Design, synthesis, protein-ligand X-ray structure, and biological evaluation of a series of novel macrocyclic human immunodeficiency virus-1 protease inhibitors to combat drug resistance

Article abstract of DOI:10.1021/jm900695w

The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic pocket in the S1′ - S2′ subsites and retain all major hydrogen bonding interactions with the protein backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were significantly more potent than their acyclic homologues, saturated rings were less active than their unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-resistant HIV-1 variants. Protein - ligand X-ray structures of inhibitors 2 and 14c provided critical molecular insights into the ligand-binding site interactions.

Full text of DOI:10.1021/jm900695w

J. Med. Chem. 2009, 52, 7689–7705 7689
DOI: 10.1021/jm900695w
Design, Synthesis, Protein-Ligand X-ray Structure, and Biological Evaluation of a Series of Novel
Macrocyclic Human Immunodeficiency Virus-1 Protease Inhibitors to Combat Drug Resistance†
Arun K. Ghosh,*,‡ Sarang Kulkarni,‡ David D. Anderson,‡ Lin Hong,§ Abigail Baldridge,‡ Yuan-Fang Wang,^
Alexander A. Chumanevich,^ Andrey Y. Kovalevsky,^ Yasushi Tojo,# Masayuki Amano,# Yasuhiro Koh,# Jordan Tang,§,
Irene T. Weber,^ and Hiroaki Mitsuya#,3
‡Departments of Chemistry and Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, §Protein Studies Program, Oklahoma
Medical Research Foundation, Oklahoma City, Oklahoma 73104, Department of Biochemistry and Molecular Biology, University of Oklahoma
Health Science Center, Oklahoma City, Oklahoma 73104, ^Department of Biology, Molecular Basis of Disease, Georgia State University,
Atlanta, Georgia 30303, #Departments of Hematology and Infectious Diseases, Kumamoto University School of Medicine, Kumamoto 860-8556,
Japan, and 3Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, Bethesda, Maryland 20892
Received May 22, 2009
The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic macrocyclic
HIV protease inhibitors are described. The inhibitors are designed to effectively fill in the hydrophobic
pocket in the S10-S20 subsites and retain all major hydrogen bonding interactions with the protein
backbone similar to darunavir (1) or inhibitor 2. The ring size, the effect of methyl substitution, and
unsaturation within the macrocyclic ring structure were assessed. In general, cyclic inhibitors were
significantly more potent than their acyclic homologues, saturated rings were less active than their
unsaturated analogues and a preference for 10- and 13-membered macrocylic rings was revealed. The
addition of methyl substituents resulted in a reduction of potency. Both inhibitors 14b and 14c exhibited
marked enzyme inhibitory and antiviral activity, and they exerted potent activity against multidrug-
resistant HIV-1 variants. Protein-ligand X-ray structures of inhibitors 2 and 14c provided critical
molecular insights into the ligand-binding site interactions.
Introduction
speculated that maximizing “backbone binding” may be an
important design strategy to combat drug resistance.7 Inhi-
bitor 1 (darunavir, TMC-114) was recently approved by the
FDA for the treatment of drug-resistant HIV strains.8 More
recently, it has been approved for all HIV/AIDS patients
including pediatric AIDS patients.9 The protein-ligand
X-ray structure of darunavir and its analogue 2 (TMC-126)
exhibited extensive hydrogen bonding interactions with the
backbone atoms throughout the active site of the HIV-1
protease.10,11
In our continuing efforts toward the conceptual design of
novel PIs, we now plan to design PIs with functionalities that
interact with the protein backbone as well as introduce flexible
macrocycles involving P10-P20 ligands for effective repacking
due to side chain mutations. The notion of such macrocyclic
design evolved from the observation that certain mutations
lead to decreased van der Waals interactions and increased
the size of the S1 hydrophobic pocket. The X-ray structure
and modeling studies of PI (2S,20S)-N,N0-((2S,3R,4R,5S)-3-
hydroxy-4-methyl-1,6-diphenylhexane-2,5-diyl)bis(3-methyl-
2-(3-methyl-3-(pyridin-2-ylmethyl)ureido)butanamide) (A-
77003)12 indicated that the V82A mutant results in decreased
van der Waals interactions withthe phenyl rings in both the S1
and S10-subsites.13 There was also evidence of repacking of
inhibitor side chains and enzyme atoms in the S1-subsite. On
the basis of this insight of enzyme flexibility in accommodat-
ing alternate packing, we planned to design flexible macro-
cycles between the P10 side chain and the P20 sulfonamide ring
to fill in the S10 and S20-subsites.13 In particular, weenvisioned
HIV/AIDS has become one of the major medical and
humanitarian challenges in the 21st century.1 Highly active
antiretroviral therapy (HAARTa), which combines protease
inhibitors along with reverse-transcriptase inhibitors, is cur-
rently used to combat this pandemic. HAART therapy has
resulted in a significant decline in the number of deaths due to
HIV infection in the developed countries.2 One of the major
challenges still faced is the emergence of drug-resistant viral
strains rendering the present drug regimens ineffective.3 There
isanurgentneed for development of antiretroviralagents with
minimal side effects and broad-spectrum activity for current
and future management of HIV/AIDS.
Recently, our structure-based design of inhibitors maximiz-
ing interactions within the active site protease backbone led to
the development of nonpeptide inhibitors (1-2) that have
shown picomolar enzyme affinity and exceptional antiviral
activity against both wild-type and drug-resistant HIV-1
strains.4-6 The X-ray crystallographic studies revealed that the
backbone conformation of mutant protease is minimally dis-
torted compared to wild-type HIV-1 proteases. We, therefore,
†The PDB accession code for 2-bound HIV-1 protease X-ray struc-
ture is 3I7E and 14c-bound HIV-1 protease X-ray structure is 3I6O.
*To whom correspondence should be addressed. Phone: (765)-494-
5323. Fax: (765)-496-1612. E-mail: akghosh@purdue.edu.
a Abbreviations: HIV, human immunodeficiency virus; bis-THF, bis-
tetrahydrofuran; PI, protease inhibitor; HAART, highly active antire-
troviral therapy; APV, amprenavir; DRV, darunavir; SQV, saquinavir;
IDV, indinavir; LPV, lopinavir; DIAD, diisopropyl azodicarboxylate.
r
2009 American Chemical Society
Published on Web 09/11/2009
pubs.acs.org/jmc

7690 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
Scheme 2. Synthesis of Compounds 13a-h
Figure 1. Structure of inhibitors 1, 2, 14c, and 15c.
Scheme 1. Synthesis of Sulfonyl Chlorides 7a-d
Chemistry
The synthesis of sulfonyl chlorides 7a-d is shown in
Scheme 1. A Mitsunobu-type reaction between 3-methoxy-
phenol and alcohols 4a-d in the presence of triphenylpho-
sphine and diisopropyl azodicarboxylate (DIAD) afforded
ethers 5a-d.14 Electrophilic aromatic substitution of ethers
5a-d with acetic anhydride and concentrated sulfuric acid in
methanol furnished a mixture of sulfonic acid regioisomers in
a 1:1 ratio that were separated by flash chromatography to
give 6a-d. Structural confirmation of the isomers was deter-
mined by extensive 2D NMR experiments (NOESY and
HMBC). Conversion to the sulfonyl chlorides 7a-d was
achieved by reaction of the sulfonic acids 6a-d with thionyl
chloride in the presence of pyridine.
The synthesis of compounds 13a-h is outlined in Scheme 2.
Nucleophilic attack of amines 9a and 9b on commercially
available epoxide 8 in the presence of isopropyl alcohol gave
hydroxy amines 10a and 10b. The conversion of amines 10a
and 10b to the sulfonamides 11a-h was realized by
coupling with sulfonyl chlorides 7a-d in the presence of
pyridine. Removal of the Boc protecting group from sul-
fonamides 11a-h using 30% trifluoroacetic acid in CH2Cl2
furnished the corresponding amines, which were then
coupled with activated bis-THF15 (12) to give acyclic inhibi-
tors 13a-h.
The acyclic compounds 13a-h thus obtained were exposed
to ring closing metathesis using Grubbs’ first- or second-
generation catalyst16 (Scheme 3) to give the unsaturated
macrocyclic inhibitors 14a-h. Interestingly, larger ring sizes
(15-13) gave a mixture of E/Z isomers, while in the case of
smaller rings (12-9), the Z isomer was obtained exclusively.
The E and Z isomers were isolated using reversed-phase
HPLC and the stereochemistry established by 2D NMR
that 11-15-membered saturated and unsaturated macro-
cycles would effectively fill in the S10-S20 hydrophobic pocket
of the enzyme active site while retaining all major interactions
of PIs 1 and 2 with the protein backbone. Conceivably, such
inhibitors will maintain potency against both wild-type and
mutant strains. On the basis of this presumption, we designed
a series of PIs that incorporated various macrocycles that
could effectively fill in the enzyme active site. Herein we report
the structure-based design, synthesis, and preliminary biolo-
gical results of these macrocylic inhibitors. Among these
compounds, 14b and 14c were the most potent, with both
excellent enzyme inhibitory and antiviral activity. Both
inhibitors exerted potent activity against multidrug-resistant
HIV-1 variants. Furthermore, protein-ligand X-ray struc-
tures of inhibitors 2- and 14c-bound HIV-1 protease have
revealed important insights regarding ligand-binding interac-
tions (Figure 1).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7691
Scheme 3. Synthesis of Macrocyclic Inhibitors 14a-h and
15a-h
Scheme 5. Synthesis of Macrocyclic Inhibitors 20a-c, 21a-c,
and 22a-c
Scheme 4. Synthesis of Compounds 19a-c
Table 1. Enzyme Inhibitory and Antiviral Activity of Inhibitors 13a-h
compd
m
n
Ki (nM)
IC50 (nM) a
ring size by RCM
13a
13b
13c
13d
13e
13f
13g
13h
4
4
4
4
1
1
1
1
4
3
2
1
4
3
2
1
16.5 ( 0.5
11.5 ( 0.4
6.9 ( 0.1
>1000
>1000
ND
15
14
13
12
12
11
10
9
10 ( 2
ND
270
1.70 ( 0.07
1.02 ( 0.08
0.63 ( 0.01
0.10 ( 0.01
290
ND
ND
a Human T-lymphoid cells, MT-2 cells (2 ꢀ 103), were exposed to
HIV-1LAI (100 TCID50), cultured in the presence of each PI, and IC50
values were determined by using the MTT assay. The IC50 values of
saquinavir (SQV) and amprenavir (APV) tested as reference agents were
16 and 27 nM, respectively. ND: not determined.
sulfonyl chloride 7d in the presence of pyridine. Removal of
the Boc protecting group from sulfonamides 18a-c using
30% trifluoroacetic acid in CH2Cl2 furnished the correspond-
ing amines, whichwerethen coupledwiththe mixedcarbonate
of activated bis-THF15 (12) to give acyclic inhibitors 19a-c. A
ring closing metathesis reaction using Grubbs’ second-gen-
eration catalyst16 (Scheme 5) provided a E/Z mixture of
unsaturated macrocyclic inhibitors 20a-c and 21a-c, which
were separated by reverse-phase HPLC and identified by 2-D
NMR (COSY and NOESY). The unsaturated compounds
(COSY and NOESY) experiments, allowing their individual
biological evaluation. The unsaturated compounds were sub-
sequently reduced using hydrogen and 10% Pd-C as the
catalyst to yield inhibitors 15a-h.
A series of selectively methylated inhibitors were prepared
in a similar fashion. Nucleophilic attack of amines 16a-c on
commercially available epoxide 8 gave hydroxy amines 17a-c
(Scheme 4). The conversion of amines 17a-c to the sulfona-
mides 18a-c was realized by coupling the amines with

7692 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
Table 2. Enzyme Inhibitory and Antiviral Activity of Inhibitors 14a-h and 15a-h
a MT-2 human T-lymphoid cells exposed to HIV-1LAI; saquinavir and amprenavir exhibited IC50 values of 16 and 27 nM, respectively. b n.t. = not
tested.
20a-c and 21a-c were subsequently hydrogenated over 10%
Interestingly, conversion of these acyclic inhibitors 13a-h to
their cyclic analogues 14a-h and 15a-h resulted in significant
improvements of enzyme inhibitory and antiviral activity as
shown in Table 2. For example, the 14-membered macrocyclic
inhibitors 14b and 15b have Ki values of less than 0.7 nM and
IC50 values less than 49 nM, whereas their corresponding acyclic
inhibitor 13b had a Ki of 11 nM and IC50 value of >1000 nM
(greater than 15-fold and 20-fold change, respectively). Another
trend observed is a preference of the S10-subsite for macrocylic
rings of size 10 and 13. This preference is consistent with our
energy-minimized model structure of a saturated 13-membered
prototype inhibitor 15c where a macrocycle is incorporated in
place of the P10-isobutyl group and attached at the ortho-
position of the sulfonamide ring of inhibitor 2 as shown in
Figure 2. The model of inhibitor 15c was created based upon the
crystal structure of inhibitor 2-bound HIV-1 protease.
Pd-C as the catalyst to yield inhibitors 22a-c.
Biological Evaluation and Discussion
The inhibitory potencies of acyclic and cyclic inhibitors
were measured by the assay protocol of Toth and Marshall.17
Compounds that showed potent enzyme inhibition Ki values
were further evaluated in an antiviral assay. Biological results
for the acyclic compounds 13a-h are shown in Table 1. In
general, elongation of the carbon chains resulted in lower
enzyme inhibitory activity. Extension of the β-hydroxyl amine
chain by three methylene groups resulted in a 10-fold loss in
activity (13a Ki=16 nM versus 13e Ki = 1.7 nM). Similarly,
extension of the ether carbon chain by three methylene groups
resulted in a 17-fold loss in activity (13h Ki=0.10 nM versus
13e Ki=1.7 nM).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7693
Figure 2. An overlay of energy-minimized macrocyclic inhibitor 15c (magenta) with the X-ray structure of inhibitor 2 (green)-bound HIV-1
protease.
Table 3. Enzyme Inhibitory and Antiviral Activity of E and Z Isomers
In comparing the potency of 14c to its unsaturated analogue
of 14a-c
15c, we observed a dramatic improvement in the Ki and IC50
values for the unsaturated cyclic inhibitor 14c. Furthermore,
the effect appeared to remain consistent throughout the variously
sized unsaturated macrocycles 14a-h as compared to their
saturated analogues 15a-h. For the 13-membered ring series,
the presence of a double bond resulted in a 10-fold increase in
both Ki and IC50 values (14c Ki = 45 pM and IC50=2 nM as
compared to 15c Ki = 470 pM and IC50 = 22 nM). Similar
differences in potency can be seen for the 11, 14, and
15-membered macrocycles, although for the smaller rings 9,
10, and 12, the effect is less pronounced. This effect may be
explained by a restriction of conformation in the molecule that
results from the presence of the double bond and leads to a
better fit in the hydrophobic pocket of the S10-subsite. These
observations led us to take a closer look at the olefinic
compounds and evaluate the importance of the stereochemistry
of this group. As shown in Table 3, only minor variations in
activity (less than 5-fold) were observed between the E and Z
isomers of the 13, 14, and 15-membered macrocycles. For the
13-membered ring system, the Z isomer was favored over the
E configuration.
On the basis of these exciting results for this series of
macrocyclic inhibitors, we began to envision possible substi-
tutions that could be made across the macrocylic ring system
in order to further enhance biological activity. The energy-
minimized structure of 15b-bound HIV-1 protease with
2-bound HIV-1 protease (Figure 2) suggested that a single
methyl substitution β to the macrocylic amine could fill an
additional hydrophobic pocket previously filled by daruna-
vir’s isobutyl group. To this end, we designed and synthesized
a series of mono- and dimethylated 14-membered macrocylic
a MT-2 human T-lymphoid cells exposed to HIV-1LAI; saquinavir
ring systems and evaluated the impact of this substitution on
and amprenavir exhibited IC50 values of 16 and 27 nM, respectively.
the biological activity. Geminal dimethyl at this location
(19a through 22a, Table 4) significantly decreased (10-fold)
the enzyme inhibitionandgreatly reduced antiviral activity. In
most cases, the addition of a single methyl group to the ring
also reduced biological activity as compared to 14b and 15b
although results varied greatly depending upon the stereo-
chemistry of the ring systems. Inhibitors 20c and 21b were the
most potent compounds from this series with Ki=0.31 and
2.8 nM and IC50 = 9.0 and 6.3 nM, respectively. In general,
antiviral potency of cyclic inhibitors was superior to that of
their acyclic homologues, unsaturated macrocyclic derivatives
Indeed, the most potent compound of the series, inhibitor
14c incorporating a 13-membered ring, showed a Ki of 45 pM
and IC50 of 2 nM. It would appear the 13-membered ring
provides an optimally sized macrocyclic ring as increasing the
ring size to 14 or 15 as well as decreasing the ring to 12 or 11
resulted inreducedenzymatic inhibitory andantiviral activity.
Interestingly, inhibitors incorporating a 10-membered ring
(14g and 15g) were also exceedingly potent, displaying a
similar activity profile as the 13-membered ring 14c (14g
Ki=51 pM; 15g Ki = 86 pM and IC50=5.5 nM).

7694 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Table 4. Enzyme Inhibitory Activity of 19a-c, 20a-c, and 21a-c
Ghosh et al.
a MT-2 human T-lymphoid cells exposed to HIV-1LAI; saquinavir and amprenavir exhibited IC50 values of 16 and 27 nM, respectively.
were more potent than the corresponding saturated derivatives,
and the addition of methyl substituents tended to decrease
potency. The two most potent macrocyclic inhibitors identified
were 14c and 14b having enzyme inhibitory Ki values of 45 and
82 pM and antiviral IC50 values of 2 and 4 nM, respectively.
The inhibitors 14b and 14c were then examined for their
activity against a clinical wild-type X4-HIV-1 isolate (HIV-
1ERS104pre) along with various multidrug-resistant clinical
X4- and R5-HIV-1 isolates using PBMCs as target cells.6b
The results are shown in Table 5. As can be seen, the potency
of inhibitors 14b and 14c against HIV-1ERS104pre (IC50=7 and
5 nM, respectively) are superior to FDA approved inhibitors
IDV, APV, and LPV. It is comparable to saquinavir but
nearly 2-fold less potent than darunavir (IC50=3 nM).6b In
these studies, both indinavir and lopinavir were unable to
suppress the replication of the multidrug-resistant clinical
agent in HAART treatment regimens, was not active against
these multidrug-resistant clinical isolates. Amprenavir dis-
played a 10-fold or greater reduction in potency except against
HIV-1MDR/MM, where it showed a 7-fold reduction in
potency. Inhibitor 14c, while less potent than darunavir,
maintained 7-fold or better potency over amprenavir against
HIV-1MDR/C,HIV-1MDR/G,HIV-1MDR/TM,andHIV-1MDR/ISL
.
It maintained over a 6-fold potency against HIV-1MDR/MM
.
