Design of HIV-1 protease inhibitors active on multidrug-resistant virus

Article abstract of DOI:10.1021/jm049454n

On the basis of structural data gathered during our ongoing HIV-1 protease inhibitors program, from which our clinical candidate TMC114 9 was selected, we have discovered new series of fused heteroaromatic sulfonamides. The further extension into the P2′

Full text of DOI:10.1021/jm049454n

J. Med. Chem. 2005, 48, 1965-1973
1965
Design of HIV-1 Protease Inhibitors Active on Multidrug-Resistant Virus
Dominique L. N. G. Surleraux,† Herman A. de Kock,† Wim G. Verschueren,† Geert M. E. Pille,†
Louis J. R. Maes,†, Anik Peeters,† Sandrine Vendeville,† Sandra De Meyer,† Hilde Azijn,† Rudi Pauwels,†
Marie-Pierre de Bethune,† Nancy M. King,‡ Moses Prabu-Jeyabalan,‡ Celia A. Schiffer,‡ and
Piet B. T. P. Wigerinck*,†
Tibotec BVBA, Generaal de Wittelaan L 11B 3, B-2800 Mechelen, Belgium, and Department of Biochemistry and
Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received July 9, 2004
On the basis of structural data gathered during our ongoing HIV-1 protease inhibitors program,
from which our clinical candidate TMC114 9 was selected, we have discovered new series of
fused heteroaromatic sulfonamides. The further extension into the P2′ region was aimed at
identifying new classes of compounds with an improved broad spectrum activity and acceptable
pharmacokinetic properties. Several of these compounds display an exceptional broad spectrum
activity against a panel of highly cross-resistant mutants. Certain members of these series
exhibit favorable pharmacokinetic profiles in rat and dog. Crystal structures and molecular
modeling were used to rationalize the broad spectrum profile resulting from the extension into
the P2′ pocket of the HIV-1 protease.
Introduction
ring, part of what is termed the bis-THF moiety, present
in 9. The structure and interactions of 9 with HIV-1
protease are shown in Figure 2. The bis-THF moiety
present in 9 allows for additional interactions with the
Asp29 of the protease,14 having a profound influence on
the antiviral activity. This observation prompted us to
extend into P2′, where the aniline nitrogen of both 9
and 5 interacts with Asp30, trying to design a next-
generation of inhibitors that incorporate structural
features that allow for comparable extra interactions
with backbone and/or with side chains of amino acids
less prone to mutate.
Besides a further advanced broad spectrum activity,
new PIs should also have an acceptable oral pharma-
cokinetic profile (PK) in order to lower pill burden,
thereby improving adherence and reducing side effects.
Within this scope we explored a large number of
annelated heterocyclic sulfonamides as P2′ moieties, but
this paper will focus only on the most promising ones,
the benzothiazole (BT) and benzoxazole (BO) sulfon-
amides. Crystal structures and calorimetric data were
obtained of complexes for one compound from each of
these classes, allowing for detailed analysis of the
interactions in the P2′ pocket.
Since their introduction in late 1995, HIV-1 protease
inhibitors (PIs) have proven to be an advantageous
extension of the existing antiretroviral armamentarium,
which at the time consisted solely of nucleoside analogue
reverse transcriptase inhibitors (NRTIs). This introduc-
tion has been marked by a profound decrease in mortal-
ity rate associated with HIV-1 infection. Today, PIs are
considered as essential components to establish highly
active antiretroviral therapy (HAART).1,2 HIV-1 pro-
tease is a prime target because it is required for HIV-1
replication. Structure-based drug design has led to the
discovery of seven approved drugs and several others
in advanced clinical trials. Despite this remarkable
success, the emergence of HIV-1 mutants that are
resistant to current drug regimens remains a critical
factor in the clinical failure of antiviral therapy. For the
seven currently approved PIs (ritonavir 1,3 saquinavir
2,4 nelfinavir 3,5 indinavir 4,6 amprenavir 5,7 atazanavir
6,8 and lopinavir 79) and one PI in development
(tipranavir 810) (Figure 1), the emergence of mutants
that confer multidrug resistance is disturbing.11,12 In-
creasing cross-resistance has necessitated further re-
search with an emphasis on broad spectrum activity
against PI-resistant mutants.
Chemistry
During our HIV-1 protease inhibitors program, efforts
directed toward extensive profiling against highly re-
sistant viruses have led to the selection of TMC114 913
as our clinical candidate (currently in phase IIB). 9 is a
next-generation inhibitor, chemically related to 5, with
an extremely low susceptibility to existing resistant
mutants.14 When 9 is structurally compared to 5, these
two compounds only differ by a second tetrahydrofuran
The first two benzothiazole compounds 10 and 11
were prepared following a described method,15 using a
p-aminophenyl precursor to build the fused heterocyclic
system via a multistep approach. For the preparation
of the benzothiazoles 20a-i a synthetic route was
sought that would give the possibility of modifying the
substitution pattern at the C2-N position of the fused
benzothiazoles in a rapid manner. In the first approach
a 2-aminobenzothiazole derivative was converted to the
2-chloro analogue using a laborious diazotation step.16a
A major improvement could be made when utilizing the
good leaving group capacity of the sulfoxide functional-
ity in compounds 18 and 21 (Scheme 1). Starting from
* To whom correspondence should be addressed. Phone: +32
(0)15292445. Fax: +32(0)15401257. Email: pwigeri1@tibbe.jnj.com.
† Tibotec BVBA.
Current address: Faculty of Pharmaceutical, Biomedical and
Veterinary Sciences, University of Antwerp, Campus Middelheim,
Groenenborgerlaan 171, B-2020 Antwerp, Belgium.
‡ University of Massachusetts Medical School.
10.1021/jm049454n CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/25/2005

1966 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Surleraux et al.
Figure 1. Chemical structure of currently approved HIV-1 protease inhibitors (1-7) and tipranavir (8).
Figure 2. Structure and binding mode of TMC 114 9. Hydrogen bond interactions are shown in P2 and P2′ pockets.
commercially available 2-SMe-benzothiazole 12a chlo-
rosulfonylation with SOCl2 and ClSO3H yielded the
desired sulfonyl chloride 13a. Coupling of 13a with the
known amine 1417 gave the intermediate 15a. Oxidation
of the thiomethyl moiety with either MCPBA or mag-
nesium monoperoxophthalate of 15a (method B) and
17a (method A) yielded 18/18′ or 21/21′, respectively,
as mixtures of sulfoxide (18, 21) and sulfone (18′, 21′).
Intermediate 18 allowed for a one-step synthesis of final
compounds (method A). Alternatively, final compounds
20a-i could be prepared via a two-step procedure
(method B).
