23 oC for 1 h. After
this period, the solvent was removed under reduced pressure to
afford the desired amine salt. Thus obtained amine salt and Et3N
(0.46 mL, 3.20 mmol) were dissolved in CH2Cl2 (5 mL), cooled to
0.41 mmol) in anhydrous THF (5 mL) at 0 oC was added EDC.HCl
(58 mg, 0.30 mmol) and stirred at 23 C for 1 h. An isostere amine
o
20 (112 mg, 0.27 mmol) and DIPEA (0.1 mL, 0.54 mmol) in THF
(3 mL) was added and resulting mixture was stirred for 8 h at 23
oC. The reaction mixture was extracted with ethyl acetate and
successively washed with 5% citric acid, sat NaHCO3, brine
solution, dried over Na2SO4 and concentrated. The crude product
was purified by column chromatography over silica gel (30%
EtOAc/hexanes) to afford inhibitor 4a (120 mg, 63%). Rf = 0.3
o
0 C and di-tert-butyldicarbonate (349 mg, 1.60 mmol) was added
o
and allowed to warm to 23 C. After stirring for 12 h, the reaction
was diluted with CH2Cl2 and washed with water, brine solution,
dried over Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
(10% EtOAc/hexanes) to afford 10 (347 mg, 99% over two steps).
23
1
(30% EtOAc/hexanes); [α]D = +22.0 (c 0.85, CHCl ); H NMR
3
(400 MHz, CDCl3) δ 7.66 (t, J = 6.7 Hz, 2H), 7.57 (d, J = 5.4 Hz,
1H), 7.29 (m, 4H), 7.21 (m, 1H), 7.10 – 7.05 (m, 1H), 6.93 (t, J =
7.5 Hz, 2H), 6.48 (d, J = 8.6 Hz, 1H), 4.79 (m, 2H), 4.40 – 4.17 (m,
2H), 3.98 (s, 1H), 3.85 (s, 3H), 3.24 – 2.90 (m, 6H), 2.89 – 2.73 (m,
2H), 2.35 (d, J = 9.5 Hz, 1H), 2.04 (d, J = 12.3 Hz, 1H), 1.91 – 1.67
(m, 2H), 1.51 – 1.34 (m, 9H), 0.85 (t, J = 6.2 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 167.52, 163.14, 154.95, 138.99, 137.99,
133.15, 129.92, 129.52, 129.00, 128.79, 126.88, 126.79, 126.08,
114.46, 80.15, 73.00, 58.93, 55.75, 54.86, 53.60, 48.10, 35.15,
28.52, 28.25, 27.34, 22.99, 20.24, 20.14; HRMS-ESI (m/z): [M+H]+
calcd for C36H48N3O7S2, 698.2928; found 698.2925.
23
1
Rf = 0.3 (10% EtOAc/hexanes); [α]D = +36.5 (c 1.0, CHCl3); H
NMR (400 MHz, CDCl3) δ 7.96 (d, J = 1.9 Hz, 1H), 7.77 (dd, J =
8.3, 2.0 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 4.89 (s, 1H), 4.75 (s,
1H), 4.35 (qd, J = 7.1, 1.4 Hz, 2H), 3.13 (td, J = 11.8, 10.6, 3.1 Hz,
1H), 3.07 – 2.98 (m, 1H), 2.39 (s, 1H), 2.09 (td, J = 10.5, 3.5 Hz,
1H), 1.49 (s, 10H), 1.38 (t, J = 7.1 Hz, 3H). LRMS-ESI (m/z): 355
[M+NH4]+.
(R)-4-((tert-Butoxycarbonyl)amino)thiochromane-6-carboxylic
acid (11)
A solution of 10 (364 mg, 1.08 mmol) in THF: MeOH (6 mL,
2:1) was treated with 1N LiOH solution (1.62 mL, 1.62 mmol). The
resulting mixture was stirred for 12 h, and then concentrated under
reduced pressure. The residue was dissolved in water and acidified
with citric acid then extracted with ethyl acetate (3×20 mL). The
combined ethyl acetate layer was dried over Na2SO4, filtered, and
concentrated to give carboxylic acid 11 (280 mg, 84%). Rf = 0.5
(10% MeOH/CH2Cl2); [α]D23 = +33.2 (c 0.8, CHCl3); 1H NMR (400
MHz, CDCl3) δ 8.03 (s, 1H), 7.82 (dd, J = 8.3, 1.9 Hz, 1H), 7.19 (d,
J = 8.3 Hz, 1H), 4.91 (s, 1H), 4.78 (s, 1H), 3.14 (t, J = 11.5 Hz,
1H), 3.04 (ddd, J = 12.7, 6.5, 3.6 Hz, 1H), 2.39 (s, 1H), 2.18 – 2.06
(m, 1H), 1.49 (s, 9H). LRMS-ESI (m/z): 332 [M+Na]+.
tert-Butyl
((R)-6-(((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamoyl)-1,1-
dioxidothiochroman-4-yl)carbamate (4b)
To a solution of inhibitor 4a (50 mg, 0.07 mmol) in
dichloromethane (1 mL), 3- chloroperbenzoic acid (26 mg, 0.15
mmol) was added slowly at 0 °C under argon atmosphere. The
o
reaction was stirred for 6 h at 23 C. The reaction mixture was
diluted with dichloromethane and washed with sat. Na2CO3
solution and brine. The organic layers were dried over Na2SO4,
filtered and concentrated in vacuo. The crude residue was purified
by flash column chromatography over silica gel to afford inhibitor
4b (48 mg, 92%). Rf = 0.3 (60% EtOAc/hexanes); [α]D = -11.0 (c
0.2, CHCl ); H NMR (400 MHz, CDCl3) δ 7.78 – 7.70 (m, 2H),
23
1
Ethyl
(S)-4-((tert-butoxycarbonyl)amino)thiochromane-6-
3
carboxylate (ent-10)
7.70 – 7.62 (m, 2H), 7.54 (d, J = 8.1 Hz, 1H), 7.29– 7.26 (m, 4H),
7.20 (m, 1H), 7.00 – 6.91 (m, 3H), 5.20 (d, J = 8.5 Hz, 1H), 4.98 (s,
1H), 4.42 (s, 1H), 4.06 (dt, J = 12.0, 4.8 Hz, 1H), 3.86 (s, 3H), 3.50
(t, J = 11.5 Hz, 1H), 3.37 (t, J = 10.9 Hz, 1H), 3.11 (dt, J = 21.0,
7.4 Hz, 4H), 2.87 (dt, J = 10.1, 5.2 Hz, 2H), 2.70 – 2.47 (m, 2H),
1.87 (m, 1H), 1.47 (s, 9H), 0.86 (dd, J = 6.7, 3.5 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 166.26, 163.24, 155.10, 138.40, 137.88,
137.05, 130.25, 129.84, 129.54, 129.42, 128.79, 128.27, 127.44,
126.86, 124.18, 114.54, 80.64, 72.86, 60.54, 58.92, 55.79, 54.92,
53.42, 48.15, 47.72, 35.03, 28.46, 27.84, 27.33, 21.19, 20.24,
20.13, 14.32; HRMS-ESI (m/z): [M+H]+ calcd for C36H48N3O9S2,
730.2827; found 730.2824.
