Structure-based design of novel HIV protease inhibitors: Sulfonamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones as potent non-peptidic inhibitors

Article abstract of DOI:10.1021/jm950888f

The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover non-peptidic HIV protease inhibitors previously identif

Full text of DOI:10.1021/jm950888f

2400
J . Med. Chem. 1996, 39, 2400-2410
Str u ctu r e-Ba sed Design of Novel HIV P r otea se In h ibitor s:
Su lfon a m id e-Con ta in in g 4-Hyd r oxycou m a r in s a n d 4-Hyd r oxy-2-p yr on es a s
P oten t Non -P ep tid ic In h ibitor s
Suvit Thaisrivongs,*,† Musiri N. J anakiraman,† Kong-Teck Chong,‡ Paul K. Tomich,§ Lester A. Dolak,§
Steve R. Turner,† J oseph W. Strohbach,† J anet C. Lynn,§ Miao-Miao Horng,§ Roger R. Hinshaw,‡ and
Keith D. Watenpaugh†
Structural, Analytical and Medicinal Chemistry, Cancer & Infectious Diseases Research, and Chemical &
Biological Screening, Pharmacia & Upjohn, Kalamazoo, Michigan 49001
Received December 5, 1995X
The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease
inhibitors have limited their utility as potential therapeutic agents. Our broad screening
program to discover non-peptidic HIV protease inhibitors previously identified compound I
(phenprocoumon, Ki ) 1 µM) as a lead template. Structure-based design of potent non-peptidic
inhibitors, utilizing crystal structures of HIV protease/inhibitor complexes, provided a rational
basis for the previously reported carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-
2-pyrones. The amino acid containing compound V (Ki ) 4 nM) provided an example of a
promising new series of HIV protease inhibitors with significantly improved enzymatic binding
affinity. In this report, further structure-activity relationship studies, in which the carboxa-
mide is replaced by a sulfonamide functionality, led to the identification of another series of
nonamino acid containing promising inhibitors with significantly enhanced enzyme binding
affinity and in vitro antiviral activity. The most active diastereomer of the sulfonamide-
containing pyrone XVIII (Ki ) 0.5 nM) shows improved antiviral activity (IC50 ) 0.6 µM) and
represents an example of a new design direction for the discovery of more potent non-peptidic
HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.
In tr od u ction
inhibitors.27,28 For our research program, the crystal
structure of inhibitor I/HIV-1 protease complex formed
the basis of iterative cycles of structure-based design
of more active analogues. From that investigation,
U-96988 (3-(R-ethylbenzyl)-6-(R-ethylphenethyl)-4-hy-
droxy-2H-pyran-2-one, compound IV in Figure 1, Ki )
38 nM) was identified as the first clinical candidate in
this promising series of non-peptidic HIV protease
inhibitors as a potential therapeutic agent for the
treatment of HIV infection.20 The present report de-
scribes our continuing structure-based design effort
which relied on the information from the crystal struc-
tures of HIV protease complexes with selected inhibi-
tors.
Recently, we reported the identification of an ap-
propriately attached carboxamide functionality to the
pyrone-based non-peptidic inhibitors which resulted in
more active inhibitors.29 In the 4-hydroxycoumarin
series, compound II (Ki ) 28 nM) was reported to be
the most active compound, while compound V (Ki ) 4
nM) was the most active compound in the 4-hydroxy-
2-pyrone series. Although these two potent inhibitors
contain amino acid residues, it was noted that com-
pound III (Ki ) 110 nM) and compound VI (Ki ) 44 nM)
in the 4-hydroxycoumarin and 4-hydroxy-2-pyrone se-
ries, respectively, did not contain any amino acid
residue, while showing reasonable enzyme inhibitory
activity, and provided a basis for further analogue
exploration. The carboxamide functional group was
evaluated, and other functionalities were also explored
in the cyclooctylpyranone template.21,30 Of particular
interest was the sulfonamide functionality which re-
sulted in compounds with improved biological activities.
In this report, we propose to evaluate a variety of
sulfonamide-containing compounds in the 4-hydroxy-
The rapid spread of the acquired immunodeficiency
syndrome (AIDS) epidemic has stimulated discovery of
therapeutic agents to arrest the replication of the
causative virus, human immunodeficiency virus (HIV).
One promising possibility to interrupt the viral life cycle
is the use of inhibitors of the virally encoded protease
which is indispensable for viral maturation.1,2 Among
the most potent inhibitors reported thus far are pepti-
domimetic compounds containing transition-state in-
serts in place of the dipeptidic cleavage sites of the
substrates.3-6 The low oral bioavailability and rapid
biliary excretion of peptide-derived compounds7 have
limited their utility as potential therapeutic agents.
Recent advances have resulted in HIV protease inhibi-
tors with reduced peptidic character and also non-
peptidic inhibitors that are more orally bioavailable, and
an increasing number of HIV protease inhibitors8-21 are
currently undergoing clinical evaluations.
In a recent communication,20 we reported the iden-
tification of phenprocoumon (3-(R-ethylbenzyl)-4-hy-
droxycoumarin, compound I in Figure 1, Ki ) 1 µM)
from a broad screening program as an active HIV
protease inhibitory template. It is noted that other
independent studies22-26 have also described 4-hydroxy-
benzopyran-2-ones and 4-hydroxypyran-2-ones as in-
hibitors of HIV protease. The increasing number of
reported crystal structures of inhibitor/HIV protease
complexes have provided numerous successful examples
of structure-based designs of potent HIV protease
† Structural, Analytical, and Medicianl Chemistry.
‡ Cancer & Infectious Diseases Research.
§ Chemical & Biological Screening.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(95)00888-0 CCC: $12.00 © 1996 American Chemical Society

Design of Novel HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2401
Ta ble 1. Sulfonamide-Containing 4-Hydroxycoumarins
trophile led to the addition of another side chain at the
C-6R position. In this particular example, the use of
ethyl bromide followed by benzyl bromide resulted in
the generation of compound 4. The carbobenzyloxy
protecting group was removed by catalytic hydrogenoly-
sis, and the resulting penultimate amine precursor 5
could be treated with a sulfonyl chloride to give the
desired inhibitor. In this particular example, the use
of 1-methylimidazole-4-sulfonyl chloride resulted in the
isolation of compound XVIII. All the sulfonamide-
containing 4-hydroxy-2-pyrones were prepared in a
manner similar to the preparation of compound XVIII.