Inhibitor 14b maintained superior potency against HIV-
1MDR/C and HIV-1MDR/G (greater than 12- and 21-fold)
compared to amprenavir. It maintained 3-fold or better
potency compared to amprenavir against all other multi-
drug-resistant clinical isolates tested. Both inhibitors 14b
and 14c have shown low cytotoxþicity (CC50 values 49 and 33
μM, respectively) in target CD4 MT-2 cells. Furthermore,
they prevented the replication of HIV-1NL4-3 variants selec-
ted against up to 5 μM of saquinavir, lopinavir, and indinavir
with IC50 values of 20-46nM. More detailed virologic studies
with inhibitors 14c and 14b will be published in due course.
isolates examined that include HIV-1MDR/B, HIV-1MDR/C
HIV-1MDR/TM HIV-1MRR/MM and HIV-1MDR/JSL Of
particular note, lopinavir which is widely used as a first-line
,
,
,
.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7695
Table 5. Antiviral Activity of Macrocyclic Inhibitors against Multidrug Resistant Clinical Isolates in PHA-PBMsa
virus
SQV
IDV
APV
LPV
DRV
14b
14c
HIV-1ERS104pre
(wild-type: X4)
0.008 ( 0.005
0.043 ( 0.004 0.030 ( 0.005
0.034 ( 0.002 0.003 ( 0.0002
0.007 ( 0.002
0.005 ( 0.003
HIV-1MDR/B (X4)
HIV-1MDR/C (X4)
HIV-1MDR/G (X4)
HIV-1MDR/TM (X4)
HIV-1MDR/MM (R5)
HIV-1MDR/JSL (R5)
0.27 ( 0.073 (34)
0.032 ( 0.002 (11)
0.030 ( 0.002 (4)
0.26 ( 0.04 (33)
0.19 ( 0.05 (24)
0.30 ( 0.02 (37)
>1 (>23)
>1 (>23)
>1 (>33)
0.37 ( 0.011 (12)
>1 (>29)
>1 (>29)
0.019 ( 0.012 (6) 0.089 ( 0.016 (13) 0.037 ( 0.016 (7)
0.008 ( 0.006 (3) 0.029 ( 0.001 (4)
0.044 ( 0.002 (9)
0.057 ( 0.012 (11)
0.34 ( 0.14 (5) 0.43 ( 0.004 (14) 0.26 ( 0.04 (8) 0.023 ( 0.006 (5) 0.028 ( 0.004 (4)
>1 (>23)
>1 (>23)
>1 (>23)
0.32 ( 0.007 (11)
0.21 ( 0.222 (7)
0.62 ( 0.02 (21)
>1 (>29)
>1 (>29)
>1 (>29)
0.004 ( 0.001 (1) 0.072 ( 0.014 (10) 0.027 ( 0.001 (6)
0.011 ( 0.002 (4) 0.055 ( 0.025 (8) 0.033 ( 0.010 (7)
0.027 ( 0.011 (9) 0.21 ( 0.032 (30) 0.073 ( 0.07 (15)
a Amino acid substitutions identified in the protease-encoding region compared to the consensus type B sequence cited from the Los Alamos database
include L63P in HIV-1ERS104pre; L10I, K14R, L33I, M36I, M46I, F53I, K55R, I62 V, L63P, A71 V, G73S, V82A, L90M, and I93L in HIV-1 MDR/B
;
L10I, I15 V, K20R, L24I, M36I, M46L, I54 V, I62 V, L63P, K70Q, V82A, and L89 M in HIV-1 MDR/C; L10I, V11I, T12E, I15 V, L19I, R41K, M46L,
L63P, A71T, V82A, and L90 M in HIV-1 MDR/G; L10I, K14R, R41K, M46L, I54 V, L63P, A71 V, V82A, L90M, and I93L in HIV-1MDR/TM; L10I,
K43T, M46L, I54 V, L63P, A71 V, V82A, L90M, and Q92K in HIV-1 MDR/MM; L10I, L24I, I33F, E35D, M36I, N37S, M46L, I54 V, R57K, I62 V, L63P,
A71 V, G73S, and V82A in HIV-1 MDR/JSL. HIV-1ERS104pre served as a source of wild-type HIV-1. IC50 values were determined by using PHA-PBMs as
target cells, and inhibition of p24 Gag protein production by each drug was used as an end point. Numbers in parentheses represent n-fold changes of
IC50 values for each isolate compared to IC50 values for wild-type HIV-1ERS104pre. All assays were conducted in duplicate or triplicate, and data shown
represent mean values ((1 standard deviation) derived from results of three independent experiments.
Figure 3. A stereoview of the X-ray structure of macrocyclic inhibitor 14c (light gray)-bound HIV-1 protease. All strong hydrogen bonding
interactions are shown as dotted lines.
The reason why these macrocyclic inhibitors maintained
impressive potency against multidrug-resistant clinical iso-
lates is possibly due to their ability to make extensive hydro-
gen bonds with protease backbone and effectively fill in the
hydrophobic pockets in the S10-S20-subsites.
the sulfonamide oxygens, the urethane carbonyl oxygen, and
the backbone amide of Ile50 and Ile500 with distances of
2.6-3.1 A. These interactions have been observed in a ma-
jority of HIV-1 protease complexes with the inhibitors18 and
substrate analogues.19 The flexible P10-P20 macrocyclic
ligand nicely packs into the hydrophobic pocket in the S10-
X-ray Crystallography
subsite. It also makes weaker C-H O interactions, which
3 3 3
play important roles as we have reported earlier.20-22 The
macrocyclic ring zigzags into a crown shape and fits well in
between the S10 and S20-pockets. Theprotein-ligand complex
shows three major interactions with the carbonyl oxygen of
backbone residues, one with Gly270 and two with Gly480, with
distances ranging from 3.0 to 3.6 A. In comparison to the
X-ray structures of the protease with 1 and 2, the P1-phenyl
ring in 14c is rotated about 30ꢀ toward Asp 290 along the
backbone. The macrocycle acts more or less like a spring that
pushes against the P1-phenyl ring, causing this rotation.
To gain molecular insights into ligand-binding site interac-
tions responsible for the potent antiviral activity of inhibitor
14c, we have determined an X-ray crystal structure of the
inhibitor complexed with wild-type protease. The crystal
structure was solved and refined at 1.17 A resolution with
anR-factor and Rfree of16.0% and 19.4%, respectively. Inthis
high resolution structure, the inhibitor 14c was bound to the
HIV-1 protease active site in the two orientations with a ratio
of 1:1. A stereoview of the X-ray structure of 14c-bound HIV-
1 protease is shown in Figure 3. As can be seen, the inhibitor
makes extensive interactions involving the P2 to P20-ligands in
the protease active site, most notably through favorable polar
interactions including hydrogen bonds and weaker
Both P2 and P20 ligands form five strong N-H O hydro-
3 3 3
gen bonds with the protease backbone. Of these, three hydro-
gen bonds are formed between the P2-bis-THF ring oxygens
and the backbone amide nitrogens of Asp29 and Asp30 with
distances of 3.1, 3.0, and 3.2 A. The fourth interaction is
between the P2-urethane NH and the carbonyl oxygen of
Gly27 with a distance of 3.0 A. The fifth backbone interaction
is between the p-methoxy group of the P20-sulfonamide and the
C-H O interactions in the active site. The transition-state
3 3 3
hydroxyl group in 14c forms asymmetric hydrogen bonding
interactions with all four carboxylate oxygen atoms of the
Asp25 and Asp250 with distances of 2.5-3.3 A. The conserved
tetrahedral water molecule forms hydrogen bonds with one of

7696 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
amide nitrogen of Asp300, with a distance of 3.0 A. All of these
ligand-backbone interactions are present in the X-ray
structure of 2-bound HIV-1 protease as well. This backbone
binding with the main chain atoms of the protease may be
responsible for both inhibitors’ (2 and 14c) abilities to maintain
robust potency against multidrug-resistant HIV-1 variants.
FT-IR spectrometer or a Perkin-Elmer spectrometer L1185247
using a NaCl plate or KBr pellet. Optical rotations were
recorded on a Perkin-Elmer 341 or Rudolph Research Autopol
III polarimeter. Low resolution mass spectra were recorded on a
FinniganMAT LCQ or Hewlett-Packard Engine mass spectro-
meter. High-resolution mass spectra were recorded on a Finni-
ganMAT XL95 mass spectrometer calibrated against PPG.
Column chromatography was performed with Whatman
240-400 mesh silica gel under low pressure of 3-5 psi. TLC
was carried out with E. Merck silica gel 60-F-254 plates. HPLC
data was collected using a system composed of an Agilent 1100
series degasser, quaternary pump, thermostatable column com-
partment, variable wavelength detector, and Agilent 1200 series
autosampler and fraction collector controlled by Chemstation
software. All chromatographic reagents used were HPLC grade.
The reported inhibitors were found to be >95% pure by
reversed-phase gradient HPLC (see Supporting Information
for specific method conditions).
1-(Hex-5-enyloxy)-3-methoxybenzene (5a). To a stirred solu-
tion of 3-methoxy phenol (1.24 g, 10 mmol), 5-hexen-1-ol, 4a
(1.4 mL, 12 mmol), and Ph3P (3.14 g, 12 mmol) in THF (20 mL)
at 0 ꢀC was added DIAD (2.3 mL, 12 mmol) dropwise. After
stirring the solution for 30 min at 0 ꢀC, the reaction mixture was
warmed to 23 ꢀC and stirred for 3 h. The reaction mixture was
concentrated in vacuo, and the residue was subjected to column
chromatography (98:2 hexanes:EtOAc) to yield 5a (1.98 g, 96%
yield) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ
1.58-1.66 (m, 2H), 1.80-1.88 (m, 2H), 2.15-2.20 (m, 2H),
3.82 (s, 3H), 3.98 (t, J = 6.4 Hz, 2H), 5.02-5.12 (m, 2H),
5.83-5.92 (m, 1H), 6.52-6.56 (m, 3H), 7.19-7.24 (m, 1H).
13C NMR (100 MHz, CDCl3) δ 25.3, 28.7, 33.4, 55.1, 67.6,
100.9, 106.0, 106.6, 114.7, 129.8, 138.5, 160.3, 160.8. FT-IR
(film, NaCl) νmax =3075, 2939, 1599, 1493, 1287, 1200, 1152,
1046 cm-1. CI LRMS (m/z): 207.25 [M þ H]þ.
Conclusions
In summary, we have designed novel macrocyclic protease
inhibitors modifying P10-P20 ligands of darunavir-like PIs
and investigated their biological activity. The inhibitors were
designed to maintain key hydrogen bonding interactions with
protease backbone, similar to darunavir. The design of macro-
cycles involving the P10-P20-ligands is based upon the premise
that a flexible macrocycle would effectively repack the hydro-
phobic pocket in the S10 to S20-subsites when it is altered by
mutations. We have investigated inhibitors containing 9-
15-membered macrocycles containing both E/Z olefins and
the corresponding saturated derivatives. Grubbs’ metathesis
reaction was the key step in building these inhibitors in very
good yields. Most remarkably, all macrocyclic inhibitors are
significantly more potent than their acyclic counterparts. The
saturated inhibitors are in general less active than the corres-
ponding unsaturated derivatives. Our investigation resulted in
the identification of inhibitors 14b and 14c, which have
displayed significantly better antiviral activity than many of
the currently FDA-approved inhibitors. Inhibitor 14b con-
tains a 14-membered ring with a Z-olefin, and 14c contains a
13-membered ring with an E-olefin. Both inhibitors exerted
potent activity against HIV-1LAI, with IC50 values of 4 and
2 nM, respectively. They have maintained excellent potency
against multidrug-resistant HIV-1 variants. These inhibitors
have shown low cytotoxicity (CC50 values 49 and 33 μM,
1-Methoxy-3-(pent-4-enyloxy)benzene (5b). Title compound
was obtained from 4-penten-1-ol 4b, as described for 5a in 95%
yield after flash-chromatography (98:2 hexanes:EtOAc) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 1.88-1.94 (m, 2H),
2.25-2.30 (m, 2H), 3.81 (s, 3H), 3.98 (t, J = 6.4 Hz, 2H),
5.03-5.13 (m, 2H), 5.85-5.95 (m, 1H), 6.51-6.56 (m, 3H),
7.20 (t, J=8.1 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 28.3,
30.0, 55.1, 67.0, 100.9, 106.0, 106.6, 115.1, 129.7, 137.7, 160.2,
160.7. FT-IR (film, NaCl) νmax=3076, 2940, 1599, 1492, 1287,
1200, 1152, 1048 cm-1. CI LRMS (m/z): 193.25 [M þ H]þ.
1-(But-3-enyloxy)-3-methoxybenzene (5c). Title compound
was obtained from 3-buten-1-ol 4c, as described for 5a in 96%
yield after flash-chromatography (98:2 hexanes:EtOAc) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 2.54-2.60 (m, 2H),
3.81 (s, 3H), 4.02 (t, J = 6.7 Hz, 2H), 5.13-5.23 (m, 2H),
5.89-5.97 (m, 1H), 6.51-6.56 (m, 3H), 7.20 (t, J = 8.1 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 33.6, 55.1, 67.1, 100.9,
106.2, 106.6, 116.9, 129.8, 134.4, 160.1, 160.8. FT-IR (film,
NaCl) νmax =3136, 2378, 1644, 1509 cm-1. CI LRMS (m/z):
179.20 [M þ H]þ.
þ
respectively) in target CD4 MT-2 cells. Furthermore, both
inhibitors 14b and 14c blocked the replication of HIV-1NL4-3
variants, selected after exposure of up to 5 μM of saquinavir,
lopinavir, and indinavir, with IC50 values of 20-46 nM. The
protein-ligand X-ray structure of 14c showed critical
ligand-binding site interactions in the protease active site.
Particularly, it maintained all key backbone hydrogen bond-
ing interactions similar to darunavir and inhibitor 2. Also, the
conformational flexibility of the P10-P20 macrocycle most
likely contributed to its impressive activity against multidrug-
resistant clinical variants. Further design and optimization of
P10-P20 macrocyclic ligands are in progress.
Experimental Section
General Experimental Methods. Chemicals and reagents were
purchased from commercial suppliers and used without further
purification. Anhydrous solvents were obtained as follows:
pyridine and dichloromethane were distilled from calcium
hydride; tetrahydrofuran and diethyl ether were distilled
from sodium with benzophenone as an indicator. All other
solvents were reagent grade. All moisture sensitive reactions
were carried out in oven-dried glassware under argon. 1H NMR
and 13C NMR spectra were recorded on a Bruker Avance ARX-
400, Bruker DRX-500, or Bruker Avance-III-800 spectrometer.
Chemical shifts are given in ppm and are referenced against the
diluting solvent. For chloroform-d: 13C triplet = 77.00 CDCl3
and 1H singlet = 7.26 ppm. For methanol-d4: 13C septuplet =
1-(Allyloxy)-3-methoxybenzene (5d). Title compound was
obtained from allyl alcohol 4d as described for 5a in 96% yield
after flash-chromatography (98:2 hexanes:EtOAc) as a colorless
oil. 1H NMR (400 MHz, CDCl3) δ 3.81 (s, 3H), 4.02 (d, J=6.7
Hz, 2H), 5.13-5.23 (m, 2H), 5.89-5.97 (m, 1H), 6.51-6.56 (m,
3H), 7.20 (t, J=8.1 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ
55.1, 67.1, 100.9, 106.2, 106.6, 116.9, 129.8, 134.4, 160.1, 160.8.
2-(Hex-5-enyloxy)-4-methoxybenzenesulfonic Acid (6a). To
6a (2 g, 9.7 mmol) was added acetic anhydride (1.4 mL, 14.5
mmol), and the resulting mixture was stirred at 0 ꢀC. To this was
then added concentrated H2SO4 (1.1 g) followed by methanol
(20 mL). The resulting solution was warmed to 23 ꢀC and stirred
for 12 h. After this time, the reaction mixture was concentrated
in vacuo and the resulting red oil was subjected to column
chromatography (88:12 CH2Cl2:MeOH) to give 6a (1.06 g,
1
49.05 and H quintuplet = 3.31 ppm. Characteristic splitting
patterns due to spin-spin coupling are expressed as follows:
br=broad, s = singlet, d=doublet, t=triplet, q = quartet, m =
multiplet, sept = septuplet. All coupling constants are measured
in hertz. FTIR spectra were recorded on a Mattson Genesis II
1
38%) as a red waxy solid. H NMR (400 MHz, D2O) δ 1.44

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7697
(quintet, J=7.4 Hz, 2H), 1.66-1.73 (m, 2H), 1.99 (q, J = 7.1 Hz,
2H), 3.70 (s, 3H), 3.98 (t, J = 6.5 Hz, 2H), 4.85-4.97 (m, 2H),
evaporated under reduced pressure, and the resulting residue
was purified by silica chromatography (5:95 MeOH:CHCl3) to
give 10a (350 mg, 97%) as a white solid. H NMR (400 MHz,
1
5.74-5.92 (m, 1H), 6.52-6.56 (m, 2H), 7.19-7.24 (m, 1H). 13
C
NMR (100 MHz, D2O) δ 25.3, 28.7, 33.4, 55.1, 67.6, 100.9,
106.0, 106.6, 114.7, 129.8, 138.5, 160.3, 160.8. ESI (m/z): 285.09
[M - H]-.
CDCl3) δ 1.34 (s, 9H), 1.39-1.50 (m,4H), 2.05 (q, J =7 Hz, 2H),
2.55-2.68 (m, 6H), 2.81-2.86 (m, 1H), 2.96 (dd, J=4.5, 14 Hz,
1H), 3.44-3.49 (m, 1H), 3.79 (br s, 1H), 4.72 (d, J = 8.7 Hz, 1H),
4-Methoxy-2-(pent-4-enyloxy)benzenesulfonic Acid (6b). Title
compound was obtained from ether 5b as described for 6a in
36% yield after flash-chromatography (88:12 CH2Cl2:MeOH)
as a white solid. 1H NMR (400 MHz, D2O) δ 1.75-1.82 (m, 2H),
2.10-2.16 (m, 2H), 3.69 (s, 3H), 3.98 (t, J = 6.4 Hz, 2H),
4.88-5.00 (m, 2H), 5.77-5.87 (m, 1H), 6.45 (dd, J=8.6, 2.2
4.92-5.02 (m, 2H), 5.74-5.84 (m, 1H), 7.17-7.28 (m, 5H). 13
C
NMR (100 MHz, CDCl3) δ 26.4, 28.2, 29.4, 33.4, 36.5, 49.6,
51.3, 54.1, 70.7, 79.3, 114.5, 126.2, 128.3, 129.4, 137.8,
138.6,155.9. CI LRMS (m/z): 363.55 [M þ H]þ.
tert-Butyl (2S,3R)-4-(allylamino)-3-hydroxy-1-phenylbutan-
2-ylcarbamate (10b). Title compound was obtained from allyla-
mine 9b and epoxide 8 as described for 10a in 99% yield after
flash-chromatography (5:95 MeOH:CHCl3) as a white solid. 1H
NMR (400 MHz, CDCl3) δ 1.35 (s, 9H), 2.60-2.86 (m, 5H), 2.96
(dd, J = 4.5, 14.1 Hz, 1H), 3.16-3.29 (m, 2H), 3.50-3.53 (m,
1H), 3.81 (br s, 1H), 4.73 (d, J=9.1 Hz, 1H), 5.10 (d, J=10.3 Hz,
1H), 5.17 (d, J=17 Hz, 1H), 5.81-5.91 (m, 1H), 7.13-7.32 (m,
5H). 13C NMR (100 MHz, CDCl3) δ 28.2, 36.5, 50.7, 52.1, 54.1,
70.9, 79.3, 116.2, 126.2, 128.3, 129.4, 136.3, 137.8, 155.9. CI
LRMS (m/z) 321.50 [M þ H]þ.