Compound ent-10 (50 mg, 68%) was synthesized from ent-8
(75 mg, 0.21 mmol) by following the procedure outlined for
compound 10. Rf = 0.2 (10% EtOAc/hexanes); [α]D23 = -37.5 (c 1.0,
1
CHCl ); H NMR (400 MHz, CDCl3) δ 7.96 (d, J = 1.9 Hz, 1H),
3
7.77 (dd, J = 8.3, 2.0 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 4.89 (s,
1H), 4.75 (s, 1H), 4.35 (q, J = 7.1, 1.4 Hz, 2H), 3.12 (td, J = 11.8,
10.6, 3.2 Hz, 1H), 3.06 – 2.97 (m, 1H), 2.38 (s, 1H), 2.18 – 2.03
(m, 1H), 1.48 (s, 9H), 1.38 (t, J = 7.1 Hz, 3H). LRMS-ESI (m/z):
360 [M+Na]+.
(S)-4-((tert-Butoxycarbonyl)amino)thiochromane-6-carboxylic
acid (ent-11)
Compound ent-10 (120 mg, 0.37 mmol) was treated with 1N
LiOH (0.55 mL, 0.55 mmol) by following the procedure outlined
for compound 11 to give compound ent-11 (75 mg, 66%). Rf = 0.5
(S)-4-Amino-N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)thiochromane-6-
carboxamide 1,1-dioxide (4c)
23
1
(10% MeOH/CH2Cl2); [α]D = -36.6 (c 0.12, CHCl3); H NMR
(400 MHz, CDCl3) δ 8.03 (s, 1H), 7.81 (dd, J = 8.3, 1.9 Hz, 1H),
7.19 (d, J = 8.3 Hz, 1H), 4.91 (s, 1H), 4.77 (s, 1H), 3.12 (d, J = 11.2
Hz, 1H), 3.07 – 2.98 (m, 1H), 2.39 (s, 1H), 2.18 – 2.06 (m, 1H),
1.49 (s, 9H); LRMS-ESI (m/z): 332 [M+Na]+.
To a stirred solution of inhibitor 4b (25 mg, 0.034 mmol) in
o
dichloromethane (1.0 mL) was added TFA (0.1 mL) at 0 C under
o
argon atmosphere. The reaction mixture was warmed to 23 C and
stirred at for 3 h. Upon completion, solvent was removed under
reduced pressure. The residue was extracted with dichloromethane
and washed with sat. NaHCO3 solution, brine, dried over Na2SO4
and concentrated in vacuo. The crude residue was purified by
tert-Butyl
((R)-6-(((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-
yl)carbamoyl)thiochroman-4-yl)carbamate (4a)
To a solution of 11 (85 mg, 0.27 mmol) and HOBt (56 mg,
column chromatography over silica gel to give inhibitor 4c (18 mg,
23
83%) as an amorphous solid. Rf = 0.2 (5% MeOH/CH2Cl2); [α]D
=
-13.6 (c 0.6, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.82 – 7.74 (m,

ACCEPTED MANUSCRIPT
2H), 7.69 – 7.63 (m, 2H), 7.56 (dd, J = 8.2, 1.7 Hz, 1H), 7.29 –
MHz, CDCl3) δ 7.80 (d, J = 8.1 Hz, 2H), 7.71 – 7.63 (m, 2H), 7.56
(d, J = 8.2 Hz, 1H), 7.27 (d, J = 4.4 Hz, 4H), 7.20 (m, 1H), 6.99 –
6.91 (m, 2H), 6.88 (d, J = 8.5 Hz, 1H), 4.46 – 4.33 (m, 1H), 4.16 (s,
1H), 4.05 (dt, J = 8.4, 4.2 Hz, 1H), 3.86 (s, 3H), 3.68 (ddd, J =
13.5, 9.6, 3.0 Hz, 1H), 3.38 – 3.24 (m, 1H), 3.13 (m, 4H), 2.88 (d, J
= 7.5 Hz, 2H), 2.65 (t, J = 12.8 Hz, 1H), 2.41 – 2.28 (m, 1H), 1.87
(m, 2H), 0.86 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
166.45, 163.24, 140.49, 138.15, 137.92, 129.84, 129.56, 129.49,
128.79, 128.09, 126.93, 126.85, 124.19, 114.54, 77.41, 72.81,
58.89, 55.80, 54.84, 53.51, 48.12, 47.53, 35.00, 30.58, 27.31,
20.24, 20.15; HRMS-ESI (m/z): [M+H]+ calcd for C31H40N3O7S2,
630.2302; found 630.2294.