Benzoxazole compounds 26a-c were prepared as
described earlier.18 To further evaluate the benzoxazole
family, a method similar to the one used for benzothi-
azole synthesis was developed (Scheme 2). The key
intermediate 15b that allowed relatively fast synthesis
was reported earlier by us.18 Substitution with ap-
propriate amines could be accomplished at elevated
temperature (e.g., refluxing acetonitrile). Subsequent
debenzylation of intermediates 24d-g with Pd/C and
H2 gas followed by coupling of the intermediate amines
25d-g with 1619 gave final compounds 26d-g in good
yield. Further synthetic improvement more resembling
the benzothiazole chemistry (Scheme 1) will be pub-
lished later.
Results and Discussion
In Vitro SAR. All compounds were tested for their
antiviral activity against a selected panel of recombi-
nant clinical isolates,20 shown in Table 1. These isolates
were selected because of their high degree of cross-
resistance to the approved and experimental PIs (Table
2) and because of the presence of mutations in the
protease gene associated with resistance to current
protease inhibitors as defined by IAS.12,21,22 Different
combinations of resistance-associated PI mutations were
found in each of the selected isolates. Comparing the
results for compounds 1-8 (Table 2) with those of our
first two benzothiazole compounds 10 and 11 (Table 3)
clearly shows a more pronounced antiviral potency for
the latter ones, both on wild type as against the selected
mutant strains. Although the presence of the exocyclic
amino group in 11 seems to lower the activity against
wild-type virus compared to 10, it clearly has a profound
influence on retaining the broad spectrum activity
across our panel. However, in vivo evaluation of 11
showed poor oral bioavailability, which may in part be
correlated with its low aqueous solubility. Another
drawback associated with this compound is the high
degree of protein binding.23
Encouraged by the exceptional broad spectrum activ-
ity of 11, we decided to further explore this class of

Design of HIV-1 Protease Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1967
Scheme 1. Synthesis of Benzothiazole (BT) 20a
i
a Reagents and conditions: (a) (1) ClSO3H, (2) SOCl2; (b) 14, Et3N, solvent; (c) (1) HCl, PrOH or TFA, DCM; (2) 16, DCM, Et3N; (d)
MCPBA, DCM or MMPP, MeOH; (e) NHR1R2 19, solvent.
Scheme 2. Synthesis of Benzoxazole (BO) Derivatives 26a
a Reagents and conditions: (a) NHR1R2 19, solvent; (b) Pd/C, H2, MeOH; (c) 16, DCM, Et3N.
Table 1. Resistant Mutant Sequences: Mutations in the Protease Gene Associated with Resistance to PIs According to IAS21 in Bold
strain
03 10 11 13 16 19 22 24 32 33 35 36 37 41 43 46 47 50 53 54 55 57 58 62 63 70 71 72 73 77 82 84 85 89 90 93
r13025
r13034
r13363
GSS004421
I
I
I
I
I
I
T
M
D
D
D
Y
T
N
N
I
R/K
K
E
P
P
P
P
T
V
V
L
V
V
V
V
I
A/V
I
I
I
R/K
V
V
V
V
I
F
F
T
I
I
V
V
V
R
R
I
A
V
M
M
L
I
V
G/A
I
L
V
C
V/I
2-amino-fused heteroaromatic sulfonamides trying to
retain broad spectrum activity while addressing oral
bioavailability and protein binding issues.
Antiviral results for 20a-i (BT) and 26a-g (BO) are
presented in Table 3. From these data a few straight-
forward SAR principles can be put forward:
(i) Replacing the 2-NH2 function present in 11 and
26a by either a 2-MeNH (20a or 26b) or a 2-Me2N (20b
or 26c) seems to have a common positive effect on wild-
type potency. However, with regard to a maintained
broad spectrum activity, the presence of a tertiary
substituted C2-amine in compounds 20b and 26c is not

1968 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Surleraux et al.
Table 2. Protease Inhibitory Activity (pEC50) and Toxicity
(TOX, pCC50) of Currently Approved HIV-1 Protease Inhibitors
(1-7) and Tipranavir (8) on Wild Type (IIIB) and Mutant
Viruses
pEC50
TOX
pCC50 IIIB r13025 r13034 r13363 GSS004421
ritonavir 1
saquinavir 2
nelfinavir 3
indinavir 4
4.53 7.19 5.99
4.68 7.58 6.83
4.75 7.15 5.31
<4.49 7.40 5.97
5.30
5.91
5.90
5.90
6.01
ND
4.89
7.54
5.82
6.39
4.95
7.23
6.28
6.15
4.97
6.30
6.28
6.47
5.75
7.32
6.02
6.73
amprenavir 5 <4.49 7.36 6.43
atazanavir 6
lopinavir 7
tipranavir 8 <4.49 6.30 5.74
4.53 7.82 5.78
4.95 8.01 7.84
6.59
ND
Table 3. Protease Inhibitory Activity (pEC50) and Toxicity
(TOX, pCC50) of Compounds of Benzoxazole and Benzothiazole
Series on Wild Type (IIIB) and Mutant Viruses
Figure 3. Scatter plot comparing BO-BT activity on wild
type and r13363. BT compounds are shown in red, while BO
compounds are shown in blue. Different side chains are
presented as different shapes. BT and BO compounds with the
same side chain are connected.
(ii) A qualitative analysis of the relationship between
the chain length and the antiviral potency showed the
ethyl spacer (compounds 20c, 20h, and 26e) to be
comparable to the propyl spacer (compounds 20e, 20i,
and 26f). The most significant difference is noted on
wild-type activity, thus slightly favoring the ethyl
spacer.
(iii) Introduction of an alkylamino functionality con-
taining a secondary amine as in compound 20d has a
deleterious impact on antiviral activity, most likely
governed by bad cell penetration rather than conflicting
interactions with the HIV-1 protease.
(iv) A direct comparison between the benzothiazole
and benzoxazole analogues is presented in Figure 3.
This graphical representation shows a nearly constant
difference in potency between BO an BT analogues, both
on wild type and on mutant strain r13363 (strain that
shows the most pronounced phenotypic impact across
both BO and BT). Making an exception to this gener-
alization are the unbranched compounds 11 and 26a,
showing less variation in activity on both strains.
Another observation is that the introduction of alkyl-
amino moieties in the BO class (comparing 26a with
26g and 26d) has a bigger impact on antiviral activity
when compared to differences observed for the BT class
(comparing 11 with 20c and 20g).
