Structure-based design of novel HIV-1 protease inhibitors to combat drug resistance

Article abstract of DOI:10.1021/jm060561m

Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b] furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[e]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 ? resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

Full text of DOI:10.1021/jm060561m

5252
J. Med. Chem. 2006, 49, 5252-5261
Structure-Based Design of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance
Arun K. Ghosh,*,† Perali Ramu Sridhar,† Sofiya Leshchenko,† Azhar K. Hussain,† Jianfeng Li,† Andrey Yu. Kovalevsky,|
D. Eric Walters,‡ Joseph E. Wedekind,‡,# Valerie Grum-Tokars,‡ Debananda Das,§ Yasuhiro Koh, Kenji Maeda,§
,§
Hiroyuki Gatanaga,§ Irene T. Weber,| and Hiroaki Mitsuya
Departments of Chemistry and Medicinal Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907; Department of Biology, Molecular
Basis of Disease, Georgia State UniVersity, Atlanta, Georgia 30303; Department of Biochemistry and Molecular Biology, Rosalind Franklin
UniVersity of Medicine and Science, North Chicago, Illinois 60064; Departments of Hematology and Infectious Diseases, Kumamoto UniVersity
School of Medicine, Kumamoto 860-8556, Japan; and Experimental RetroVirology Section, HIV and AIDS Malignancy Branch, National
Cancer Institute, Bethesda, Maryland 20892
ReceiVed May 11, 2006
Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are
designed specifically to interact with the backbone of HIV protease active site to combat drug resistance.
Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this
inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-
resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]-
furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active
(3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of
meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction
as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Å
resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding
interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug
resistance.
Introduction
The AIDS epidemic is one of the most challenging problems
in medicine in the 21st century.1 Among many strategies to
combat this disease, highly active antiretroviral therapy (HAART)
with HIV protease inhibitors (PI) in combination with reverse
transcriptase inhibitors continues to be the first line treatment
for control of HIV infection.2 This treatment regimen has
definitely improved quality of life, enhanced HIV management,
and halted the progression of the disease. Despite these
impressive successes, there are serious limitations including
major toxicity and complexity of these treatment regimens.
Perhaps the most serious problem is that a growing number of
patients are developing multi-drug-resistant strains of HIV, and
there is ample evidence that these strains can be transmitted.3,4
Thus far, no effective treatment options exist for these patients.
In this context, our research emphasis has been to design
nonpeptidyl inhibitors and optimize their potency against mutant
strains resistant to currently approved PIs.
We recently designed and developed a number of protease
Figure 1. Structure of inhibitors 1-4.
inhibitors with remarkable antiviral potency and drug-resistance
profiles.5,6 As shown in Figure 1, inhibitor 1 (now known as
tion for treatment of drug-resistant HIV.9 A high-resolution
X-ray crystal structure of 1-bound HIV protease has now
revealed critical molecular insight into interactions responsible
TMC-114, or Darunavir) has shown unprecedented picomolar
enzyme inhibitory activity and antiviral potency, favorable drug
resistance profiles against multi-drug-resistant HIV and encour-
for the observed resistance profiles.10 Structural analysis revealed
aging pharmacokinetic properties.7,8 This inhibitor has recently
that close contact of inhibitor 1 with the main chains of the
protease active site amino acids (Asp-29 and Asp-30) is critical
been approved by the United States Food and Drug Administra-
to its potency and wide-spectrum activity against multi-PI-
resistant HIV-1 variants. It appears that both P2- and P2′-ligands
* Corresponding author. Phone (765)-494-5323. Fax (765) 496-1612.
E-mail: akghosh@purdue.edu.
of inhibitor 1 are involved in extensive hydrogen bonding with
the protein backbone. Interestingly, examination of X-ray
structures of 1-bound protein-ligand complexes of wild-type
HIV protease and mutant HIV proteases also revealed only a
small distortion in the backbone conformations.10 This is also
evident in the X-ray structures of a number of other HIV
† Purdue University.
| Georgia State University.
‡ Rosalind Franklin University of Medicine and Science.
Kumamoto University School of Medicine.
§ HIV and AIDS Malignancy Branch, National Cancer Institute.
# Present address: Department of Biochemistry and Biophysics University
of Rochester School of Medicine and Dentistry Rochester, NY 14642.
10.1021/jm060561m CCC: $33.50 © 2006 American Chemical Society
Published on Web 08/02/2006

HIV-1 Protease Inhibitors To Combat Drug Resistance
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5253
Scheme 2. Synthesis of P2-ligand and Active Carbonate
Scheme 1. Synthesis of Hexahydrocyclopenta[b]furanyl
Carbonate
protease-inhibitor and related complexes.11,12 Our working
hypothesis to combat drug-resistance is to design inhibitors that
make maximum interactions in the active site of HIV protease,
particularly extensive hydrogen bonding interactions with the
protein backbone in both S2- and S2′-sites. Conceivably,
inhibitors exhibiting maximum hydrogen bonding interactions
with the backbone of the wild-type enzyme may also retain
potency against the mutant strains.
Upon the basis of this presumption, we have now designed
and evaluated protease inhibitors (Figure 1) incorporating a
novel ligand that can extensively interact with the backbone
residues. The inhibitors incorporate a stereochemically defined
bicyclic hexahydrocyclo-pentanofuran as the P2-ligand where
the cyclic ether oxygen is positioned to hydrogen bond with
the backbone Asp-29 NH. The bicyclic ligand is also expected
to fill in the S2-subsite effectively. Furthermore, a 4-hydroxy-
methylphenylsulfonamide is introduced as a P2′-ligand so that
the hydroxyl group will be optimally positioned to hydrogen
bond with the backbone residues in the S2′-subsite. Herein we
report structure-based design, synthesis, preliminary biological
evaluation, and X-ray crystal structure of inhibitor 3-bound
HIV-1 protease. The inhibitor has shown remarkable enzyme
inhibitory and antiviral potency. Preliminary drug resistance
profiles also indicated that the inhibitor maintains impressive
potency against multi-PI-resistant clinical HIV-1 variants iso-
lated from patients with drug-resistant HIV.
pentafuran-5-ol (9) in 88% yield. Treatment of 9 with N,N′-
disuccinimidyl carbonate in the presence of Et3N afforded mixed
carbonate 10.17
For the synthesis of inhibitor 4, the corresponding P2-ligand,
endo-cis-bicyclo[3.3.0]octan-3-ol was synthesized according to
Scheme 2. Benzyl ether 11 was synthesized according to the
literature procedure.18 Exposure of 11 to iodine in the presence
of silver acetate in acetic acid afforded the corresponding
iodoacetate derivative.19 Reduction of the resulting iodoacetate
with LAH furnished hydroxy benzyl ether 1220 in 50% yield in
two steps. The hydroxyl group in 12 was protected as TBS-
ether by reaction with TBSCl and imidazole.21 Subsequent
removal of the benzyl ether with sodium in liquid ammonia22
furnished hydroxyl TBS ether 13 in 89% yield. Removal of
hydroxyl group in 13 was effected by using Barton-McCombie
deoxygenation23 reaction. Thus, alcohol 13 was reacted with
N,N′-thiocarbonyldiimidazole in a mixture of (2:1) toluene and
pyridine at 55 °C for 12 h. The resulting thiocarbonylimidazoyl
derivative was treated with nBu3SnH in toluene at reflux to
provide TBS ether 14 in 66% yield in two steps. Deprotection
of TBS with TBAF in THF21 furnished endo-cis-bicyclo[3.3.0]-
octan-3-ol (15).24 Treatment of alcohol 15 with N,N′-disuccin-
imidyl carbonate in the presence of Et3N in acetonitrile afforded
succinimidyl carbonate 16 in excellent yield.17
The synthesis of inhibitor 2 with hexahydrocyclopenta[b]-
furanyl urethane as the P2-ligand and 4-aminosulfonamide as
the P2′-ligand is shown in Scheme 3. Nitrosulfonamide deriva-
tive 18 was prepared from commercially available epoxide 17
as described previously.25 Catalytic hydrogenation of 18 over
10% Pd/C in ethyl acetate for 11 h afforded the corresponding
aminosulfonamide derivative.
Subsequent removal of Boc-group was effected by exposure
of the resulting aminosulfonamide to trifluoroacetic acid in CH2-
Cl2 to furnish the corresponding diamine. Selective alkoxycar-
bonylation of the aliphatic amine with succinimidyl carbonate
10 provided inhibitor 2 in 87% yield in the three-step sequence.