7.23 (m, 4H), 7.19 (m, 1H), 6.95 (dd, J = 8.4, 5.4 Hz, 3H), 4.40 (tt,
J = 9.4, 5.0 Hz, 1H), 4.15 (dd, J = 7.1, 4.3 Hz, 1H), 4.05 (dt, J =
8.3, 4.3 Hz, 1H), 3.85 (s, 3H), 3.67 (ddd, J = 13.4, 9.8, 3.0 Hz, 1H),
3.37 – 3.24 (m, 1H), 3.22 – 2.99 (m, 4H), 2.87 (d, J = 7.5 Hz, 2H),
2.65 (dt, J = 20.3, 6.8 Hz, 1H), 2.38 – 2.26 (m, 2H), 1.87 (m, 1H),
0.85 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 166.71,
163.13, 142.56, 138.11, 137.26, 130.04, 129.54, 129.47, 128.68,
126.71, 114.46, 72.90, 60.51, 58.73, 55.75, 54.95, 53.48, 48.12,
44.11, 34.91, 28.56, 28.47, 27.28, 22.25, 21.73, 20.24, 20.13;
HRMS-ESI (m/z): [M+H]+ calcd for C31H40N3O7S2, 630.2302;
found 630.2298.
tert-Butyl
((S)-6-(((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
(R,E)-N-(7-Bromo-3,4-dihydronaphthalen-1(2H)-ylidene)-2-
methylpropane-2-sulfinamide (13)
methoxyphenyl)sulfonamido)-1-phenylbutan-2-
A mixture of 5-bromo-1-tetralone 12 (1.5 g, 6.66 mmol), (R)-
(+)-2-methyl-2 propanesulfinamide (1.21 g, 9.99 mmol) and
titanium (IV) ethoxide (3.01 g, 13.32 mmol) were dissolved in
yl)carbamoyl)thiochroman-4-yl)carbamate (4d)
Compound ent-11 (65 mg, 0.21 mmol) was treated with
isostere amine 20 (85 mg, 0.21 mmol) by following the procedure
outlined for inhibitor 4a to give inhibitor 4d (117 mg, 80%) as an
o
anhydrous THF (15 mL) and stirred at 66 C for 10 h under argon
23
o
amorphous solid. Rf = 0.2 (30% EtOAc/hexanes); [α]D = -6.5 (c
atmosphere. The reaction mixture was cooled to 23 C and ethyl
1
0.49, CHCl ); H NMR (400 MHz, CDCl3) δ 7.66 (d, J = 8.5 Hz,
acetate and aq sodium bicarbonate was added. The mixture was
filtered through a pad of celite and the aqueous layer was extracted
with ethyl acetate. The combined organic phase were dried over
Na2SO4 and concentrated under reduced pressure. The crude
residue was purified by column chromatography over silica gel (5%
EtOAc/hexanes) to afford 13 (1.9 g, 87%). Rf = 0.5 (40%
3
2H), 7.54 (s, 1H), 7.27 (d, J = 4.6 Hz, 3H), 7.24 (s, 1H), 7.19 (m,
1H), 7.06 (d, J = 8.3 Hz, 1H), 6.92 (d, J = 8.6 Hz, 2H), 6.43 (d, J =
8.4 Hz, 1H), 4.78 (s, 2H), 4.32 (m, 1H), 3.96 (s, 1H), 3.84 (s, 3H),
3.22 – 2.91 (m, 6H), 2.85 (d, J = 7.5 Hz, 1H), 2.33 (s, 1H), 2.03 (d,
J = 9.7 Hz, 1H), 1.83 (m, 1H), 1.72 (m, 1H), 1.45 (s, 9H), 0.84 (t, J
= 5.7 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 167.80, 163.44,
155.25, 139.27, 138.28, 133.42, 130.32, 129.88, 129.10, 127.21,
127.13, 126.50, 114.78, 80.51, 73.21, 59.24, 56.08, 55.04, 53.96,
48.40, 35.43, 28.87, 28.53, 27.67, 23.27, 20.56, 20.44; HRMS-ESI
(m/z): [M+H]+ calcd for C36H48N3O7S2, 698.2928; found 698.2920.
1
EtOAc/hexanes); H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 2.2
Hz, 1H), 7.48 (dd, J = 8.2, 2.2 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H),
3.27 (ddd, J = 17.6, 9.3, 4.8 Hz, 1H), 3.05 (ddd, J = 17.6, 7.5, 4.5
Hz, 1H), 2.81 (t, J = 6.2 Hz, 2H), 1.99 – 1.87 (m, 2H), 1.33 (d, J =
2.3 Hz, 9H).
(R)-N-((R)-7-Bromo-1,2,3,4-tetrahydronaphthalen-1-yl)-2-
methylpropane-2-sulfinamide (14)
tert-Butyl
((S)-6-(((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)carbamoyl)-
1,1-dioxidothiochroman-4-yl)carbamate (4e)
Inhibitor 4d (25 mg, 0.035 mmol) was treated with mCPBA
(13 mg, 0.075 mmol) by following the procedure outlined for
compound 4b to give compound inhibitor 4e (26 mg, 96%) as an
Compound 13 (175 mg, 0.53 mmol) was dissolved in
THF/H2O (2 mL, 98:2) and cooled to -50 °C. To the mixture was
then added NaBH4 (61 mg, 1.59 mmol), and the resulting solution
o
was warmed to 23 C over a period of 3 h. The solvent was then
23
removed in vacuo, and the resulting residue was triturated with
dichloromethane, dried over Na2SO4, filtered, and concentrated
under reduced pressure.The crude product was purified by column
chromatography over silica gel (30% EtOAc/hexanes) to afford 14
amorphous solid. Rf = 0.2 (60% EtOAc/hexanes); [α]D = -8.0 (c
0.1, CHCl ); 1H NMR (400 MHz, CDCl3) δ 7.70 (s, 1H), 7.67 (d, J
3
= 8.8 Hz, 2H), 7.58 (dd, J = 15.8, 8.3 Hz, 1H), 7.44 – 7.36 (m, 1H),
7.30 – 7.26 (m, 4H), 7.19 (m, 1H), 7.07 (d, J = 8.6 Hz, 1H), 6.94 (d,
J = 8.8 Hz, 2H), 5.34 (s, 1H), 4.97 (s, 1H), 4.40 (dq, J = 9.6, 4.8
Hz, 1H), 4.06 (dt, J = 8.3, 4.6 Hz, 1H), 3.85 (s, 3H), 3.53 (ddd, J =
12.5, 8.8, 3.2 Hz, 1H), 3.36 (dd, J = 13.3, 8.0 Hz, 1H), 3.18 – 3.01
(m, 4H), 2.89 (dd, J = 7.7, 2.1 Hz, 2H), 2.70 – 2.49 (m, 3H), 1.88
(m, 1H), 1.49 (d, J = 5.0 Hz, 9H), 0.86 (d, J = 6.6 Hz, 6H); 13C
NMR (100 MHz, CDCl3) δ 166.24, 163.21, 155.40, 141.03, 138.30,
137.96, 137.02, 133.68, 129.95, 129.56, 129.50, 128.75, 128.29,
127.45, 126.90, 124.00, 114.52, 80.96, 77.43 72.86, 58.84, 55.79,
54.85, 53.49, 48.04, 47.58, 34.94, 28.51, 27.32, 20.26, 20.11;
HRMS-ESI (m/z): [M+H]+ calcd for C36H48N3O9S2, 730.2827;
found 730.2822.