Resu lts a n d Discu ssion
Tables 1-7 display the HIV-1 protease inhibitory
activity (Ki values) and the cell-culture (MT4/HIV-1IIIB
)
antiviral activity (IC50 or IC90 values). These com-
pounds were also tested for cytotoxicity in MT4 cells,
and their CCTD50 values were greater than 30 µM, and
any observed antiviral activity of these compounds was
not compromised by any significant residual cytotoxic-
ity. In the 4-hydroxycoumarin template (see Table 1),
compounds VII-X contain a group of representative
aryl sulfonamides. On the basis of the previous re-
search21 on sulfonamide-containing compounds, these
aryl groups are expected to bind into the S3′ pocket31 of
the enzyme active site. Compound VIII incorporated
the previously reported p-cyanophenyl group21 and
exhibited good enzyme inhibitory activity (Ki ) 5.8 nM)
when compared to the carboxamide-containing com-
pound III (Ki ) 110 nM) or even to the amino acid-
containing compound II (Ki ) 28 nM). The N-meth-
ylimidazole-containing32 compound X showed even higher
enzyme inhibitory activity with a Ki value of 0.7 nM.
Unfortunately, these compounds with potent enzyme
inhibitory activity showed very poor in vitro antiviral
activity, and a concentration of 10 µM failed to show
noticeable inhibition of viral replication in the cell
culture assay.
F igu r e 1. Structures of HIV protease inhibitors.
coumarin and the 4-hydroxy-2-pyrone series. We present
these findings that result in the important identification
of inhibitors with significantly improved enzyme binding
affinity and in vitro antiviral activity and point to an
additional new direction of research in the design of
potent non-peptidic HIV protease inhibitors.
Ch em istr y
The sulfonamide-containing 4-hydroxycoumarins
VII-X (shown in Table 1) were prepared by the reaction
of appropriate sulfonyl chlorides with the same penul-
timate amine precursor which was used in the prepara-
tion of the previously described compound II.29 The
preparation of the representative sulfonamide-contain-
ing 4-hydroxy-2-pyrone XVIII is shown in Scheme 1.
The commercially available 4-hydroxy-6-methyl-2-py-
rone (1) was condensed with the previously described
benzylic alcohol 229 under acidic catalysis to give the
adduct 3. Treatment with 3 equiv of lithium diisopro-
pylamide and an electrophile resulted in an alkylation
reaction which added a side chain at the C-6R position.
Repeating the alkylation reaction with a second elec-
For the 4-hydroxy-2-pyrone series, the template of the
carboxamide-containing compound VI was used as a
starting point in which the carboxamide functionality
was replaced with the N-methylimidazole-containing

2402 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Sch em e 1. Preparation of Compound XVIIIa
Thaisrivongs et al.
Ta ble 3. Sulfonamide-Containing 4-Hydroxy-2-pyrones
Ta ble 4. Sulfonamide-Containing 4-Hydroxy-2-pyrones
though they showed some antiviral activity at 3 µM, the
inhibition of viral replication was less than 50%.
Table 3 shows a few representative compounds in the
N-methylimidazolesulfonamide containing 4-hydroxy-
2-pyrones in which the C-6R position contains two
identical substituents. Compounds XV and XVI showed
potent inhibitory activity (Ki ) 0.5-1 nM). Although
this enzyme inhibitory activity did not improve over that
for compounds in Table 2, compound XVI showed
significant in vitro antiviral activity (IC50 ) 1.2 µM).
The dioligoether-containing compound XVII showed
reduced enzyme inhibitory activity and no in vitro
antiviral activity.
Table 4 shows representative compounds in the
N-methylimidazolesulfonamide containing 4-hydroxy-
2-pyrones in which there are two substituents at the
C-6R position and one of which is an ethyl group. Even
though compound XVIII showed enzyme inhibitory
activity comparable to that of compound XV, the benzyl
group in compound XVIII resulted in a compound with
significant in vitro antiviral activity (IC50 ) 1.3 µM).
Attempt to increase hydrophilicity in compounds XIX
and XX, while maintaining the enzyme inhibitory
activity, led to some loss of antiviral activity.
a
(a) TsOH,CH2Cl2; (b) 3 equiv of LiNPri2, CH3CH2Br; 3 equiv
of LiNPri , C6H5CH2Br; (c) (NH4)(HCO2), Pd/C, CH3OH; (d) 2 equiv
of pyridine, 1 equiv of 1-methylimidazole-4-sulfonyl chloride.
2
Ta ble 2. Sulfonamide-Containing 4-Hydroxy-2-pyrones
compd
R
Ki (nM)
IC50 (µM)
XI
H
14
.3
.3
.3
.3
XII
XIII
XIV
CH3CH2
C6H5CH2
H3C(OCH2CH2)2
1.4
0.97
1.6
sulfonamide functionality. Table 2 shows a series of
compounds in which there is no R-branching at the C-6
position. The simplest inhibitor XI, with a methyl group
at C-6, showed good inhibitory activity (Ki ) 14 nM).
Extending the substitution at C-6 with an alkyl or aryl
group led to compounds with significantly improved
inhibitory activity as represented by compounds XII and
XIII (Ki ≈ 1 nM). The oligoether-containing compound
XIV was prepared to enhance water solubility and
maintained enzyme inhibitory potency. Also unfortu-
nately, these compounds with high enzyme inhibitory
activity showed poor in vitro antiviral activity. Even
Since compound XVI showed antiviral activity among
the analogues in Table 3, further evaluation of other
arenesulfonamides was explored. Some representative
compounds are shown in Table 5. However, compounds
XXI-XXIV all showed weaker enzyme inhibitory activ-

Design of Novel HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2403
Ta ble 5. Sulfonamide-Containing 4-Hydroxy-2-pyrones
Ta ble 7. Individual Isomers of Sulfonamide-Containing
4-Hydroxy-2-pyrones
Ta ble 6. Sulfonamide-Containing 4-Hydroxy-2-pyrones
Since compounds XVI and XVIII emerged as com-
pounds of interest, and, as with all other compounds
discussed so far, they are mixture of stereoisomers, we
had interest in studying the activity of individual
isomers of these two compounds. A chiral HPLC
technique was utilized in the separation and prepara-
tion of the individuals isomers of compounds XVI and
XVIII. The components were studied for their enzyme
inhibitory and in vitro antiviral activities, and the
results are summarized in Table 7. For compound XVI,
the two individual enantiomers were shown to be very
close in activity and differed by less than a factor of 2.