Hz, 1H), 6.51 (d, J=2.1 Hz, 1H), 7.58 (d, J=8.8 Hz, 1H). 13
C
NMR (100 MHz, D2O) δ 28.0, 29.9, 56.1, 68.7, 100.5, 104.9,
115.5, 123.7, 130.2, 139.4, 157.8, 163.5. ESI (m/z): 271.07 [M - H]-.
2-(But-3-enyloxy)-4-methoxybenzenesulfonic Acid (6c). Title
compound was obtained from ether 5c as described for 6a in
30% yield after flash-chromatography (88:12 CH2Cl2:MeOH)
as a white solid. 1H NMR (400 MHz, D2O) δ 2.38-2.43 (m, 2H),
3.62 (s, 3H), 3.98 (t, J = 6.8 Hz, 2H), 4.93-5.05 (m, 2H),
5.77-5.87 (m, 1H), 6.37 (dd, J=8.6, 2.2 Hz, 1H), 6.43 (d, J =
2.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H) 13C NMR (100 MHz, D2O)
δ 33.3, 56.1, 68.8, 100.6, 105.0, 117.5, 123.7, 130.2, 135.4, 157.5,
163.4. ESI (m/z): 257.10 [M - H]-.
2-(Allyloxy)-4-methoxybenzenesulfonic Acid (6d). Title com-
pound was obtained from ether 5d as described for 6a in 35%
yield after flash-chromatography (88:12 CH2Cl2:MeOH) as a
white solid. 1H NMR (400 MHz, D2O) δ 3.71 (s, 3H), 4.58-4.75
(m, 2H), 5.16-5.38 (m, 2H), 5.92-5.99 (m, 1H), 6.47-6.57 (m,
2H), 7.58 (d, J=8.8. Hz, 1H). 13C NMR (100 MHz, D2O) δ 56.1,
69.7, 101.1, 105.3, 118.0, 123.7, 130.6, 133.2, 157.1, 163.5. ESI
(m/z): 243.13 [M - H]-.
2-(Hex-5-enyloxy)-4-methoxybenzene-1-sulfonyl Chloride (7a).
To a stirring solution of sulfonic acid 6a (266 mg, 0.9 mmol) in
pyridine (2 mL) was added thionyl chloride (0.2 mL, 2.8 mmol)
dropwise. The resulting solution was allowed to stir for 4 h and
then the reaction mixture concentrated in vacuo. The resulting
residue was purified using column chromatography (5:1 hexanes:
EtOAc) to give 7a (140 mg, 50%) as a colorless oil. 1H NMR (400
MHz, CDCl3) δ 1.63-1.70 (m, 2H), 1.87-1.93 (m, 2H),
2.10-2.16 (m, 2H), 3.88 (s, 3H), 4.14 (t, J = 6.2 Hz, 2H),
4.95-5.05 (m, 2H), 5.76-5.86 (m, 1H), 6.51-6.54 (m, 2H), 7.84
(d, J=9.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 24.9, 28.1,
33.1, 55.9, 69.2, 99.9, 104.6, 114.7, 124.3, 131.7, 138.3, 158.7, 166.8.
4-Methoxy-2-(pent-4-enyloxy)benzene-1-sulfonyl Chloride (7b).
Title compound was obtained from ether 6b as described for 7a in
48% yield after flash-chromatography (6:1 hexanes:EtOAc) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 1.96-1.2.03 (m, 2H),
2.31-2.37 (m, 2H), 3.88 (s, 3H), 4.15 (t, J = 6.2 Hz, 2H), 4.99-
5.09 (m, 2H), 5.79-5.90 (m, 1H), 6.51-6.54 (m, 2H), 7.84 (d, J=
9.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 27.8,29.7, 55.9, 68.4.
99.9, 104.6, 115.6, 124.3, 131.7, 137.3, 158.6, 166.8.
tert-Butyl-(2S,3R)-4-(N-(hex-5-enyl)-2-(hex-5-enyloxy)-4-eth-
oxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate
(11a). To a stirring solution of 10a (50 mg, 0.14 mmol) in
pyridine (2 mL) was added 7a (64 mg, 0.20 mmol), and the
resulting solution was allowed to stir for 2 h at 23 ꢀC. The
reaction mixture was concentrated under reduced pressure, and
the resulting residue was purified by flash column chromatog-
raphy (3:1 hexanes:EtOAc) to yield 11a (74 mg 84% yield) as a
colorless oil. [R]D20 -1.4 (c 1.00, CHCl3). 1H NMR (400 MHz,
CDCl3) δ 1.25-1.34 (m, 11 H), 1.47-1.50 (m, 2H), 1.57-1.60
(m, 2H), 1.82-1.90 (m, 2H), 1.97 (q, J=7.1 Hz, 2H), 2.11 (q, J =
7.0 Hz, 2H), 2.92-2.95 (m, 2H), 3.10-3.17 (m, 1H), 3.30 (br s,
3H), 3.76 (br s, 2H), 3.84 (s, 3H), 3.90-3.92 (m, 1H) 4.03 (t, J=
6.7 Hz, 2H), 4.65 (d, J = 7.1 Hz, 1H), 4.89-5.04 (m, 4H),
5.66-5.82 (m, 2H), 6.46 (d, J = 2 Hz, 1H), 6.49 (dd, J=8.8,
2.3 Hz, 1H), 7.17-7.29 (m, 5H), 7.83 (d, J=7.8 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ 24.9, 25.7, 27.9, 28.2, 28.3, 33.1,
33.2, 35.1, 49.9, 52.3, 54.5, 55.6, 69.1, 72.2, 79.4, 100.2, 104.1,
114.7, 115.0, 119.4, 126.2, 128.3, 129.5, 133.5, 137.8, 138.1,
138.2, 156.0, 157.6, 164.7. FT-IR (film, NaCl) νmax=3395, 2935,
1705, 1597, 1325 cm-1. ESI (þ) LRMS (m/z): 653.13 [M þ Na]þ.
tert-Butyl-(2S,3R)-4-(N-(hex-5-enyl)-4-methoxy-2-(pent-4en-
yloxy)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarba-
mate (11b). Title compound was obtained from 10a and 7b, as
described for 11a in 79% yield after flash-chromatography (3:1
hexanes:EtOAc) as a colorless oil. [R]D20 -1.6 (c 1.20, CHCl3).
1H NMR (400 MHz, CDCl3) δ 1.25-1.37 (m, 12H), 1.43-1.51
(m, 2H), 1.88-1.98 (m, 4H), 2.27 (q, J=7.1 Hz, 2H), 2.86-2.97
(m, 2H), 3.10-3.17 (m, 1H), 3.25-3.31 (m, 3H), 3.76 (br s, 2H),
3.84 (s, 3H), 4.04 (t, J=6.6 Hz, 2H), 4.66 (d, J=7.1 Hz, 1H),
4.88-5.10 (m, 4H), 5.65-5.72 (m, 1H), 5.78-5.85 (m, 1H), 6.46
(d, J = 2 Hz, 1H), 6.49 (dd, J = 8.8, 2.0 Hz, 1H), 7.17-7.29 (m,
5H), 7.83 (d, J = 8.7 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ
25.7, 27.9, 28.2, 29.7, 33.1, 35.2, 49.8, 52.2, 54.6, 55.6, 68.4,
72.2, 79.4, 100.2, 104.1, 114.7, 115.7, 119.4, 126.2, 128.3, 129.5,
133.5, 137.0, 137.8, 138.2, 156.0, 157.6, 164.7. FT-IR (film,
NaCl) νmax = 3398, 2931, 1706, 1596, 1325 cm-1. ESI (þ)
LRMS (m/z): 639.06 [M þ Na]þ.
2-(But-3-enyloxy)-4-methoxybenzene-1-sulfonyl Chloride (7c).
Title compound was obtained from ether 6c as described for 7a
in 52% yield after flash-chromatography (6:1 hexanes:EtOAc) as
a colorless oil. 1H NMR (300 MHz, CDCl3) δ 2.62-2.70 (m, 2H),
3.88 (s, 3H), 4.19 (t, J=6.2 Hz, 2H), 5.12-5.25 (m, 2H), 5.91-6.05
(m, 1H), 6.52-6.56 (m, 2H), 7.86 (d, J=9.2 Hz, 1H).
2-(Allyloxy)-4-methoxybenzene-1-sulfonyl Chloride (7d). Title
compound was obtained from ether 6d as described for 7a in
58% yield after flash-chromatography (6:1 hexanes:EtOAc) as a
colorless oil. 1H NMR (400 MHz, CDCl3) δ 3.88 (s, 3H), 4.72 (d,
J=4.4 Hz, 2H), 5.33 (d, J=10.6 Hz, 1H), 5.57 (d, J=17.3 Hz,
1H), 5.99-6.07 (m, 1H), 6.52-6.55 (m, 2H), 7.83 (d, J=8.7 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 55.9, 69.7, 100.5, 105.0,
118.1, 124.4, 131.0, 131.7, 158.0, 166.7.
tert-Butyl-(2S,3R)-4-(2-(but-3-enyloxy)-N-(hex-5-enyl)-4-meth-
oxyphenylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate
(11c). Title compound was obtained from 10a and 7c, as
described for 11a in 50% yield after flash-chromatography
20
(3:1 hexanes:EtOAc) as a colorless oil. [R]D þ0.6 (c 2.00,
1
CHCl3). H NMR (500 MHz, CDCl3) δ 1.28-1.38 (m, 12H),
1.48-1.52 (m, 2H), 2.00 (q, J=6.7 Hz, 2H), 2.65 (q, J=6.7, 2H),
2.96 (br s, 2H), 3.14-3.18 (m, 1H), 3.30-3.39 (m, 3H), 3.79 (br s,
2H), 3.88 (s, 3H), 4.13 (t, J=7.1 Hz, 2H), 4.68 (d, J=5.1 Hz, 1H),
4.92-4.98 (m, 2H), 5.17-5.24 (m, 2H), 5.68-5.76 (m, 1H),
5.91-5.99 (m, 1H), 6.51 (d, J=2 Hz, 1H), 6.54 (dd, J=8.8, 2.0
tert-Butyl (2S,3R)-4-(hex-5-enylamino)-3-hydroxy-1-phenyl-
butan-2-ylcarbamate (10a). A solution of hex-5-en-1-amine 9a
(297 mg, 3 mmol) and epoxide 8 (263 mg, 1 mmol) was heated to
60 ꢀC in isopropyl alcohol (4 mL) for 4 h. The solvent was then

7698 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
Hz, 1H), 7.21-7.32 (m, 5H), 7.88 (d, J = 8.8 Hz, 1H). 13C NMR
(125 MHz, CDCl3) δ 25.9, 28.2, 28.4, 29.9, 33.4, 35.4, 50.1, 52.5,
54.8, 55.9, 68.7, 72.5, 79.7, 100.6, 104.5, 114.9, 118.0, 119.8,
126.5, 128.6, 129.8, 133.7, 133.9 138.1, 138.5, 156.3, 157.7, 165.0.
compound was obtained from 10b and 7d, as described for 11a
in 80% yield after flash-chromatography (3:1 hexanes:EtOAc) as
a colorless oil. 1H NMR (400 MHz, CDCl3) δ 1.33 (s, 9H),
2.83-2.96 (m, 2H), 3.25-3.36 (m, 2H), 3.77 (br s, 2H), 3.83 (s,
3H), 3.87-3.94 (m, 2H), 4.56-4.67 (m, 3H), 5.07-5.14 (m, 2H),
5.33 (d, J=10.5Hz, 1H), 5.44(d, J = 17.2 Hz, 1H), 5.57-5.64 (m,
1H), 6.00-6.10 (m, 1H), 6.47(d, J=1.9 Hz, 1H), 6.51 (dd, J =1.9,
8.9 Hz, 1 H) 7.16-7.28 (m, 5H), 7.85 (d, J=8.7 Hz, 1H). 13C
NMR (100 MHz, CDCl3) δ 28.2, 35.3, 51.3, 52.1, 54.5, 55.6, 69.9,
71.8, 79.3, 100.6, 104.5, 119.0, 119.4, 119.8, 126.2, 128.2, 129.5,
FT-IR (film, NaCl) νmax=3394, 2931, 1704, 1596, 1325 cm-1
ESI (þ) (m/z): 625.05 [M þ Na]þ.
.
tert-Butyl(2S,3R)-4-(2-(allyloxy)-N-(hex-5-enyl)-4-methoxyphe-
nylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (11d). Ti-
tle compound was obtained from 10a and 7d, as described for 11a
in 64% yield after flash-chromatography (3:1 hexanes:EtOAc) as a
20
1
colorless oil. [R]D þ1.1 (c 2.80, CHCl3). H NMR (400 MHz,
CDCl3) δ 1.25-1.34 (m, 11H), 1.39-1.46 (m, 2H), 1.96 (q, J = 7.0
Hz, 2H), 2.89-2.96 (m, 2H), 3.08-3.15 (m, 1H), 3.24-3.30 (m,
3H), 3.77 (br s, 2H), 3.84 (s, 3H), 4.60 (d, J = 5.4 Hz, 2H), 4.66 (d,
J=7.2 Hz, 1H), 4.88-4.94 (m, 2H), 5.33 (d, J = 10.4, 2H), 5.43 (d,
J = 17.4 Hz, 1H), 5.63-5.73 (m, 1H), 6.00-6.10 (m, 1H), 6.47 (d,
J= 1.8 Hz, 1H), 6.54 (dd, J=8.9, 1.9Hz, 1H), 7.18-7.29 (m, 5H),
7.85 (d, J=8.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 25.7, 27.8,
28.2, 33.1, 35.2, 49.9, 52.5, 54.5, 55.6, 69.9, 72.2, 79.4, 100.6, 104.4,
114.7, 119.4, 119.7 126.2, 128.3, 129.5, 131.9, 133.5, 137.9, 138.2,
155.9, 157.0, 164.6. FT-IR (film, NaCl) νmax=3400, 2929, 1704,
1596, 1324 cm-1. ESI (þ) LRMS (m/z): 611.03 [M þ Na]þ.
tert-Butyl-(2S,3R)-4-(N-allyl-2-(hex-5-enyloxy)-4-methoxyphe-
nylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (11e).
Title compound was obtained from 10b and 7a, as described
for 11a in 77% yield after flash-chromatography (3:1 hexanes:
EtOAc) as a colorless oil. [R]D20 -6.6 (c 1.96, CHCl3). 1H NMR
(500 MHz, CDCl3) δ 1.36 (s, 9H), 1.60-1.66 (m, 2H), 1.87-1.97
(m, 2H), 2.13-2.17 (m, 2H), 2.91-2.98 (m, 2H), 3.27-3.39 (m,
2H), 3.79 (br s, 2H), 3.87 (s, 3H), 3.91-3.99 (m, 2H), 4.08 (t, J=
6.7 Hz, 2H), 4.65 (d, J=7.1 Hz, 1H), 4.98 (d, J=10.1, 1H), 5.04
(d, J = 17.1, 1H), 5.13-5.19 (m, 2H), 5.63-5.73 (m, 1H),
5.78-5.86 (m, 1H), 6.50-6.53 (m, 2H), 7.21-7.31 (m, 5H),
7.88 (d, J = 8.7 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 25.0,
28.3, 33.3, 35.3, 51.2, 52.2, 54.6, 55.7, 69.2, 71.8, 79.5, 100.3,
104.2, 115.1, 119.0, 119.6, 126.3, 128.4, 129.6, 133.6, 137.9, 138.2,
156.1, 157.7, 164.8. FT-IR (film, NaCl) νmax=3392, 2932, 1702,
1595, 1324 cm-1. ESI (þ) LRMS (m/z): 611.04 [M þ Na]þ.
tert-Butyl-(2S,3R)-4-(N-allyl-4-methoxy-2-(pent-4-enyloxy)phe-
nylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (11f). Ti-
tle compound was obtained from 10b and 7b, as described for 11a
in 86% yield after flash-chromatography (3:1 hexanes:EtOAc) as a
131.8, 133.4, 137.9, 155.9, 157.0, 164.7. FT-IR (film, NaCl) νmax=
3390, 2976, 1710, 1597, 1325 cm-1. ESI (þ) LRMS (m/z): 569.06
[M þ Na]þ.
Compound 13a. To a stirring solution of 11a (63 mg, 0.1
mmol) in CH2Cl2 (2 mL) was added a solution of 30% trifluoro-
acetic acid in CH2Cl2, and the resulting mixture was stirred for
30 min. The solvent was evaporated under reduced pressure, and
the residue was dissolved in CH3CN (2 mL). To this solution was
added 12 (31 mg, 0.11 mmol), followed by i-Pr2NEt. After
stirring for 24 h, the reaction mixture was concentrated in vacuo
and the resulting residue was subjected to flash-chromatogra-
phy (1:1 hexanes:EtOAc) to give 13a (36 mg, 53% yield) as a
colorless oil. [R]D20 -13.1 (c 1.50, CHCl3). 1H NMR (500 MHz,
CDCl3) δ 1.28-1.31 (m, 6H), 1.47-1.51 (m, 3H), 1.58-1.61 (m,
3H), 1.84-1.88 (m, 2H), 1.96-1.99 (m, 2H), 2.01-2.13 (m, 2H),
2.79-2.84 (m, 1H), 2.88-2.92 (m, 1H), 3.09-3.15 (m, 1H),
3.24-3.40 (m, 3H), 3.61 (br s, 1H), 3.64-3.72 (m, 2H),
3.83-3.92 (m, 4H), 3.93-3.96 (m, 1H), 4.05 (t, J = 6.7 Hz,
2H), 4.90-5.03 (m, 4H), 5.64 (d, J=5 Hz, 1H), 5.66-5.82 (m,
2H), 6.46 (s, 1H), 6.51 (d, J = 8.8 Hz, 1H), 7.17-7.26 (m, 5H),
7.83 (d, J=8.8 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 27.9,
28.2, 28.1, 29.4, 31.7, 32.9, 33.1, 35.1, 45.1, 49.8, 52.1, 54.8, 55.5,
68.9, 69.3, 70.5, 72.0, 73.1, 100.1, 103.9, 109.0, 114.6, 114.9,
118.9, 126.3, 128.2, 129.1, 133.4, 137.4, 137.8, 138.0, 155.2,
157.4, 164.6. FT-IR (film, NaCl) νmax = 3343, 2928, 1721,
1595, 1325 cm-1. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for
C36H50N2O9S, 709.3135; found, 709.3136.
Compound 13b. Title compound was obtained from 11b and
12 as described for 13a in 55% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (500 MHz,
1
CDCl3) δ 1.27-1.32 (m, 3H), 1.41-1.53 (m, 3H), 1.57-1.62 (m,
1H), 1.92-1.99 (m, 4H), 2.27 (q, J=7.05, 2H), 2.80 (dd, J=10,
14 Hz, 1H), 2.86-2.91 (m, 1H), 3.01 (dd, J=4, 14 Hz, 1H),
3.10-3.15 (m, 1H), 3.27-3.33 (m, 2H), 3.38 (dd, J=8.6, 15.2 Hz,
1H), 3.61 (br s, 1H), 3.64-3.70 (m, 2H), 3.81-3.84 (m, 5H),
3.88-3.95 (m, 2H), 4.05 (t, J=6.6 Hz, 2H), 4.89-5.09 (m, 5H),
5.63 (d, J=5.2 Hz, 1H), 5.65-5.73 (m, 1H), 5.77-5.85 (m, 1H),
6.46 (d, J=2 Hz, 1H), 6.51 (dd, J = 2.1, 8.8 Hz, 1H), 7.17-7.26
(m, 5H), 7.83 (d, J=8.8 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ
25.7, 27.9, 29.7, 33.1, 35.4, 45.1, 49.8, 52.1, 55.1, 55.7, 68.5, 69.5,
70.7, 72.2 73.2, 100.3, 104.2, 109.2, 114.8, 115.7, 119.2, 126.4,
128.4, 129.3, 133.6, 137.1, 137.6, 138.2, 155.4, 157.5, 164.8. ESI
(þ) HRMS (m/z): [M þ H]þ calcd for C35H48N2O9S, 673.3159;
found, 673.3153.