Because both the benzoxazole and the benzothiazole
series, despite the differences mentioned above, show
promising broad spectrum antiviral activity, a subset
of compounds was selected and their main properties
that could contribute to an improved oral bioavailability
were evaluated (in silico/in vitro). The in vitro assays
used are well-known and deal with all phases of the
ADME process. We have measured the solubility at
different pH, the permeability in Caco-2 assay, and the
metabolic stability in the presence of rat, dog, and
human liver microsomes. Results are presented in Table
4.
well tolerated. An analogous trend can be observed for
the alkylamino-substituted series when comparing the
antiviral activity on mutant strains of compounds 20e,
20g, and 26d with 20f, 20h, and 26e, respectively. The
preference of the secondary amines can be explained by
the presence of a strong hydrogen bond interaction with
the side chain of Asp30′ shown in the crystal structures
of 20g and 26b (see also next paragraph).
Although the cLogP values are well within an accept-
able range (2.7-3.9), the calculated polar surface area

Design of HIV-1 Protease Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1969
Table 4. Results of in Vitro Assays in Comparison with
Calculated Propertiesa
Caco-2
Papp Papp
metabolic
stability
in silico
solubility
compd CLogP PSA pH 7 pH 5 RLM HLM DLM Ap-Bl Bl-Ap
11
3.26 189.9 <0.01 <0.01 77
3.34 175.9 <0.01 <0.01 72
3.47 179.2 0.04 0.41 19
3.74 170.4 1.18 1.64 47
3.94 179.2 <0.01 0.09 56
65
63
35
35
26
62
37
74
80
30
52
46
67
69
9.9 27.3
19.3 14.2
6.1 26.6
20a
20c
20f
20g
26a
26b
2.0
8.5
3.5 21.7
3.8 23.8
17.6 24.0
2.70 174.8 ND
ND
90
2.78 160.8 <0.01 0.01 70
a The log P and polar surface area (PSA) have been calculated
using Scitegic’s Pipeline Pilot software. Solubility is determined
at pH 7 and pH 5 and expressed in mg/mL. Metabolic stability
has been measured in three different species as the percent
remaining parent compound after 30 min of incubation with liver
microsomes of rat (RLM), dog (DLM), and human (HLM) origin.
Apparent permeability in Caco-2 assay is presented in cm/s (×106).
(PSA) values exceed those that are generally accepted
(which is around 80 for commercially available orally
administered compounds). Starting from compound 11
or 26a, the introduction of either alkyl (compound 20a
or 26b) or alkylamino (e.g., compound 20g) compounds
slightly lowers PSA values; however, the introduction
of a basic nitrogen (alkylamino series) was mainly
directed toward improving aqueous solubility. Compar-
ing the solubility of compounds 20g, 20f, and 20c with
that of 11 shows an increased aqueous solubility that
is influenced by the substitution pattern around the
basic nitrogen. This is illustrated by the solubility at
pH 7, which ranges from <0.01 to 1.18 mg/mL going
from [2-(1-pyrrolidinyl)ethyl]amino in 20g to [2-(dimeth-
ylamino)ethyl]amino in 20c to [3-(dimethylamino)pro-
pyl]methylamino in 20f. These findings have been
further substantiated by more recent findings for both
the BT and BO series (data not shown). Metabolic
stability in the presence of liver microsomes seems to
decrease when introducing the alkylamino moieties,
metabolic degradation being the most extensive for
human liver microsomes. From the Caco-2 data it is
apparent that introduction (starting from compound 11
or 26a) of a methyl group (compounds 20a and 26b)
markedly increases the permeability, increasing Papp
(apical to basolateral) from 9.9 to 19.3 for 20a and from
3.8 to 17.6 for 26b. However, the introduction of an
alkylamino side chain substantially lowers the apparent
permeability (e.g., 3.5 for compound 20g) besides induc-
ing more pronounced efflux properties.
Figure 4. Side view and top view of hydrogen bond interac-
tions between 20g and the enzyme in the P2′ pocket.
Crystal Structures of HIV-1 Protease Com-
plexes. For one benzothiazole (20g) and one benzox-
azole (26b) compound presented here, the crystal struc-
ture in complex with wild-type HIV-1 protease has been
determined to a resolution of 1.83 and 2.00 Å, respec-
tively. The most important hydrogen bond interactions
are shown in Figures 4 (20g) and 5 (26b). The refine-
ment statistics are presented in Table 5. The high
resolution of the structures allows for a detailed analysis
of the intermolecular interactions present in these
complexes.
Hydrogen bonds were calculated for both complexes
(Table 6) after the addition of hydrogen atoms. The
central water molecule present in the flap region makes
hydrogen bonds similar to what has been observed for
other HIV-1 protease inhibitors. With the bis-THF
moiety present in the P2 pocket, two strong hydrogen
Figure 5. Side view and top view of hydrogen bond interac-
tions between 26b and the enzyme in the P2′ pocket.
bonds can be accepted from the backbone of Asp29 and
Asp30. The importance of this group has already been
reported before. In a recent paper24 the crystal structure
of 5 (mono-THF) has been compared to that of 9 (bis-

1970 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Surleraux et al.
Table 7. Thermodynamics of Inhibitor Bindinga
Table 5. Crystallographic Data Collection and Refinement
Statistics for the Complexes of HIV-1 Protease with
Compounds 20g and 26b
Kd
∆H
-T∆S
∆G
HB NROT
5b
9b
3.9 × 10-10
-7.3 -5.3
-12.6
-15.2
-15.6
8
10
8
12
12
13
16
13
20g
26b
4.5 × 10-12
-12.1 -3.1
data collection
resolution (Å)
a (Å)
TMC126c 3.9 × 10-12
-12
-3.6
1.83
50.6
57.8
61.5
P212121
4
2
50.7
58
62
P212121
4
7
93.5
210
20g
26b
6.37 × 10-11 -11.7 -1.97 -13.67 11
9.29 × 10-10
-7.3 -4.84 -12.11
7
b (Å)
a Dissociation constant Kd is expressed in M; ∆H, -T∆S, and
∆G are given in kcal/mol. The number of hydrogen bond interac-
tions with a donor-acceptor distance of <3.5 Å (HB) and the
number of rotatable bonds (NROT) are also shown. b Reference
24. c Reference 36.
c (Å)
space group
Z
Rmerge (%)
completeness (%)
temp (K)
5.3
98.7
210
Table 8. In Vivo Assaysa
unique reflections
total reflections
I(σ)
16 311
69 567
10.5
12 027
30 716
10.1
compd species Cmax (ng/mL) Tmax (h) t1/2 (h) AUC (ng‚h/mL)
20f
dog
dog
dog
rat
rat
1680
1972
986
1.0
0.5
2.0
1.0
1.4
2.9
1.66
0.9
1.6
ND
6592
6048
2034
448
refinement
R factor (%)
R free (%)
20g
26b
26a
26b
18.1
22.8
18.2
23.8
260
multiple conformations Q18, E21, D30, D35, I50, Q18, E35,
927
1196
G51, Q61, P81, E21′,
D30′, I50′, G51′, V75′
K7′, E34′
a Pharmacokinetics in Wistar rats was determined in a single
dose study at dose levels of 20 mg/kg. Both compounds were
formulated in PEG400. The studies in dog were carried out in
coadministration with ritonavir. Data of oral administration to dog
were obtained at 20 mg/kg as solution in either PEG400 (20f and
26b) or CD40 + CA (20g) (40% w/v hydroxypropyl-â-cyclodextrin
with 2% citric acid).
accuracy
bond length rmsd (Å)
0.03
0.022
1.347
bond angle rmsd (deg) 1.423
Table 6. Hydrogen Bond Distances (in Å) with HIV-1 Protease
for 20g and 26ba
20g
26b
of 3 (in vitro and in vivo). However, an asparagine
should still be able to form the same hydrogen bonds to
these compounds. Therefore, in terms of the impact of
resistance mutations, this interaction can be considered
equivalent to a backbone interaction.