Synthesis of inhibitors 3 and 4 was carried out as shown in
Scheme 4. For the preparation of hydroxyethylamine sulfona-
mide inhibitor scaffold, commercially available epoxide 17 was
reacted with isobutylamine in 2-propanol. Treatment of the
resulting amino alcohol with p-(diacetoxymethyl)phenylsulfonyl
Chemistry
For synthesis of target inhibitors 2 and 3, we devised an
efficient synthetic route to (3aS,5R,6aR)-5-hydroxy-hexahydro-
cyclopenta[b]furan in optically active form. As shown in Scheme
1, enzymatic asymmetrization of meso-diacetate with acetyl
cholinesterase provided the monoacetate 5 in multigram scale
(85% yield).13 Formation of Mosher ester of 5 revealed that
enantiomeric purity of 5 was 95% ee.14 Protection of hydroxyl
group with TBSCl in the presence of imidazole in THF afforded
TBS-ether 615 in 98% yield. Hydrolysis of 6 with potassium
carbonate in methanol gave the alcohol. Treatment of the
resulting alcohol with ethyl vinyl ether and NBS in CH2Cl2
furnished bromo acetal 7. Radical cyclization16 of 7 with n-Bu3-
SnH in the presence of AIBN in benzene under reflux furnished
bicyclic acetal 8 in excellent yield. Reduction of 8 with Et3SiH
in the presence of BF3.OEt2 followed by removal of TBS group
with TBAF in THF afforded optically active hexahydrocyclo-

5254 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Scheme 3. Synthesis of Inhibitor 2
Ghosh et al.
Table 1. Enzyme Inhibitory and Antiviral Activity of Inhibitors
inhibitora
Ki (nM)
IC50 (nM)
2
3
4
0.14 (0.02
0.0045 ( 0.001
5.3 ( 0.3
8
1.8
>1000
aInhibitor 1 has exhibited a Ki value of 14 pM and antiviral IC50 of 3
nM.
Table 2. Antiviral Data (IC50) of 3 in PBMC and MT-2 Cells (nM)
virus
SQV
RTV
INV
NFV
APV
3
a
HIV-1LAI
14
18
24
1.9
43
36
34
32
24
26
13
14
7
10
20
34
29
24
1.8
2.0
1.8
a
HIV-1BA-L
b
HIV-1LAI
b
HIV-2EHO
290
440
21
a PBMC. b MT-2 cells; SQV (saquinavir), RTV (ritonavir), INV (indi-
navir), NFV (nelfinavir), APV (amprenavir); data represent the mean value
of three determinations.
Scheme 4. Synthesis of Inhibitors 3 and 4
and the Ki-values denote the mean values of at least four
determinations. As can be seen, inhibitor 2 which incorporates
a stereochemically defined hexahydrocyclopenta[b]furanyl ure-
thane as the P2-ligand and 4-aminosulfonamide as the P2′-ligand
has shown enzymatic Ki value of 0.14 nM. As described
previously, the X-ray crystal structure of protein-ligand
complex of 1 and HIV-1 protease revealed that structural
changes on the P2′-aryl ring could lead to improved interaction
with the Asp-29 and Asp 30 NH.10 In an effort to make the
compound interact with backbone residues in the S2′-pocket
more effectively, we incorporated a hydroxymethylsulfonamide
derivative as the P2′-ligand in inhibitor 3. This inhibitor
exhibited a very impressive Ki value of 4.5 pM. To examine
the importance of the ring oxygen of (3aS,5R,6aS)-5-hydroxy-
hexahydro-cyclopenta[b]furanyl ligand in inhibitor 3, the cor-
responding inhibitor 4 with an endo-3-bicyclo[3.3.0]octanyl
urethane as the P2-ligand was evaluated. As shown, inhibitor 4
has shown an enzymatic Ki value of 5.3 nM, a >1100-fold
reduction in potency compared to 3. This marked difference in
enzyme inhibitory potency is also reflected in their antiviral
potency. Inhibitor 3 has shown an antiviral IC50 value of 1.8
nM in MT-2 human T-lymphoid cells exposed to HIV-1LAI
.
Consistent with its enzyme inhibitory potency, inhibitor 2 has
also shown good antiviral activity. In comparison, compound
4 has exhibited antiviral IC50 value of >1 µM, a drastic >500-
fold reduction with respect to inhibitor 3.
We have compared antiviral potency of inhibitor 3 against
various FDA approved protease inhibitors. It has maintained a
remarkable potency in MT-2 cells exposed to HIV-1LAI com-
pared to other FDA approved protease inhibitors. Antiviral
activity against three different HIV isolates was determined in
PHA-PBMC and MT-2 cells. The results are shown in Table
2. This inhibitor exerted far more potent activity against two
HIV-1 isolates (HIV-1LAI and HIV-1Ba-L) in both MT-2 cells
and PHA-PBMC than all currently available approved protease
inhibitors examined. In addition, it was as potent against HIV-
2EHO as indinavir and nelfinavir with an IC50 value of 21 nM.
In vitro cytotoxicity of inhibitor 3 was minimal and its
concentration that reduced the viability of target cells by 50%
(CC50) was greater than 100 µM.
chloride26 in the presence of diisopropylethylamine furnished
sulfonamide derivative 19. It was converted to hydroxymeth-
ylsulfonamide derivative 20 by deprotection of the acetates with
K2CO3 and subsequent reduction of the resulting aldehyde with
NaBH4 in methanol. Sulfonamide derivative 20 was converted
to inhibitor 3 by removal of Boc group by exposure to
trifluoroacetic acid and reaction of the resulting amine with
succinimidyl carbonate 10 and diisopropylethylamine in CH2-
Cl2. Inhibitor 2 was obtained in 80% yield. Similarly, reaction
of the resulting amine with succinimidyl carbonate 16 afforded
inhibitor 4 in 87% yield.
We also examined inhibitor 3 for its antiviral activity against
a panel of multi-drug-resistant HIV-1 variants as shown in Table
3. An HIV-1 clinical strain HIV-1ET, isolated from a drug-na¨ıve
patient with HIV-1 infection, was sensitive to all protease
inhibitors examined, among which inhibitor 3 was most potent
with the lowest IC50 value of 3 nM. In contrast, each of six
drug-resistant clinical strains containing 10-12 protease inhibi-
Results and Discussion
Inhibitors with hexahydrocyclopenta[b]furanyl urethane as the
P2-ligand have shown impressive in vitro potency. Inhibitors
2-4 were evaluated in enzyme inhibitory and antiviral assays
and the results are shown in Table 1. For enzyme inhibitory
assay, we utilized the protocol described by Toth and Marshall27

HIV-1 Protease Inhibitors To Combat Drug Resistance
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5255
Table 3. Activity of 3 against a Wide Spectrum of HIV-1 Variantsa
IC50 (nM) values
virus
mutationsa
SQV
17
RTV
15
>1000
IDV
NFV
APV
23
1
3
1 (ET)
2 (B)
L10I
30
>1000
32
>1000
nd
10.2
3
15
L10I,K14R,L33I,M36I,M46I,F53L,K55R,I62V,L63P,A71V,
G73S,V82A,L90M,I93L
230
290
3 (C)
I10L,I15V,K20R,M36I,M46L,I54V,K55R,I62V,L63P,K70Q,
V82A,L89M
100
>1000
500
310
300
3.5
5
4 (G)
5 (TM)
6 (EV)
L10I,V11I,T12E,I15V,L19I,R41K,M46L,L63P,A71T, V82A,L90M
L10I,K14R,R41K,M46L, I54V,L63P,A71V,V82A,L90M, I93L
L10V,K20R,L33F,M36I, M46I,I50V,I54V,D60E,L63P,A71V,
V82A,L90M
59
250
>1000
>1000
>1000
>1000
500
>1000
>1000
170
>1000
>1000
310
220
>1000
3.7
3.5
nd
20
4
52
7 (ES)
8 (K)
L10I,M46L,K55R,I62V,L63P, I72L,G73C,V77I,I84V,L90M
L10F/D30N/K45I/A71V/T74S
>1000
>1000
>1000
>1000
>1000
>1000
nd
3
31
3
20
57
260
68
a Amino acid substitutions identified in the protease-encoding region of HIV-1ET (ET), HIV-1B (B), HIV-1C (C), HIV-1G (G), HIV-1TM (TM), HIV-1EV
(EV), HIV-1ES (ES), HIV-1K (NFVR) as compared to consensus B sequence cited from the Los Alamos database. All values were determined in triplicate.
The IC50 values were determined by employing PHA-PBMC as target cells and the inhibition of p24Gag protein production as the endpoint.
Figure 2. Stereoview of compound 3 bound to the active site of wild-type HIV-1 protease. The protein backbone is illustrated as a ribbon structure.
Hydrogen bonds to the active site Asp-25 and Asp-25′, Gly-27, backbone NH of Asp-29, and Asp-30′, and to two bound water molecules are
shown.
tor resistance-associated amino acid substitutions (HIV-1B, HIV-
1C, HIV-1G, HIV-1TM, HIV-1EV, and HIV-1ES), isolated from
patients with HIV-1 infection having received 7-11 different
antiviral agents for 24 to 81 months,5,7 was highly resistant to
all the currently available protease inhibitors tested (Table 3).