(110 mg, 63%) and 15 (55 mg, 31%). Rf
= 0.4 (30%
1
EtOAc/hexanes); H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 2.1
Hz, 1H), 7.30 (dd, J = 8.2, 2.1 Hz, 1H), 6.98 (d, J = 8.2 Hz, 1H),
4.52 (q, J = 4.2 Hz, 1H), 3.21 (d, J = 3.7 Hz, 1H), 2.76 (dt, J =
17.0, 5.2 Hz, 1H), 2.70 – 2.57 (m, 1H), 2.08 – 1.97 (m, 1H), 1.96 –
1.81 (m, 2H), 1.79 – 1.70 (m, 1H), 1.22 (s, 9H).
(R)-N-((S)-7-Bromo-1,2,3,4-tetrahydronaphthalen-1-yl)-2-
methylpropane-2-sulfinamide (15)
Compound 13 (275 mg, 0.83 mmol) was dissolved in
anhydrous THF (3 mL) and cooled to 0 °C. To this solution was
then added L-Selectride (2.5 mL, 1.0 M in THF, 2.51 mmol) and
the resulting solution was allowed to warm to 23 oC over period of
3 h. The solution was then concentrated under vacuo to furnish
crude product. The crude residue was purified by column
chromatography over silica gel (30% EtOAc/hexanes) to give 15
(S)-4-Amino-N-((2S,3R)-3-hydroxy-4-((N-isobutyl-4-
methoxyphenyl)sulfonamido)-1-phenylbutan-2-
yl)thiochromane-6-carboxamide 1,1-dioxide (4f)
Inhibitor 4f (20 mg, 0.027 mmol) was treated with TFA (0.1
mL) by following the procedure outlined for inbitor 4c to give
inhibitor 4f (16 mg, 93%) as an amorphous solid; Rf = 0.1 (5%
1
(265 mg, 96%). Rf = 0.3 (30% EtOAc/hexanes); H NMR (400
MHz, CDCl3) δ 7.54 (d, J = 2.1 Hz, 1H), 7.28 (dd, J = 8.1, 2.1 Hz,
1H), 6.96 (d, J = 8.2 Hz, 1H), 4.42 (q, J = 8.0, 7.4 Hz, 1H), 3.36 (d,
J = 10.1 Hz, 1H), 2.80 – 2.56 (m, 2H), 2.40 – 2.24 (m, 1H), 1.96 –
23
1
MeOH/CH2Cl2); [α]D = +13.7 (c 0.35, CHCl3); H NMR (400

ACCEPTED MANUSCRIPT
1.73 (m, 3H), 1.28 (s, 9H).
1.52 (s, 9H); LRMS-ESI (m/z): 342 [M+Na]+.
tert-Butyl
yl)carbamate (16)
(S)-(7-bromo-1,2,3,4-tetrahydronaphthalen-1-
(5R,8S)-8-((tert-Butoxycarbonyl)amino)-5-hydroxy-5,6,7,8-
tetrahydronaphthalene-2-carboxylic acid (18)
A solution of 15 (1.2 g, 3.63 mmol) in MeOH (20 mL) was
Argon was bubbled through a solution of 17 (150 mg, 0.46
mmol) and RuCl[(R,R)-TsDPEN](mesitylene) (9.0 mg, 0.014
mmol) in dry DMF (2 mL) for 10 min. A premixed combination of
formic acid (35 µL, 0.938 mmol) and Et3N (135 µL, 0.938 mmol)
was added and the mixture stirred at 60 °C for 12 h. The mixture
was cooled to 23 oC and diluted with CH2Cl2 and successively
washed with water, brine and dried over anhydrous Na2SO4. The
solvent was removed in vacuo to give the crude product. The crude
residue was purified by column chromatography over silica gel
(30% EtOAc/hexanes) to afford the desired alcohol (142 mg, 94%)
treated with 6 N HCl in isopropanol (5 mL). After 2.5 h, solvent
was removed in vacuo, and the residue was co-evaporated with
ethyl acetate. The residue obtained was dried under high vacuum to
provide
(S)-7-bromo-1,2,3,4-tetrahydronaphthalen-1-amine
hydrochloride (0.94 g).
To a stirred solution of above amine hydrochloride (0.94 g, 3.6
mmol) in CH2Cl2 (30 mL) was consecutively added triethylamine
(1.5 mL, 10.89 mmol) and di-tert-butyl-dicarbonate (1.17 g, 5.44
o
o
mmol) at 0 C. The reaction mixture was stirred at 23 C for 12 h.
The reaction mixture was then diluted with CH2Cl2 and washed
with water and brine solution, dried over Na2SO4 and concentrated.