20
1
colorless oil. [R]D -5.6 (c 1.10, CHCl3). H NMR (400 MHz,
CDCl3) δ 1.32 (s, 9H), 1.94 (quintet, J=7 Hz, 2H), 2.65 (q, J=7
Hz, 2H), 2.86-2.96 (m, 2H), 3.27 (dd, J = 7.4, 14.8 Hz, 1H),
3.34-3.38 (m, 1H), 3.76 (br s, 2H), 3.82 (s, 3H), 3.87-3.92 (m,
2H), 4.04 (t, J = 6.5 Hz, 2H), 4.65 (d, J = 8.2 Hz, 1H), 4.98-5.15
(m,4H),5.57-5.63(m,1H),5.75-5.86(m,1H),6.46-6.49(m,2H),
7.12-7.27 (m, 5H), 7.83 (d, J=8.5 Hz, 1H). 13C NMR (100 MHz,
CDCl3) δ 27.8, 28.1, 29.7, 35.3, 51.0, 51.9, 54.5, 55.6, 68.4, 71.8, 79.3,
100.2, 104.2, 115.7, 119.0, 119.5, 126.1, 128.2, 129.5, 133.4, 137.2,
137.9, 155.9, 157.6, 164.7. FT-IR (film, NaCl) νmax =3390, 2976,
1710, 1597, 1325 cm-1. ESI (þ) LRMS (m/z): 597.13 [M þ Na]þ.
tert-Butyl-(2S,3R)-4-(N-allyl-2-(but-3-enyloxy)-4-methoxyphe-
nylsulfonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (11g).
Title compound was obtained from 10b and 7c, as described for
11a in 78% yield after flash-chromatography (3:1 hexanes:
Compound 13c. Title compound was obtained from 11c and
12 as described for 13a in 81% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (500 MHz,
1
1
EtOAc) as a colorless oil. H NMR (500 MHz, CDCl3) δ 1.34
CDCl3) δ 1.31-1.35 (m, 3H), 1.50-1.54 (m, 3H), 1.62-1.67 (m,
1H), 2.00 (q, J=7 Hz, 2H), 2.65 (q, J=6.7 Hz, 2H), 2.82-2.87
(m, 1H), 2.91-2.94 (m, 1H), 3.06 (dd, J=4.1, 14.2 Hz, 1H),
3.13-3.19 (m, 1H), 3.30-3.43 (m, 3H), 3.62 (br s, 1H),
3.68-3.74 (m, 2H), 3.85-3.88 (m, 4H), 3.92-3.99 (m, 2H),
4.13 (t, J=6.9 Hz, 2H), 4.93-5.05 (m, 2H), 5.07-5.09 (m, 2H),
5.17-5.24 (m, 2H), 5.66 (d, J=5.1 Hz, 1H), 5.69-5.77 (m, 1H),
5.90-5.99 (m, 1H), 6.52 (s, 1H), 6.55 (dd, J = 2.05, 8.8 Hz, 1H),
7.22-7.30 (m, 5H), 7.87 (d, J=8.8 Hz, 1H). 13C NMR (125
MHz, CDCl3) δ 25.8, 25.9, 28.1, 29.8, 33.3, 35.6, 45.4, 50.0, 52.4,
55.2, 55.8, 68.7, 69.7, 70.8, 72.4, 73.4, 100.6, 104.5, 109.4, 114.9,
118.0, 119.5, 126.6, 128.6, 129.5, 133.5, 137.8, 138.3, 155.9,
157.6, 164.9. FT-IR (film, NaCl) νmax = 3339, 2929, 1719,
(s, 9H), 2.62 (q, J=7 Hz, 2H), 2.88-2.96 (m, 2H), 3.27 (dd, J =
7.8, 15.1 Hz, 1H), 3.34-3.38 (m, 1H), 3.76 (br s, 2H), 3.85 (s, 3H),
3.87-3.99 (m, 2H), 4.10 (t, J = 6.5 Hz, 2H), 4.65 (br s, 1H),
5.10-5.22 (m, 4H), 5.57-5.63 (m, 1H), 5.87-5.95 (m, 1H), 6.49
(d, J=2.2 Hz, 1H), 6.51 (dd, J=2.3, 8.7 Hz 1 H) 7.18-7.37 (m,
5H), 7.85 (d, J=8.7Hz, 1H). 13C NMR(100 MHz, CDCl3) δ27.8,
28.1, 35.3, 51.0, 51.9, 54.5, 55.6, 68.4, 71.8, 79.3, 100.2, 104.2,
115.7, 119.0, 119.5, 126.2, 128.2, 129.5, 133.4, 137.2, 137.9, 155.9,
157.6, 164.7. FT-IR (film, NaCl) νmax = 3390, 2976, 1710, 1597,
1325 cm-1. ESI LRMS (m/z): 582.95 [M þ Na]þ.
tert-Butyl-(2S,3R)-4-(N-allyl-2-(allyloxy)-4-methoxyphenylsul-
fonamido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (11h). Title

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7699
1596, 1324 cm-1. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for
C34H46N2O9S, 681.2822; found, 681.2812.
Compound 13h. Title compound was obtained from 11h and
12 as described for 13a in 52% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (400 MHz,
1
Compound 13d. Title compound was obtained from 11d and
12 as described for 13a in 55% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (500 MHz,
CDCl3) δ 1.37-1.43 (m, 1H), 1.54-1.65 (m, 1H), 2.73-2.80 (m,
1H), 2.85-2.90 (m, 1H), 3.02 (dd, J = 2.9, 13.7 Hz, 1H),
3.28-3.39 (m, 2H), 3.51 (br s, 1H), 3.63-3.70 (m, 2H),
3.77-3.95 (m, 10H), 4.62 (d, J=5.2 Hz, 2H), 4.96-5.06 (m,
2H), 5.10-5.16 (m, 2H), 5.33 (d, J=10.4 Hz, 1H), 5.45 (d, J=
17.2 Hz, 1H), 5.62-5.66 (m, 2H), 6.01-6.10 (m, 1H), 6.49 (s,
1H), 6.52 (d, J = 8.9 Hz, 1H), 7.18-7.33 (m, 5H), 7.85 (d, J=8.8
Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 25.7, 35.4, 45.3, 51.4,
52.2, 54.9, 55.7, 69.6, 70.0, 70.7, 71.8, 73.1, 100.7, 104.5, 109.2,
119.1, 119.4, 119.5, 126.4, 128.4, 129.3, 131.8, 133.5, 137.7,
155.3, 157.0, 164.8. FT-IR (film, NaCl) νmax = 3351, 1717,
1596, 1324 cm-1. ESI (þ) HRMS (m/z): [M þ H]þ calcd for C30-
H38N2O9S, 603.2376; found, 603.2375.
Inhibitor 14a. To stirring solution of 13a (32 mg, 0.047 mmol)
in CH2Cl2 (15 mL) was added Grubbs’ first-generation catalyst
(4 mg, 0.0046 mmol). After stirring at 23 ꢀC for 16 h, the solvent
was evaporated under reduced pressure and the residue was
subjected to flash column chromatography to yield 14a (27 mg,
88% yield) as a white solid and E/Z mixture (3:1, determined by
HPLC). The isomers were isolated by reverse-phase HPLC
using the following conditions: YMC Pack ODS-A column
(250 mm ꢀ 100 mm, 5 μm); flow rate =2.75 mL/min; isocratic
60:40 CH3CN:H2O; T = 35 ꢀC; λ = 215 nm; E isomer Rt = 16.5
min; Z isomer Rt = 14.5 min.
1
CDCl3) δ 1.24-1.30 (m, 2H), 1.43-1.51 (m, 3H), 1.57-1.63 (m,
1H), 1.96 (q, J = 7 Hz, 2H), 2.80 (dd, J = 10, 14 Hz, 1H),
2.86-2.91 (m, 1H), 3.01 (dd, J = 4.5, 14.5 Hz, 1H), 3.08-3.14
(m, 1H), 3.25-3.30 (m, 2H), 3.40 (dd, J = 8.5, 15 Hz, 1H) 3.58
(br s, 1H), 3.65-3.72 (m, 2H), 3.80-3.82 (m, 2H), 3.87 (s, 3H),
3.88-3.96 (m, 2H), 4.60 (d, J = 5.5 Hz, 2H), 4.90-4.95 (m, 2H),
4.98-5.06 (m, 2H), 5.33 (d, J = 10 Hz, 1H), 5.45 (d, J = 18 Hz,
1H), 5.64 (d, J = 5.5 Hz, 1H), 5.65-5.72 (m, 1H), 6.02-6.10 (m,
1H), 6.47 (d, J = 2 Hz, 1H), 6.53 (dd, J = 2.5, 9 Hz, 1H),
7.17-7.27 (m, 5H), 7.85 (d, J = 9 Hz, 1H). 13C NMR (125 MHz,
CDCl3) δ 25.7, 27.8, 33.0, 35.3, 45.2, 49.9, 52.4, 55.0, 55.6, 69.5,
69.9, 70.6, 72.2, 73.3, 100.6, 104.5, 109.2, 114.7, 119.4, 126.4,
128.4, 129.3, 131.9, 133.5, 137.6, 138.1, 155.4, 157.0, 164.7. FT-
IR (film, NaCl) νmax =3339, 1718, 1594, 1324 cm-1. ESI (þ)
HRMS (m/z): [M þ Na]þ calcd for C33H44N2O9S, 667.2665;
found, 667.2661.
Compound 13e. Title compound was obtained from 11e and
12 as described for 13a in 68% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (400 MHz,
1
CDCl3) δ 1.52-1.61 (m, 3H), 1.81-1.93 (m, 2H), 2.09 (q, J = 7
Hz, 2H), 2.76 (dd, J = 10, 13.7 Hz, 1H), 2.83-2.88 (m, 1H), 3.01
(dd, J = 3.6, 14.2 Hz, 1H), 3.28-3.32 (m, 2H), 3.61-3.69 (m,
2H), 3.78-3.85 (m, 6H), 3.87-3.93 (m, 4H), 4.04 (t, J = 6.5 Hz,
2H), 4.92-5.01 (m, 3H), 5.09-5.16 (m, 3H), 5.60-5.71 (m, 2H),
5.72-5.81 (m, 1H), 6.47-6.51 (m, 2H), 7.14-7.26 (m, 5H), 7.82
(d, J = 8.6 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ 24.9, 25.7,
28.2, 33.2, 35.4, 45.3, 51.0, 52.1, 54.9, 55.7, 69.1, 69.5, 70.7, 71.8,
73.1, 100.3, 104.2, 109.2, 115.0, 119.0, 119.2, 126.3, 128.3, 129.3,
133.4, 133.6, 137.5, 138.0, 155.4, 157.6, 164.8. FT-IR (film,
NaCl) νmax = 3368, 1720, 1596, 1325 cm-1. ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C33H44N2O9S, 667.2665; found,
667.2668.
Compound 14aE. 1H NMR (800 MHz, CDCl3) δ 1.39-1.44
(m, 2H), 1.48-1.53 (m, 1H), 1.57-1.64 (m, 2H), 1.68-1.74 (m,
3H), 1.77-1.82 (m, 1H), 1.83-1.88 (m, 1H), 1.94-1.98 (m, 1H),
2.10-2,14 (m, 3H), 2.71 (dd, J=9.6, 14.1 Hz, 1H), 2.86-2.89 (m,
1H), 2.90 (dd, J = 4.3, 14.1 Hz, 1H), 2.91-2.99 (m, 2H),
3.31-3.33 (m, 1H), 3.61-3.64 (m, 1H), 3.65-3.70 (m, 2H),
3.73 (br s, 1H), 3.76-3.78 (m, 1H), 3.79-3.85 (m, 5H), 3.93 (dd,
J =6.6, 9.4 Hz, 1H), 3.96-3.98 (m, 1H), 4.02-4.05 (m,1H), 4.87
(d, J = 9.1 Hz, 1H), 4.97-5.00 (m, 1H), 5.44-5.48 (m, 1H),
5.54-5.56 (m, 1H), 5.63 (d, J=5.1 Hz, 1H), 6.44 (d, J = 2 Hz,
1H), 6.49 (dd, J = 2.2, 8.8 Hz, 1H), 7.13-7.24 (m, 5H), 7.84 (d,
J = 8.8 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 25.1, 25.3, 25.7,
26.8, 29.8, 30.4, 32.5, 35.5, 45.2, 50.6, 51.6, 54.7, 55.6, 68.8, 69.5,
70.7, 71.6, 73.3, 99.9, 103.9, 109.2, 118.1, 126.4, 128.4, 129.3, 130.5,
132.1, 134.0, 137.4, 155.2, 157.9, 164.9. ESI (þ) HRMS (m/z): [Mþ
Na]þ calcd for C34H46N2O9S, 681.2822; found, 681.2815.
Compound 14aZ. 1H NMR (800 MHz, CDCl3) δ 1.28-1.33
(m, 2H), 1.38-1.47 (m, 2H), 1.52-1.64 (m, 5H), 1.84-1.90 (m,
2H), 2.04-2.08 (m, 2H), 2.16-2.22 (m, 2H), 2.74 (dd, J=9.5,
14.1 Hz, 1H), 2.88-2.90 (m, 1H), 3.00 (dd, J=4.5, 14.1 Hz, 1H),
3.05-3.14 (m, 2H), 3.15 (dd, J=9.4, 15.1 Hz, 1H), 3.44-3.48 (m,
1H), 3.61 (br s, 1H), 3.66-3.71 (m, 2H), 3.76-3.89 (m, 6H), 3.95
(dd, J = 6.2, 9.6 Hz, 1H), 4.10-4.15 (m, 1H), 4.87 (d, J =9.1 Hz,
1H), 5.00-5.03 (m, 1H), 5.35-5.39 (m, 1H), 5.49-5.52 (m, 1H),
5.63 (d, J = 5.2 Hz, 1H), 6.50 (s, 1H), 6.49 (dd, J = 2.3, 8.8 Hz,
1H), 7.17-7.26 (m, 5H), 7.84 (d, J=8.8 Hz, 1H). 13C NMR (125
MHz, CDCl3) δ 25.0, 25.1, 25.7, 26.0, 26.6, 27.8, 29.6, 35.5, 45.2,
48.5, 52.0, 54.7, 55.6, 68.8, 69.5, 70.6, 71.9, 73.3, 100.7, 104.2,
109.2, 117.8, 126.4, 128.4, 129.2, 129.8, 130.0, 134.1, 137.5,
155.2, 158.0, 165.0. ESI (þ) HRMS (m/z): [M þ Na]þ calcd
for C34H46N2O9S, 681.2822; found, 681.2819.
Compound 13f. Title compound was obtained from 11f and 12
as described for 13a in 64% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (400 MHz,
1
CDCl3) δ 1.37-1.43 (m, 1H), 1.54-1.62 (m, 1H), 1.91-1.97 (m,
2H), 2.27 (q, J = 7 Hz, 2H), 2.76 (dd, J = 10.1, 13.9 Hz, 1H),
2.84-2.89 (m, 1H), 3.02 (dd, J = 4, 14.1 Hz, 1H), 3.30-3.37 (m,
2H), 3.54 (br s, 1H), 3.62-3.69 (m, 2H), 3.79-3.87 (m, 5H),
3.88-3.95 (m, 4H), 4.06 (t, J=6.7 Hz, 2H), 4.96-5.00 (m, 2H),
5.04-5.16 (m, 4H), 5.59-5.67 (m, 2H), 5.76-5.85 (m, 1H), 6.47
(d, J = 1.9 Hz, 1H), 6.50 (dd, J = 2.1, 8.9 Hz, 1H), 7.16-7.26
(m, 5H), 7.82 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
δ 25.7, 27.9, 29.7, 35.5, 45.3, 51.0, 52.1, 54.9, 55.7, 68.5, 69.5,
70.7, 71.8, 73.2, 100.3, 104.2, 109.2, 115.7, 119.1, 119.3, 126.4,
128.3, 129.3, 133.5, 137.1, 137.7, 155.4, 157.6, 164.8. FT-IR
(film, NaCl) νmax=3350, 1720, 1596, 1325 cm-1. ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C32H42N2O9S, 653.2509; found,
653.2509.
Compound 13g. Title compound was obtained from 11g and
12 as described for 13a in 68% yield after flash-chromatography
(1:1 hexanes:EtOAc) as a colorless oil. H NMR (400 MHz,
1
CDCl3) δ 1.39-1.45 (m, 1H), 1.56-1.63 (m, 1H), 1.81 (br s, 1H),
2.61 (q, J = 6.6 Hz, 2H), 2.75-2.80 (m, 1H), 2.86-2.91 (m, 1H),
3.02 (dd, J = 4.1, 14.1 Hz, 1H), 3.32 (d, J = 5.8 Hz, 2H), 3.53 (br
s, 1H), 3.64-3.70 (m, 2H), 3.81-3.95 (m, 8H), 4.10 (t, J=6.7 Hz,
2H), 4.97-5.01 (m, 2H), 5.12-5.20 (m, 4H), 5.62-5.66 (m, 2H),
5.86-5.94 (m, 1H), 6.49 (s, 1H), 6.51 (d, J = 9.1 Hz, 1H),
7.18-7.26 (m, 5H), 7.85 (d, J = 8.8 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 25.8, 33.2, 35.6, 45.4, 51.2, 52.3, 55.0, 55.8, 68.7,
69.6, 70.8, 71.9, 73.3, 100.5, 104.5, 109.3, 117.9, 119.2, 119.4,
126.5, 128.5, 129.4, 133.5, 133.7, 137.8, 155.4, 157.5, 164.9. FT-
IR (film, NaCl) νmax =3350, 1722, 1596, 1325 cm-1. ESI (þ)
HRMS (m/z): [M þ H]þ calcd for C31H40N2O9S, 617.2533;
found, 617.2540.
Inhibitor 14b. The title compound was obtained from a ring
closing metathesis reaction of 13b using Grubbs’ first-genera-
tion catalyst as described for 14a. The crude material was
purified by silica gel chromatography (60:40 EtOAc:hexanes)
to give the desired product (50% yield) as a mix of E/Z isomers
(27:73 by HPLC). The isomers were isolated by chiral HPLC
using the following conditions: Chiralpak IA column (250 mm ꢀ
4.6 mm, 5 μm); flow rate = 0.75 mL/min; isocratic 60:40 IPA:
hexanes; T=25 ꢀC; λ=215 nm; E isomer Rt=7.56 min; Z-isomer
Rt=8.89 min.