However, inhibitor 3 exerted highly potent activity against all
of these six variants with IC50 values ranging 4-52 nM. An
HIV-1 variant, HIV-1K, which was selected in vitro in the
presence of up to 5 µM concentrations of nelfinavir and contains
five amino acid substitutions including D30N,7 was also tested
against inhibitor 3. It was found that inhibitor 3 was highly
potent against HIV-1K with an IC50 value of as low as 3 nM
(Table 3). These data indicate that inhibitor 3 is highly active
against a wide spectrum of drug-resistant variants. Overall, the
potency of inhibitor 3 against the HIV-1 strains tested in the
present study was comparable to that of inhibitor 1.
2.6-3.2 Å. An oxygen of the sulfonamide group and the
carbamate carbonyl oxygen form hydrogen bonds (2.8 and 2.9
Å, respectively) to the conserved water molecule that is
hydrogen bonded to the backbone NH groups of Ile50 and Ile50′,
as is commonly seen in HIV-1 protease/inhibitor complexes.29
The oxygen of the P2 hexahydrocyclopentafuran forms a
hydrogen bond with the backbone NH of Asp29 with the
distance between heavy atoms of 2.8 Å.
This interaction cannot occur for inhibitor 4 which lacks the
ring oxygen. The P2 hexahydrocyclopentafuranyl group also
makes good C-H‚‚‚O contacts with the main-chain carbonyl
of Gly48. The amide nitrogen of the carbamate moiety has a
3.2 Å hydrogen bond with the main chain carbonyl oxygen of
Gly27. The hydroxyl of the P2′ benzyl alcohol group forms a
hydrogen bond to the backbone NH (3.1 Å) and a water-
mediated contact with the side chain oxygen of Asp30 (OHinh
‚
To gain molecular insight into the ligand-binding site
interactions responsible for its potent antiviral activity against
a wide spectrum of multi-PI-resistant HIV-1 variants, we have
solved the X-ray crystal structure of the inhibitor complex with
wild-type protease at 1.35 Å resolution.28 A stereoview of the
inhibitor 3-bound structure is shown in Figure 2. The central
hydroxyl group of the inhibitor forms four hydrogen bonds to
the active site Asp25 and Asp25′ side chains, with the distances
‚‚H2O‚‚‚OOC distances are 2.5 and 2.3 Å, respectively).
Therefore, the inhibitor 3 forms three direct hydrogen bonds
and three water-mediated contacts to the protease residues,
excluding contacts with catalytic aspartates. The important
hydrophobic contacts include C-H‚‚‚π interactions. The P1
benzyl group makes C-H‚‚‚π interactions with Pro81′ and
Val82′ (3.6-3.8 Å). The P1′ isobutyl group lies in a hydro-
phobic pocket formed by Pro81, Val82, Ile84, Gly49′, and Ile-

5256 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Ghosh et al.
Table 4. Comparison of the Crystal Structure of Protease and Inhibitor 3 with Multi-PI-resistant Protease Crystal Structuresa
PDB code
mutations
RMSD (Å)
2FDD30
1SGU31
1HSH32
2AZC33
1B6K34
2AVV35
L10I, K20R, M36I, M46I, I50V, I54V, I62V, L63P, A71V, V82A, L90M
K20R, L33F, M36I, I54V, L63P, A71V, V82A, I84V, L90M
L10V, M36I, M46I, A71V, I93L
M46I, F53L, L63P, V77I
K14R, R41K, L63P
0.7
0.5
1.1
0.8
0.5
0.5
L33I, G73S
a RMSD is the root-mean-square deviation of alpha-carbon positions for protease backbones.
Figure 3. Stereoview of inhibitor 3 bound to the active site of wild-type HIV-1 protease, superimposed upon the structures of the three most highly
mutated drug-resistant proteases of Table 3. Protein backbones are represented as alpha-carbon traces. Red ) wild-type; blue ) 2FDD; green )
1SGU; yellow ) 1HSH. Note that, despite the multiple mutations, backbone positions change very little, especially in the active site, and hydrogen
bonds to backbone NHs are not disrupted. Also, the hydroxymethyl is able to hydrogen bond to both the backbone and the side chain of Asp-30′.
50′. The P2′ group also forms a number of C-H‚‚‚π contacts
with Ala28′, Val32′, Ile47′, and Ile50, with distances of 3.6-
3.9 Å with the closest interaction involving the side chain of
Val32′. Inhibitor 3 participates in extensive interactions in the
S2 to S2′ subsites, including tight hydrogen bonding with the
protein backbone. Other PIs typically show fewer interactions
with the main chain atoms. These interactions of inhibitor 3
with the protease backbone may explain its superior antiviral
property and its potent activity against a wide spectrum of multi-
PI-resistant HIV-1 strains.
To evaluate interactions of inhibitor 3 with the multi-drug-
resistant variants of HIV-1 protease listed in Table 3, we have
compared our crystal structure with several published crystal
structures containing multiple mutations.30-35 The structures
used for comparison, and the relevant mutations, are listed in
Table 4. None of these structures corresponds exactly to
proteases of the multi-drug-resistant HIV-1 variants illustrated
in Table 3, but these structures incorporate 21 of the 33
mutations listed in Table 3. The other mutations in Table 3
(V11I, T12E, I15V, L19I, M46L, K55R, D60E, K70Q, A71T,
I72L, G73C, and L89M) have not, to our knowledge, been
present in published crystal structures, with the exception of
M46L. This mutation has been published only in unliganded
crystal structures, in which the active site is open and dis-
torted.36,37 Residues 11, 12, 15, 19, 46, 55, 60, 70, 71, 72, 73,
and 89 are all far from the active site, so that the effects of
these mutations on drug resistance must be indirect and,
therefore, difficult to model or predict. A stereoview of inhibitor
3 bound to the active site of wild-type HIV-1 protease,
superimposed upon the structures of the three most highly
mutated drug-resistant proteases (from Table 3), is shown in
Figure 3.
protease were examined. In all six structures, the following
interactions were maintained: (a) hydrogen bonding of the
secondary OH of the inhibitor to Asp-25 and Asp-25′ of the
active site; (b) hydrogen bonding of the tetrahydrofuran oxygen
of the inhibitor to the backbone NH of Asp-29, and a possible
hydrogen bond to the backbone NH of Asp-30; (c) hydrogen
bonding of the CH2OH of the inhibitor to the backbone NH of
Asp-30′; (d) hydrogen bonding of the CH2OH of the inhibitor
to the side chain carboxylate of Asp-30′ (requires ∼10-20°
rotation of the side chain around the C-alpha:C-beta bond to
optimize hydrogen bond distance; (e) in P2, van der Waals
contact with Gly-48; (f) in P1, van der Waals contact with Gly-
49, Ile-50, Leu-23′, and Pro-81′; (g) in P1′, van der Waals
contact with Ile-84; (h) in P2′, van der Waals contact with Ile-
50, Asp-30′, Val-32′, and Ile-47′.
Furthermore, many other binding interactions were also
retained. These include: (a) in P2, van der Waals contact of
Ile-47 was retained in all structures except 1HSH, which has
the I47V mutation; van der Waals contact of Ile-50′ was retained
in four of the structures, but was reduced in 2FDD (which has
the I50V mutation) and in 2AZC; (b) in P1, van der Waals
contact of Val-82′ was reduced in 2FDD, 1SGU, and 2AZC,
all of which have the V82A mutation; van der Waals contact
of Ile-84′ was retained in all structures except 1SGU, which
has the I84V mutation; (c) in P1′, van der Waals contact of
Pro-81 was reduced in 1HSH, 2AZC, and 1B6K; van der Waals
contact of Val-82 was reduced in 2FDD, 1SGU, and 2AZC,
all of which have the V82A mutation. All six were able to
maintain binding to the water molecule in the binding site,
with the exception of the 2AZC structure; as reported,33 one of
the flaps of this structure is distorted and a second water
molecule is required for inhibitor binding to one of the Ile50
NH groups. It is noteworthy that, even in multi-drug-resistant
In each case, a least-squares fit of protease alpha-carbons was
carried out, and the interactions of inhibitor 3 with the mutant

HIV-1 Protease Inhibitors To Combat Drug Resistance
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5257
Hz, J ) 9.0 Hz, 1H), 5.77 (dd, J ) 1 Hz, J ) 9 Hz, 1H), 4.56 (t,
J ) 9 Hz, 1H), 4. 48 (t, J ) 9 Hz, 1H), 2.59 (dt, J ) 12 Hz, J )
22 Hz, 1H), 2.40 (bs, 1H), 1.42 (dt, J ) 7.5 Hz, J ) 22 Hz, 1H).
13C NMR (CDCl3, 75 MHz): δ 137.0, 136.1, 75.6, 75.4, 44.9, 26.2,
18.5, -4.2.
proteases with a number of mutations, the hydrogen bonding
interactions of inhibitor 3 with the protease backbone are well
maintained.