The racemic XVI was, therefore, not experimentally
distinguishable in activity from the “more active” enan-
tiomer. For compound XVIII, two diastereomers (1 and
2) were found to be slightly more active than the
remaining two diastereomers (3 and 4). Diastereomers
1 and 2 possess the same absolute stereochemistry at
C-6R. Compound XVIII, which is a mixture of four
diastereomers, showed experimentally similar activity
to the “most active” diastereomer (1). From these two
sets of compounds, it is suggested that HIV protease
does not have high preference for the absolute stereo-
chemistry at the two chiral centers in these series of
inhibitors.
Cr ysta llogr a p h ic Stu d y
It is important to obtain crystal structures of the
enzyme complexes of some representative compounds
to evaluate whether there has been any significant
change in the binding conformation of a new class of
compounds. Crystallization experiments with a few
compounds against both HIV-1 and HIV-2 proteases
ity and poorer antiviral activity as compared to com-
pound XVI. Similar study for the active compound
XVIII is summarized in Table 6, where a variety of
arenesulfonamides were assessed. Compounds XXV-
XXXI showed weaker activity than compound XVIII.

2404 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Thaisrivongs et al.
were attempted, and the crystal of the HIV-2 protease
complexed with the diastereomer 1 of compound XVIII
was suitable for a crystallographic study. Even though
HIV-1 and HIV-2 proteases show 50% sequence homol-
ogy, with homology being much higher in the inhibitor-
binding regions, we realize that these can be subtle
differences in the binding conformation of an inhibitor
in HIV-1 or HIV-2 proteases. The diastereomer 1 of
compound XVIII inhibited HIV-2 protease with a Ki
value of 1.0 nM, which is comparable to its inhibition
of HIV-1 protease (Ki ) 0.52 nM). The crystal structure
revealed that this ligand bound to HIV-2 protease in
two orientations related by the pseudo 2-fold symmetry
axis of the protease dimer; equal occupancies to the
atoms of the inhibitor in the two orientations were
observed. Figure 2 displays diastereomer 1 of compound
XVIII in only one of the two possible orientations.
Figure 2a shows the ligand occupying the S2′ to the S3
subsites of the enzyme active site. The C-3R cyclopropyl
group was positioned in the S1 pocket, while the C-3R
phenyl group was directed toward the S2 pocket. The
meta substituent on the C-3R phenyl group, viz., the
N-methylimidazolesulfonamide group, was located in
the S3 pocket, as was seen previously with the p-
cyanobenzenesulfonamide group.21 Free rotation about
the C-6/C-6R inhibitor bond allowed for two possible
orientations of the groups attached to the C-6R atom.
However, the difference electron density map favored
the conformation in which the ethyl group was placed
in the S1′ pocket while the benzyl group spanned the
S2′ pocket. Figure 2b highlights the notable hydrogen-
bonding arrangements between the ligand and the
enzyme active site. The C-4 hydroxyl group of com-
pound XVIII was positioned within hydrogen-bonding
distance to the two catalytic aspartate groups (2.8 and
3.0 Å, D25a and D25b, respectively). The lactone
carbonyl oxygen atom of the ligand replaced the ubiq-
uitous water molecule found in most complexes of
peptide-derived inhibitors;27,28 this oxygen atom was
positioned within hydrogen-bonding distance (3.0 Å) to
the amide nitrogen atom of the flap residue Ile 50b.
These results compared well with those previously
observed for the HIV-1 protease/inhibitor I complex.20
One of the sulfonamide oxygen atoms exhibited bifur-
cated hydrogen-bonding interactions with Asp30b (3.1
Å for O2[SO2]-HN[D30b] and 2.9 Å for O2[SO2]-OD-
[D30b]). The unsubstituted nitrogen of the imidazole
ring had a strong hydrogen-bonding interaction (2.7 Å)
with the amide nitrogen of aspartic acid D29b, which
was not possible for the phenyl moiety of the p-
cyanobenzenesulfonamide.21 This additional interaction
may partially explain the increase of enzymatic potency
of compound XVIII (Ki ) 1.3 nM) compared to inhibitor
XXIX (Ki ) 5.7 nM). Even though the observation of
this latter interaction in the crystal structure was with
an inhibitor in HIV-2 protease, we also observed the
same hydrogen-bonding interaction in the crystal struc-
tures of HIV-1 protease complexed with other imidazole-
containing pyrone-based inhibitors.
(compound IV, Ki ) 38 nM). Molecular modeling
studies based on crytal structures of HIV protease/
inhibitor complexes suggested the incorporation of a
carboxamide functionality at the meta position of the
benzyl side chain at C-3. Carboxamide-containing 4-hy-
droxycoumarin (compound II, Ki ) 28 nM) and 4-hy-
droxy-2-pyrone (compound V, Ki ) 4 nM) were shown
to have improved inhibitory activity. In this study, the
replacement of the carboxamide group with a sulfona-
mide functionality resulted in non amino acid containing
compounds with improved activity. The most active
diastereomer of compound XVIII showed potent HIV
protease inhibitory activity with a Ki value of 0.5 nM.
A crystal structure of this compound in the enzyme
complex was determined and confirmed the expected
conformation of the ligand in the enzyme active site.