O7‚‚‚H-N (Asp29)
2.1
2.2
2.1
1.5
1.7
1.9
2.1
2.5
2.0
1.9
2.9
2.1
2.7
1.8
2.0
2.1
2.0
2.2
3.1
2.6
2.3
O6‚‚‚H-N (Asp30)
CdO2‚‚‚H-O-H (flap H2O)
SdO5‚‚‚H-O-H (flap H2O)
O3-H‚‚‚-OOC (Asp25)
(flap H2O) O‚‚‚H-N (Ile50)
(flap H2O) O‚‚‚H-N (Ile50′)
N1-H‚‚‚OdC (Gly27)
N(thia/oxazole)‚‚‚H-N (Asp30′)
N3-H‚‚‚OOC (Asp30′)
N29(pyrrolidine)‚‚‚OOC (Asp29′)
For compound 20g an additional interaction between
pyrrolidine and the side chain of Asp29′ has been
observed. The distance between the pyrrolidine N atom
and the closest O atom of the carboxylic acid side chain
of Asp29′ is 2.9 Å. The pyrrolidine N is likely to be
protonated because its estimated pKa is 8.8. Therefore,
20g and Asp29′ likely form a strong hydrogen bond.
Thermodynamics of Inhibitor Binding. In addi-
tion, thermodynamic data were collected with isother-
mal titration calorimetry for the binding of 20g and 26b
(Table 7). Correlating the thermodynamics of inhibitor
binding with crystal structures is very difficult because
the binding free energy is an equilibrium between the
bound and free forms of both the protease and the
inhibitor as well as their changing interactions with the
solvent. Nevertheless, we will note a few patterns that
we observed between these similar types of inhibitors.
The binding free enthalpy (∆H) of 20g and 26b is -11.7
and -7.3 kcal/mol, respectively. The higher binding
enthalpy measured for 20g is close to the one reported
for 924 (-12.1 kcal/mol), which is in agreement with it
forming more hydrogen bonds in the crystal structures.
This may be explained by the fact that 9 is also capable
of making a water-mediated hydrogen bond interaction
with the side chain of Asp30′ in the P2′ pocket.24 In
addition, inhibitor 20g, which contains more degrees of
freedom and more rotatable bonds, appears to have the
least favorable interaction entropy (Table 7).
a Atom numbering for 20g and 26b as in Figures 4 and 5,
respectively.
THF). For 9, hydrogen bond distances of 2.0 Å (Asp29)
and 2.1 Å (Asp30) were observed. For compound 26b,
the interaction with Asp30 is significantly weaker. For
this compound also, the N1-H does not interact with
the backbone of Gly27. This is caused by a change in
the position of the Gly27 carbonyl group due to an
intramolecular hydrogen bond (1.9 Å) formed between
the adjacent N-H of Ala28 with one of the catalytic
aspartate residues (Asp25). This intramolecular interac-
tion is less pronounced in other crystal structures of
HIV-1 protease.
The hydrogen bonds formed with Asp29′ and Asp30′
are very important because this research effort is
focused on optimizing interactions in the P2′ pocket. The
N atom of the thiazole ring in 20g interacts with the
backbone N-H of Asp30′. The oxazole ring of 26b is in
a similar position as the thiazole ring in 20g, but the
interaction with the backbone of Asp30′ is less favorable.
Perhaps the presence of a larger sulfur atom in 20g
forces this compound to be located a little lower in the
pocket, enhancing the interaction of the N atom with
Asp30′. The secondary amine present in both structures
forms a strong hydrogen bond interaction with the side
chain of Asp30′. This interaction is stronger for 20g than
for 26b (Table 6, 1.9 and 2.3 Å, respectively). Asp30 is
fairly conserved, only mutating to Asn in the presence
In Vivo Results. A limited in vivo evaluation in rat
and dog is summarized in Table 8. The pharmacokinet-
ics of 26a and 26b in Wistar rats was determined in a
single dose study at dose levels of 20 mg/kg. Both
compounds were formulated in PEG400. After oral
administration, 26b showed a higher Cmax than 26a by

Design of HIV-1 Protease Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1971
tions: d for doublet, t for a triplet, m for a multiplet, etc. For
the sake of brevity, it was opted to completely characterize
(NMR included) one representative example of each subset of
compounds. Low-resolution mass spectra (LRMS) were ob-
tained from a single quadrupole (Waters ZMD), ion trap
(ThermoFinnigan LCQ Deca), or time-of-flight (Waters LCT)
mass spectrometer using electrospray ionization (ESI) in
positive or negative mode. All reagents were purchased from
commercial sources (Acros, Aldrich, etc.) and were used as
received. Column chromatography was carried out on silica
gel 60 Å, 60-200 µm (ROCC). Thin-layer chromatography was
performed on silica gel 60 F254 plates (Merck). Optical rotations
were measured at the sodium D line, using a Propol automatic
polarimeter. Analytical HPLC was done on a Waters Alliance
2690 (pump + autosampler) system equipped with a Waters
996 photodiode array detector. To check the purity of the end
products (20 and 26), two independent chromatographic
systems were used.
The first system consisted of the following: column, Waters
Xterra MS C18, (3.5 µm, 4.60 mm × 100 mm); mobile phase
A, 10 mM CH3COONH4 in H2O; mobile phase B, CH3CN.
Analyses were run at 30 °C using a flow rate of 1 mL/min and
applying the following gradient: 0 min, 5% mobile phase B;
10 min, 95% mobile phase B; 12 min, 95% mobile phase B. In
every case, 10 µL of a 1 mM solution was injected. The
equilibration time between two runs was 3 min.