The crude product was purified by column chromatography over
silica gel (20% EtOAc/hexanes) to give 16 (1.0 g, 85% over two
steps) as an amorphous solid. Rf = 0.7 (20% EtOAc/hexanes); [α]D-
1
as an amorphous white solid. Rf = 0.3 (30% EtOAc/hexanes); H
NMR (400 MHz, CDCl3) δ 8.04 (s, 1H), 7.92 (d, J = 7.9 Hz, 1H),
7.51 (d, J = 8.0 Hz, 1H), 4.85 (s, 1H), 4.77 (m, 2H), 3.91 (s, 3H),
2.09 – 1.93 (m, 5H), 1.49 (s, 9H); LRMS-ESI (m/z): 643 [2M+H]+.
To a solution of above methyl ester (80 mg, 0.248 mmol) in
THF: MeOH (1.5 mL, (2:1) was added 1N LiOH (0.37 mL, 0.373
mmol) at 23 °C. The reaction mixture was stirred at 23 °C for 12 h.
Solvent was removed under reduced pressure, acidified with aq.
saturated citric acid to pH 3-4 and the product was extracted with
ethyl acetate, dried over Na2SO4 and concentrated to afford 18 (68
mg, 89 %) as an amorphous solid. Rf = 0.3 (10% MeOH/CH2Cl2);
[α]D23 = -38.6 (c 0.29, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.98
(s, 1H), 7.89 (dd, J = 8.0, 1.9 Hz, 1H), 7.52 (d, J = 8.1 Hz, 1H),
7.08 (d, J = 9.1 Hz, 1H), 4.71 (s, 2H), 2.05 – 1.89 (m, 4H), 1.50 (d,
J = 4.7 Hz, 9H); LRMS-ESI (m/z): 615 [2M+H]+.
23
1
= -9.32 (c 1.0, CHCl ); H NMR (400 MHz, CDCl3) δ 7.54 –
3
7.39 (m, 1H), 7.33 – 7.09 (m, 1H), 6.94 (d, J = 8.1 Hz, 1H), 4.78 (q,
J = 10.6, 8.8 Hz, 2H), 2.88 – 2.50 (m, 2H), 2.02 (td, J = 10.9, 9.9,
4.9 Hz, 1H), 1.78 (ddp, J = 23.3, 11.4, 4.2 Hz, 3H), 1.49 (s, 9H);
13C NMR (100 MHz, CDCl3) δ 155.55, 139.66, 136.43, 131.38,
130.84, 130.31, 119.73, 79.77, 48.67, 30.45, 28.89, 28.57, 20.05.
Methyl
(S)-8-((tert-butoxycarbonyl)amino)-5-oxo-5,6,7,8-
tetrahydronaphthalene-2-carboxylate (17)
Boc-amine derivative 16 (950 mg, 2.91 mmol) was dissolved
in acetone (55 mL) and cooled to 0 °C. MgSO4 (837 mg, 6.99
mmol) and water (23 mL) were added to the solution. KMnO4 (2.38
g, 15.15 mmol) was added to this mixture in small portions over 1 h
(5S,8S)-8-((tert-Butoxycarbonyl)amino)-5-hydroxy-5,6,7,8-
tetrahydronaphthalene-2-carboxylic acid (19)
Argon was bubbled through a solution of 17 (150 mg, 0.469
mmol) and RuCl(p-cymene)[(S,S)-Ts-DPEN] (9.0 mg, 0.014 mmol)
in dry DMF (1.5 mL) for 10 min. A premixed combination of
formic acid (35 µL, 0.938 mmol) and Et3N (135 µL, 0.938 mmol)
was added and the mixture was stirred at 60 °C for 12 h. The
mixture was cooled to 23 oC and diluted with CH2Cl2 and
successively washed with water, brine and dried over anhydrous
Na2SO4. The solvent was removed in vacuo to give the crude
product. The crude product was purified by column
chromatography over silica gel (30% EtOAc/ hexanes) to give the
desired alcohol (140 mg, 93%) as an amorphous white solid. Rf =
o
and stirred for 8 h at 23 C. The solid was filtered off and the
filtrate was treated with a saturated solution of sodium sulfite. The
resulting mixture was filtered and the acetone was removed from
the filtrate in vacuo. The remaining aqueous residue was extracted
with dichloromethane. The combined organic phases were washed
with water, brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The crude residue was purified by column
chromatography over silica gel (20% EtOAc/hexanes) to afford
bromo ketone derivative (600 mg, 61%) as an amorphous solid. 1H
NMR (400 MHz, CDCl3) δ 7.88 (d, J = 8.4 Hz, 1H), 7.62 (s, 1H),
7.53 (dd, J = 8.4, 2.0 Hz, 1H), 5.03 (s, 1H), 4.82 (d, J = 9.0 Hz,
1H), 2.81 (dt, J = 17.4, 5.3 Hz, 1H), 2.66 (ddd, J = 17.1, 11.5, 4.7
Hz, 1H), 2.49 – 2.32 (m, 1H), 2.18 – 1.99 (m, 2H), 1.51 (s, 9H).
A solution of above bromo ketone (600 mg, 1.76 mmol) in
triethylamine (5.3 mL) and methanol (1 mL) was degassed with
argon and palladium (II) acetate (8.0 mg, 0.035 mmol) and
Xantphos (41 mg, 0.070 mmol) were added. The solution was
degassed again and CO gas was bubbled through the solution for
approximately 2 min. The reaction flask was fitted with a
condenser and a CO balloon and the reaction mixture was heated at
1
0.3 (30% EtOAc/hexanes); H NMR (400 MHz, CDCl3) δ 8.01 (s,
1H), 7.94 – 7.88 (m, 1H), 7.57 (d, J = 8.1 Hz, 1H), 4.88 (s, 1H),
4.83 – 4.59 (m, 2H), 3.90 (s, 3H), 2.38 – 2.16 (m, 2H), 2.07 – 1.94
(m, 1H), 1.89 – 1.66 (m, 2H), 1.49 (s, 9H); LRMS-ESI (m/z): 643
[2M+H]+.