Conclusion
To a solution of above hydroxyl ether (400 mg, 1.86 mmol) and
N-bromosuccinimide (330 mg, 1.86 mmol) in CH2Cl2 (10 mL) at
-10 °C was added ethyl vinyl ether (0.27 mL, 2.8 mmol). The
reaction mixture was allowed to warm to 23 °C. After 12 h aqueous
ammonium chloride (15 mL) was added and the layers were
separated. The aqueous layer was extracted with CH2Cl2 (2×).
Combined organic layers were washed with brine, dried over
anhydrous sodium sulfate, and then concentrated in vacuo. The
crude product was purified by flash column chromatography to
afford compound 7 (660 mg, 97%) as a colorless liquid. 1H NMR
(CDCl3, 300 MHz): 5.82 (d, 2H, J ) 2.7 Hz), 4.70 (q, 1H, J )
5.7 Hz), 4.50-4.58 (m, 2H), 3.50-3.62 (m, 2H), 3.27-3.30 (m,
2H), 2.59-2.64 (m, 1H), 1.53-1.60 (m, 1H), 1.16-1.18 (m, 3H),
0.83 (s, 9H), 0.04 (s, 6H). 13C NMR (CDCl3, 75 MHz): 138.1,
138.0, 133.4, 133.0, 101.2, 101.0, 79.7, 79.6, 75.2, 75.1, 62.1, 61.9,
42.8, 42.3, 32.5, 32.4, 26.2, 26.0, 18.5, 15.6, 15.5, -4.1, -4.2.
MS (CI): m/z 365.1 [M + H]+; HRMS calcd for C15H29BrO3Si
[M + H]+ 365.1148; found 365.1145.
In summary, we have reported here the structure-based design
of novel HIV-1 protease inhibitors incorporating a stereochemi-
cally defined 5-hexahydrocyclopenta[b]furanyl urethane as the
P2-ligand and a 4-hydroxymethylsulfonamide as the P2′-ligand.
The inhibitors are designed with the purpose of making
extensive interactions including hydrogen bonding with the
protein backbone of HIV-1 protease active site. One such
inhibitor (3) has exhibited exceedingly potent antiviral activity
and superior activity against multi-PI-resistant variants compared
to other FDA approved PIs. The synthesis of P2-ligand alcohol,
(3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was car-
ried out enantioselectively by an enzymatic asymmetrization
with acetyl cholinesterase as one of the key steps. A protein-
ligand crystal structure of 3-bound HIV-1 protease (1.35 Å
resolution) revealed extensive interactions in the enzyme active
site. Of particular note, both P2 and P2′-ligands are involved
in hydrogen bonding with the backbone of both S2- and S2′-
subsites. Comparison of protein-ligand X-ray structure of 3
with others structures of mutant proteases clearly indicated that
the backbone interactions are maintained. The design of an
inhibitor to specifically interact with the backbone may serve
as an important guide to combat drug resistance. Further design
and chemical modifications are currently underway.
(3aR,5R,6aR)-2-Ethoxy-5-tert-butyldimethylsiloxy-hexahydro-
cyclopenta[b]furan (8). The bromo derivative 7 (600 mg, 1.64
mmol), n-tributyltin hydride (0.6 mL, 2.14 mmol), and AIBN (10
mg) in benzene (15 mL) were refluxed for 4 h. Then it was cooled
to 23 °C, and benzene was removed under reduced pressure. The
crude product was chromatographed on silica gel to obtain bicyclic
1
ether 8 (410 mg, 87%) as a viscous liquid. H NMR (CDCl3, 300
MHz): 5.86 (dd, 0.5 H, J ) 2.4 Hz, J ) 9.3 Hz), 5.12 (dd, 0.5 H,
J ) 4.5 Hz, J ) 7.2 Hz), 3.97-4.75 (m, 2H), 3.63-3.74 (m, 1H),
3.32-3.40 (m, 1H), 2.22-2.78 (m, 1H), 1.52-2.04 (m, 4H). 13C
NMR (CDCl3, 75 MHz): 106.1, 105.7, 83.1, 75.1, 73.6, 62.8, 62.6,
43.9, 42.6, 42.0, 41.5, 40.9, 40.2, 38.9, 38.9, 28.6, 27.1, 26.2, 26.1,
18.5, 18.3, 15.6, 15.5, -4.2, -4.4, -4.5. MS (CI): m/z 287.2 [M
+ H]+; HRMS calcd for C15H30O3Si [M + H]+ 287.2043: found
287.2041.
(3aS,5R,6aR)-5-Hydroxy-hexahydro-cyclopenta[b]furan (9).
To a cold (0 °C) solution of bicyclic ether 8 (400 mg, 1.4 mmol)
and triethylsilane (0.9 mL, 5.6 mmol) in CH2Cl2 (10 mL) was added
BF3‚Et2O (320 µL, 2.8 mmol), and the reaction mixture was stirred
for 10 min. Saturated aqueous sodium bicarbonate solution (10 mL)
was added, and the mixture was extracted with CH2Cl2 (3×). The
combined extracts were dried over anhydrous sodium sulfate and
concentrated in vacuo. Purification by flash column chromatography
Experimental Section
General. All moisture sensitive reactions were carried out under
nitrogen or argon atmosphere. Anhydrous solvents were obtained
as follows: THF, diethyl ether and benzene, distilled from sodium
and benzophenone; dichloromethane, pyridine, triethylamine, and
diisopropylethylamine, distilled from CaH2. All other solvents were
HPLC grade. Column chromatography was performed with What-
man 240-400 mesh silica gel under low pressure of 5-10 psi.
TLC was carried out with E. Merck silica gel 60-F-254 plates. 1H
and 13C NMR spectra were recorded on Varian Mercury 300 and
Bruker Avance 400 and 500 spectrometers. Infrared spectra were
recorded on a Mattson Genesis II FTIR instrument. Optical rotations
were measured using a Perkin-Elmer 341 polarimeter.
(1R,4S)-(+)-4-(tert-Butyldimethylsilyloxy)-2-cyclopentenyl Ac-
etate (6). To a stirred solution of alcohol 5 (1.1 g, 7.6 mmol) in
tetrahydrofuran (20 mL) was added imidazole (779 mg, 11.4 mmol)
followed by tert-butyldimethylsilyl chloride (1.54 g, 9.5 mmol).
The reaction mixture was stirred at 23 °C for 24 h after which
solids were removed by filtration, and the filtrate was concentrated
to dryness. The residue was dissolved in ethyl acetate and washed
with 1 N hydrochloric acid (3×), saturated aqueous sodium
bicarbonate (2×), and brine solution. The organic phase was dried
over anhydrous magnesium sulfate, filtered, and concentrated to
give compound 6 (1.9 g, 99%) as a colorless liquid. 1H NMR
(CDCl3, 300 MHz): δ 6.03 (m, 1H), 5.95 (m, 1H), 5.51 (m, 1H),
4.77 (m, 1H), 2.86 (dt, 1H, J ) 7.5 Hz, J ) 13.5 Hz), 2.10 (s, 3H),
1.66 (dt, 1H, J ) 5 Hz, J ) 14 Hz), 0.99 (s, 9H), 0.15 (s, 3H),
0.14 (s, 3H). 13C NMR (CDCl3, 75 MHz): δ 171.3, 139.3, 131.6,
77.4, 75.3, 41.6, 26.3, 21.6, 18.6, -4.1, -4.2.
provided cyclopentanofuran derivative (300 mg, 90%) as a colorless
1
liquid. [R]20 8.6 (c 1, CHCl3); H NMR (CDCl3, 300 MHz): δ
D
4.36 (m, 1H), 4.03 (m, 1H), 3.85 (m, 1H), 3.73 (m, 1H), 2.50 (m,
1H), 2.1-1.9 (m, 3H), 1.70 (m, 1H), 1.57 (m, 1H), 1.39 (m, 1H),
0.84 (s, 9H), 0.00 (s, 6H). 13C NMR (CDCl3, 75 MHz): δ 82.9,
73.6, 67.2, 42.8, 41.7, 40.9, 33.5, 26.2, 18.5, -4.3, -4.4. MS
(CI): m/z 243.1 [M + H]+; HRMS calcd for C13H26O2Si [M +
H]+ 243.1781; found 243.1779.