3.5-fold. These results correlate nicely with the observed
difference between the apparent permeability of 26a
(Papp ) 3.8) and 26b (Papp ) 17.6). The studies in dog
were carried out in coadministration with ritonavir
because the systemic availability can be “boosted”
considerably through inhibition of P450 metabolism
(e.g., first-pass effect and metabolic clearance), a prin-
ciple used with lopinavir in the commercial preparation
Kaletra. While compound 11 showed low systemic
availability after oral dosing, we hoped to improve on
oral bioavailability with limited impact on the antiviral
spectrum. Data of oral administration to dog at 20 mg/
kg as a solution in either PEG400 (20f and 26b) or CD40
+ CA (20g) (40% w/v hydroxypropyl-â-cyclodextrin with
2% citric acid) are presented in Table 8. The results
clearly indicate that for both BO and BT families,
acceptable oral PK can be attained. Peak plasma levels
as high as 1972 and 1680 ng/mL were observed for 20g
and 20f, respectively. These data warranted a further
exploration of both families. Moreover, as mentioned
before, the substitution pattern around the basic nitro-
gen has a significant influence on the aqueous solubility
and hence also influences the systemic availability.
Recent findings show that further improvement seems
possible. These will be published in due course.
The second system (polar) consisted of the following: col-
umn, Alltech Prevail C18 (3.0 µm, 4.6 mm × 100 mm); mobile
phase A, 10 mM CH3COONH4 in H2O; mobile phase B, CH3-
CN. Analyses were run at 30 °C using a flow rate of 1 mL/min
and applying the following gradient: 0 min, 0% mobile phase
B; 1 min, 0% mobile phase B; 9 min, 95% mobile phase B; 12
min, 95% mobile phase B. In every case, 10 µL of a 1 mM
solution was injected. The equilibration time between two runs
was 3 min.
Conclusion
In summary, using structural information obtained
during our continuing HIV-1 protease program, we have
designed and synthesized novel series of fused hetero-
cyclic sulfonamides. The pronounced broad spectrum
activity of one of our first compounds in the benzothi-
azole series 11 encouraged us to further explore fused
heterocyclic sulfonamide inhibitors that extend further
into the P2′ region of the HIV-1 protease. Synthetic
improvements that allow for fast preparation of a
diverse set of compounds were developed for both the
benzothiazole and benzoxazole series. In vitro results
demonstrate that incorporation of a heterocyclic part in
P2′ can result in a substantial increase in the broad
spectrum against highly PI-resistant mutants. Against
r13363, containing no less than seven resistance-associ-
ated protease mutations, several of our compounds
maintain an activity in the range of 7.5-8 (pEC50).
Crystal structures and molecular modeling were used
to rationalize the broad spectrum profile resulting from
the extension into the P2′ pocket of the HIV-1 protease.
In vitro and in vivo evaluation of ADME properties
was done in parallel with our synthesis program and
delivered a set of compounds with better physical
properties (i.e., solubility, protein binding). Peak plasma
levels as high as 1972 and 1680 ng/mL were observed
for 20g and 20f, respectively. Despite the need for
boosting, these compounds proved to be well absorbed
in animal species and could be candidates for further
development.
Eluted peaks were detected at a single wavelength (λmax
)
for both chromatographic systems. The retention time for each
compound is given for both systems and is reported in minutes.
A full table with retention time and purity (%) is in Supporting
Information.
Synthesis of Compound 20g. To a 0.5 M solution of
sulfoxide 18 (22.5 mmol) in DCM was added 3 equiv of
aminoethylpyrrolidine. This solution was refluxed for 1.5 h,
washed with a saturated NaHCO3 solution, dried over MgSO4,
and filtered over dicalite. Evaporation of solvent and purifica-
tion on silica provided compound 20g (12.02 g, 76%). MS (m/z):
702 (MH+). 1H NMR and 13C NMR spectra of 20g can be found
in Supporting Information.
Synthesis of Compound 26d. To a solution of amine 25d
(10 mmol) in DCM (20 mL) at 0 °C was added 13 mmol of
triethylamine and 12 mmol of 2,5-pyrrolidinedione-1-[[(3R,-
3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy] 16. The reaction
mixture was then stirred at room temperature for 3 h, then
washed with a saturated NaHCO3 solution. The organic phase
was dried over MgSO4 and evaporated. Residue was then
purified by column chromatography. Upon concentrating the
suitable fractions, compound 26d was isolated (3.56 g, 52%).
1
MS (m/z): 686 (MH+). The H NMR spectrum of 26d can be
found in Supporting Information.
Virology. Cell-Based Anti-HIV and Toxicity Assay. The
antiviral activity has been determined with a cell-based
replication assay. This assay directly measures the ongoing
replication of virus in MT4 cells via the specific interaction of
HIV-tat with LTR sequences coupled to GFP. In the toxicity
assay, a reduced expression of the GFP reporter protein serves
as a marker for cellular toxicity of a compound. Briefly, various
concentrations of the test compounds are brought into a 384-
well microtiter plate. Subsequently, MT4 cells and HIV-1/LAI
(wild type) are added to the plate at a concentration of 150 000
cells/mL and 200 cell culture infectious doses 50% (CCID50).
To determine the toxicity of the test compound, mock-infected
cell cultures containing an identical compound concentration
range are incubated for 3 days (37 °C, 5% CO2) in parallel with
the HIV-infected cell cultures. On the basis of the calculated
Experimental Section
Chemistry. General Experimental Procedures. NMR
spectra were recorded on a Bruker Avance 400 spectrometer,
1
operating at 400 MHz for H and 100 MHz for 13C and with
DMSO as solvent unless otherwise stated. In every case
tetramethylsilane (TMS) was used as an internal standard.
Chemical shifts are given in ppm, and J values are given in
Hz. Multiplicity is indicated using the following abbrevia-

1972 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6
Surleraux et al.
percent inhibition for each compound concentration, dose
response curves are plotted and EC50, pEC50, CC50, and pCC50
values are calculated.
optimizations. The refinement was finished when the largest
derivative was smaller than 0.01.
Acknowledgment. M.P.-J., N.M.K., and C.A.S.
were supported by the National Institutes of Health
(Grant P01-GM66524) and Tibotec/COSAT. We thank
Liesbet J. Smeulders and Inge M. J. Vereycken for
performing the antiviral assays, Marleen J. M. Van
Dooren for analytical support, and Vincent Chou for
assistance with the crystallographic refinement.
Activity in the Presence of Human Serum and Human
Serum Proteins. MT4-LTR-EGFP cells were infected with
HIV-1/LAI at a MOI of 0.001-0.01 CCID50 per cell. After 1 h
of incubation, cells were washed and plated into a 384-well
plate containing serial dilutions of the compound in the
presence of 10% FCS or 10% FCS + 50% human serum (HS).
After 3 days of incubation, the relative fluorescence of treated
cultures was measured and compared with the relative
fluorescence of untreated cultures. The EC50 of a compound
was defined as the concentration that inhibited the relative
fluorescence by 50%.