Compound 14bE. 1H NMR (800 MHz, CDCl3) δ 1.60-1.20
(m, 4H), 2.30-1.90 (m, 7H), 3.10-2.80 (m, 5H), 3.32 (m, 1H),

7700 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
4.00-3.50 (m, 12 H), 4.15 (m, 2H), 4.98 (m, 2H), 5.45 (m, 1H),
5.63 (m, 2H), 6.51 (m, 2H), 7.25-7.15 (m, 5H), 7.76 (d, J = 19.2
Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 23.1, 24.8, 25.0, 25.7,
26.2, 26.6, 29.0, 35.5, 45.2, 48.2, 52.0, 54.8, 55.6, 67.1, 69.5, 70.7,
71.6, 73.3, 100.1, 104.0, 109.2, 114.6, 118.4, 126.4, 128.4, 128.9,
129.3, 130.6, 133.8, 137.4, 155.3, 157.7, 164.9.; ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C33H44N2O9S, 667.2665; found,
667.2660.
140.4, 155.4, 156.4, 164.3. ESI (þ) HRMS (m/z): [M þ H]þ
calcd for C31H40N2O9S, 617.2533; found, 617.2534.
Inhibitor 14e. Title compound was obtained from 13e and
Grubbs’ second-generation catalyst as described for 14a in 52%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid. 1H NMR (500 MHz, CDCl3) δ 1.46-1.51 (m,
1H), 1.60-1.76 (m, 4H), 1.88-1.92 (m, 2H), 2.23-2.37 (m,
2H), 2.79 (dd, J=9, 14 Hz, 1H), 2.88-3.01 (m, 2H), 3.10-3.13
(m, 2H), 3.57 (br s, 1H), 3.66-3.72 (m, 2H), 3.72-3.89 (m, 5H),
3.92-3.99 (m, 2H), 4.07-4.15 (m, 2H), 4.94 (d, J = 8.5 Hz, 1H),
5.00-5.04 (m, 1H), 5.44-5.54 (m, 3H), 5.64 (d, J = 5.1 Hz, 1H),
6.43 (d, J = 2 Hz, 1H), 6.49-6.51 (m, 1H), 7.16-7.28 (m, 5H),
7.81 (d, J=9 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 25.7, 26.3,
26.5, 27.4, 29.6, 35.5, 43.9, 45.2, 50.7, 54.7, 55.6, 69.5, 69.7, 70.7,
73.3, 100.0, 103.9, 109.2, 117.7, 122.9, 126.4, 128.4, 129.3, 133.7,
135.1, 137.4, 155.3, 157.7, 164.9. ESI (þ) HRMS (m/z): [M þ
Na]þ calcd for C31H40N2O9S, 639.2352; found, 639.2345.
Inhibitor 14f. Title compound was obtained from 13f and
Grubbs’ second-generation catalyst as described for 14a in 81%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid. 1H NMR (500 MHz, CDCl3) δ 1.40-1.47 (m,
1H), 1.58-1.62 (m, 2H), 1.92-1.95 (m, 1H), 2.11-2.15 (m,
1H), 2.28-2.39 (m, 2H), 2.73-2.78 (m, 1H), 2.80-3.05 (m, 5H),
3.64-3.70 (m, 2H), 3.80-3.89 (m, 6H), 3.93-3.96 (m, 2H),
4.12-4.15 (m, 1H), 4.62 (br s, 1H), 4.97-4.99 (m, 1H), 5.11 (d,
J = 9.2 Hz, 1H), 5.52-5.54 (m, 2H), 5.61 (d, J=5.1 Hz, 1H),
6.46-6.49 (m, 2H), 7.18-7.26 (m, 5H), 7.80 (d, J = 9.4 Hz, 1H).
13C NMR (125 MHz, CDCl3) δ 22.7, 25.1, 25.7, 29.6, 35.5, 41.2,
45.3, 47.8, 54.8, 55.6, 65.9, 69.5, 70.7, 73.2, 101.0, 104.3, 109.2,
118.7, 123.3, 126.4, 128.4, 129.3, 133.4, 133.6, 137.5, 155.4,
157.8, 164.7. ESI (þ) HRMS (m/z): [M þ H]þ calcd for
C30H38N2O9S, 603.2376; found, 603.2369.
Inhibitor 14g. Title compound was obtained from 13g and
Grubbs’ second-generation catalyst as described for 14a in 81%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid. 1H NMR (500 MHz, CDCl3) δ 1.47-1.55 (m,
1H), 1.64-1.70 (m, 2H), 2.51-2.54 (m, 1H), 2.73-2.78 (m,
1H), 2.84-3.94 (m, 2H), 3.04-3.11 (m, 2H), 3.19 (d, J=13.6 Hz,
1H), 3.70-3.75 (m, 2H), 3.86 (s, 4H), 3.94-3.99 (m, 4H),
4.07-4.08 (m, 1H), 4.18 (br s, 1H), 4.58 (s, 1H), 5.02-5.06 (m,
1H), 5.12 (s, 1H), 5.61 (d, J=5 Hz, 1H), 5.76-5.82 (m, 1H),
5.88-5.93 (m, 1H), 6.35 (d, J=1.1 Hz, 1H), 6.51 (dd, J = 1.5, 8.7
Hz, 1H), 7.20-7.31 (m, 5H), 7.79 (d, J = 8.8 Hz, 1H). 13C NMR
(125 MHz, CDCl3) δ 25.8, 26.7, 35.4, 43.6, 45.3, 47.1, 55.1, 55.7,
67.4, 69.6, 70.5, 70.8, 73.3, 99.4, 104.1, 109.2, 120.5, 126.5, 126.9,
128.5, 129.4, 131.2, 132.0, 137.6, 155.6, 157.4, 164.7. ESI (þ)
HRMS (m/z): [M þ Na]þ calcd for C29H36N2O9S, 611.2039;
found, 611.2040.
Inhibitor 14h. Title compound was obtained from 13h and
Grubbs’ second-generation catalyst as described for 14a in 79%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid. 1H NMR (400 MHz, CDCl3) δ 1.45-1.51 (m,
1H), 1.60-1.68 (m, 2H), 2.80-2.90 (m, 2H), 3.00-3.12 (m,
3H), 3.53 (br s, 1H), 3.66-3.71 (m, 2H), 3.83 (s, 4H), 3.92-3.95
(m, 3H), 4.25 (br s, 1H), 4.88-5.01 (m, 3H), 5.13 (d, J = 8.3 Hz,
1H), 5.63 (d, J = 5.1 Hz, 1H), 5.72-5.76 (m, 2H), 6.59-6.64 (m,
2H), 7.19-7.26 (m, 5H), 7.74 (d, J=8.7 Hz, 1H). 13C NMR (125
MHz, CDCl3) δ 25.7, 29.6, 35.3, 43.9, 45.2, 47.8, 55.1, 55.7, 69.5,
70.7, 71.1, 73.3, 104.5, 107.2, 109.2, 123.4, 126.1, 126.4, 128.4,
129.3, 131.3, 131.5, 137.6, 155.6, 157.7, 164.5. ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C28H34N2O9S, 597.1883; found,
597.1887.
Inhibitor 15a. To a stirring solution of 14a (10 mg, 0.015
mmol) in EtOAc (2 mL) was added 10% Pd on carbon and the
reaction was stirred under H2 atmosphere for 12 h. After this
time, the reaction was filtered through a pad of celite and solvent
was evaporated under reduced pressure. The residue was then
purified by flash-chromatography to give 15a (9 mg, 93% yield)
as a white solid. 1H NMR (500 MHz, CDCl3) δ 0.87-0.92 (m,
2H), 1.25-1.28 (m, 8H), 1.38-1.49 (m, 5H), 1.60-1.64 (m, 3H),
Compound 14bZ. 1H NMR (800 MHz, CDCl3) δ 1.60-1.0 (m,
7H), 1.72 (m, 2H), 1.90 (m, 3H), 2.21 (m, 2H), 2.43 (m, 1H), 2.55
(m, 1H), 2.67 (m, 1H), 2.76 (m, 1H), 2.84 (m, 1H), 3.01 (m, 1H),
3.26 (m, 1H), 3.41 (m, 3H), 3.60 (m, 4H), 3.69 (m, 1H), 3.86 (m,
2H), 4.43 (d, J = 15.2 Hz, 1H), 4.83 (m, 1H), 5.24 (m, 1H), 5.33
(m, 1H), 5.51 (d, J=8.8 Hz, 1H), 6.45 (m, 2H), 7.17 (m, 3H), 7.24
(m, 2H), 7.87 (d, J=13.6 Hz, 1H). 13C NMR (125 MHz, CDCl3)
δ 24.7, 25.7, 27.3, 28.6, 30.9, 32.1, 35.5, 45.2, 49.3, 51.8, 54.8,
55.6, 69.5, 70.7, 71.0, 71.9, 73.3, 101.2, 104.3, 109.2, 119.3, 126.4,
128.4, 128.9, 129.3, 130.8, 130.9, 133.4, 137.5, 155.3, 158.2,
164.5. ESI (þ) HRMS (m/z): [MþNa]þ calcd for C33H44N2O9S,
667.2665; found, 667.2667.
Inhibitor 14c. Title compound was obtained from 13c and
Grubbs’ first-generation catalyst as described for 14a in 89%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid and E/Z mixture (3:1, determined by HPLC). The
isomers were isolated using reversed-phase HPLC under the
following conditions: YMC Pack ODS-A column (250 mm ꢀ
100 mm, 5 μm); flow rate = 2.75 mL/min; isocratic 60:40
CH3CN:H2O; T=35 ꢀC; λ=215 nm; E isomer Rt=13.43 min;
Z isomer Rt=11.76 min.
1
Compound 14cE. H NMR (800 MHz, CDCl3) δ 1.45-1.54
(m, 3H), 1.56-1.64 (m, 2H), 1.68-1.72 (m, 1H), 2.11-2.20 (m,
2H), 2.52-2.62 (m, 2H), 2.79 (dd, J=9.6, 14 Hz, 1H), 2.87-2.90
(m, 1H), 2.96-3.00 (m, 2H), 3.04 (dd, J=4.2, 14.2 Hz, 1H),
3.40-3.44 (m, 1H), 3.53-3.56 (m, 1H), 3.66-3.70 (m, 3H),
3.82-3.89 (m, 6H), 3.94 (dd, J = 6.3, 9.5 Hz, 1H), 4.07-4.14 (m,
2H), 4.96 (d, J = 9.4 Hz, 1H), 4.98-5.01 (m, 1H), 5.53-5.56 (m,
1H), 5.64-5.68 (m, 2H), 6.52-6.53 (m, 2H), 7.18-7.27 (m, 5H),
7.76 (d, J = 9.4 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 24.4,
25.7, 28.3, 32.3, 32.4, 35.6, 45.2, 49.6, 51.9, 54.9, 55.6, 69.5, 69.9,
70.7, 71.9, 73.2, 101.9, 104.9, 109.2, 119.3, 126.4, 128.0, 128.4,
129.3, 133.4, 133.9, 137.6, 155.3, 158.0, 164.5. ESI (þ) HRMS
(m/z): [M þ H]þ calcd for C32H42N2O9S, 631.2689; found,
631.2698.
1
Compound 14cZ. H NMR (500 MHz, CDCl3) δ 1.48-1.52
(m, 3H), 1.57-1.72 (m, 3H), 2.10-2.14 (m, 1H), 2.28-2.32 (m,
1H), 2.47-2.51 (m, 1H), 2.78 (dd, J = 9, 14 Hz, 2H), 2.87-2.91
(m, 1H), 2.98-3.08 (m, 3H), 3.45-3.60 (m, 2H), 3.65-3.71 (m,
3H), 3.80-3.90 (m, 6H), 3.95 (dd, J = 6, 9.5 Hz, 1H), 4.07-4.10
(m, 1H), 4.18-4.21 (m, 1H), 4.95 (d, J = 9.5 Hz, 1H), 4.98-5.02
(m, 1H), 5.44-5.49 (m, 2H), 5.63 (d, J=5.5 Hz, 1H), 6.51 (dd,
J = 2.5, 9.8 Hz, 1H), 6.53 (d, J = 2 Hz, 1H), 7.18-7.27 (m, 5H),
7.81 (d, J=9 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 24.7, 24.8,
25.7, 26.3, 27.7, 35.5, 45.2, 46.9, 50.2, 54.9, 55.6, 69.5, 69.9, 70.6,
70.7, 73.2, 101.9, 104.8, 109.1, 120.5, 126.4, 127.6, 128.4, 129.3,
129.6, 132.5, 133.2, 137.6, 155.3, 158.1, 164.6. ESI (þ) HRMS
(m/z): [M þ H]þ calcd for C32H42N2O9S, 631.2689; found,
631.2706.
Inhibitor 14d. Title compound was obtained from 13d and
Grubbs’ first-generation catalyst as described for 14a in 71%
yield after flash-chromatography (2:3 hexanes:EtOAc) as a
white solid. 1H NMR (500 MHz, CDCl3) δ 1.40-1.47 (m,
1H), 1.58-1.62 (m, 2H), 1.92-1.95 (m, 1H), 2.11-2.15 (m,
1H), 2.28-2.39 (m, 2H), 2.73-2.78 (m, 1H), 2.80-3.05 (m, 6H),
3.64-3.70 (m, 3H), 3.80-3.89 (m, 6H), 3.93-3.96 (m, 2H),
4.12-4.15 (m, 1H), 4.62 (br s, 1H), 4.97-4.99 (m, 1H), 5.11 (d,
J=9.2 Hz, 1H), 5.52-5.54 (m, 2H), 5.61 (d, J=5.1 Hz, 1H),
6.46-6.49 (m, 2H), 7.18-7.26 (m, 5H), 7.80 (d, J=9.4 Hz, 1H).
13C NMR (125 MHz, CDCl3) δ 22.6, 22.9, 23.4, 25.7, 25.9, 29.6,
35.4, 45.1, 45.2, 47.9, 55.0, 55.6, 62.0, 69.5, 70.7, 73.2, 100.2,
103.6, 109.2, 119.9, 123.5, 126.4, 128.4, 129.3, 132.6, 137.6,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7701
1.71-1.83 (m, 4H), 2.32-2.38 (m, 1H), 2.75 (dd, J = 9.5, 13.7
Hz, 1H), 2.86-2.91 (m, 1H), 3.01-3.10 (m, 3H), 3.64-3.71 (m,
2H), 3.85 (s, 4H), 3.94 (dd, J = 6.4, 9.5 Hz, 1H), 4.01-4.04
(m, 1H), 4.10-4.16 (m,1H), 4.94 (d, J = 9.2 Hz, 1H), 5.01-5.15
(m, 1H), 5.67 (d, J = 5.1 Hz, 1H), 6.52-6.54 (m, 2H), 7.19-7.29
(m, 5H), 7.85 (d, J=9 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ
23.8, 24.2, 25.5, 26.3, 26.4, 28.2, 29.1, 29.4, 35.3, 45.1, 51.3, 52.8,
54.6, 55.4, 69.2, 69.3, 69.5, 70.5, 71.9, 73.1, 100.2, 103.9, 109.0,
118.7, 126.3, 128.2, 129.1, 133.5, 137.3, 155.1, 157.9, 164.6. ESI
(þ) HRMS (m/z): [M þ Na]þ calcd for C34H48N2O9S, 683.2978;
found, 683.2984.
Inhibitor 15g. Title compound was obtained from 14g as
described for 15a in 90% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.46-1.50 (m, 3H), 1.59-1.67 (m, 4H), 1.96-2.04 (m,
2H), 2.81-2.91 (m, 2H), 3.03 (dd, J = 3, 14 Hz, 1H), 3.07-3.12
(m, 2H), 3.56 (br s, 1H), 3.57-3.71 (m, 2H), 3.83-3.91 (m, 6H),
3.93-4.02 (m, 3H), 4.25 (br s, 1H), 4.98-5.02 (m, 2H), 5.63 (d,
J = 5 Hz, 1H), 6.40 (d, J = 2 Hz, 1H), 6.49 (dd, J = 2, 9 Hz, 1H),
7.18-7.28 (m, 5H), 7.76 (d, J = 9 Hz, 1H). 13C NMR (125 MHz,
CDCl3): δ 22.4, 24.4, 25.3, 25.7, 35.4, 43.1, 45.2, 47.3, 54.9, 55.6,
69.5, 70.1, 70.6, 70.7, 73.2, 100.5, 104.2, 109.2, 121.4, 126.4, 128.4,
129.3, 131.5, 137.5, 155.4, 157.7, 164.5. ESI (þ) HRMS (m/z):
[M þ H]þ calcd for C29H38N2O9S, 591.2376; found, 591.2381.
Inhibitor 15h. Title compound was obtained from 14h as
described for 15a in 92% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.39-1.51 (m, 3H), 1.60-1.68 (m, 3H), 1.72-1.81 (m,
2H), 2.82-2.92 (m, 2H), 3.00-3.06 (m, 3H), 3.66-3.71 (m, 3H),
3.80-3.87 (m, 6H), 3.95 (dd, J=6, 9.5 Hz, 1H), 3.97-4.02 (m,
1H), 4.25 (br s, 1H), 4.98-5.02 (m, 2H), 5.63 (d, J =5 Hz, 1H),
6.62-6.64 (m, 2 H), 7.18-7.28 (m, 5H), 7.77 (d, J=10.5 Hz, 1H).
13C NMR (125 MHz, CDCl3) δ 24.3, 25.0, 25.7, 35.3, 44.5, 45.2,
46.5, 55.0, 55.6, 69.5, 70.6, 73.2, 73.9, 105.6, 107.3, 109.1, 126.4,
128.4, 129.3, 130.9, 137.3, 155.5, 157.4, 164.4. ESI (þ) HRMS
(m/z): [M þ H]þ calcd for C28H36N2O9S, 577.2220; found,
577.2222.
Inhibitor 15b. Title compound was obtained from 14b as
described for 15a in 90% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.30-1.47 (m, 8H), 1.57-1.70 (m, 5H), 1.72-1.79 (m,
1H), 1.82-1.87 (m, 2), 2.77 (dd, J=10, 14 Hz, 1H), 2.86-2.91
(m, 1H),3.00 (dd, J = 4.5, 14 Hz, 1H), 3.05 (d, J=2.5, 15 Hz,
1H), 3.14-3.23 (m, 2H), 3.56-3.62 (m, 2H), 3.65-3.72 (m, 2H),
3.81-3.90 (m, 6H), 3.94 (dd, J = 6.5, 10 Hz, 1H), 4.05-4.15 (m,
2H), 4.96 (d, J = 9.5 Hz, 1H), 4.99-5.02 (m, 1H), 5.64 (d, J=5.5
Hz, 1H), 6.49-6.52 (m, 2H), 7.17-7.27 (m, 5H), 7.82 (d, J =9.5
Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 23.2, 24.2, 24.9, 25.2,
25.7, 26.1, 27.6, 28.9, 35.4, 45.2, 49.2, 52.7, 54.8, 55.6, 68.9, 69.5,
70.7, 71.7, 73.2, 100.5, 104.2, 109.2, 118.5, 126.4, 128.4, 129.3,
133.8, 137.5, 155.3, 158.1, 164.8. ESI (þ) HRMS (m/z): [M þ
Na]þ calcd for C33H46N2O9S, 669.2822; found, 669.2828.