The above cyclopentanofuran (200 mg, 0.82 mmol) was dis-
solved in tetrahydrofuran (5 mL) and treated with tetrabutylam-
monium fluoride (1.2 mL, 1 M solution in tetrahydrofuran, 1.23
mmol). The mixture was stirred for 2 h at 23 °C. Solvent was
removed under reduced pressure, and the crude product was purified
by flash column chromatography to afford alcohol 9 (101 mg, 97%)
1
as a viscous oil. [R]20 13.0 (c 1, CHCl3); H NMR (CDCl3, 300
D
MHz): δ 4.62 (dt, 1H, J ) 1.2 Hz, J ) 6.3 Hz), 4.47 (m, 1H),
4.27 (m, 1H), 3.85 (m, 1H), 2.90 (m, 1H), 1.5-2.6 (m, 7H). 13C
NMR (CDCl3, 75 MHz): δ 86.0, 75.1, 68.6, 42.9, 41.8, 41.8, 35.4.
MS (CI): m/z 129.1 [M + H]+; HRMS calcd for C7H12O2 [M +
H]+ 129.0916; found 129.0915.
(3aS,5R,6aR)-[Carbonic Acid 2′,5′-Dioxo-pyrrolidin-1-yl-
ester]-hexahydro-cyclopenta[b]furan-5-yl Ester (10). A solution
of alcohol 9 (50 mg, 0.39 mmol), N,N′-disuccinimidyl carbonate
(122 mg, 0.47 mmol), and triethylamine (82 µL, 0.59 mmol) in
acetonitrile (2 mL) was stirred at 23 °C for 12 h. After this period,
1R,4S-[4-(2-Bromo-1-ethoxy-ethoxy)-cyclopent-2-enyloxy]-
tert-butyl-dimethyl-silane (7). TBS-ether 6 (2 g, 7.8 mmol) was
dissolved in methanol (50 mL) and treated with potassium carbonate
(1.7 g, 12.5 mmol). The mixture was stirred for 20 min at 23 °C;
solvent was evaporated under reduced pressure. The product was
extracted with ethyl acetate (3×), dried over anhydrous sodium
sulfate, and concentrated. The crude product was purified by flash
column chromatography to provide hydroxyl ether (1.6 g, 97%) as
a colorless oil. [R]20 -21.6 (c 1, CHCl3) [lit.,38 [R]20 -21.2 (c
D
D
1
0.89, CHCl3)]; H NMR (CDCl3, 300 MHz): δ 5.84 (dd, J ) 1

5258 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Ghosh et al.
the reaction mixture was treated with saturated aqueous sodium
bicarbonate (2 mL). The resulting mixture was extracted with ethyl
acetate (3×). The combined organic extracts were dried over sodium
sulfate. Evaporation of the solvent followed by flash column
chromatography furnished mixed carbonate 10 (80 mg, 78%) as
viscous oil. 1H NMR (CDCl3, 300 MHz): δ 5.23 (dt, 1H, J ) 1.5
Hz, J ) 5.7 Hz), 4.58 (m, 1H), 4.08 (m, 1H), 3.84 (m, 1H), 2.93
(s, 4H), 2.84 (m, 1H), 1.9-2.39 (m, 6H). 13C NMR (CDCl3, 75
MHz): δ 169.1, 151.3, 84.6, 83.5, 68.1, 41.9, 39.5, 38.4, 34.1, 25.8.
(()-4-(Benzyloxy)-octahydropentalen-2-ol (12). To a suspen-
sion of benzyl ether 11 (500 mg, 2.47 mmol) and silver acetate
(495 mg, 2.96 mmol) in acetic acid (6 mL) at 23 °C was added
iodine (688 mg, 2.72 mmol) slowly for a period of 10 min. After
being stirred for 2 h the reaction mixture was filtered and the filter
cake was washed with CH2Cl2. The filtrate was concentrated under
reduced pressure. The dark brown solution was diluted with CH2-
Cl2 washed with water, 2 N sodium carbonate solution, 5 N sodium
thiosulfate solution, water, and brine solution, and dried over
anhydrous sodium sulfate. The solvent was evaporated, and the
crude product was purified by flash column chromatography to
raphy of the crude product furnished alcohol 13 (61 mg, 99%) as
colorless liquid. 1H NMR (CDCl3, 300 MHz): δ 4.22 (m 1H), 3.97
(bs, 1H), 3.84 (bd, 1H, J ) 9.3 Hz), 2.57-2.60 (m, 1H), 2.39-
2.42 (m, 1H), 1.39-1.94 (m, 8H), 0.80 (s, 9H), 0.00 (s, 6H). 13C
NMR (CDCl3, 75 MHz): δ 77.0, 74.4, 48.4, 43.4, 41.4, 38.1, 35.7,
31.7, 26.1, 18.4, -4.2, -4.8. MS (CI): m/z 257.1 [M + H]+; HRMS
calcd for C14H28O2Si [M + H]+ 257.1936, found 257.1933.
(()-tert-Butyldimethyl-(octahydropentalen1yloxy)silane (14).
A solution of alcohol 13 (61 mg, 0.24 mmol) and N,N′-thiocarbo-
nyldiimidazole (128 mg, 0.72 mmol) in 2:1 toluene-pyridine (6
mL) was heated at 55 °C for 12 h. After this period, the reaction
mixture was concentrated in vacuo to a yellow residue which was
dissolved in CH2Cl2 (20 mL) and washed with 0.1 N hydrochloric
acid, saturated sodium bicarbonate solution, and then with water.
The organic layer was dried over anhydrous sodium sulfate.
Evaporation of solvent, followed by flash column chromatography
yielded 1-O-thiocarbonyl imidazoyl derivative (70 mg, 82%) as
colorless semisolid. 1H NMR (CDCl3, 300 MHz): δ 8.32 (s, 1H),
7.61 (s, 1H), 6.99 (s, 1H), 5.52-5.57 (m, 1H), 4.02-4.08 (m, 1H),
2.73-2.79 (m, 1H), 2.36-2.40 (m, 1H), 2.02-2.12 (m, 3H), 1.18-
1.80 (m, 5H), 0.83 (s, 9H), 0.00 (s, 6H). 13C NMR (CDCl3, 75
MHz): δ 184.2, 137.3, 131.0, 118.3, 86.8, 74.8, 43.7, 42.0, 38.8,
36.1, 29.6, 28.5, 26.3, 18.6, -4.3, -4.2.
1
afford iodoacetate (690 mg, 70%) as a colorless liquid. H NMR
(CDCl3, 300 MHz): δ 7.45-7.59 (m, 5H), 5.31-5.40 (dt, 1H, J
) 9.9 Hz), 4.72-4.85 (ABq, 2H, υ ) 20.7 Hz, J ) 11.7 Hz, J )
29.7 Hz), 4.54 (t, 1H, J ) 9 Hz), 4.09-4.15 (m, 1H), 3.07-3.15
(m, 1H), 2.65-2.70 (m, 1H), 2.24 (s, 3H), 2.20-2.23 (m, 1H),
1.78-1.90 (m, 2H), 1.56-1.63 (m, 1H), 1.26-1.37 (dt, 1H, J )
9.6 Hz). 13C NMR (CDCl3, 75 MHz): δ 170.9, 139.1, 128.7, 127.9,
127.8, 83.6, 80.2, 72.5, 54.4, 39.8, 37.0, 31.0, 28.9, 24.8, 21.4. MS
(CI): m/z 401.0 [M + H]+; HRMS calcd for C7H21IO3 [M + H]+
401.0614; found 401.0611.
To a refluxing solution of tributyltinhydride (288 µL, 0.99 mmol)
in dry toluene (5 mL) under argon atmosphere was added above
thiocarbonylimidazoyl derivative (70 mg, 0.19 mmol) in dry toluene
(5 mL) in a dropwise manner over 10 min. The reaction was
refluxed for an additional 30 min, and the solvent was removed in
vacuo to give colorless oil. The crude product was purified on a
silica gel column to afford TBS ether 14 (40 mg, 80%) as a colorless
1
oil. H NMR (CDCl3, 300 MHz): δ 3.95 (m, 1H), 2.24 (m, 2H),
A solution of above iodoacetate (500 mg, 1.25 mmol) in diethyl
ether (10 mL) was added to a suspension of lithium aluminum
hydride (95 mg, 2.5 mmol) in diethyl ether (20 mL) at 0 °C. The
mixture was heated at reflux for 24 h and then quenched by the
addition of aqueous 4% sodium hydroxide solution (0.4 mL). The
reaction mixture was stirred at room temperature for another 2 h.
The white precipitate was filtered off, and the filtrate was evaporated
to dryness in vacuo to give an oily residue which was purified by
silica gel column chromatography to afford alcohol 12 (210 mg,
72%) as colorless oil. 1H NMR (CDCl3, 300 MHz): δ 7.43-7.47
(m, 5H), 4.73 (ABq, 2H, υ ) 25.6 Hz, J ) 11.7 Hz, J ) 39.3 Hz),
4.32 (m, 1H), 4.11 (m, 1H), 3.92 (bs, 1H), 2.70-2.85 (m, 1H),
2.64-2.69 (m, 1H), 1.96-2.19 (m, 5H), 1.65-1.78 (m, 3H). 13C
NMR (CDCl3, 75 MHz): δ 138.4, 128.8, 128.1, 82.0, 75.1, 71.6,
46.7, 42.4, 42.1, 35.5, 32.2, 31.2. MS (CI): m/z 233.1 [M + H]+;
HRMS calcd for C15H20O2 [M + H]+ 233.1542; found 233.1540.