This inhibitor also showed improved in vitro antiviral
activity (IC50 ) 0.6 µM) over previously reported car-
boxamide-containing 4-hydroxy-2-pyrones which exhib-
ited IC50 values above 3 µM. This finding of novel
inhibitors with improved enzyme binding affinity and
antiviral activity provides a new direction for the
preparation of a new promising series of potent and non-
peptidic HIV protease inhibitors for the treatment of
HIV infection.
Exp er im en ta l Section
Ch em istr y. Mass spectra, infrared spectra, and combus-
tion analyses were obtained by the Structural, Analytical and
Medicinal Chemistry Department of Pharmacia & Upjohn. 1H
NMR spectra were recorded at 300 MHz with a Bruker Model
AM-300 spectrometer. Chemical shifts were reported as δ
units relative to tetramethylsilane as internal standard. Thin-
layer chromatography was conducted with Analtech 0.25-mm
glass plates precoated with silica gel GF. Chromatography
used E. Merck silica gel 60 (70-230 mesh for column chro-
matography and 230-400 mesh for flash chromatography). All
solvents for chromatography were reagent grade.
Reagents were from commercial sources and used without
further purification unless otherwise noted. Diethyl ether was
Mallinkrodt anhydrous grade. Dichloromethane was dried
over 4A molecular sieves. Diisopropylethylamine and benzene
were distilled from calcium hydride. Diethyl cyanophospho-
nate was distilled before use. Tetrahydrofuran was distilled
under argon from sodium metal in the presence of benzophe-
none.
N-[3-[Cyclop r op yl(4-h yd r oxy-7-m et h oxy-2-oxo-2H -1-
ben zopyr an -3-yl)m eth yl]ph en yl]-4-flu or oben zen esu lfon a-
m id e (VII). To a cold (0 °C), stirred solution of 51 mg (0.15
mmol) of 3-[(3-aminophenyl)cyclopropylmethyl]-4-hydroxy-7-
methoxybenzopyran-2-one29 and 24 µL (0.30 mmol) of pyridine
in 0.5 mL of dichloromethane was added 30 mg (0.15 mmol)
of 4-fluorobenzenesulfonyl chloride. The solution was stirred
for 18 h, diluted with ethyl acetate, washed with dilute
aqueous HCl and brine, and then dried (MgSO4). Following
removal of solvent in vacuo, the residue was flash chromato-
graphed with elution using 1-4% methanol in dichloromethane
to afford 64.6 mg (0.13 mmol, 86%) of N-[3-[cyclopropyl(4-
hydroxy-7-methoxy-2-oxo-2H-1-benzopyran-3-yl)methyl]phenyl]-
4-fluorobenzenesulfonamide (VII): 1H NMR δ 0.18 (m, 1H),
0.43 (m, 1H), 0.56 (m, 1H), 0.70 (m, 1H), 1.46 (m, 1H), 3.85 (s,
3H), 3.85 (d, 1H), 6.72 (d, 1H), 6.83 (dd, J ) 2.4 Hz, J ) 8.9
Hz, 1H), 6.9-7.0 (m, 3H), 7.2-7.3 (m, 4H), 7.34 (s, 1H), 7.6-
7.7 (m, 3H); MS (EI) m/z 495 (calcd 495 for C26H22NO6FS); TLC
Rf 0.25 (3% methanol in dichloromethane).
Su m m a r y
Compounds VIII-X were prepared in a manner similar to
that of the preparation of compound VII; their physical data
are summarized as follows.
N-[3-[Cyclop r op yl(4-h yd r oxy-7-m et h oxy-2-oxo-2H -1-
ben zopyr an -3-yl)m eth yl]ph en yl]-4-cyan oben zen esu lfon a-
m id e (VIII): 1H NMR δ 0.18 (m, 1H), 0.38 (m, 1H), 0.55 (m,
From a broad screening program to discover non-
peptidic HIV protease inhibitors, phenprocoumon (com-
pound I, Ki ) 1 µM) was previously identified as a lead
template, and a structure-based design effort led to the
identification of the first-generation drug candidate

Design of Novel HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2405
F igu r e 2. (a) Crystal structure of the diastereomer 1 of compound XVIII/HIV-2 protease complex. The dot surface is a smoothed
representation of the van der Waals surface of the HIV-2 protease binding site, as it appears in complex with compound XVIII.
The labels S3′ through S3 approximately locate the six subsites commonly filled by peptidic inhibitors. (b) Interatomic distances
of potential hydrogen bonding atoms in the HIV-2 protease/XVIII complex. See description in the text.
1H), 0.68 (m, 1H), 1.55 (m, 1H), 3.79 (d, J ) 8.8 Hz, 1H), 3.85
(s, 3H), 6.71 (d, J ) 2.4 Hz, 1H), 6.86 (dd, J ) 2.4 Hz, J ) 8.9
Hz, 1H), 7.01 (m, 1H), 7.20 (t, J ) 7.8 Hz, 1H), 7.3 (m, 1H),
7.53 (d, 2H), 7.69 (m, 2H), 7.78 (d, 2H); HRMS (EI) m/z
502.1192 found (calcd 502.1198 for C27H22N2O6S); TLC Rf 0.24
(3% methanol in dichloromethane).
N-[3-[Cyclop r op yl(4-h yd r oxy-7-m et h oxy-2-oxo-2H -1-
b e n zop yr a n -3-yl)m e t h yl]p h e n yl]-8-q u in olin e su lfon a -
m id e (IX): 1H NMR -0.03 (m, 1H), 0.33 (m, 1H), 0.5 (m, 2H),
1.24 (m, 1H), 3.70 (d, J ) 8.8 Hz, 1H), 3.88 (s, 3H), 6.76 (d, J
) 2.4 Hz, 1H), 6.85 (dd, J ) 2.4 Hz, J ) 8.8 Hz, 1H), 6.92 (br,
1H), 7.1-7.3 (m, 4H), 7.44 (m, 1H), 7.54 (d, 8.8 Hz, 1H), 7.82

2406 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Thaisrivongs et al.
(dd, J ) 1.5 Hz, J ) 8.3 Hz, 1H), 8.05 (dd, J ) 1.8 Hz, J ) 8.4
Hz, 1H), 8.22 (dd, J ) 1.5 Hz, J ) 7.3 Hz, 1H), 9.04 (dd, J )
1.8 Hz, J ) 4.3 Hz, 1H); HRMS (EI) m/z 528.1345 found (calcd
528.1355 for C29H24N2O6S); TLC Rf 0.36 (3% methanol in
dichloromethane).