Inhibitor 15c. Title compound was obtained from 14c as
described for 15a in 90% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.35-1.39 (m, 3H), 1.40-1.54 (m, 4H), 1.60-1.66 (m,
6H), 1.85-1.98 (m, 2H), 2.78 (dd, J=9.1, 14 Hz, 1H), 2.88-2.91
(m, 1H),2.97-3.03 (m, 1H), 3.06-3.14 (m, 1H), 3.39-3.43 (m,
1H), 3.50-3.56 (m, 1H), 3.66-3.72 (m, 3H), 3.82-3.85 (m, 6H),
3.95 (dd, J = 6.3, 9.6 Hz, 1H), 4.07-4.10 (m, 2H), 4.95-5.03 (m,
2H), 5.64 (d, J=5 Hz, 1H), 6.50-6.53 (m, 2H), 7.17-7.27 (m,
5H), 7.82 (d, J=9 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 22.6,
23.5, 24.0, 24.6, 25.3, 25.7, 26.1, 35.5, 44.8, 45.2, 46.5, 51.0, 54.8,
55.6, 69.5, 70.1, 70.7, 70.8, 73.2, 100.9, 104.3, 109.2, 118.5, 126.4,
128.4, 129.4, 133.9, 137.5, 155.2, 158.2, 164.8. ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C32H44N2O9S, 655.2668; found,
655.2667.
Inhibitor 15d. Title compound was obtained from 14d or 14e
as described for 15a in 94% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.33-1.54 (m, 6H), 1.57-1.74 (m, 3H), 1.86-1.94 (m,
2H), 2.77 (dd, J=9.5, 14 Hz, 1H), 2.86-2.90 (m, 1H), 2.96-3.08
(m, 3H), 3.24-3.29 (m, 1H), 3.64-3.72 (m, 2H), 3.74-3.86 (m,
7H), 3.94-4.00 (m, 2H), 4.15-4.22 (m, 2H), 4.92 (d, J = 9.2 Hz,
1H), 4.97-5.02 (m, 1H), 5.62 (d, J = 5.2 Hz, 1H), 6.48 (d, J=2.1
Hz, 1H), 6.52 (dd, J=2, 8.9 Hz, 1H), 7.17-7.27 (m, 5H), 7.87 (d,
J = 8.6 Hz, 1H) 13C NMR (125 MHz, CDCl3) δ 23.2, 25.0, 25.6,
26.1, 26.3, 35.4, 42.9, 45.2, 48.5, 54.7, 55.6, 69.3, 69.5, 70.7, 73.3,
99.9, 103.8, 109.1, 117.1, 126.4, 128.4, 129.3, 134.6, 137.4, 155.1,
157.7, 165.2. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for
C31H42N2O9S, 641.2509; found, 641.2512.
tert-Butyl-(2S,3R)-4-(2,2-dimethylpent-4-enylamino)-3-hydroxy-
1-phenylbutan-2-ylcarbamate (17a). A stirring solution of amine
16a (1.58 g, 4.33 mmol) and epoxide 8 (304 mg, 1.15 mmol) was
heated to 60 ꢀC for 4 h and allowed to stir at 23 ꢀC overnight. The
reaction mixture was concentrated and purified by silica chroma-
tography (3:97 MeOH:CH2Cl2) to give 370 mg (98% yield) of
20
product as a clear oil that solidified upon refrigeration. [R]D þ0.9
(c 0.14, CHCl3). 1H NMR (CDCl3, 400 MHz) δ 0.90 (s, 6H), 1.36
(s, 9H), 2.01 (d, J = 7.6 Hz, 2H), 2.35 (s, 2H), 2.69 (d, J = 4.8 Hz,
2H), 2.87 (dd, J = 8, 14.8 Hz, 1H), 2.98 (dd, J = 4.8, 9.2 Hz, 1H),
3.45 (m, 1H), 3.82 (m, 1H), 4.79 (d, J = 9.2 Hz, 1H), 5.03 (m, 2H),
5.81 (m, 1H), 7.26 (m, 5H). 13C NMR (CDCl3, 100 MHz) δ 25.4,
25.4, 29.6, 34.3, 36.8, 44.5, 52.1, 54.3, 60.1, 70.3, 79.1, 116.9, 126.2,
128.3, 129.4, 135.2, 137.9, 155.8. FTIR (NaCl) νmax = 3349, 3064,
2928, 1693, 1391, 1366, cm-1. ESI LRMS (m/z): 376.06 [M þ H]þ.
tert-Butyl-(2S,3R)-4-(N-(2,2-dimethylpent-4-enyl)-2-(hex-5-
enyloxy)-4-methoxyphenylsulfonamido)-3-hydroxy-1-phenylbu-
tan-2-ylcarbamate (18a). To a mixture of amine 17a (188 mg,
0.5 mmol) and sulfonyl chloride 7d (183 mg, 0.6 mmol) at 0 ꢀC
under argon atmosphere was added pyridine (8 mL, freshly
distilled over KOH), and the reaction was allowed to warm to
23 ꢀC while stirring. The reaction turned orange and was
allowed to stir overnight. The reaction was condensed under
reduced pressure, washed with saturated CuSO4, and the
product extracted with dichloromethane. The organic layer
was washed with H2O, brine, dried over sodium sulfate, and
purified by silica chromatography (20:80 EtOAc:hexanes) to
20
give 18a (180 mg, 56% yield) as a clear oil. [R]D þ18.3 (c 0.12,
1
CHCl3). H NMR (CDCl3, 400 MHz) δ 0.90 (s, 3H), 0.92 (s,
3H), 1.25 (m, 1H), 1.31 (s, 9H), 1.59 (m, 2H), 1.86 (quintet, J =
8 Hz, 2H), 1.99 (d, J = 7.2 Hz, 2H), 2.12 (q, J = 6.8 Hz, 2H),
2.77 (dd, J=8, 13.2 Hz, 1H), 2.86 (dd, J = 4.8, 14.4 Hz, 1H),
3.20 (m, 4H), 3.66 (m, 1H), 3.80 (m, 1H), 3.83 (s, 3H), 4.00 (m,
2H), 4.40 (d, J = 8.8 Hz, 1H), 5.00 (m, 4H), 5.79 (m, 2H), 6.47
(m, 2H), 7.20 (m, 5H),7.78 (d, J = 8.4 Hz, 1H). 13C NMR
(CDCl3, 100 MHz) δ 25.0, 25.5, 25.6, 28.0, 28,2, 28.4, 33.2, 35.8,
45.2, 54.2, 55.5, 55.6, 62.4, 69.1, 72.0, 79.3, 100.1, 104.2, 115.1,
117.5, 118.5, 126.2, 128.3, 129.4, 134.0, 134.7, 137.6, 138.0,
155.5, 157.5, 164.8. FTIR (NaCl) νmax=3412, 2852, 1701, 1596,
1496, 1456, 1391, 1367 cm-1. ESI (þ) HRMS (m/z): [M þ Naþ]
calcd for C35H52N2O7S 667.3393; found, 667.3399.
Inhibitor 15f. Title compound was obtained from 14f as
described for 15a in 93% yield after flash-chromatography
(2:3 hexanes:EtOAc) as a white solid. 1H NMR (500 MHz,
CDCl3) δ 1.33-1.48 (m, 4H), 1.56-1.66 (m, 3H), 1.67-1.71 (m,
2H), 1.83-1.86 (m, 1H), 2.04-2.12 (m, 2H), 2.74 (dd, J = 9.7,
14 Hz, 1H), 2.83-2.99 (m, 4H), 3.18 (br s, 1H), 3.64-3.71 (m,
2H), 3.76-3.85 (m, 6H), 3.89-3.99 (m, 3H), 4.08 (br s, 1H),
4.19-4.23 (m, 1H), 4.96-5.00 (m, 2H), 5.63 (d, J=5 Hz, 1H),
6.45 (d, J=2 Hz, 1H), 6.50 (dd, J=2.5, 9 Hz, 1H), 7.15-7.26
(m, 5H), 7.88 (d, J=9 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ
22.2, 24.4, 24.6, 25.7, 25.8, 35.4, 45.2, 46.8, 51.8, 54.7, 55.7,
69.2, 69.6, 70.5, 70.8, 73.3, 99.8, 103.9, 109.2, 117.5, 126.5,
128.4, 129.3, 134.3, 137.4, 155.2, 157.8, 165.3. ESI (þ) HRMS
(m/z): [M þ Na]þ calcd for C30H40N2O9S, 605.2533; found,
605.2526.
(3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl (2S,3R)-4-(N-(2,2-
Dimethylpent-4-enyl)-2-(hex-5-enyloxy)-4-methoxyphenylsulfona-
mido)-3-hydroxy-1-phenylbutan-2-ylcarbamate (19a). To a stirring

7702 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
solution of 18a (180 mg, 0.28 mmol) in CH2Cl2 (5 mL) at 0 ꢀC was
added trifluoroacetic acid (1.5 mL). The reaction was allowed to
warm to 23 ꢀC and stirred overnight. Solvents were removed
under reduced pressure, and saturated aqueous NaHCO3 and 1N
NaOH (1 mL) was added and extracted with ether. Solvents were
removed under reduced pressure to afford the crude amine
product.
2H), 7.81 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ
164.9, 157.9, 155.1, 137.5, 134.8, 131.0, 129.3, 128.4, 126.7,
126.5, 117.3, 109.3, 104.1, 100.2, 73.3, 72.6, 70.7, 69.6, 68.8,
64.1, 56.1, 55.7, 54.7, 45.3, 40.5, 36.1, 35.6, 29.7, 27.4, 26.7, 26.1,
25.8, 25.3. FTIR (film, NaCl) νmax =3344, 2925, 1718, 1595,
1575, 1388 cm-1. [M þ Na]þ calcd for C35H48N2O9S 695.2978;
found, 695.2970.
To a solution of above amine (0.28 mmol) in MeCN (20 mL)
was added carbonate 12 (92 mg, 0.31 mmol) under argon
followed by dropwise addition of i-Pr2NEt (1 mL) and pyridine
(1 mL), and the reaction was allowed to stir overnight at 23 ꢀC.
After stirring for 2 days, the solvent was removed under reduced
pressure and the crude material purified by silica gel column
chromatography (50:50 EtOAc:hexanes) to give 19a (144 mg,
Compound 22a. The corresponding olefin 20a or 21a (22 mg,
0.032 mmol) was dissolved in ethyl acetate (5 mL). Pd/C (10%)
was added and the reaction flask flushed with H2 gas. After
stirring overnight, the reaction was filtered over celite and
purified by silica chromatography (50:50 f 75:25 EtOAc:
hexanes) to give 19 mg (90% yield) of the desired product.
[R]D20 -2.9 (c 0.70, CHCl3). 1H NMR (CDCl3, 800 MHz) δ 0.97
(s, 3H), 1.03 (s, 3H), 1.18 (m, 1H), 1.27 (m, 2H), 1.38 (m, 3H),
1.45 (m, 4H), 1.54-1.64 (m, 3H), 1.69 (m, 1H), 1.81 (m, 2H),
2.71 (dd, J = 9.6, 14.4 Hz, 1H), 2.87 (m, 1H), 2.98 (dd, J=4, 14.4
Hz, 1H), 3.01 (dd, J = 1.6, 15.2 Hz, 1H), 3.19 (dd, J=9.6, 15.2
Hz, 1H), 3.63-3.70 (m, 2H), 3.80 (m, 1H), 3.82 (dt, J = 1.6, 8
Hz, 1H), 3.86 (s, 3H), 3.93 (dd, J=6.4, 9.6 Hz, 1H), 3.94 (m, 1H),
4.07 (m, 1H), 4.10 (m, 1H), 4.75 (d, J = 9.6 Hz, 1H), 4.98 (q, J =
5.6 Hz, 1H), 5.63 (d, J=4.8 Hz, 1H), 6.51 (m, 2H), 7.15 (d, J =
7.2 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.24 (t, J = 8 Hz, 2H), 7.82
(d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ 19.6, 22.3,
25.0, 25.4, 25.8, 26.2, 27.4, 28.3, 35.7, 35.8, 39.8, 45.3, 54.8, 55.7,
56.1, 61.8, 68.9, 69.6, 70.7, 72.5, 73.3, 100.2, 104.0, 109.3, 117.7,
126.5, 128.4, 129.3, 135.6, 137.5, 155.1, 158.1, 164.9. FTIR
(NaCl) νmax = 3449, 2930, 1718, 1596, 1534 cm-1. ESI (þ)
HRMS (m/z): [MþH]þ calcd for C35H50N2O9S 675.3315; found,
675.3318.
20
73% yield over two steps) as a white solid; mp=49-52 ꢀC; [R]D
1
þ3.9 (c 1.03, CHCl3). H NMR (CDCl3, 400 MHz) δ 0.90 (s,
3H), 0.91 (s, 3H), 1.35 (m, 1H), 1.58 (m, 3H), 1.86 (m, 2H), 2.02
(m, 3H), 2.11 (m, 2H), 2.69 (dd, J = 9.6, 14 Hz, 1H), 2.84 (m,
1H), 2.94 (dd, J = 4, 14.4 Hz, 1H), 3.08 (m, 2H), 3.29 (m, 2H),
3.63 (m, 2H), 3.78 (m, 2H), 3.83 (s, 3H), 3.91 (m, 3H), 4.03 (m,
2H), 4.87 (d, J = 9.6 Hz, 1H), 5.02 (m, 4H), 5.60 (d, J = 5.2 Hz,
1H), 5.77 (m, 2H), 6.48 (m, 2H), 7.18 (m, 5H), 7.79 (d, J = 8.4
Hz, 1H). 13C NMR (CDCl3, 100 MHz) δ 24.9, 25.4, 25.5, 25.7,
28.3, 33.2,35.5, 35.7, 45.1, 45.2, 54.8, 55.6, 55.6, 62.6, 69.2, 69.5,
70.6, 72.3, 73.1, 100.1, 104.3, 109.2, 115.0, 117.6, 118.0, 126.3,
128.3, 129.2, 134.0, 134.5, 137.5, 137.9, 155.0, 157.6, 164.9.
FTIR (NaCl) νmax =1323, 1595, 1722, 2922, 3487 cm-1. ESI
(þ) HRMS (m/z): [M þ H]þ calcd for C37H52N2O9S 701.3472;
found, 701.3473.
Inhibitors 20a and 21a. Compound 19a (100 mg, 0.14 mmol)
was dissolved in CH2Cl2 (90 mL). Grubbs’ second-generation
catalyst (12 mg, 0.014 mmol) was added, and the reaction was
allowed to stir overnight at 23 ꢀC. Solvent was removed under
reduced pressure and the material purified by silica gel column
chromatography (50:50 f 75:25 EtOAc:hexanes) to give 92 mg
(96% yield) of product as a mixture of stereoisomers (31:69 Z/E
by HPLC) as a white solid. The individual stereoisomers were
isolated by reversed-phase HPLC YMC-Pack ODSA (250 mm
ꢀ 10 mm, 5 μm); flow rate=1.5 mL/min; isocratic 80:20 MeOH:
H2O; T=25 ꢀC; λ = 210 nm, Rt Z=17 min, Rt E = 18 min).
tert-Butyl-(2S,3R)-3-hydroxy-4-(2-methylpent-4-enylamino)-
1-phenylbutan-2-ylcarbamate (17b and 17c). A mixture of 2-
methylpent-4-en-1-amine (460 mg, 4.6 mmol) and epoxide 8
(361.5 mg, 1.3 mmol) was allowed to stir at 60 ꢀC overnight
under argon atmosphere. The crude material was purified by
silica gel column chromatography (3:97 MeOH:CH2Cl2) to give
17b and 17c (470 mg, 95%) as a white solid as a mix of
20
diastereomers (50:50 by NMR); mp 89-90 ꢀC; [R]D þ5.4
(c 2.58, CHCl3). 1H NMR (CDCl3, 400 MHz) δ 0.91 (d, J = 2
Hz, 3H), 0.92 (d, J=2.4 Hz, 3H), 1.35 (s, 18H), 1.67 (m, 2H), 1.92
(m, 2H), 2.13 (m, 2H), 2.40 (dd, J=6.8, 11.6 Hz, 2H), 2.52 (dd,
J = 6, 12 Hz, 2H), 2.60-3.01 (m, 10H), 3.45 (m, 2H), 3.59-3.45
(m, 4H), 4.55-4.77 (m, 2H), 5.04 (m, 4H), 5.77 (m, 2H), 7.22 (m,
6H), 7.29 (m, 4H). 13C NMR (CDCl3, 100 MHz) δ 17.8, 28.3,
33.2, 36.7, 39.2, 39.2, 51.4, 51.4, 54.1, 55.7, 70.6, 79.3, 116.0,
Compound 21a. [R]D -0.8 (c 2.36, CHCl3). 1H NMR
20
(CDCl3, 800 MHz) δ 1.05 (s, 3H), 1.12 (s, 3H), 1.24 (t, J=6.4
Hz, 1H), 1.25 (s, 1H), 1.39 (m, 1H), 1.58 (m, 1H), 1.90-1.72 (m,
4H), 1.94-2.10 (m, 4H), 2.68 (dd, J = 9.6, 14.4 Hz, 1H), 2.86 (q,
J = 7.2 Hz, 1H), 2.92 (dd, J = 4, 14.4 Hz, 1H), 2.97 (dd, J = 2.4,
15.2 Hz, 1H), 3.11 (dd, J = 9.6, 15.2 Hz, 1H), 3.64 (m, 1H), 3.68
(m, 1H), 3.71 (dd, J = 6.4, 13.6 Hz, 1H), 3.77 (m, 1H), 3.82 (m,
1H), 3.84 (s, 3H), 4.04-3.88 (m, 4H), 4.71 (d, J = 9.6 Hz, 1H),
4.98 (q, J = 7.2 Hz, 1H), 5.60 (d, J = 5.6 Hz, 1H), 5.62 (d, J=5.6
Hz, 1H), 5.68 (m, 1H), 6.47 (m, 1H), 6.50 (dd, J = 1.6, 8.8 Hz,
1H), 7.12 (d, J = 7.2 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.24 (t,
J = 8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 125
MHz) δ 25.7, 26.1, 27.6, 29.2, 29.6, 29.7, 35.6, 35.8, 45.3, 46.1,
54.7, 55.7, 56.1, 63.0, 69.6, 69.7, 70.7, 72.2, 73.3, 100.6, 104.4,
109.3, 117.8, 126.5, 128.4, 128.5, 129.3, 133.6, 134.5, 137.5,
155.1, 158.1, 164.9. FTIR (film, NaCl) νmax = 3445, 2926,
1720, 1596, 1575, 1469, 1369 cm-1. ESI (þ) HRMS (m/z): [M
þ Na]þ calcd for C35H48N2O9S 695.2978; found, 695.2989.
126.3, 128.4, 129.5, 137.0, 137.9, 155.9. FTIR (NaCl) νmax
=
3365, 1683, 1520, 1455, 1391, cm-1. ESI (þ) LRMS m/z (relative
intensity): 363.03 [M þ H]þ.