(()-5-(tert-Butyldimethylsilyloxy)-octahydropentalen-1-ol (13).
To a solution of alcohol 12 (120 mg, 0.51 mmol) and imidazole
(80 mg, 1.24 mmol) in dimethylformamide (3 mL) at 0 °C was
added tert-butyldimethylsilyl chloride (92 mg, 0.61 mmol). The
resulting mixture was stirred for 12 h at 23 °C. The reaction was
quenched by addition of cold water (30 mL) and extracted with
diethyl ether (3×). The combined organic extracts were dried over
anhydrous sodium sulfate and filtered. Evaporation of the solvent
followed by flash column chromatography of the crude product
1.91-1.96 (m, 2H), 1.11-1.59 (m, 8H), 0.83 (s, 9H), -0.01 (s,
6H). 13C NMR (CDCl3, 75 MHz): δ 75.0, 43.0, 40.1, 34.0, 29.0,
27.5, 26.3, 18.6, 16.2, 14.0, -4.3, -4.3. MS (CI): m/z 241.2 [M
+ H]+; HRMS calcd for C14H28OSi [M + H]+ 241.2069; found
241.2061.
(()-Octahydropentalen-2-ol (15). To a solution of TBS-ether
14 (30 mg, 0.11 mmol) in dry THF (1 mL) was added a 1 M
solution of tetrabutylammonium fluoride (440 µL, 0.44 mmol), and
the mixture was stirred at 23 °C for 10 h. After this period, solvent
was removed under reduced pressure and the crude product was
purified by flash chromatography to afford alcohol 15 (14 mg, 99%)
as a colorless liquid. 1H NMR (CDCl3, 300 MHz): δ 4.06 (m, 1H),
2.33-2.36 (m, 2H), 2.11-2.16 (m, 2H), 1.54-1.66 (m, 4H), 1.42-
1.45 (m, 2H), 1.14-1.18 (m, 2H). 13C NMR (CDCl3, 75 MHz): δ
74.3, 42.4, 40.0, 33.5, 29.6. MS (CI): m/z 127.1 [M + H]+; HRMS
calcd for C8H14O [M + H]+ 127.1124; found 127.1123.
Succinimidyl Carbonate 16. A solution of alcohol 15 (10 mg,
0.07 mmol), N,N′-disuccinimidyl carbonate (24.3 mg, 0.09 mmol),
and triethylamine (14 µL, 0.1 mmol) in acetonitrile (2 mL) was
stirred at 23 °C for 12 h. After this period, the solvent was
evaporated and the residue was purified by flash column chroma-
tography to afford the mixed carbonate 16 (15.3 mg, 89%) as a
1
white solid. H NMR (CDCl3, 300 MHz): δ 4.88-4.95 (m, 1H),
2.76 (s, 4H), 2.35-2.37 (m, 2H), 2.13-2.23 (m, 2H), 1.59-1.66
(m, 2H), 1.35-1.49 (m, 6H). 13C NMR (CDCl3, 75 MHz): δ 169.1,
84.9, 40.6, 38.7, 34.1, 25.8.
1
furnished TBS ether (160 mg, 90%) as colorless liquid. H NMR
(CDCl3, 300 MHz): δ 7.18-7.28 (m, 5H), 4.43 (ABq, 2H, υ )
18.7 Hz, J ) 12.0 Hz, J ) 25.8 Hz), 3.95 (m, 1H), 4.77 (q, 1H, J
) 7.2 Hz), 2.39 (m, 1H), 2.23 (m, 1H), 2.03 (m, 1H), 1.07-1.79
(m, 7H), 0.83 (s, 9H), 0.02 (s, 6H). 13C NMR (CDCl3, 75 MHz):
δ 139.4, 128.7, 127.9, 127.7, 82.1, 74.8, 71.7, 43.8, 41.5, 38.1,
35.8, 29.2, 28.6, 26.3, 18.6, -4.2, -4.2. MS (CI): m/z 346.2 [M
+ H]+; HRMS (m/z) C21H34O2Si [M + H]+ 346.2328; found
346.2325.
To a stirred solution of sodium in liquid ammonia was added a
solution of above TBS-ether (85 mg, 0.24 mmol) in THF (5 mL)
dropwise for 5 min. The reaction was quenched by addition of
excess solid ammonium chloride and allowed to warm to 23 °C.
The mixture was diluted with water and extracted with CH2Cl2 (3×).
The combined organic extracts were dried over anhydrous sodium
sulfate. Evaporation of the solvent and flash column chromatog-
Preparation of 4-Diacetoxytoluenesulfonyl Chloride. A solu-
tion of p-toluenesulfonyl chloride (4.02 g, 21.1 mmol) in a mixture
(1:1) of acetic acid and acetic anhydride (80 mL) was treated with
concentrated sulfuric acid (6.4 mL, 105.5 mmol) at 0 °C. Chromium
trioxide (8 g, 84.4 mmol) was added slowly to maintain the reaction
temperature below 10 °C. The mixture was stirred at 5 °C for 30
min. The reaction was quenched with ice water and filtered, and
the solid filter cake was washed with water. The solid product was
then suspended in saturated sodium bicarbonate solution, and the
mixture was stirred for 2 h. The reaction mixture was extracted
with ethyl acetate (3×). The combined organic layers were dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by purification of the resulting crude product by flash column
chromatography provided the title compound (2.4 g, 38%) as a solid.

HIV-1 Protease Inhibitors To Combat Drug Resistance
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17 5259
1H NMR (CDCl3, 300 MHz): δ 7.92 (d, 2H, J ) 8.4 Hz), 7.79 (d,
2H, J ) 8.4 Hz), 7.75 (s, 1H), 2.16 (s, 6H).
Inhibitor 3. A solution of compound 20 (30 mg, 0.06 mmol) in
30% trifluoroacetic acid in CH2Cl2 (3 mL) was stirred at 23 °C for
30 min. After this period, the reaction mixture was concentrated
under reduced pressure, and the residue was dissolved in toluene
and evaporated at reduced pressure. The residue was dissolved in
acetonitrile (2 mL) and cooled to 0 °C. To this solution were added
N,N′-diisopropylethylamine (41 µL, 0.24 mmol) and mixed carbon-
ate 10 (16.8 mg, 0.06 mmol). The resulting mixture was stirred at
23 °C for 6 h. The reaction mixture was concentrated under reduced
pressure, and the residue was purified by flash chromatography
over silica gel to furnish inhibitor 3 (28 mg, 87%) as an amorphous
Compound 19. To a stirred solution of (1-oxiranyl 2-phenyl-
ethyl)-carbamate 17 (200 mg, 0.76 mmol) in 2-propanol (6 mL)
was added isobutylamine (340 µL, 4.55 mmol). The resulting
mixture was heated at reflux for 6 h. After this period, the reaction
mixture was concentrated under reduced pressure and the residue
was purified by flash column chromatography to provide the
corresponding secondary amine (268 g, 99%) as a white solid. Mp
145 °C (decomposed); 1H NMR (CDCl3, 300 MHz): δ 7.20-7.33
(m, 5H), 4.69 (d, 1H, J ) 8.8 Hz), 3.84-3.88 (m, 1H), 3.48-3.53
(m, 1H), 3.04 (dd, 1H, J ) 4.5 Hz, J ) 14.2 Hz), 2.90 (dd, 1H, J
) 3.8 Hz, J ) 7.8 Hz), 2.84 (dd, 1H, J ) 3.1 Hz, J ) 12.4 Hz),
2.76 (dd, 1H, J ) 5.8, 12.3), 2.57 (dd, 1H, J ) 6.6 Hz, J ) 11.4
Hz), 2.44 (dd, 1H, J ) 7 Hz, J ) 11.7 Hz), 1.85-1.89 (m, 1H),
1.35 (s, 9H), 0.96 (d, 3H, J ) 4.3 Hz), 0.95 (d, 3H, J ) 4.3 Hz).13C
NMR (75 MHz, CDCl3): δ 156.8, 137.8, 130.0, 128.9, 126.9, 80.3,
70.7, 57.9, 54.1, 52.2, 36.9, 28.7, 28.1, 20.9, 20.9. MS (ESI): m/z
359.2 [M + Na]+; HRMS calcd for C19H32N2O3 [M + Na]+;
359.2311; found 359.2306.