Above methyl ester (75 mg, 0.233 mmol) was treated with 1N
LiOH (0.46 mL, 0.466 mmol) by following the procedure outlined
for 18 to give the titled compound 19 (60 mg, 85%) as an
23
amorphous solid; Rf = 0.3 (10% MeOH/CH2Cl2); [α]D = -6.0 (c
0.5, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H), 7.85
(dd, J = 8.2, 1.8 Hz, 1H), 7.56 (d, J = 8.1 Hz, 1H), 4.79 – 4.67 (m,
2H), 2.24 – 2.11 (m, 3H), 1.74 (td, J = 12.1, 8.8 Hz, 2H), 1.46 (s,
9H); LRMS-ESI (m/z): 615 [2M+H]+.
o
70 °C for 3.5 h. The reaction mixture was cooled to 23 C, diluted
with EtOAc and filtered through a pad of celite. The filtrate was
evaporated and the residue was purified by silica gel column
chromatography (20% EtOAc/hexanes) to afford titled compound
23
(5S,8R)-8-((tert-Butoxycarbonyl)amino)-5-hydroxy-5,6,7,8-
tetrahydronaphthalene-2-carboxylic acid (ent-18)
Argon was bubbled through a solution of ethyl (R)-8-((tert-
butoxycarbonyl)amino)-5-oxo-5,6,7,8-tetrahydronaphthalene-2-
carboxylate (100 mg, 0.299 mmol) and RuCl(p-cymene)[(S,S)-Ts-
DPEN] (6.0 mg, 0.0089 mmol) in dry DMF (1.0 mL) for 10 min. A
17 (450 mg, 80 %). Rf = 0.3 (20% EtOAc/hexanes); [α]D = -10.7
(c 1.28, CHCl ); 1H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H), 8.09
3
(d, J = 8.1 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 5.09 (s, 1H), 4.83 (s,
1H), 3.95 (s, 3H), 2.87 (dt, J = 17.4, 5.0 Hz, 1H), 2.71 (ddd, J =
16.9, 11.4, 4.7 Hz, 1H), 2.50 – 2.32 (m, 1H), 2.22 – 2.03 (m, 1H),

ACCEPTED MANUSCRIPT
premixed combination of formic acid (22 µL, 0.598 mmol) and
114.41, 77.48, 72.92, 67.88, 58.60, 55.71, 54.61, 53.42, 49.18,
34.97, 28.64, 28.50, 27.18, 20.20, 20.11; HRMS-ESI (m/z): [M+H]+
calcd for C32H42N3O6S, 596.2789; found 596.2785.
Et3N (83 µL, 0.598 mmol) was added and the mixture stirred at 60
°C for 12 h. The mixture was cooled to room temperature and
diluted with CH2Cl2 and successively washed with water, brine and
dried over anhydrous Na2SO4. The solvent was removed in vacuo
to give the crude product. The crude product was purified by
column chromatography over silica gel (30% EtOAc/ hexanes) to
give desired alcohol (87 mg, 86%) as an amorphous white solid. Rf
tert-Butyl
((1S,4S)-4-hydroxy-7-(((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-
yl)carbamoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
(4i)
1
= 0.3 (30% EtOAc/hexanes); H NMR (400 MHz, CDCl3) δ 8.03
Carboxylic acid 19 (40 mg, 0.13 mmol) was treated with
isostere amine 20 (68 mg, 0.13 mmol) by following the procedure
outlined for inhibitor 4a to give inhibitor 4i (85mg, 94%) as an
(s, 1H), 7.94 (d, J = 7.9 Hz, 1H), 7.57 (d, J = 8.1 Hz, 1H), 4.90 (s,
1H), 4.84 – 4.67 (m, 2H), 4.37 (q, J = 7.1 Hz, 2H), 2.46 – 2.12 (m,
2H), 1.90 – 1.67 (m, 3H), 1.50 (s, 9H), 1.39 (t, J = 7.2 Hz, 3H).
LRMS-ESI (m/z): 353 [M+NH4]+.
Above ethyl ester (47 mg, 0.11 mmol) was treated with 1N
LiOH (0.2 mL, 0.21 mmol) by following the procedure outlined for
18 to give the titled compound ent-18 (40 mg, 89%) as an
amorphous solid. Rf = 0.3 (10% MeOH/CH2Cl2); LRMS-ESI (m/z):
615 [2M+H]+.
amorphous solid. Rf = 0.5 (80% EtOAc/hexanes); [α]D23 = +15.0 (c
1
0.22, CHCl ); H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.9 Hz,
3
2H), 7.53 (s, 1H), 7.46 – 7.43 (m, 1H), 7.41 (d, J = 8.1 Hz, 1H),
7.25 – 7.20 (m, 4H), 7.15 (td, J = 6.2, 2.9 Hz, 2H), 6.90 (d, J = 8.9
Hz, 2H), 5.03 (d, J = 8.9 Hz, 1H), 4.68 (dd, J = 16.6, 10.3 Hz, 2H),
4.28 (d, J = 20.1 Hz, 1H), 3.96 (dt, J = 8.6, 4.3 Hz, 1H), 3.82 (s,
3H), 3.22 – 2.92 (m, 4H), 2.84 (h, J = 7.6, 6.8 Hz, 2H), 2.22 (m,
6H), 1.86 (dt, J = 13.6, 6.7 Hz, 1H), 1.77 – 1.55 (m, 2H), 1.46 (s,
9H), 0.83 (d, J = 6.6 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ
168.13, 163.05, 156.10, 143.27, 138.26, 137.31, 133.64, 129.93,
129.49, 128.58, 128.46, 126.57, 114.40, 80.15, 72.86, 67.63, 58.61,
55.70, 54.74, 53.42, 49.80, 49.58, 49.37, 49.03, 34.94, 30.22,
28.49, 27.73, 27.18, 20.11, 20.03; HRMS-ESI (m/z): [M+H]+ calcd
for C37H50F2N3O8S, 696.3313; found 696.3305.