Metabolic Stability. The metabolic stability was deter-
mined in dog, human, and rat liver microsomes (10 µM
compound, 30 and 120 min incubation at 37 °C). The degree
of metabolism is determined by direct measurement of the
residual parent compound in the reaction mixture using LC-
MS (liquid chromatography-mass spectrometry).
Protein Crystallography. Protein expression, isolation,
and purification were carried out as previously described.25
The protein used for crystallizing the inhibitors was further
purified using a Pharmacia Superdex 75 FPLC column.
Crystal screens were set up by the hanging drop vapor
diffusion method as described elsewhere.26 The inhibitors were
dissolved in dimethyl sulfoxide (DMSO). A protein concentra-
tion of 1 mg/mL was used for crystallization by adding 3 molar
excess of the inhibitors. The reservoir and the precipitant
solutions contained 126 mM sodium phosphate at pH 6.2, 63
mM sodium citrate, and 27-35% AMSO4. Crystals of longest
dimension, 0.2 mm, appeared in 2-3 days.
Supporting Information Available: Experimental de-
tails for the synthetic procedures and characterization data
for intermediate and final compounds. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
References
(1) Bartlett, J. A.; DeMasi, R.; Quinn, J.; Moxham, C.; Rousseau,
F. Overview of the effectiveness of triple combination therapy
in antiretroviral-naive HIV-1 infected adults. AIDS 2001, 15,
1369-1377.
(2) Gulick, R. M.; Mellors, J. W.; Havlir, D.; Eron, J. J.; Meibohm,
A.; Condra, J. H.; Valentine, F. T.; McMahon, D.; Gonzalez, C.;
Jonas, L.; Emini, E. A.; Chodakewitz, J. A.; Isaacs, R.; Richman,
D. D. 3-year suppression of HIV viremia with indinavir, zidovu-
dine, and lamivudine. Ann. Intern. Med. 2000, 133, 35-39.
(3) Xiaoqi, C.; Lin, L.; Kempf, D. J.; Hing, S.; Wideburg, N. E.;
Saldivar, A.; Vasavanonda, S.; Marsh, K. C.; McDonald, E.;
Norbeck, D. W. Evaluation of furofuran as a P2 ligand for
symmetry-based HIV protease inhibitors. Bioorg. Med. Chem.
Lett. 1996, 6, 2847-2852.
(4) Roberts, N. A.; Martin, J. A.; Kinchington, D.; Broadhurst, A.
V.; Craig, J. C.; Duncan, I. B.; Galpin, S. A.; Handa, B. K.; Kay,
J.; Krohn, A. Rational design of peptide-based HIV proteinase
inhibitors. Science 1990, 248, 358-361.
(5) Kaldor, S. W.; Kalish, V. J.; Davies, J. F.; Shetty, B. V.; Fritz, J.
E.; Appelt, K.; Burgess, J. A.; Campanale, K. M.; Chirgadze, N.
Y.; Clawson, D. K.; Dressman, B. A.; Hatch, S. D.; Khalil, D. A.;
Kosa, M. B.; Lubbehusen, P. P.; Muesing, M. A.; Patick, A. K.;
Reich, S. H.; Su, K. S.; Tatlock, J. H. Viracept (Nelfinavir
Mesylate, AG1343): A Potent, Orally Bioavailable Inhibitor of
HIV-1 Protease. J. Med. Chem. 1997, 40, 3979-3985.
(6) Dorsey, B. D.; Levin, R. B.; McDaniel, S. L.; Vacca, J. P.; Guare,
J. P.; Darke, P. L.; Zugay, J. A.; Emini, E. A.; Schleif, W. A.;
Quintero, J. C.; Lin, J. H.; Chen, I.-W.; Holloway, M. K.;
Fitzgerald, P. M. D.; Axel, M. G.; Ostovic, D.; Anderson, P. S.;
Huff, J. R.L-735,524: the design of a potent and orally bioavail-
able HIV protease inhibitor. J. Med. Chem. 1994, 37, 3443-
3451.
(7) Kim, E. E.; Baker, C. T.; Dwyer, M. D.; Murcko, M. A.; Rao, B.
G.; Tung, R. D.; Navia, M. A. Crystal structure of HIV-1 protease
in complex with VX-478, a potent and orally bioavailable
inhibitor of the enzyme. J. Am. Chem. Soc. 1995, 117, 1181-
1182.
(8) Robinson, B. S.; Riccardi, K. A.; Gong, Y. F.; Guo, Q.; Stock, D.
A.; Blair, W. S.; Terry, B. J.; Deminie, C. A.; Djang, F.; Colonno,
R. J.; Lin, P. F. BMS-232632, a highly potent human immuno-
deficiency virus protease inhibitor that can be used in combina-
tion with other available antiretroviral agents. Antimicrob.
Agents Chemother. 2000, 44, 2093-2099.
(9) Sham, H. L.; Kempf, D. J.; Molla, A.; Marsh, K. C.; Kumar, G.
N.; Chen, C. M.; Kati, W.; Stewart, K.; Lal, R.; Hsu, A.;
Betebenner, D.; Korneyeva, M.; Vasavanonda, S.; McDonald, E.;
Saldivar, A.; Wideburg, N.; Chen, X.; Niu, P.; Park, C.; Jayanti,
V.; Grabowski, B.; Granneman, G. R.; Sun, E.; Japour, A. J.;
Norbeck, D. W. ABT-378, a highly potent inhibitor of the human
immunodeficiency virus protease. Antimicrob. Agents Chemother.
1998, 42, 3218-3224.
(10) Turner, S. R.; Strohbach, J. W.; Tommasi, R. A.; Aristoff, P. A.;
Johnson, P. D.; Skulnick, H. I.; Dolak, L. A.; Seest, E. P.; Tomich,
P. K.; Bohanon, M. J.; Horng, M. M.; Lynn, J. C.; Chong, K. T.;
Hinshaw, R. R.; Watenpaugh, K. D.; Janakiraman, M. N.;
Thaisrivongs, S. Tipranavir (PNU-140690): a potent, orally
bioavailable nonpeptidic HIV protease inhibitor of the 5,6-
dihydro-4-hydroxy-2-pyrone sulfonamide class. J. Med. Chem.
1998, 41, 3467-3476.
(11) Condra, J. H.; Schleif, W. A.; Blahy, O. M.; Gabryelski, L. J.;
Graham, D. J.; Quintero, J. C.; Rhodes, A.; Robbins, H. L.; Roth,
E.; Shivaprakash, M. In vivo emergence of HIV-1 variants
resistant to multiple protease inhibitors. Nature 1995, 374, 569-
571.
(12) Clavel, F.; Hance, A. J. HIV drug resistance. Structural and
thermodynamic basis of resistance to HIV-1 protease inhibi-
tion: Implications for inhibitor design. N. Engl. J. Med. 2004,
350, 1023-1035.