1
solid. H NMR (CDCl3, 300 MHz): δ 7.91 (d, 2H, J ) 8.4 Hz),
7.66 (d, 2H, J ) 8.4 Hz), 7.35-7.39 (m, 5H), 5.01 (t, 1H, J ) 4.2
Hz), 4.93 (s, 2H), 4.89 (bs, 1H), 3.95-4.03 (m, 3H), 3.80-3.87
(m, 1H), 2.95-3.32 (m, 6H), 2.75-2.81 (m, 1H), 2.11-2.19 (m,
4H), 1.92-2.04 (m, 2H), 1.69-1.75 (m, 1H), 1.58 (dt, 1H, J )
4.5 Hz, J ) 14.4 Hz), 1.08 (d, 3H, J ) 6.6 Hz), 1.05 (d, 3H, J )
6.6 Hz). 13C NMR (CDCl3, 75 MHz): δ 156.6, 146.7, 137.9, 137.4,
129.8, 128.9, 127.9, 127.5, 126.9, 84.0, 77.4, 72.8, 68.1, 64.5, 59.1,
55.2, 54.1, 41.8, 39.7, 38.7, 36.2, 34.2, 27.6, 20.5, 20.2. MS (ESI):
m/z 583.2 [M + Na]+; HRMS calcd for C29H40N2O7S [M + Na]+
583.2454; found 583.2465.
To a solution of above secondary amine (97 mg, 0.29 mmol)
and 4-diacetoxytoluenesulfonyl chloride (103 mg, 0.35 mmol) in
THF (5 mL) at 0 °C was added N,N′-diisopropylethylamine (78
µL, 0.45 mmol) followed by 4-(dimethylamino)pyridine (4 mg, 0.03
mmol). The resulting mixture was stirred at 23 °C for 4 h.
Evaporation of the solvent under reduced pressure, followed by
flash column chromatography over silica gel yielded compound
Inhibitor 4. A solution of compound 20 (23.4 mg, 0.05 mmol)
in 30% trifluoroacetic acid in CH2Cl2 (3 mL) was stirred at 23 °C
for 30 min. After this period, the reaction mixture was concentrated
under reduced pressure and the residue was dissolved in toluene
and evaporated. The residue was dissolved in acetonitrile (2 mL)
and cooled to 0 °C. N,N′-Diisopropylethylamine (41 µL, 0.24 mmol)
and mixed carbonate 16 (10.0 mg, 0.04 mmol) were then added.
The resulting mixture was stirred for 6 h at 23 °C. The reaction
mixture was then concentrated under reduced pressure, and the
residue was purified by chromatography over silica gel to furnish
inhibitor 4 (18 mg, 80%) as an amorphous solid. 1H NMR (CDCl3,
1
19 (151 mg, 88%) as an amorphous solid. H NMR (CDCl3, 300
MHz): δ 7.92 (d, 2H, J ) 8.4 Hz), 7.81 (s, 1H), 7.76 (d, 2H, J )
8.4 Hz), 7.30-7.43 (m, 5H), 4.73 (d, 1H, J ) 7.5 Hz), 3.84-3.92
(m, 2H), 3.20 (d, 2H, J ) 6 Hz), 2.92-3.14 (m, 4H), 2.26 (s, 6H),
1.92-2.01 (m, 1H), 1.45 (s, 9H), 1.02 (d, 3H, J ) 6.6 Hz), 0.98
(d, 3H, J ) 6.6 Hz). 13C NMR (75 MHz, CDCl3): δ 169.0, 156.5,
140.4, 140.0, 138.1, 129.9, 128.9, 128.1, 128.0, 126.9, 88.9, 73.1,
58.8, 55.1, 54.0, 35.8, 28.6, 27.5, 21.2, 20.5, 20.2. MS (ESI): m/z
630.2 [M + Na]+; HRMS calcd for C30H42N2O9S [M + Na]+
630.2509: found 630.2504.
1
300 MHz): δ H NMR (CDCl3, 300 MHz): δ 8.00 (d, 2H, J )
8.4 Hz), 7.76 (d, 2H, J ) 8.4 Hz), 7.45-7.57 (m, 5H), 5.04 (s,
2H), 4.96-5.00 (m, 1H), 4.06-4.14 (bm, 2H), 3.01-3.41 (m, 4H),
2.59 (m, 2H), 2.25-2.36 (m, 3H), 2.04-2.13 (m, 1H), 1.34-1.87
(m, 10H), 1.16 (d, 3H, J ) 6.6 Hz), 1.12 (d, 3H, J ) 6.6 Hz). 13
C
Compound 20. A solution of compound 19 (151 mg, 0.25 mmol)
in methanol (10 mL) was treated with potassium carbonate (51 mg,
0.37 mmol). The mixture was stirred for 20 min at 23 °C. The
solvent was evaporated under reduced pressure, and the product
was extracted with ethyl acetate, dried over anhydrous sodium
sulfate and concentrated. The crude product was purified by flash
chromatography to provide the corresponding aldehyde (110 mg,
NMR (CDCl3, 75 MHz): δ 156.5, 146.5, 138.0, 137.6, 129.9, 128.9,
127.9, 127.5, 126.9, 73.0.4, 64.6, 59.0, 55.3, 54.0, 40.5, 39.2, 35.7,
34.1, 27.6, 25.7, 20.5, 20.2. MS (ESI): m/z 581.2 [M + Na]+;
HRMS calcd for C30H42N2O6S [M + Na]+ 581.2656; found
581.2663.
Inhibitor 2. To a solution of compound 18 (128 mg, 0.24 mmol)
in ethyl acetate (15 mL) was added 10% Pd/C (10 mg). The mixture
was stirred at 23 °C under an H2-filled balloon for 11 h. The reaction
mixture was filtered through a bed of Celite, and the filter cake
was washed with ethyl acetate. Evaporation of solvent under
reduced pressure, followed by flash chromatography on silica gel,
afforded the corresponding aromatic amine (122 mg, 95%) as a
1
95%). H NMR (CDCl3, 300 MHz): 10.1 (s, 1H), 8.05 (d, 2H, J
) 8.5 Hz), 7.99 (d, 2H, J ) 8.5 Hz), 7.2-7.36 (m, 5H), 4.75 (d,
1H, J ) 8.5 Hz), 3.81-3.94 (m, 3H), 3.22 (t, 2H, J ) 3.5 Hz),
2.94-3.06 (m, 4H), 1.93 (m, 1H), 1.40 (s, 9H), 0.93 (d, 3H, J )
6.5 Hz), 0.91 (d, 3H, J ) 6.5 Hz). 13C NMR (75 MHz, CDCl3): δ
191.2, 156.6, 144.4, 139.2, 138.1, 130.6, 129.9, 128.8, 128.4, 123.1,
80.3, 72.9, 58.3, 55.4, 53.4, 36.0, 28.6, 27.4, 20.4, 20.3. MS (ESI):
m/z 527.2 [M + Na]+; HRMS calcd for C26H36N2O6S [M + Na]+
527.2192; found 527.2188.
1
white solid. M.p.: 60-63 °C; H NMR (CDCl3, 300 MHz): δ
7.54 (d, 2H, J ) 8.5 Hz), 7.19-7.30 (m, 5H), 6.68 (d, 2H, J ) 8.5
Hz), 4.60 (d, 1H, J ) 8.4 Hz), 3.75-3.80 (m, 2H), 2.99-3.11 (m,
3H), 2.89-2.92 (m, 2H), 2.77 (dd, 1H, J ) 6.7 Hz, J ) 13.2 Hz),
1.80-1.86 (m, 1H), 1.34 (s, 9H), 0.89 (d, 3H, J ) 6.6 Hz), 0.86
(d, 3H, J ) 6.6 Hz). 13C NMR (CDCl3, 75 MHz): δ 156.0, 150.3,
137.9, 129.5, 128.4, 126.4, 114.3, 79.6, 72.8, 58.7, 54.6, 53.8, 35.4,
29.7, 28.2, 27.2, 20.2, 19.9. MS (ESI): m/z 514.2 [M + Na]+;
HRMS calcd for C25H37N3O5S [M + Na]+ 514.2352; found
514.2349.