tert-Butyl
((1S,4R)-4-hydroxy-7-(((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-
yl)carbamoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
(4g)
Carboxylic acid 18 (40 mg, 0.130 mmol) was treated with
isostere amine 20 (68 mg, 0.130 mmol) by following the procedure
outlined for inhibitor 4a to give inhibitor 4g (85 mg, 94%) as an
23
amorphous white solid. Rf = 0.5 (80% EtOAc/hexanes); [α]D = -
(5S,8S)-8-Amino-5-hydroxy-N-((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphnyl)sulfonamido)-1-phenylbutan-2-yl)-
5,6,7,8-tetrahydronaphthalene-2-carboxamide (4j)
Inhibitor 4i (50 mg, 0.071 mmol) was treated with TFA (0.2
mL) by following the procedure outlined for inbitor 4h to give
3.2 (c 0.62, CHCl ); 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 8.5
3
Hz, 2H), 7.57 (d, J = 17.2 Hz, 1H), 7.40 (m, 1H), 7.31 – 7.18 (m,
4H), 7.18 – 7.07 (m, 2H), 6.952 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.5
Hz, 1H), 5.35 – 5.24 (m, 1H), 4.66 (m, 1H), 4.56 (m, 1H), 4.35 (m,
2H), 4.10 (q, J = 7.1 Hz, 1H), 3.97 (s, 1H), 3.83 (s, 3H), 3.27 – 2.93
(m, 5H), 2.85 (d, J = 7.6 Hz, 2H), 1.89 (m, 2H), 1.46 (s, 9H), 0.84
(m, 6H); 13C NMR (100 MHz, CDCl3) δ 167.75, 163.09, 155.79,
142.78, 138.07, 137.74, 133.70, 129.94, 129.52, 128.86, 128.65,
126.90, 126.65, 126.10, 114.44, 79.83, 73.00, 67.38, 58.70, 55.73,
54.57, 53.53, 48.80, 35.08, 29.79, 29.20, 28.56, 27.24, 25.96,
20.21, 20.06; HRMS-ESI (m/z): [M+Na]+ calcd for
C37H49N3O8SNa, 718.3133; found 718.3124.
compound inhibitor 4j (40 mg, 94%) as an amorphous solid. Rf =
23
0.15 (10% MeOH/CH2Cl2); [α]D = +35.6 (c 0.8, CHCl ); 1H
3
NMR (400 MHz, CDCl3) δ 7.67 (d, J = 8.8 Hz, 2H), 7.62 (s, 1H),
7.38 – 7.31 (m, 2H), 7.30 – 7.24 (m, 4H), 7.22 – 7.14 (m, 1H), 6.98
– 6.86 (m, 2H), 6.83 (d, J = 8.3 Hz, 1H), 4.68 (dd, J = 7.6, 4.3 Hz,
1H), 4.37 (dd, J = 9.4, 4.9 Hz, 1H), 4.04 (m, 1H), 3.84 (s, 3H), 3.23
(dd, J = 15.0, 4.2 Hz, 1H), 3.16 – 2.97 (m, 4H), 2.87 (m 3H), 2.26 –
2.10 (m, 2H), 1.87 (dq, J = 13.8, 6.8 Hz, 2H), 1.69 (q, J = 9.6, 8.2
Hz, 2H), 1.52 (q, J = 11.3, 9.9 Hz, 1H), 0.84 (dd, J = 6.6, 2.5 Hz,
6H); 13C NMR (100 MHz, CDCl3) δ 168.10, 163.09, 142.51,
140.87, 138.30, 133.51, 130.08, 129.55, 128.68, 128.33, 126.68,
125.50, 114.44, 72.92, 68.03, 58.72, 55.74, 54.77, 53.50, 49.34,
35.00, 31.05, 30.20, 29.89, 27.22, 20.22, 20.14; HRMS-ESI (m/z):
[M+H]+ calcd for C32H42N3O6S, 596.2789; found 596.2784.
(5R,8S)-8-Amino-5-hydroxy-N-((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-
5,6,7,8-tetrahydronaphthalene-2-carboxamide (4h)
To a solution of 4g (80 mg, 0.114 mmol) in CH2Cl2 (1 mL)
was added TFA (0.3 mL) and resulting solution was stirred at 23 oC
for 3 h. Then reaction mixture concentrated under vacuo and
extracted with ethyl acetate and washed with sat. NaHCO3, water,
brine, dried over Na2SO4, filtered and evaporated under reduced
pressure. The crude residue was purified by column
chromatography over silica gel (10% MeOH/NH3 in CH2Cl2) to
give inhibitor 4h (60 mg, 88%) as an amorphous white solid. Rf =
tert-Butyl
((1R,4S)-4-hydroxy-7-(((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-
yl)carbamoyl)-1,2,3,4-tetrahydronaphthalen-1-yl)carbamate
(4k)
Carboxylic acid ent-18 (41 mg, 0.10 mmol) was treated with
isostere amine 20 (52 mg, 0.10 mmol) by following the procedure
outlined for compound 4a to give compound inhibitor 4k (75 mg,
23
1
0.15 (10% MeOH/CH2Cl2); [α]D = +12.2 (c 0.63, CHCl ); H
3
NMR (400 MHz, CDCl3) δ 7.66 (dd, J = 9.1, 2.8 Hz, 2H), 7.58 –
7.53 (m, 1H), 7.36 – 7.29 (m, 1H), 7.29 – 7.24 (m, 3H), 7.21 (t, J =
7.4 Hz, 2H), 7.17 – 7.07 (m, 2H), 6.91 (dd, J = 9.5, 2.6 Hz, 2H),
4.54 (d, J = 5.3 Hz, 1H), 4.38 (dp, J = 9.2, 4.8 Hz, 2H), 4.03 (dt, J
= 8.4, 4.2 Hz, 2H), 3.86 (d, J = 6.0 Hz, 1H), 3.82 (s, 3H), 3.23 (dd,
J = 15.1, 4.0 Hz, 1H), 3.15 – 2.96 (m, 4H), 2.88 (td, J = 13.9, 6.9
Hz, 3H), 1.97 – 1.61 (m, 6H), 0.83 (t, J = 5.6 Hz, 6H); 13C NMR
(100 MHz, CDCl3) δ 168.11, 163.05, 143.02, 140.41, 138.27,
133.62, 130.06, 129.51, 128.60, 128.46, 126.94, 126.56, 125.91,
23
94%) as an amorphous solid. Rf = 0.5 (80% EtOAc/hexanes); [α]D
= +5.9 (c 0.57, CHCl ); 1H NMR (400 MHz, CDCl3) δ 7.65 (dd, J
3
= 8.5, 6.4 Hz, 3H), 7.34 (d, J = 7.7 Hz, 1H), 7.28 (m, 4H), 7.23 –
7.15 (m, 1H), 6.99 – 6.87 (m, 2H), 6.70 (d, J = 8.7 Hz, 1H), 5.06 (d,
J = 8.9 Hz, 1H), 4.71 (m, 1H), 4.64 (m, 1H), 4.47 – 4.27 (m, 2H),
3.99 (s, 1H), 3.85 (s, 3H), 3.21 – 2.99 (m, 4H), 2.85 (d, J = 7.5 Hz,
2H), 2.62 (s, 1H), 1.97 (m, 3H), 1.86 (m, 3H), 1.47 (s, 9H), 0.85
(dd, J = 6.6, 3.3 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 167.89,

ACCEPTED MANUSCRIPT
163.15, 155.72, 142.88, 138.06, 137.88, 133.87, 129.95, 129.55,
Acknowledgements
128.96, 128.76, 127.01, 126.75, 126.08, 114.47, 79.93, 73.04,
67.50, 58.87, 55.76, 54.87, 53.62, 48.89, 35.02, 29.83, 29.33,
28.57, 27.34, 26.07, 20.24, 20.13; HRMS-ESI (m/z): [M+H]+ calcd
for C37H50N3O8S, 696.3313; found 696.3310.