Intensity data were collected at the in-house (University of
Massachusetts Medical School) R-axis IV image plate, mounted
on a Rigaku X-ray generator delivering X-rays at a power
rating of 50 kV/100 mA. Data collection was carried out at -80
°C. Data processing was carried out using Denzo and
ScalePack.27,28 Data collection statistics are listed in Table 5.
Structure determination was carried out by AMoRe29 using
the CCP4i interface.30 Crystallographic refinement included
water picking using ARP/wARP31 and positional refinement
using Crystallography and NMR System (CNS)32 and Refmac
5.33 Model building was performed using the interactive
graphics program CHAIN.34 The final refinement statistics are
provided in Table 5.
Isothermal Titration Calorimetry. Thermodynamic pa-
rameters of inhibitor binding were determined using an
isothermal titration calorimeter, VP-ITC (MicroCal Inc.,
Northampton, MA). The buffer used for all protease and
inhibitor solutions consisted of 10 mM sodium acetate, pH 5.0,
2% DMSO, and 2 mM tris(2-carboxyethyl)phosphine (TCEP).
The binding affinities of 20g and 26b were obtained by the
displacement titration method, using acetylpepstatin (300 µM)
as the weaker binder.35,36 For experiments with 20g, 32 or 24
injections of 9 µL each of pepstatin were injected into the
calorimetric cell containing 13 or 9 µM protease, respectively.
This was followed by 9 or 7 µL injections of 125 µM 20g to
saturation. For experiments with 26b, 36 injections of 8 µL
each were injected into the cell containing protease in the
range of 18-21 µM, followed by 6 µL injections of 200 µM 26b
to saturation. Each experiment was performed at least twice.
Heats of dilution were subtracted from the corresponding heats
of reaction to obtain the heat due solely to inhibitor binding
to the enzyme. Data were processed using the Origin 7
software package from Microcal.
Modeling. The crystal structures have some missing and
disordered side chains and do not contain information on the
position of the hydrogen atoms. Therefore, the structures were
first completed by adding the missing hydrogen atoms and side
chains using the Biopolymer module in InsightII (Accelrys Inc).
This was followed by an optimization of the hydrogen atoms
using the conjugate gradient approach. The CVFF force field37
was used in the Discover module of InsightII to perform these

Design of HIV-1 Protease Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 6 1973
(13) Koh, Y.; Nakata, H.; Maeda, K.; Ogata, H.; Bilcer, G.; Devasam-
udram, 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. Novel bis-tetrahydrofuranylurethane-contain-
ing nonpeptidic protease inhibitor (PI) UIC-94017 (TMC114)
with potent activity against multi-PI-resistant human immu-
nodeficiency virus in vitro. Antimicrob. Agents Chemother. 2003,
47, 3123-3129.
(14) Ghosh, A. K.; Kincaid, J. F.; Walters, D. E.; Chen, Y.; Chaudhuri,
N. C.; Thompson, W. J.; Culberson, C.; Fitzgerald, P. M. D.; Lee,
H. Y.; McKee, S. P.; Munson, P. M.; Duong, T. T.; Darke, P. L.;
Zugay, J. A.; Schleif, W. A.; Axel, M. G.; Lin, J.; Huff, J. R.
Nonpeptidal P2 Ligands for HIV Protease Inhibitors: Structure-
Based Design, Synthesis, and Biological Evaluation. J. Med.
Chem. 1996, 39, 3278-3290.
(15) (a) Nagarajan, S. R.; De Crescenzo, G. A.; Getman, D. P.; Lu,
H.-F.; Sikorski, J. A.; Walker, J. L.; McDonald, J. J.; Houseman,
K. A.; Kocan, G. P.; Kishore, N.; Mehta, P. P.; Funkes-Shippy,
C. L.; Blystone, L. Discovery of novel benzothiazolesulfonamides
as potent inhibitors of HIV-1 protease. Bioorg. Med. Chem. 2003,
11, 4769-4777. (b) Kunda, S. A.; Letendre, L. J.; De Crescenzo,
G. A. Synthesis of benzo-fused heterocyclic sulfonyl chlorides for
preparation of amino acid hydroxyethylamine sulfonamide ret-
roviral protease inhibitors. U.S. Patent 6140505, 2000; CAN 133:
322130, 2000.
(20) Hertogs, K.; de Bethune, M. P.; Miller, V.; Ivens, T.; Schel, P.;
Van Cauwenberge, A.; Van Den Eynde, C.; Van Gerwen, V.;
Azijn, H.; Van Houtte, M.; Peeters, F.; Staszewski, S.; Conant,
M.; Bloor, S.; Kemp, S.; Larder, B.; Pauwels, R. A rapid method
for simultaneous detection of phenotypic resistance to inhibitors
of protease and reverse transciptase in recombinant human
immunodeficiency virus type 1 isolates from patients treated
with antiretroviral drugs. Antimicrob. Agents Chemother. 1998,
42, 269-276.
(21) Johnson, V. A.; Brun-Ve´zinet, F.; Clotet, B.; Conway, B.;
D’Aquila, R. T.; Demeter, L. M.; Kuritzkes, D. R.; Pillay, D.;
Schapiro, J. M.; Telenti, A.; Richman, D. D. Update of the Drug
Resistance Mutations in HIV-1: 2004. Top. HIV Med. 2004, 12,
119-124.
(22) Velazquez-Campoy, A.; Muzammil, S.; Ohtaka, H.; Schoen, A.;
Vega, S.; Freire, E. Structural and thermodynamic basis of
resistance to HIV-1 protease inhibition: Implications for inhibi-
tor design. Curr. Drug Targets: Infect Disord. 2003, 3, 311-
328.
(23) (a) Koeplinger, K. A.; Zhao, Z. Chromatographic measurement
of drug-protein interaction: determination of HIV protease
inhibitor-serum albumin association. Anal. Biochem. 1996, 243,
66-73. (b) Schon, A.; Ingaramo, M. d. M.; Freire, E. The binding
of HIV-1 protease inhibitors to human serum proteins. Biophys.
Chem. 2003, 105, 221-230.
(16) (a) Surleraux, D. L. N. G.; Wigerinck, P. T. B. P.; Getman, D. P.