The layers were separated, and the aqueous phase was
extracted twice with additional portions of diethyl ether. The
combined organic extract was dried over sodium sulfate and
concentrated under reduced pressure. The resulting residue
was purified by flash column chromatography eluting with
5-10% ethyl acetate in dichloromethane to afford 153 mg (0.29
mmol, 60%) of 3-[R-cyclopropyl-m-[(benzyloxycarbonyl)amino]-
benzyl]-6-(R-ethylphenethyl)-4-hydroxy-2H-pyran-2-one (4) as
a white solid: 1H NMR (CDCl3) δ 0.26 (m, 2H), 0.46 (m, 1H),
0.66 (m, 1H), 0.78 (2t, J ) 7.4 Hz, 3H), 1.6 (m, 2H), 1.8 (m,
1H), 2.4 (m, 1H), 2.7 (m, 1H), 2.9 (m, 1H), 3.45 (d, J ) 9.9 Hz,
1H), 5.12 (s, 2H), 5.85 (s, 1H), 6.79 (br, 1H), 6.9-7.4 (m, 14H),
9.56 (br, 1H); EI HRMS m/z ) 523.2350 (calcd for C33H33NO5
523.2359); TLC Rf 0.27 (5% ethyl acetate in dichloromethane).
3-(r-Cyclopr opyl-m-am in oben zyl)-6-(r-eth ylph en eth yl)-
4-h yd r oxy-2H-p yr a n -2-on e (5). A mixture of 2.43 g (4.64
mmol) of 3-[R-cyclopropyl-m-[(benzyloxycarbonyl)amino]ben-
zyl]-6-(R-ethylphenethyl)-4-hydroxy-2H-pyran-2-one (4), 2.93
g (46 mmol) of ammonium formate, and 250 mg of 5%
palladium on carbon in 20 mL of methanol was stirred under
argon for 1.5 h. An additional 250 mg of catalyst was added,
and stirring was continued for another hour, at which time
TLC indicated completion of reaction. The mixture was
filtered through Celite and the filtrate concentrated under
reduced pressure. Following trituration with dichloromethane
to separate product from ammonium salts, the crude amine
was flash chromatographed on silica gel 60 (230-400 mesh)
using 35-50% ethyl acetate in dichloromethane to give 1.65
g (91%) of 3-(R-cyclopropyl-m-aminobenzyl)-6-(R-ethylphen-
ethyl)-4-hydroxy-2H-pyran-2-one (5): 1H NMR (CDCl3) δ 0.3
(m, 2H), 0.5 (m, 1H), 0.65 (m, 1H), 0.81 (2t, J ) 7.3 Hz, 3H),
1.6 (m, 3H), 2.5 (m, 1H), 2.7 (m, 1H), 2.9 (m, 1H), 3.46 (2d, J
) 9.8 Hz, 1H), 5.81 (2s, 1H), 6.49 (d, J ) 7.9 Hz, 1H), 6.80 (s,
1H), 6.9-7.3 (m, 7H); TLC Rf 0.38 (30% ethyl acetate in
dichloromethane); EI MS m/z 389, 361 (C24H27NO2), 360
(C23H22NO3).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H -p yr a n -3-yl]m et h yl]p h en yl]-1-m et h yl-1H -
im id a zole-4-su lfon a m id e (XVIII). To a cold (0 °C), stirred
solution of 78 mg (0.20 mmol) of 3-(R-cyclopropyl-m-aminoben-
zyl)-6-(R-ethyl-phenethyl)-4-hydroxy-2H-pyran-2-one (5) and
32 µL (0.40 mmol) of pyridine in 1 mL dichloromethane was
added 38 mg (0.21 mmol) of 1-methylimidazole-4-sulfonyl
chloride. The mixture was stirred for several hours and then
flash column chromatographed using 5% methanol in dichlo-
romethane to provide 82.8 mg (0.16 mmol, 77%) of N-[3-
[cyclopropyl[4-hydroxy-2-oxo-6-[1-(phenylmethyl)propyl]-2H-
pyran-3-yl]methyl]phenyl]-1-methyl-1H-imidazole-4-sulfon-
amide (XVIII): 1H NMR δ 0.19 (m, 2H), 0.46 (m, 1H), 0.65
(m, 1H), 0.86 (t, J ) 7.4 Hz, 3H), 1.5-1.9 (m, 3H), 2.51 (m,
1H), 2.8 (2dd, 1H), 2.9 (2dd, 1H), 3.28 (d, J ) 10.3 Hz, 1H),
3.61 (s, 3H), 5.74 (s, 1H), 6.9-7.4 (m, 12H); HRMS (EI)
533.1988 found (calcd 533.1984 for C29H31N3O5S); TLC Rf 0.29
(5% methanol in dichloromethane).
N-[3-[Cyclopr op yl(4-h yd r oxy-7-m eth oxy-2-oxo-2H-ben -
zop yr a n -3-yl)m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-
su lfon a m id e (X): 1H NMR δ 0.18 (m, 1H), 0.35 (m, 1H), 0.50
(m, 1H), 0.63 (m, 1H), 1.6 (m, 1H), 3.51 (s, 3H), 3.67 (d, J )
9.3 Hz, 1H), 3.84 (s, 3H), 6.74 (d, J ) 2.4 Hz, 1H), 6.80 (dd, J
) 2.5 Hz, J ) 8.9 Hz, 1H), 7.1-7.4 (m, 7H), 7.71 (d, J ) 8.8
Hz, 1H); HRMS (EI) m/z 481.1301 found (calcd 481.1307 for
C24H23N3O6S); TLC Rf 0.29 (8% methanol in dichloromethane).