Pure 17c was prepared in a similar fashion by using enantio-
pure (S)-2-methylpent-4-en-1-amine. 1H NMR (CDCl3, 400
MHz) δ 0.99 (d, J = 6 Hz, 3H, 1.32 (s, 9H), 1.99 (m, 2H), 2.12
(m, 1H), 2.70-2.95 (m, 4H), 3.08 (m, 2H), 3.80 (m, 2H), 5.04 (s,
1H), 5.07 (m, 1H), 5.71 (m, 1H), 6.41 (br s, 3H),7.15-7.34 (m, 5H).
tert-Butyl-(2S,3R)-4-(2-(hex-5-enyloxy)-4-methoxy-N-(2-meth-
ylpent-4-enyl)phenylsulfonamido)-3-hydroxy-1-phenylbutan-2-yl-
carbamate (18b and 18c). A mixture of amines 17b and 17c (166
mg, 0.46 mmol) and sulfonyl chloride 7d (167 mg, 0.55 mmol)
were cooled to 0 ꢀC. Pyridine (5 mL) was added and the solution
stirred overnight. Solvents were removed by reduced pressure
and the crude material purified by silica gel column chromatog-
raphy (20:80 EtOAc:hexanes) to give a mixture of 18b and 18c
(148 mg, 51%yield) asa colorless oil. [R]D20 -1.8(c 0.67, CHCl3).
1H NMR (CDCl3, 300 MHz) δ 0.80 (d, J=6 Hz, 3H), 0.88 (d, J =
6 Hz, 3H), 1.33 (s, 18H), 1.52-1.65 (m, 4H), 1.70-1.92 (m, 9H),
2.06-2.26 (m, 6H), 3.36-2.80 (m, 12H), 3.73 (br, 4H), 3.84 (s,
6H), 4.06-3.94 (m, 5H), 4.59 (m, 2H), 4.90-5.06 (m, 8H),
5.58-5.87 (m, 4H), 6.44-6.54 (m, 4H), 7.16-7.30 (m, 10H),
7.83 (d, J=8.7 Hz, 2H). 13C NMR (CDCl3, 75 MHz) δ 17.1, 25.0,
28.2, 28.3, 31.7, 31.9, 33.2, 35.3, 38.7, 53.2, 53.3, 54.4, 54.6, 55.6,
56.8, 57.1, 69.1, 2.3, 72.7, 79.4, 100.2, 104.1, 115.1, 116.3, 119.0,
Compound 20a. [R]D -0.5 (c 0.99, CHCl3). 1H NMR
20
(CDCl3, 800 MHz) δ 1.07 (s, 3H), 1.13 (s, 3H), 1.25 (s, 3H),
1.38 (m, 1H), 1.57 (m, 1H), 1.62-1.76 (m, 2H), 1.90 (br s, 2H),
1.98-2.20 (m, 4H), 2.70 (dd, J = 9.6, 14.4 Hz, 1H), 2.86 (m, 1H),
2.91 (dd, J = 0.8, 15.2 Hz, 1H), 2.93 (dd, J = 4, 14.4 Hz, 1H),
3.08 (dd, J = 8.8, 15.2 Hz, 1H), 3.65 (m, 1H), 3.68 (dd, J = 6.4,
9.6 Hz, 1H), 3.79 (m, 1H), 3.82 (dt, J = 2.4, 8.8 Hz, 1H), 3.85 (s,
3H), 3.93 (dd, J = 6.4, 9.6 Hz, 1H), 3.96 (m, 1H), 4.08 (t, J = 4.8
Hz, 2H), 4.74 (d, J = 9.6 Hz, 1H), 4.98 (q, J = 5.6 Hz, 1H), 5.53
(q, J = 8.8 Hz, 1H), 5.62 (d, J = 5.6 Hz, 1H), 5.65 (q, J = 8.8 Hz,
1H), 6.47 (d, J = 2.4 Hz, 1H), 6.51 (dd, J = 2.4, 8.8 Hz, 1H), 7.13
(d, J = 7.2 Hz, 2H), 7.18 (d, J = 7.2 Hz, 1H), 7.23 (t, J = 8 Hz,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7703
Compound 20b. [R]D -29 (c 0.60, CHCl3). 1H NMR
20
126.3, 128.3, 129.5, 133.7, 136.3, 137.7, 137.9, 138.1, 155.9, 157.6,
164.7. FTIR (NaCl) νmax=3392, 2929, 1702, 1640, 1445, 1391,
1366, cm-1. ESI (þ) HRMS (m/z): [M þ H]þ calcd for
C34H50N2O7S 631.3417; found, 631.3408.
(CDCl3, 800 MHz) δ 1.12 (d, J = 6.4 Hz, 3H) 1.50-1.62(m,
2H), 1.73 (m, 2H), 1.86 (m, 1H), 1.95 (m, 1H), 2.01-2.11 (m,
3H), 2.18 (m, 1H), 2.74 (dd, J = 9.6, 14.4 Hz, 1H), 2.81 (d, J =
15.2 Hz, 1H), 2.87 (m, 1H), 2.99 (dt, J=4, 13.6 Hz, 2H), 3.16 (dd,
J = 9.6, 15.2 Hz, 1H), 3.65-3.75 (m, 5H), 3.79-3.85 (m, 2H),
3.85 (s, 3H), 3.93 (dd, J = 6.4, 9.6 Hz, 1H), 4.11 (m, 2H), 4.25 (m,
1H), 4.84 (d, J = 9.6 Hz, 1H), 4.99 (q, J = 6.4 Hz, 1H), 5.44 (q,
J=9.6 Hz, 1H), 5.57 (q, J=7.2 Hz, 1H), 5.63 (d, J = 4.8 Hz, 1H),
6.48 (d, J = 1.6 Hz, 1H), 6.50 (dd, J = 2.4, 8.8 Hz, 1H), 7.17 (m,
3H), 7.24 (m, 2H), 7.86 (d, J=8.8 Hz, 1H). 13C NMR (CDCl3,
125 MHz) δ 17.4, 25.4, 25.8, 26.1, 27.5, 32.2, 32.8, 35.7, 45.3,
53.9, 54.7, 55.7, 59.1, 68.4, 69.6, 70.7, 73.0, 73.3, 100.3, 103.9,
109.3, 118.1, 126.5, 127.2, 128.4, 129.4, 131.1, 134.4, 137.5,
155.2, 157.8, 164.9. FTIR (film, NaCl) νmax = 3467, 2923,
1717, 1595, 1444, 1384, 1256 cm-1. ESI (þ) HRMS (m/z):
[M þ Na]þ calcd for C34H46N2O9S 681.2822; found, 681.2829.
Pure 18c was prepared in a similar fashion using pure 17c. 1H
NMR (CDCl3, 400 MHz) δ 0.80 (d, J = 6 Hz, 3H), 1.34 (s, 9H),
1.59 (m, 2H), 1.74 (m, 2H), 1.87 (m, 2H), 2.12 (q, J=7.2 Hz, 2H),
2.19 (m, 1H), 2.94 (m, 3H), 3.25 (m, 3H), 3.74 (m, 2H), 3.85 (s,
3H), 3.96 (m, 1H), 4.03 (t, J=6.8 Hz, 2H), 4.57 (d, J=7.2 Hz,
1H), 4.93-5.05 (m, 4H), 5.66 (m, 1H), 5.80 (m, 1H), 6.46 (d, J=
2.4 Hz, 1H), 6.50 (dd, J = 2.4, 8.8 Hz, 1H), 7.20 (m, 3H), 7.27
(m, 2H), 7.84 (d, J = 8.4 Hz, 1H). 13C NMR (CDCl3, 100 MHz)
δ 17.1, 25.0, 28.2, 28.4, 31.8, 33.3, 35.2, 38.1, 53.2, 54.5, 55.7,
56.9, 9.1, 72.3, 79.5, 100.2, 104.1, 115.1, 116.3, 119.1, 126.3,
128.4, 129.6, 133.7, 136.4, 137.7, 138.1, 155.9, 157.6, 164.7.
(3R,3aS,6aR)-Hexahydrofuro[2,3-b]furan-3-yl (2S,3R)-4-(2-(hex-
5-enyloxy)-4-methoxy-N-(2-methylpent-4-enyl)phenylsulfonamido)-
3-hydroxy-1-phenylbutan-2-ylcarbamate (19b and 19c). To a stirred
solution of 18b and 18c (140 mg, 0.22 mmol) in CH2Cl2 (4.5 mL)
was added TFA (1.5 mL). The resulting mixture was stirred for 4 h
and then quenched with saturated aqueous NaHCO3 (1.5 mL).
Then 2N NaOH was added until the solution turned basic. The
aqueous layer was extracted with ether, washed with brine, and
dried over Na2SO4. Solvents were removed under reduced pressure
to give crude N-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyl)-
2-(hex-5-enyloxy)-4-methoxy-N-(2-methylpent-4-enyl)benzensul-
fonamide.
Compound 20c. [R]D -30.6 (c 0.83, CHCl3). 1H NMR
20
(CDCl3, 800 MHz) δ 1.13 (d, J = 7.2 Hz, 3H) 1.61 (m, 1H),
1.69 (m, 1H), 1.78-1.90 (m, 4H), 1.94-2.09 (m, 5H), 2.18 (m,
1H), 2.74 (dd, J=8.8, 13.6 Hz, 1H), 2.89 (m, 2H), 2.96 (dd, J=
3.2, 13.6 Hz, 1H), 3.11 (dd, J=8, 14.4 Hz, 1H), 3.65-3.74 (m,
3H), 3.78-3.86 (m, 6H), 3.88-4.02 (m, 4H), 4.84 (d, J=8.8 Hz,
1H), 5.00 (q, J = 5.6 Hz, 1H), 5.52 (m, 1H), 5.64 (d, J = 4.8 Hz,
1H), 5.66 (m, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.50 (dd, J=1.6, 8.8
Hz, 1H), 7.17 (m, 3H), 7.25 (m, 2H), 7.83 (d, J = 8.8 Hz, 1H).
13C NMR (CDCl3, 125 MHz) δ 18.4, 25.8, 26.2, 28.6, 29.7, 30.0,
32.3, 35.4, 37.9, 45.3, 54.4, 54.8, 55.7, 58.1, 58.5, 69.6, 70.8, 72.6,
73.4, 100.7, 104.3, 109.3, 118.8, 126.5, 128.1, 128.5, 129.3, 133.3,
134.0, 137.6, 155.3, 158.1, 164.8. FTIR (NaCl) νmax = 3442,
3339, 2924, 1717, 1595, 1495, 1444, 1325, 1256, 1206 cm-1. ESI
(þ) HRMS (m/z): [M þ Na]þ calcd for C34H46N2O9S 681.2822;
found, 681.2825.
To the above crude amine in MeCN (5 mL) were added
(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl
4-nitrophenyl
carbonate 12 (71 mg, 0.24 mmoL) and i-Pr2NEt (0.75 mL)
under argon atmosphere and the reaction stirred overnight.
Solvents were removed under reduced pressure and the crude
material purified by silica gel column chromatography (50:50
EtOAc:hexanes) to give 68 mg (45% yield) of product as a clear
oil. [R]D20 -5.7 (c 0.17, CHCl3). 1H NMR (CDCl3, 400 MHz) δ
0.80 (d, J = 6.4 Hz, 3H) 0.90 (d, J = 6.4 Hz, 3H), 1.40-2.24 (m,
22H), 2.70-3.40 (m, 14H), 3.62-4.06 (m, 24H), 4.92-5.05 (m,
12H), 5.60-5.85 (m, 6H), 6.50 (m, 4H), 7.18 (m, 6H), 7.24 (m,
4H), 7.83 (d, J=8.8 Hz, 2H). 13C NMR (CDCl3, 100 MHz) δ
17.1, 25.0, 25.7, 28.4, 31.9, 32.1, 33.2, 35.4, 38.1, 38.7, 45.3, 53.2,
53.4, 54.9, 55.0, 55.7, 56.9, 57.3, 69.2, 69.6, 70.7, 72.3, 72.8, 73.3,
100.3, 104.2, 109.2, 115.1, 116.5, 118.7, 118.8, 126.5, 128.4,
129.3, 133.8, 136.2, 137.5, 137.6, 138.0, 155.3, 157.6, 164.8.
FTIR (film, NaCl) νmax=3436, 2970, 2927, 1718, 1600, 1458,
1374 cm-1. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for
C36H50N2O9S 709.3135; found, 709.3131.
20
Compound 21b. [R]D þ27.6 (c 0.36, CHCl3). 1H NMR
(CDCl3, 800 MHz) δ 1.09 (d, J=6.4 Hz, 3H) 1.50-1.60 (m,
2H), 1.72 (m, 1H), 1.86 (m, 2H), 1.94-2.12 (m, 4H), 2.18 (m,
1H), 2.78 (dd, J = 10.4, 14.4 Hz, 1H), 2.90 (m, 2H), 3.02 (dd,
J=4.8, 14.4 Hz, 1H), 3.12 (dd, J = 4, 14.4 Hz, 1H), 3.17 (m, 2H),
3.46 (m, 1H), 3.64-3.71 (m, 3H), 3.84-3.90 (m, 5H), 3.96 (m,
2H), 4.12 (m, 1H), 4.15 (m, 1H), 4.88 (d, J = 8.8 Hz, 1H), 5.00 (q,
J = 8 Hz, 1H), 5.47 (q, J = 8.8 Hz, 1H), 5.57 (q, J=7.2 Hz, 1H),
5.64 (d, J=5.6 Hz, 1H), 6.49 (m, 2H), 7.20 (m, 3H), 7.27 (m, 2H),
7.83 (d, J = 8.8 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ 18.0,
25.3, 25.8, 26.0, 27.3, 32.2, 32.7, 35.4, 45.3, 53.3, 55.1, 55.7, 58.0,
68.4, 69.6, 70.9, 72.3, 73.4, 100.2, 104.0, 109.3, 118.3, 126.5,
127.6, 128.5, 129.3, 130.8, 134.3, 137.7, 155.6, 157.8, 164.8.
FTIR (film, NaCl) νmax=3467, 2927, 1717, 1595, 1456, 1325,
cm-1. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for C34H46N2O9S
681.2822; found, 681.2819.
Pure 19c was prepared in a similar fashion using pure 18c. 1H
NMR (CDCl3, 400 MHz) δ 0.81 (d, J=6 Hz, 3H), 1.45-1.90 (m,
8H), 2.12 (m, 2H), 2.23 (m, 1H), 2.80 (dd, J=9.2, 14 Hz, 1H),
2.93 (m, 3H), 3.15 (dd, J = 2.4, 15.2 Hz, 1H), 3.24 (dd, J=8, 14
Hz, 1H), 3.38 (dd, J=9.2, 15.6 Hz, 1H), 3.65-3.75 (m, 3H),
3.77-3.89 (m, 6H), 3.95 (dd, J = 6.4, 9.6 Hz, 1H), 4.05 (t, J=6.8
Hz, 2H), 4.85-5.05 (m, 6H), 5.62-5.85 (m, 3H), 6.48 (d, J=2.4
Hz, 1H), 6.52 (dd, J = 2, 8.8 Hz, 1H), 7.20 (m, 3H), 7.26 (m, 2H),
7.84 (d, J = 8.8 Hz, 1H). ESI (þ) HRMS (m/z): [M þ Na]þ calcd
for C36H50N2O9S 709.3135; found, 709.3139.
Inhibitors 20b, 20c, 21b, and 21c. To a solution of carbamates
19b and 19c (119 mg, 0.17 mmol) in CH2Cl2 (125 mL) was
added Grubb’s second-generation catalyst (15 mg, 0.02
mmol). The resulting solution was stirred overnight. The
solvent was removed under reduced pressure and the crude
material purified by silica gel chromatography (50:50 EtOAc:
hexanes) to give 110 mg (97% yield) of product as an oil.
Reversed-phase HPLC (Waters Sunfire C18 50 mm ꢀ 4.6 mm,
5 μm coupled to Agilent Eclipse XDB C18 150 mm ꢀ 4.6 mm,
5 μm and YMC-Pack C8 250 mm ꢀ 4.6 mm, 5 μm, flow rate =
0.95 mL/min, λ = 215 nm, T = 30 ꢀC, isocratic 60:40
MeCN:H2O) was used to isolate the individual isomers: Rt
(RZ)=22 min, Rt (SZ)=24 min, Rt (RE)=25.3 min, Rt (SE)=
26.8 min.
20
Compound 21c. [R]D þ20.5 (c 1.17, CHCl3). 1H NMR
(CDCl3, 800 MHz) δ 1.06 (d, J=6.4 Hz, 3H), 1.47 (m, 1H),
1.63 (m, 1H), 1.70-1.82 (m, 4H), 1.87 (m, 1H), 1.93 (m, 1H),
2.17 (m, 2H), 2.26 (m, 1H), 2.47 (br, 1H), 2.76 (dd, J = 8.8, 14.4
Hz, 1H), 2.91 (m, 2H), 2.98 (m, 2H), 3.55 (m, 1H), 3.69 (m, 2H),
3.75 (m, 1H), 3.80 (m, 1H), 3.85 (m, 4H), 3.88 (t, J = 8.8 Hz,
1H), 3.94 (dd, J = 6.4, 9.6 Hz, 1H), 4.11 (m, 1H), 4.17 (br, 1H),
4.76 (d, J = 9.6 Hz, 1H), 5.00 (q, J = 8 Hz, 1H), 5.53 (m, 1H),
5.64 (d, J = 4.8 Hz, 1H), 5.67 (m, 1H), 6.47 (d, J = 2.4 Hz, 1H),
6.50 (dd, J=2.4, 8.8 Hz, 1H), 7.14 (d, J=7.2 Hz, 2H), 7.18 (m,
1H), 7.24 (t, J = 7.2 Hz, 2H),7.83 (d, J = 8.8 Hz, 1H). 13C NMR
(CDCl3, 125 MHz) δ 19.0, 25.8, 26.2, 28.1, 29.8, 30.3, 33.6, 35.7,
38.2, 45.3, 54.6, 54.9, 55.7, 58.6, 69.6, 70.8, 71.5, 73.4, 100.5,
104.2, 109.3, 119.0, 126.5, 128.5, 129.4, 129.6, 132.5, 134.2,
137.4, 155.2, 158.1, 164.8. FTIR (film, NaCl) νmax = 3463,
3339, 1717, 1595, 1495, 1387, 1326 cm-1. ESI(þ) HRMS (m/z):
[M þ Na]þ calcd for C34H46N2O9S 681.2822; found, 681.2812.
Compound 22b. To a stirred solution of the corresponding
(R)-E olefin (8 mg, 0.013 mmol) was dissolved into EtOAc
(5 mL) a spatula tip of Pd on carbon (10 wt %) and the mixture
was stirred under H2 atmosphere. The reaction stirred overnight.

7704 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Ghosh et al.
Solvents were removed under reduced pressure, and the crude
material was purified by silica gel column chromatograph (70:30
Et2O:hexanes f 100% Et2O) to give 22b (8 mg, quantitative
The crystal structure and structure factors have been deposited in the
RCSB Protein Data Bank,34 with PDB code 3I6O for inhibitor 14c.
X-ray Structure for 2. The wild-type HIV-1 protease was
expressed and purified as described previously.35 After purifica-
tion, the protease was concentrated to 4.0 mg/mL. Inhibitor 2, at
a 10-fold molar excess, was mixed with the protease. The
mixture was incubated at room temperature for 2 h and then
clarified with a 0.2 mm spin filter. The crystallization trials
employed the hanging drop method using equal volumes of
enzyme-inhibitor and well solution. The reservoir contained
20% (NH4)2SO4, 200 mM NaPO4/citric acid buffer, pH 5.5. The
well solutions also included 10% dimethyl sulfoxide, 30 mM
β-mercaptoethanol, and 4% isopropyl alcohol.