Broad-spectrum 2-aminobenzothiazole sulfonamide HIV pro-
tease inhibitors. PCT Int. Appl. WO 2004014371, 2004; CAN 140:
193033, 2004. (b) Surleraux, D. L. N. G.; Wigerinck, P. T. B. P.;
Getman, D.; Verschueren, W. G.; Vendeville, S.; de Bethune, M.-
P.; De Kerpel, J. O. A.; Moors, S. L. C.; de Kock, H. A.; Voets,
M. C. J. Broad-spectrum 2-(substituted-amino)-benzothiazole-
sulfonamide HIV protease inhibitors. PCT Int. Appl. WO
2002083657, 2002; CAN 137:325410, 2002. (c) Vazques, M. L.;
Mueller, R. A.; Talley, J. J.; Getman, D. P.; Decrescenzo, G. A.;
Freskos, J. N.; Bertenshaw, D. E.; Heintz, R. M. Amino acid
hydroxyethylamino sulfonamides useful as retroviral protease
inhibitors. U.S. Patent 5968942, 1999; CAN 131:295568, 1999.
(d) Sikorski, J. A.; Getman, D. P.; Decrescenzo, G. A.; Devadas,
B.; Freskos, J. N.; Lu, H.-F.; McDonald, J. J. Preparation of
substituted sulfonylalkanoylamino hydroxyethylamino sulfon-
amide retroviral protease inhibitors. U.S. Patent 5753660, 1998;
CAN 129:16390, 1998. (e) Sikorski, J. A.; Getman, D. P.;
Decrescenzo, G. A.; Devadas, B.; Freskos, J. N.; Lu, H.-F.;
McDonald, J. J. Preparation of N-[2-hydroxy-4-phenyl-3-(sulfo-
nylalkanoylamino)butyl]arylsulfonamides and analogs as ret-
roviral protease inhibitors. PCT Int. Appl. WO 9718205, 1997;
CAN 127:65754, 1997.
(17) (a) Kunda, S. A.; Letendre, L. J.; De Crescenzo, G. A. Benzo fused
heterocyclo sulfonyl halide intermediates for the preparation of
amino acids as HIV protease inhibitors. PCT Int. Appl. WO
9959989, 1999; CAN 132:6692, 1999. (b) Getman, D. P.; De-
crescenzo, G. A.; Freskos, J. N.; Vazquez, M. L.; Sikorski, J. A.;
Devadas, B.; Nagarajan, S.; Brown, D. L.; McDonald, J. J.
Preparation of N-heterocyclecarbonyl amino acid hydroxyethy-
lamino sulfonamide as retroviral protease inhibitors. PCT Int.
Appl. WO 9628465, 1996; CAN 126:31657, 1996. (c) Getman, D.
P.; Descrescenzo, G. A.; Freskos, J. N.; Vasquez, M. L.; Sikorski,
J. A.; Devadas, B.; Nagarajan, S.; Brown, D. L.; McDonald, J. J.
Preparation of bisamino acid hydroxyethylaminosulfonamide
retroviral protease inhibitors. PCT Intl. Appl. WO 9628464,
1996; CAN 126:8710, 1996.
(18) (a) Surleraux, D. L. N. G.; Vendeville, S. M. H.; Verschueren,
W. G.; de Bethune, M.-P. T. M. M. G.; de Kock, H. A.; Tahri, A.
Preparation of 2-amino-benzoxazole sulfonamide as broad-spec-
trum HIV protease inhibitors. PCT Int. Appl. WO 2002092595,
2002; CAN 137:384835, 2002. (b) Surleraux, D. L. N. G.;
Vendeville, S. M. H.; Verschueren, W. G.; de Bethune, M.-P. T.
M. M. G.; de Kock, H. A.; Tahri, A.; Erra S. M. Preparation of
2-(substituted-amino)benzoxazole sulfonamides as broad spec-
trum HIV protease inhibitors. PCT Int. Appl. WO 2002081478,
2002; CAN 137:310904, 2002.
(19) Takeda, K.; Akagi, Y.; Saiki, A.; Tsukahara, T.; Ogura, H.
Studies on activating methods of functional groups. Part X.
Convenient methods for syntheses of active carbamates, ureas
and nitrosoureas using N,N′-disuccinimido carbonate (DSC).
Tetrahedron Lett. 1983, 24, 4569-4572.
(24) King, N. M.; Prabu-Jeyabalan, M.; Nalivaika, E. A.; Wigerinck,
P.; de Be´thune, M.-P.; Schiffer, C. A. The structural and
thermodynamic basis for the binding of TMC114, a next-
generation HIV-1 protease inhibitor. J. Virol. 2004, 78, 12012-
12021.
(25) King, N. M.; Melnick, L.; Prabu-Jeyabalan, M.; Nalivaika, E.
A.; Yang, S.-S.; Gao, Y.; Nie, X.; Zepp, C.; Heefner, D. L.; Schiffer,
C. A. Lack of synergy for inhibitors targeting a multi-drug-
resistant HIV-1 protease. Protein Sci. 2002, 11, 418-429.
(26) Prabu-Jeyabalan, M.; Nalivaika, E. A.; King, N. M.; Schiffer, C.
A. Viability of a drug-resistant HIV-1 protease variant: struc-
tural insights for better anti-viral therapy. J. Virol. 2003, 77,
1306-1315.
(27) Minor, W. XDISPLAYF (program); Purdue University: West
Lafayette, IN, 1993.
(28) Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data
collected in oscillation mode. Macromolecular Crystallography
Part A; Academic Press: New York, 1997; pp 307-326.
(29) Navaza, J. AmoRe: an automated package for molecular re-
placement. Acta Crystallogr. 1994, A50, 157-163.
(30) Collaborative-Computational-Project, N. The CCP4 suite: pro-
grams for protein crystallography. Acta Crystallogr. 1994, D50,
760-763.
(31) Morris, R. J.; Perrakis, A.; Lamzin, V. S. ARP/wARP’s model-
building algorithms. I. The main chain. Acta Crystallogr. 2002,
D58, 968-975.
(32) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges,
M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren,
G. L. Crystallography & NMR system: A new software suite
for macromolecular structure determination. Acta Crystallogr.
1998, D54, 905-921.
(33) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method.
Acta Crystallogr. 1997, D53, 240-255.
(34) Sack, J. S. CHAINsa crystallographic modeling program. J. Mol.
Graphics 1988, 6, 224-225.
(35) Sigurskjold, B. Exact analysis of competition ligand binding by
displacement isothermal titration calorimetry. Anal. Biochem.
2000, 277, 260-266.
(36) Velazquez-Campoy, A.; Kiso, Y.; Freire, E. The binding energetics
of first- and second-generation HIV-1 protease inhibitors: im-
plications for drug design. Arch. Biochem. Biophys. 2001, 390,
169-175.
(37) Dauber-Osguthorpe, P.; Roberts, V. A.; Osguthorpe, D. J.; Wolff,
J.; Genest, M.; Hagler, A. T. Structure and energetics of ligand
binding to proteins: E. coli dihydrofolate reductase-trimethop-
rim, a drug-receptor system. Proteins: Struct., Funct., Genet.
1988, 4, 31-47.
JM049454N