3-[r-Cyclop r op yl-m -[(ben zyloxyca r bon yl)a m in o]ben -
zyl]-4-h yd r oxy-6-m eth ylp yr a n -2-on e (3). A mixture of 493
mg (3.91 mmol) of 4-hydroxy-6-methyl-2-pyrone (1), 56 mg
(0.29 mmol) of p-toluenesulfonic acid monohydrate, and 592
mg (1.99 mmol) of m-[(benzyloxycarbonyl)amino]phenyl-
cyclopropylcarbinol29 (2) in 20 mL of dichloromethane was
refluxed through an addition funnel containing 3 Å molecular
sieves under an argon atmosphere for 6 h. The mixture was
cooled to room temperature and concentrated under reduced
pressure. The residue was adsorbed onto silica gel and
purified by flash column chromatography eluting with 30-
50% ethyl acetate in dichloromethane to afford 470 mg (1.16
mmol, 58%) of 3-[R-cyclopropyl-m-[(benzyloxycarbonyl)amino]-
benzyl]-4-hydroxy-6-methylpyran-2-one (3) as a tan foam: TLC
1
Rf 0.52 (ethyl acetate); H NMR (CDCl3) δ 0.23 (m, 2H), 0.43
(m, 1H), 0.66 (m, 1H), 1.78 (m, 1H), 2.04 (s, 3H), 3.41 (d, J )
10.2 Hz, 1H), 5.09 (m, 2H), 5.89 (s, 1H), 7.00 (s, 1H), 7.14 (m,
2H), 7.3 (m, 5H), 7.37 (s, 1H), 10.1 (br s, 1H); MS (EI) m/z )
405 (M+). Anal. (C24H23NO5) C, H, N.
3-[r-Cyclop r op yl-m -[(ben zyloxyca r bon yl)a m in o]ben -
zyl]-4-h yd r oxy-6-p r op ylp yr a n -2-on e (3a ). To a stirred
solution of 0.45 mL (3.21 mmol) of diisopropylamine in 3 mL
of anhydrous tetrahydrofuran at -78 °C under an argon
atmosphere was added 2.0 mL (3.2 mmol) of a 1.6 M solution
of n-butyllithium in hexane. The mixture was allowed to
warm to 0 °C for 15 min and then recooled to -78 °C. The
resulting solution was treated with 405 mg (1.00 mmol) of 3-[R-
cyclopropyl-m-[(benzyloxycarbonyl)amino]benzyl]-4-hydroxy-
6-methylpyran-2-one (3) as a solution in 4 mL of anhydrous
tetrahydrofuran. After 1 h at -78 °C, the mixture was treated
with 85 µL (1.14 mmol) of ethyl bromide. The reaction mixture
was allowed to stir for 3 h at -78 °C, and then the reaction
was quenched with excess 1 N aqueous hydrochloric acid. The
mixture was partioned between ethyl acetate and aqueous pH4
phosphate buffer. The layers were separated, and the aqueous
phase was extracted twice with additional portions of ethyl
acetate. The combined organic extract was dried over mag-
nesium sulfate and concentrated under reduced pressure. The
resulting residue was purified by flash column chromatogra-
phy eluting with 10% to 20% ethyl acetate in dichloromethane
to afford 277 mg (0.64 mmol, 64%) of 3-[R-cyclopropyl-m-
[(benzyloxycarbonyl)amino]benzyl]-4-hydroxy-6-propylpyran-
2-one (3a ) as a pale yellow foam: Rf 0.33 (10% ethyl acetate
in dichloromethane); 1H NMR (CDCl3) δ 0.24 (m, 2H), 0.45 (m,
1H), 0.65 (m, 1H), 0.88 (t, J ) 7.4 Hz, 3H), 1.54 (hextet, J )
7.5 Hz, 2H), 1.8 (m, 1H), 2.28 (t, J ) 7.6 Hz, 2H), 3.42 (d, J )
10.2 Hz, 1H), 5.1 (m, 2H), 5.95 (s, 1H), 6.89 (s, 1H), 7.15 (m,
2H), 7.3 (m, 5H), 7.35 (s, 1H), 10.0 (br s, 1H); MS (EI) m/z )
433 (M+). Anal. (C26H27NO5) C, H, N.
3-[r-Cyclop r op yl-m -[(ben zyloxyca r bon yl)a m in o]ben -
zyl]-6-(r-eth ylp h en eth yl)-4-h yd r oxy-2H-p yr a n -2-on e (4).
To a stirred solution of 210 mg (0.48 mmol) of the 3-[R-
cyclopropyl-m-[(benzyloxycarbonyl)amino]benzyl]-4-hydroxy-
6-propylpyran-2-one (3a ) in 3 mL of anhydrous tetrahydrofu-
ran at -78 °C under an argon atmosphere was added 1.0 mL
(1.5 mmol) of a 1.5 M solution of lithium diisopropylamide
mono(tetrahydrofuran) in cyclohexane. The mixture was
warmed to -20 °C over 45 min and then recooled to -78 °C.
The resulting orange suspension was treated with 60 µL (0.50
mmol) of benzyl bromide and was warmed to -20 °C over 1 h
to afford a yellow solution. The reaction was then quenched
with excess 1 N aqueous hydrochloric acid and partioned
between diethyl ether and aqueous pH ) 4 phosphate buffer.
Compounds XI-XVII and XIX-XXXI were prepared in a
manner similar to that of the preparation of compound XVIII;
their physical data are summarized as follows.
N-[3-[Cyclop r op yl(4-h yd r oxy-6-m eth yl-2oxo-2H-p yr a n -
3-yl)m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-su lfon a -
m id e (XI): 1H NMR (CDCl3-CD3OD) δ 0.19 (m, 2H), 0.43 (m,
1H), 0.67 (m, 1H), 1.82 (m, 1H), 2.18 (s, 3H), 3.29 (d, J ) 10.4
Hz, 1H), 3.67 (s, 3H), 5.92 (s, 1H), 6.93 (m, 1H), 7.10 (t, J )
8.0 Hz, 1H), 7.2 (m, 2H), 7.37 (s, 1H), 7.47 (s, 1H); HRMS (FAB)
416.1296 found (calcd 416.1280 for C20H22N3O5S); TLC Rf 0.13
(5% methanol in dichloromethane). Anal. (C20H22N3O5S) C,
H, N.