This research was supported by the National Institutes of Health
(Grant GM53386 to A.K.G. and Grant GM62920 to I.T.W.). 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 Infectious Diseases (Renkei
Jigyo) of Monbu-Kagakusho.
(5S,8R)-8-Amino-5-hydroxy-N-((2S,3R)-3-hydroxy-4-((N-
isobutyl-4-methoxyphenyl)sulfonamido)-1-phenylbutan-2-yl)-
5,6,7,8-tetrahydronaphthalene-2-carboxamide (4l)
Inhibitor 4k (30 mg, 0.043 mmol) was treated with TFA (0.1
mL) by following the procedure outlined for inbitor 4h to give
inhibitor 4l (23 mg, 89%) as an amorphous solid. Rf = 0.15 (10%
23
MeOH/CH2Cl2); [α]D = -9.5 (c 0.2, CHCl ); 1H NMR (400 MHz,
3
CDCl3) δ 7.73 – 7.59 (m, 3H), 7.48 – 7.40 (m, 1H), 7.38 – 7.25 (m,
4H), 7.19 (t, J = 7.0 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 6.81 (d, J =
8.3 Hz, 1H), 4.66 (t, J = 4.7 Hz, 1H), 4.38 (d, J = 8.7 Hz, 1H), 4.01
(dt, J = 10.4, 4.5 Hz, 2H), 3.85 (s, 3H), 3.22 (dd, J = 15.0, 4.3 Hz,
1H), 3.14 – 3.01 (m, 4H), 2.85 (dt, J = 13.5, 7.4 Hz, 2H), 1.96 –
1.76 (m, 6H), 0.85 (d, J = 6.7 Hz, 6H); 13C NMR (100 MHz,
CDCl3) δ 168.03, 163.12, 143.32, 140.01, 138.25, 133.71, 130.09,
130.08, 129.56, 128.69, 126.82, 126.68, 126.30, 114.45, 73.02,
68.02, 58.67, 55.75, 54.85, 53.40, 49.38, 35.05, 29.83, 28.66,
28.09, 27.24, 20.23, 20.15; HRMS-ESI (m/z): [M+H]+ calcd for
C32H42N3O6S, 596.2789; found 596.2786.
5. References and notes
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For X-ray crystallographic studies, HIV-1 protease was
expressed and purified as described.45 The protease-inhibitor
complex was crystallized by the hanging drop vapor diffusion
method with well solutions of 0.9M NaCl, 0.1M Sodium
Cacodylate, pH 6.4 for PR/GRL-02815A (4d) complex, and 0.95M
NaCl, 0.1M Sodium Acetate, pH 5.5 for PR/GRL-043-15A (4k)
complex. Diffraction data were collected on a single crystal cooled
to 90 K at SER-CAT (22-ID beamline), Advanced Photon Source,
Argonne National Lab (Chicago, USA) with X-ray wavelength of
1.0 Å. X-ray data were processed by HKL-200046 to give Rmerge
values of 8.5% and 7.8% for inhibitors 4d- and 4k-bound HIV-1
protease complexes, respectively. The crystal structures were
solved by PHASER47 in CCP4i Suite48-50 using one of the
previously reported isomorphous structures51 as the initial model,
and refined by SHELX-201452,53 with X-ray data at 1.20 Å
resolution for inhibitor 4d and HIV-1 protease complex and 1.14 Å
for inhibitor 4k and HIV-1 protease complex. PRODRG-254 was
used to construct the inhibitor and geometric restraints for
refinement. COOT55,56 was used for modification of the model.
Alternative conformations were modeled, and isotropic atomic
displacement parameters (B factors) were applied for all atoms
including solvent molecules. The final refined solvent structure
comprised one Na+ ion, two Cl- ions, one glycerol molecules and
209 water molecules for inhibitor 4d and HIV-1 protease complex
and Na+ ion, two Cl- ions, one acetate ion, one glycerol molecules
and 142 water molecules for for inhibitor 4k and HIV-1 protease
complex. The crystallographic statistics are listed in Table 1 (Please
see, supporting information). The coordinates and structure factors
of the protease complexes with inhibitors 4d and 4k have been
deposited in the Protein Data Bank57 with accession codes of 6DV0
and 6DV4, respectively.
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Highlights
•
•
•
•
•
Design and synthesis of novel HIV-1 protease inhibitors are reported
Aminothiochromane and aminotetrahydronaphthalene derivatives were synthesized
The synthesis of ligands utilized reduction of chiral sulfinamide derivatives
The X-ray crystal structures of inhibitor-bound HIV-1 protease were determined
Hydrogen bonding and van der Waals interactions were observed in the active site