To a stirred solution of above aldehyde (110 mg, 0.22 mmol) in
methanol (2 mL) at 0 °C was added sodium borohydride (9.5 mg,
0.25 mmol), and the mixture was stirred for 15 min. The reaction
was quenched with saturated ammonium chloride solution. The
solvent was evaporated under reduced pressure, and the product
was extracted with ethyl acetate (3×). The combined organic
extracts were dried over anhydrous sodium sulfate, and solvent was
evaporated. The crude product was purified by flash chromatog-
raphy to provide compound 20 (96 mg, 90%) as an amorphous
A solution of above amine (22 mg, 0.04 mmol) in 30%
trifluoroacetic acid in CH2Cl2 (3 mL) was stirred at 23 °C for 40
min. After this period, the reaction mixture was concentrated under
reduced pressure and the residue was dissolved in acetonitrile (2
mL). The solution was cooled to 0 °C, and mixed carbonate 10
(11.5 mg, 0.04 mmol) and N,N′-diisopropylethylamine (43.8 µL,
0.25 mmol) were added. The resulting mixture was stirred at 23
°C for 8 h. The reaction mixture was then concentrated under
reduced pressure, and the residue was purified by flash column
chromatography over silica gel to provide inhibitor 2 (18.5 mg,
75%) as an amorphous solid. 1H NMR (CDCl3, 300 MHz): δ 7.58
1
solid. H NMR (CDCl3, 300 MHz): δ 7.67 (d, 2H, J ) 8.4 Hz),
7.43 (d, 2H, J ) 8.4 Hz), 7.12-7.25 (m, 5H), 4.71 (s, 2H), 4.57
(d, 1H, J ) 7.8 Hz), 3.67-3.76 (m, 3H), 2.72-3.03 (m, 6H), 1.78
(m, 1H), 1.27 (s, 9H), 0.83 (d, 3H, J ) 6.6 Hz), 0.79 (d, 3H, J )
6.6 Hz). 13C NMR (CDCl3, 75 MHz): δ 156.4, 146.5, 138.1, 137.7,
129.9, 128.9, 127.9, 127.5, 126.8, 80.1, 73.1, 64.6, 59.0, 55.1, 54.0,
35.8, 28.6, 27.5, 20.5, 20.2. MS (ESI): m/z 529.2 [M + Na]+;
HRMS calcd for C26H38N2O6S [M + Na]+ 529.2349; found
529.2344.

5260 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 17
Ghosh et al.
(d, 2H, J ) 8.6 Hz), 7.22-7.33 (m, 5H), 6.71 (d, 2H, J ) 8.6 Hz),
4.91 (m, 1H), 4.79 (m, 1H), 4.43 (m, 1H), 3.87 (m, 2H), 3.71 (q,
1H, J ) 7H), 3.01-3.16 (m, 2H), 2.93 (dd, 1H, J ) 8.2 Hz), 2.86
(dd, 1H, J ) 8.6 Hz, J ) 14.1 Hz), 2.80 (dd, 1H, J ) 6.1 Hz),
2.66 (m, 1H), 2.04 (m, 3H), 1.87 (m, 2H), 1.65 (m, 3H), 1.48 (m,
1H), 0.95 (d, 3H, J ) 6.5 Hz), 0.90 (d, 3H, J ) 6.5 Hz). 13C NMR
(CDCl3, 75 MHz): δ 150.3, 137.4, 129.2, 128.1, 126.1, 125.9,
113.8, 83.4, 72.3, 67.4, 58.6, 54.5, 53.6, 41.2, 39.0, 38.1, 35.5, 33.6,
29.4, 27.0, 19.9, 19.6. MS (ESI): m/z 568.2 [M + Na]+; HRMS
calcd for C28H39N3O6S [M + Na]+ 568.2458; found 568.2461.
Cells, Viruses, and Antiviral Agents. MT-2 cells were grown
in an RPMI-1640-based culture medium supplemented with 15%
fetal calf serum (HyClone Laboratories, Logan, UT), 50 unit/mL
penicillin, and 50 µg/mL of streptomycin. The following HIV-1
viruses were used for the drug susceptibility assay: HIV-1LAI, HIV-
structure was refined using SHELX9742 and refitted using O 10.43
Alternate conformations were modeled for the protease residues
when obvious in the electron density maps. Anisotropic atomic
displacement parameters (B-factors) were refined for all atoms
including solvent molecules. Stereochemical parameters for the
inhibitor were created using GAUSSIAN03 with the DFT quantum-
chemical method. Hydrogen atoms were added at the final stages
of the refinement. The identity of ions and other solvent molecules
from the crystallization conditions was deduced based on the shape
and peak height of the 2Fo - Fc and Fo - Fc electron density, the
potential hydrogen bond interactions and interatomic distances. The
crystal structure was refined with three chloride anions, two sodium
cations, one glycerol molecule, and 205 water molecules including
partial occupancy sites. The final Rwork (Rfree) values were 14.7%
(19.2%) for all data between 10 and 1.35 Å resolution. The rmsd
values from ideal bonds and angles were 0.012 Å and 3.0°. The
average B-factor for all atoms was 20.9 Å2; the average B-factor
for atoms of the inhibitor was 12.7 Å.
1
Ba-L, and HIV-2EHO.
7,9 3′-Azido-2′,3′-dideoxythymidine (AZT or
zidovudine) was purchased from Sigma (St. Louis, MO). Saquinavir
(SQV) and ritonavir (RTV) were kindly provided by Roche Products
Ltd. (Welwyn Garden City, U.K.) and Abbott Laboratories (Abbott
Park, Ill.), respectively. Indinavir (IDV) and Nelfinavir (NFV) were
kindly provided by Japan Energy Inc., Tokyo. Amprenavir (APV)
was a kind gift from Glaxo-Wellcome, Research Triangle, NC.
Drug Susceptibility Assay. The susceptibility of HIV to various
drugs was determined as previously described7,9 with minor
modifications. Briefly, MT-2 cells (2 × 104/mL) were exposed to
100 50% tissue culture infectious doses (TCID50) of HIV in the
presence of various concentrations of drugs in 96-well microculture
plates and were incubated at 37 °C for 7 days. After 100 µL of the
medium was removed from each well, 3-(4,5 dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) solution (10 µL, 7.5
mg/mL in phosphate-buffered saline) was added to each well in
the plate, followed by incubation at 37 °C for 2 h. After incubation,
to dissolve the formazan crystals, 100 µL of acidified 2-propanol
containing 4% (v/v) Triton X-100 was added to each well and the
optical density measured in a kinetic microplate reader (Vmax,
Molecular Devices, Sunnyvale, CA). All assays were performed
in duplicate or triplicate. In determining the sensitivty of HIV
isolates to drugs, phytohemagglutinin-activated peripheral blood
mononuclear cells (PHA-PBM) (1 × 106/mL) were exposed to 50
TCID50 of each isolate and cultured in the presence or absence of
various concentrations of drugs in 10-fold serial dilutions in 96-
well microculture plates. On day 7 of culture, the supernatant was
harvested and the amount of p24 Gag protein was determined by
using a fully automated chemiluminescent enzyme immunoassay
system (Lumipulse F; Fujirebio Inc., Tokyo).7,9 Drug concentrations
that suppressed the production of p24 Gag protein by 50% (IC50)
were determined by comparison with the p24 production level in
drug-free control cell culture. All assays were performed in
triplicate.
Acknowledgment. Financial support by the National Insti-
tutes of Health (GM 53386, A.K.G. and GM62920, I.T.W.) is
gratefully acknowledged. This work was also supported in part
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 Cooperative Research Project on
Clinical and Epidemiological Studies of Emerging and Re
emerging Infectious Diseases (Renkei Jigyo: No. 78, Kumamoto
University) of Monbu-Kagakusho. The work was supported by
the Georgia Research Alliance and the Molecular Basis of
Disease program of Georgia State University. We thank Annie
Heroux for measuring the X-ray diffraction data at beamline
X26C of the National Synchrotron Light Source. Use of the
National Synchrotron Light Source, Brookhaven National
Laboratory, was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under
Contract No. DE-AC02-98CH10886.
Supporting Information Available: HPLC and HRMS data
for compounds 2-4. This material is available free of charge via
the Internet at http://pubs.acs.org.
Determination of X-ray Structure of Inhibitor 3-Bound HIV
ProteaseWT. The X-ray crystal structure of inhibitor 3 bound to
HIV ProteaseWT has been deposited in the Protein Databank with
access code 2HB3. The crystals of the PRWT complexed with GRL-
06579A, which was dissolved in dimethyl sulfoxide, were grown
by the hanging-drop vapor diffusion method using 10:1 molar ratio
of the inhibitor to protein. The well solution contained sodium
acetate buffer (pH ) 4.2) and 1.4 M NaCl. Crystals were transferred
into a cryoprotectant solution containing the reservoir solution plus
20-30% (v/v) glycerol, mounted on a nylon loop, and flash-frozen
in liquid nitrogen. X-ray diffraction data were collected on the ×26C
beamline of the National Synchrotron Light Source, Brookhaven
National Laboratory at 90 K using 0.96 Å wavelength. Data were
processed using HKL2000.39 A medium-sized platelike crystal, with
dimensions of 0.2 × 0.2 × 0.1 mm, diffracted to 1.35 Å resolution
with mosaicity of 0.4° and produced an Rsym value of 5.0% (52%)
for data between 50 and 1.35 Å resolution. These data were reduced
in space group P21212 with unit cell dimensions of a ) 58.1 Å, b
) 86.5, c ) 45.9 Å with one dimer per asymmetric unit. The CPP4i
suite of programs40,41 was used to obtain a molecular replacement
solution using as the starting model the PRL90M complex with
TMC114 (PDB code 2F81), which is in the same space group. The
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