N-[3-[Cyclopr opyl(4-h ydr oxy-2-oxo-6-pr opyl-2H-pyr an -
3-yl)m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-su lfon a -
m id e (XII): 1H NMR (CDCl3-CD3OD) δ 0.2 (m, 2H), 0.45 (m,
1H), 0.65 (m, 1H), 0.97 (t, J ) 7.4 Hz, 3H), 1.65 (m, 2H), 1.8
(m, 1H), 2.39 (t, J ) 7.6 Hz, 2H), 3.28 (d, J ) 10.4 Hz, 1H),
3.66 (s, 3H), 5.89 (s, 1H), 6.93 (m, 1H), 7.10 (t, J ) 7.8 Hz,
1H), 7.2 (m, 2H), 7.34 (s, 1H), 7.44 (s, 1H); HRMS (FAB)
444.1589 found (calcd 444.1593 for C22H26N3O5S); TLC Rf 0.17
(5% methanol in dichloromethane).

Design of Novel HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2407
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-(2-p h en yleth yl)-
2H-p yr a n -3-yl]m eth yl]p h en yl]-1-m eth yl-1H-im id a zole-4-
su lfon a m id e (XIII): 1H NMR δ 0.21 (m, 2H), 0.45 (m, 1H),
0.63 (m, 1H), 1.80 (m, 1H), 2.69 (m, 2H), 2.92 (m, 2H), 3.30 (d,
J ) 10.3 Hz, 1H), 3.59 (s, 3H), 5.89 (s, 1H), 6.9 (m, 1H), 7.08
(t, 1H), 7.1-7.4 (m, 9H); HRMS (EI) m/z 505.1672 found (calcd
505.1671 for C27H27N3O5S); TLC Rf 0.27 (5% methanol in
dichloromethane).
N-[3-[Cyclop r op yl[6-[3-(2-m et h oxyet h oxy)p r op yl]-4-
h yd r oxy-2-oxo-2H-p yr a n -3-yl]m eth yl]p h en yl]-1-m eth yl-
1H-im id a zole-4-su lfon a m id e (XIV): 1H NMR (CDCl3) δ 0.10
(m, 1H), 0.26 (m, 1H), 0.44 (m, 1H), 0.60 (m, 1H), 1.75 (m,
1H), 1.86 (quintet, J ) 7.5 Hz, 2H), 2.47 (t, J ) 7.5 Hz, 2H),
3.32 (d, J ) 10.2 Hz, 1H), 3.35 (s, 3H), 3.4-3.6 (m, 4H), 3.59
(s, 3H), 5.96 (s, 1H), 6.98 (m, 1H), 7.11 (t, J ) 7.8 Hz, 1H), 7.2
(m, 2H), 7.28 (s, 1H), 7.44 (s, 1H); HRMS (EI) m/z 517.1874
found (calcd 517.1883 for C25H31N3O7S); TLC Rf 0.20 (5%
methanol in dichloromethane).
N-[3-[Cyclop r op yl[6-(1-eth ylp r op yl)-4-h yd r oxy-2-oxo-
2H-p yr a n -3-yl]m eth yl]p h en yl]-1-m eth yl-1H-im id a zole-4-
su lfon a m id e (XV): 1H NMR (CDCl3) δ 0.14 (m, 2H), 0.39 (m,
1H), 0.58 (m, 1H), 0.79 (t, J ) 7.3 Hz, 6H), 1.55 (m, 4H), 1.75
(m, 1H), 2.1 (m, 1H), 3.22 (d, J ) 10.3 Hz, 1H), 3.58 (s, 3H),
5.85 (s, 1H), 6.85 (m, 1H), 7.02 (t, J ) 7.8 Hz, 1H), 7.15 (m,
2H), 7.29 (s, 1H), 7.38 (s, 1H); HRMS (FAB) 472.1924 found
(calcd 472.1906 for C24H30N3O5S); TLC Rf 0.76 (20% methanol
in dichloromethane).
XVIII): 1H NMR δ 0.17 (m, 2H), 0.43 (m, 1H), 0.62 (m, 1H),
0.83 (t, J ) 7.4 Hz, 3H), 1.6 (m, 2H), 1.75 (m, 1H), 2.49 (m,
1H), 2.78 (dd, J ) 7.0 Hz, J ) 13.5 Hz, 1H), 2.90 (dd, J ) 8.0
Hz, J ) 13.5 Hz, 1H), 3.28 (d, J ) 10.2 Hz, 1H), 3.55 (s, 3H),
5.75 (s, 1H), 6.9-7.4 (m, 11H); HRMS (EI) m/z 533.1983 found
(calcd 533.1984 for C29H31N3O5S); TLc f 0.32 (5% methanol in
dichloromethane).
N-[3-[[S or R]-Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1(R)-
(p h en ylm eth yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]-1-
m et h yl-1H -im id a zole-4-su lfon a m id e (diastereomer 2 of
XVIII): 1H NMR δ 0.19 (m, 2H), 0.42 (m, 1H), 0.62 (m, 1H),
0.84 (t, J ) 7.4 Hz, 3H), 1.6 (m, 2H), 1.75 (m, 1H), 2.50 (m,
1H), 2.78 (dd, J ) 7.0 Hz, J ) 13.5 Hz, 1H), 2.91 (dd, J ) 7.9
Hz, J ) 13.5 Hz, 1H), 3.28 (d, J ) 10.2 Hz, 1H), 3.55 (s, 3H),
5.74 (s, 1H), 6.9-7.4 (m, 11H); HRMS (EI) m/z 533.1993 found
(calcd 533.1984 for C29H31N3O5S); TLC Rf 0.30 (5% methanol
in dichloromethane).