Diffraction data were collected at room temperature with an
Raxis-IV image plate and integrated and reduced with the HKL
program package.25 The diffraction data set has a resolution of
1.7 A with an Rmerge value of 0.052. Data completeness was
95.5%. The space group was determined to be P212121 with unit
cell dimensions of a = 51.9 A, b=59.0 A, c = 62.6 A, with one
dimer in the asymmetric unit. The initial phase was solved by
molecular replacement using the program AMoRe.36 A pro-
tease dimer (PDB code 2AID)37 from a previously determined
HIV-1 protease crystal structure was used as the search model.
Crystallographic refinement was carried out using X-PLOR 3.1.
Molecular graphics program O31 was used for map display and
model building. Water molecules were added to the structure as
identified in the |Fo| - |Fc| map contoured at the 3σ level. Data
collection and refinement statistics are listed in Table 2
(Supporting Information). The final Rwork was 21.6% and Rfree
was 28.9% for data between the resolution of 20 and 1.7 A
(Table 1). The rmsd values from ideal bond distances and angles
were 0.009 A and 1.5ꢀ, respectively. The average B factor was
28.3 and 19.6 A2 for protein and inhibitor atoms, respectively,
and 41.0 A2 for solvent atoms. The crystal structures and
structure factors have been deposited in the RCSB Protein Data
Bank,34 with PDB code 3I7E for inhibitor 2.
23
yield) as a white solid. TLC 100% Et2O. [R]D -7.8 (c 0.84,
CHCl3). 1H NMR (CDCl3, 500 MHz) δ 0.92 (d, J=7 Hz, 3H)
1.16-1.84 (m, 15H), 2.74 (dd, J = 9.5, 14 Hz, 1H), 2.89 (m, 1H),
3.05 (m, 3H), 3.28 (m, 2H), 3.65-3.87 (m, 9H), 3.94 (m, 1H),
4.10 (m, 1H), 4.21 (m, 1H), 4.84 (d, J = 9.5 Hz, 1H), 5.01 (q, J=7
Hz, 1H), 5.64 (d, J=5.5 Hz, 1H), 6.51 (m, 2H), 7.19 (m, 3H), 7.26
(m, 2H),7.88 (d, J=9.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ
17.8, 23.4, 23.5, 25.5, 25.8, 26.1, 27.5, 31.8, 31.9, 35.7, 45.3, 52.5,
54.8, 55.2, 55., 68.8, 69.6, 70.7, 71.3, 73.3, 100.4, 104.0, 109.3,
118.0, 126.5, 128.5, 129.4, 134.4, 137.6, 155.2, 158.0, 165.0.
FTIR (NaCl) νmax=3463, 2924, 2853, 1721, 1596, 1323, 1256
cm-1. ESI(þ) HRMS (m/z): [M þ Na]þ calcd for C34H48N2O9S
683.2978; found, 683.2987.
Compound 22c. To a solution of the corresponding (S)-E olefin
(12 mg, 0.018 mmol) in EtOAc (5 mL) a spatula tip of Pd on
carbon (10 wt %) was added and the mixture was stirred under H2
atmosphere. The reaction was stirred overnight. Solvents were
removed under reduced pressure and the crude material purified
by silica chromatograph (70:30 Et2O:hexanes f 100% Et2O) to
give 22c (9 mg, 78% yield) as a white solid. [R]D23 -6.6 (c 0.91,
CHCl3). 1H NMR (CDCl3, 800 MHz) δ 0.90 (d, J = 6.4 Hz, 3H)
1.20-1.90 (m, 15H), 2.73 (m, 1H), 2.79 (dd, J=8.8, 13.6 Hz, 1H),
2.90 (m, 1H), 2.96 (m, 1H), 3.08 (m 1H), 3.33 (dd, J =9.6, 15.2 Hz,
1H), 3.47 (m, 1H), 3.61 (m, 1H), 3.70 (m, 2H), 3.85 (m, 6H), 3.95
(m, 1H), 4.10 (m, 1H), 4.25 (m, 1H), 4.86 (d, J =8.8 Hz, 1H), 5.03
(q, J = 7.2 Hz, 1H), 5.65 (d, J = 4.8 Hz, 1H), 6.52 (m, 2H), 7.18
(m, 3H), 7.25 (m, 2H), 7.87 (d, J=9.6 Hz, 1H). 13C NMR (CDCl3,
200 MHz) δ 18.4, 23.4, 24.3, 25.0, 25.8, 26.5, 27.2, 32.8, 33.1, 35.4,
45.3, 53.5, 54.8, 55.7, 57.0, 69.3, 69.6, 70.8, 72.2, 73.3, 100.6, 104.2,
109.3, 118.0, 126.5, 128.5, 129.4, 134.5, 137.5, 155.3, 158.1, 165.1.
FTIR (NaCl) νmax = 3463, 2854, 1717, 1596, 1444, 1323, 1256
cm-1. ESI (þ) HRMS (m/z): [M þ Na]þ calcd for C34H48N2O9S
683.2978; found, 683.2976.
Determination of X-ray Structures of HIV-1 Protease
(wt)-Inhibitor Complexes for Inhibitors 14c and 2. X-ray Struc-
ture for 14c. The HIV-1 protease construct with the substitutions
Q7K, L33I, L63I, C67A, and C95A to optimize protein stability
without altering protease structure and activity was expressed
and purified as described.23,24 Crystals were grown by the
hanging drop vapor diffusion method using 1:15 molar ratio
of protease at 2.0 mg/mL and inhibitor 14c dissolved in
dimethylsulfoxide. The reservoir contained 5% glycerol, 0.5 M
NaI in 0.2 M MES buffer with pH 6.0. Subsequently, crystals
were mounted on nylon loops in liquid nitrogen with additional
28% glycerol (v/v) as cryoprotectant. X-ray diffraction data
were collected on SER-CAT 22-ID beamline of the Advanced
Photon Source, Argonne National Laboratory, with wave-
Acknowledgment. The research was supported by grants
from the National Institutes of Health (GM53386, A.K.G.,
and GM062920, I.T.W.). This workwas 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 (Kosei
Rohdosho: H15-AIDS-001), and the Grant to the Coopera-
tive Research Project on Clinical and Epidemiological Studies
of Emerging and Reemerging Infectious Diseases (Renkei
Jigyo: no. 78, Kumamoto University) of Monbu-Kagakusho.
Wethank thestaff at the SER-CAT beamlineatthe Advanced
Photon Source, Argonne National Laboratory, for assistance
during X-ray data collection. Use of the Advanced Photon
Source was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under
contract no. DE-AC02-06CH11357. We thank Dr. John
Harwood, Purdue University, for many helpful discussions
surrounding NMR spectroscopy.
ꢀ
length 0.8 A under the stream of liquid nitrogen at 90 K. Data
were processed by HKL2000,25 resulting in Rmerge of 10.8% for
14c, and the structure was solved by molecular replacement with
our previous structure (PDB code 3B7V) using PHASER26 in
CCP4i Suite,27,28 refined by SHELX-97,29,30 and refitted manu-
ally with the graphic programs O10,31 and COOT.32 The
inhibitor structure was modeled by PRODRG-2,33 which also
provided restraints for refinement. The crystal structure of HIV-
1 protease with inhibitor 14c was determined at 1.17 A resolu-
tion. The rmsd for CR atoms in comparison with our wild type
protease with DRV (PDB code 2IEN19) is only 0.33 A . Alter-
nate conformations were modeled for the protease residues
when obvious in the electron density maps. Anisotropic atomic
displacement parameters (B factors) were applied for all atoms
including solvent molecules. The occupancies of iodine atoms were
refined and varied from 0.24 to 0.60. Hydrogen atoms were added
at the final stages of the refinement. The final structure includes
2 Naþ ions, 19 I- ions, 2 glycerols, and 181 waters in PR-14c
complex. The R factor and Rfree are 16.0 and 19.4%. The crystal-
lographic statistics are listed in Table 1 (Supporting Information).
Supporting Information Available: HPLC and HRMS data of
all inhibitors; crystallographic data collection and refinement
statistics. This material is available free of charge via the Internet
at http://pubs.acs.org.
References
(1) 2007 AIDS Epidemic Update; UNAIDS, WHO, December, 2007.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7705
(2) Spekowitz, K. A. AIDS;The First 20 Years. Engl. J. Med. 2001,
344, 1764–1772.
(3) Hertogs, K.; Bloor, S.; Kemp, S. D.; Van den Enyde, C.; Alcorn,
T. M.; Pauwels, R.; Van Houtte, M.; Staszewski, S.; Miller, V.;
Larder, B. A Phenotypic and genotypic analysis of clinical HIV-1
isolated reveals extensive protease inhibitor cross-resistance: a
survey of over 6000 samples. AIDS 2000, 14, 1203–1210.
Inhibitors That Combat Drug Resistance. ChemMedChem 2006,
1, 937–950.
(16) (a) Schwab, P.; France, M. B.; Ziller, J. W.; Grubbs, R. H. A
Series of Well-Defined Metathesis Catalysts;Synthesis of
[RuCl2(dCHR0)(PR3)2] and Its Reactions. Angew. Chem., Int.
Ed. Engl. 1995, 34, 2039–2041. (b) Scholl, M.; Ding, S.; Lee, C.;
Grubbs, R. H. Synthesis and Activity of a NewGeneration ofRuthenium-
Based Olefin Metathesis Catalysts Coordinated with 1,3-Dimesityl-4,5-
dihydroimidazol-2-ylidene Ligands. Org. Lett. 1999, 1, 953–956.
(17) Toth, M. V.; Marshall, G. R. A simple, continuous fluorometric
assay for HIV protease. Int. J. Pept. Protein Res. 1990, 36, 544–550.
(18) Gustchina, A.; Sansom, C.; Prevost, M.; Richelle, J.; Wodak, S.;
Wlodawer, A.; Weber, I. Energy calculations and analysis of HIV-1
protease-inhibitor crystal structures. Protein Eng. 1994, 7, 309–317.
(19) Tie, Y.; Boross, P. I.; Wang, Y. F.; Gaddis, L.; Liu, F.; Chen, X.;
Tozser, J.; Harrison, R. W.; Weber, I. T. Molecular basis for
substrate recognition and drug resistance from 1.1 to 1.6 A resolu-
tion crystal structures of HIV-1 protease mutants with substrate
analogs. FEBS J. 2005, 272, 5265–5277.
(4) (a) Ghosh, A. K.; Kincaid, J. F.; Cho, W.; Walters, D. E.;
Krishnan, K.; Hussain, K. A.; Koo, Y.; Cho, H.; Holland, J.;
Buthod, J. Potent HIV-1 inhibitors incorporating high-affinity P2-
ligands and (R)-(hydroxyethylamino)sulofonamide isostere.
Bioorg. Med. Chem. Lett. 1998, 8, 687–690. (b) Koh, Y.; Nakata,
H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J. F.;
Boross, P.; Wang, Y.-F.; Tie, Y.; Volarath, P.; Gaddis, L.; Harrison,
R. W.; Weber, I. T.; Ghosh, A. K.; Mitsuya, H. A novel bis-tetrahy-
drofuranylurethane-containing non-peptide protease inhibitor (PI) UIC-
94017 (TMC-114) potent against multi-PI-resistant HIV in vitro.
Antimicrob. Agents Chemother. 2003, 47, 3123–3129. (c) Ghosh,
A. K.; Pretzer, E.; Cho, H.; Hussain, K. A.; Duzgunes, N. Antiviral
activity of UIC-PI, a novel inhibitor of human immunodeficiency virus
type 1 protease. Antiviral Res. 2002, 54, 29–36.
(5) Surleraux, D. L. N. G.; Tahri, A.; Verchueren, W. G.; Pille, G. M.
E.; de Kock, H. A.; Jonckers, T. H. M.; Peeters, A.; De Meyer, S;
Azijn, H.; Pauwels, R.; de Bethune, M. -P.; King, N. M.; Prabu-
Jeyabalan, M.; Schiffer, C. A.; Wigerinck, P. B. T. P. Discovery and
selection of TMC-114, a next generation of HIV-1 protease in-
hibitor. J. Med. Chem. 2005, 48, 1813–1822.
(6) (a) Yoshimura, K.; Kato, R.; Kavlick, M. F.; Nguyen, A.; Maroun,
V.; Maeda, K.; Husssin, K. A.; Ghosh, A. K.; Gulnik, S. V.;
Erickson, J. W.; Mitsuya, H. A. A potent human immunodefi-
ciency virus type 1 protease inhibitor, UIC-94003 (TMC-126), and
selection of a novel (A28S) mutation in the protease active site.
J. Virol. 2002, 76, 1349–1358. (b) Koh, Y.; Nakata, H.; Maeda, K.;
Ogata, H.; Bilcer, G.; Devasamudram, T.; Kincaid, J. F.; Boross, P.;
Wang, Y.-F.; Tie, Y.; Volarath, P.; Gaddis, L.; Harrison, R. W.; Weber,
I. T.; Ghosh, A. K.; Mitsuya, H. A novel bis-tetrahydrofuranylurethane-
containing non-peptide protease inhibitor (PI) UIC-94017 (TMC-114)
potent against multi-PI-resistant HIV in vitro. Antimicrob. Agents
Chemother. 2003, 47, 3123–3129.
(20) Panigrahi, S.; Desiraju, G. Strong and weak hydrogen bonds in the
protein-ligand interface. Proteins 2007, 67, 128–141.
(21) Steiner, T. Hydrogen bonds from water molecules to aromatic
acceptors in very high-resolution protein crystal structures. Bio-
phys. Chem. 2002, 95, 195–201.
(22) Wang, Y. F.; Tie, Y.; Boross, P. I.; Tozser, J.; Ghosh, A. K.;
Harrison, R. W.; Weber, I. T. Potent new antiviral compound
shows similar inhibition and structural interactions with drug
resistant mutants and wild type HIV-1 protease. J. Med. Chem.
2007, 50, 4509–4515.
(23) Louis, J. M.; Clore, G. M.; Gronenborn, A. M. Autoprocessing of
HIV-1 protease is tightly coupled to protein folding. Nat. Struct.
Biol. 1999, 6, 868–875.
(24) Mahalingam, B.; Louis, J. M.; Hung, J.; Harrison, R. W.; Weber,
I. T. Structural implications of drug resistant mutants of HIV-1
protease: high resolution crystal structures of the mutant protease/
substrate analog complexes. Proteins 2001, 43, 455–464.
(25) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
Collected in Oscillation Mode. Methods in Enzymology, 276:
Macromolecular Crystallography, Part A; Carter, C.W., Jr., Sweet,
R. M., Eds.; Academic Press: New York, 1997; pp 307-326.
(26) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software.
J. Appl. Crystallogr. 2007, 40, 658–674.
(7) Ghosh, A. K.; Chapsal, B. D.; Weber, I. T.; Mitsuya, H. Design of
HIV protease inhibitors targeting protein backbone: an effective
strategy for combating drug resistance. Acc. Chem. Res. 2008, 41,
78–86.
(8) On June 23, 2006, FDA approved new HIV treatment for patients
who do not respond to existing drugs. Please see: http://www.fda.
gov/bbs/topics/NEWS/2006/NEW01395.html.
(9) On October 21, 2008, FDA granted traditional approval to Prezista
(darunavir), coadministered with ritonavir and with other antire-
troviral agents, for the treatment of HIV-1 infection in treatment-
experienced adult patients. In addition to the traditional approval,
(27) Collaborative Computational Project, Number 4 The CCP4 Suite:
Programs for Protein Crystallography. Acta Crystallogr., Sect. D:
Biol. Crystallogr., 1994, 50, 760-763.
(28) Potterton, E.; Briggs, P.; Turkenburg, M.; Dodson, E. A graphical
user interface to the CCP4 program suite. Acta Crystallogr., Sect.
D: Biol. Crystallogr. 2003, 59, 1131–1137.
(29) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr.,
a new dosing regimen for treatment-naıve patients was approved.
¨
Sect. A: Found. Crystallogr. 2008, 64, 112–122.
(10) Tie, Y.; Boross, P. I.; Wang, Y. F.; Gaddis, L.; Hussain, A. K.;
Leshchenko, S.; Ghosh, A. K.; Louis, J. M.; Harrison, R. W.;
Weber, I. T. High resolution crystal structures of HIV-1 protease
with a potent non-peptide inhibitor (UIC-94017) active against
multidrug-resistant clinical strains. J. Mol. Biol. 2004, 338,
341–352.
(11) Kovalevsky, A. Y.; Tie, Y.; Liu, F.; Boross, P. I.; Wang, Y. F.;
Leshchenko, S.; Ghosh, A. K.; Harrison, R. W.; Weber, I. T.
Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1
protease with highly drug resistant mutations D30N, I50 V, and
L90M. J. Med. Chem. 2006, 49, 1379–1387.
(12) Kempf, D. J.; Marsh, K. C.; Paul, D. A.; Knige, M. F.; Norbeck, D.
W.; Kohlbrenner, W. E.; Codacovi, L.; Vasavanonda, S.; Bryant,
P.; Wang, X. C.; Wideburg, N. E.; Clement, J. J.; Plattner, J. J.;
Erickson, J. Antiviral and Pharmacokinetic Properties of C2-
Symmetric Inhibitors of the Human Immunodeficiency Virus
Type 1 Protease. Antimicrob. Agents Chemother. 1991, 35, 2209–
2214.
(13) Baldwin, E. T.; Bhat, T. N.; Liu, B.; Pattabriaman, N.; Erickson,
J. W. Structural Basis of Drug Resistance for V82A mutant of HIV-
1 Proteinase. Nat. Struct. Biol. 1995, 2, 244–49.
(30) Sheldrick, G. M.; Schneider, T. R. SHELXL: high resolution
refinement. Methods Enzymol. 1997, 277, 319–343.
(31) Jones, T. A.; Zou, J. Y.; Cowan, S. W.; Kjeldgaard, M. Improved
methods for building protein models in electron density maps and
the location of errors in these models. Acta Crystallogr., Sect. A:
Found. Crystallogr. 1991, 47, 110–119.
(32) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for Molecular
Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60,
2126–2132.
(33) Schuettelkopf, A. W.; van Aalten, D. M. F. PRODRG;a tool for
high-throughput crystallography of protein-ligand complexes.”.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, 1355–1363.
(34) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data
Bank. Nucleic Acids Res. 2000, 28, 235–242.
(35) Hong, L.; Treharne, A.; Hartsuck, J. A.; Foundling, S.; Tang, J.
Crystal Structures of Complexes of a Peptidic Inhibitor with Wild-
Type and Two Mutant HIV-1 Protease. Biochemistry 1996, 35,
10627–10633.
(36) Navaja, J. A. MoRe: An Automated Package for Molecular
Replacement. Acta Crystallogr., Sect. A: Found. Crystallogr.
1994, 50, 157–163.
(37) Rutenber, E.; Fauman, E. B.; Keenan, R. J.; Fong, S.; Furth, P. S.;
Ortiz de Montellano, P. R.; Meng, E.; Kuntz, I. D.; DeCamp, D.
L.; Salto, R. Structure of a non-peptide inhibitor complexed with
HIV-1 protease. Developing a cycle of structure-based drug design.
J. Biol. Chem. 1993, 268, 15343–15346.
(14) Mitsunobu, O. The use of diethyl azodicarboxylate and triphenyl-
phosphine in synthesis and transformation of natural products.
Synthesis 1981, 1–28.
(15) Ghosh, A. K.; Sridhar, P. R.; Kumaragurubaran, N.; Koh, Y.;
Weber, I. T.; Mitsuya, H. Bis-Tetrahydrofuran: a Privileged Li-
gand for Darunavir and a New Generation of HIV Protease