N-[3-[[R or S]-Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1(S)-
(p h en ylm eth yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]-1-
m et h yl-1H -im id a zole-4-su lfon a m id e (diastereomer 3 of
XVIII): 1H NMR δ 0.19 (m, 2H), 0.43 (m, 1H), 0.62 (m, 1H),
0.84 (t, J ) 7.4 Hz, 3H), 1.6 (m, 2H), 1.75 (m, 1H), 2.50 (m,
1H), 2.78 (dd, J ) 7.0 Hz, J ) 13.5 Hz, 1H), 2.91 (dd, J ) 7.9
Hz, J ) 13.5 Hz, 1H), 3.28 (d, J ) 10.2 Hz, 1H), 3.58 (s, 3H),
5.74 (s, 1H), 6.9-7.4 (m, 11H); HRMS (EI) m/z 533.1993 found
(calcd 533.1984 for C29H31N3O5S); TLC Rf 0.30 (5% methanol
in dichloromethane).
N -[3-[Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclop r op yl-
m et h yl)et h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]-
p h en yl]-1-m eth yl-1H-im id a zole-4-su lfon a m id e (XVI): 1H
NMR (CDCl3-CD3OD) δ -0.02 (m, 4H), 0.21 (m, 2H), 0.4 (m,
5H), 0.65 (m, 3H), 1.4 (m, 2H), 1.6 (m, 2H), 1.8 (m, 1H), 2.6
(m, 1H), 3.31 (d, J ) 10.8 Hz, 1H), 3.67 (s, 3H), 6.02 (s, 1H),
6.89 (m, 1H), 7.07 (t, J ) 7.7 Hz, 1H), 7.12 (m, 1H), 7.25 (s,
1H), 7.44 (s, 1H), 7.57 (s, 1H); HRMS (EI) 523.2142 found
(calcd 523.2141 for C28H33N3O5S); TLC Rf 0.32 (5% methanol
in dichloromethane).
N-[3-[[S or R]-Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1(S)-
(p h en ylm eth yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]-1-
m et h yl-1H -im id a zole-4-su lfon a m id e (diastereomer 4 of
XVIII): 1H NMR δ 0.19 (m, 2H), 0.44 (m, 1H), 0.62 (m, 1H),
0.83 (t, J ) 7.4 Hz, 3H), 1.6 (m, 2H), 1.75 (m, 1H), 2.50 (m,
1H), 2.79 (dd, J ) 7.0 Hz, J ) 13.5 Hz, 1H), 2.91 (dd, J ) 7.9
Hz, J ) 13.5 Hz, 1H), 3.30 (d, J ) 10.2 Hz, 1H), 3.53 (s, 3H),
5.80 (s, 1H), 6.9-7.4 (m, 11H); HRMS (EI) m/z 533.1990 found
(calcd 533.1984 for C29H31N3O5S).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-[(tetr a h yd r o-
2H -p yr a n -4-yl)m et h yl]p r op yl]-2H -p yr a n -3-yl]m et h yl]-
p h en yl]-1-m eth yl-1H-im id a zole-4-su lfon a m id e (XIX): 1H
NMR δ 0.12 (m, 1H), 0.26 (m, 1H), 0.45 (m, 1H), 0.60 (m, 1H),
0.82 (t, J ) 7.4 Hz, 3H), 1.1-1.7 (m, 9H), 1.77 (m, 1H), 2.35
(m, 1H), 3.3 (m, 3H), 3.58 (s, 3H), 3.86 (m, 2H), 6.00 (s, 1H),
6.9-7.5 (m, 5H), 7.59 (m, 1H); HRMS (EI) m/z 541.2238 found
(calcd 541.2246 for C28H35N3O6S); TLC Rf 0.24 (5% methanol
in dichloromethane).
N-[3-[Cyclop r op yl[6-[1-eth yl-3-(2-m eth oxyeth oxy)p r o-
p yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]p h en yl]-1-
m eth yl-1H-im idazole-4-su lfon am ide (XX): 1H NMR (CDCl3)
δ 0.05 (m, 1H), 0.3 (m, 1H), 0.5 (m, 1H), 0.6 (m, 1H), 0.83 (t,
J ) 7.3 Hz, 3H), 1.5-1.9 (m, 5H), 2.45 (m, 1H), 3.31 and 3.33
(2 s, 3H total), 3.3-3.6 (m, 7H), 3.62 (s, 3H), 6.01 (s, 1H), 6.98
(m, 1H), 7.1-7.3 (m, 4H), 7.48 (s, 1H); HRMS (EI) m/z 545.2186
found (calcd 545.2196 for C27H35N3O7S); TLC Rf 0.24 (5%
methanol in dichloromethane); Anal. (C27H35N3O7S) C, H, N.
N -[3-[Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclop r op yl-
m et h yl)et h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]-
p h en yl]ben zen esu lfon a m id e (XXI): 1H NMR (CDCl3) δ
-0.06 (m, 4H), 0.11 (m, 1H), 0.3-0.7 (m, 9H), 1.3 (m, 2H), 1.4
(m, 2H), 1.65 (m, 1H), 2.55 (m, 1H), 3.43 (d, J ) 9.7 Hz, 1H),
6.06 (s, 1H), 6.9 (m, 1H), 7.1-7.5 (m, 7H), 7.72 (m, 2H); HRMS
(EI) 519.2078 found (calcd 519.2079 for C30H33NO5S); TLC Rf
The two individual enantiomers of compound XVI were
obtained via chiral HPLC preparative work; their physical data
are summarized as follows.
N-[3-[[R or S]-Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclo-
p r op ylm e t h yl)e t h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]-
m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-su lfon a m id e
(enantiomer 1 of XVI): 1H NMR (CDCl3-CD3OD) δ -0.07 (m,
4H), 0.15 (m, 2H), 0.35 (m, 5H), 0.57 (m, 3H), 1.35 (m, 2H),
1.55 (m, 2H), 1.75 (m, 1H), 2.52 (m, 1H), 3.24 (d, J ) 10.2 Hz,
1H), 3.60 (s, 3H), 5.88 (s, 1H), 6.85 (m, 1H), 7.04 (t, J ) 7.9
Hz, 1H), 7.15 (m, 2H), 7.27 (s, 1H), 7.37 (s, 1H); HRMS (EI)
523.2149 found (calcd 523.2141 for C28H33N3O5S); TLC Rf 0.32
(5% methanol in dichloromethane).
N-[3-[[S or R]-Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclo-
p r op ylm e t h yl)e t h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]-
m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-su lfon a m id e
(enantiomer 2 of XVI): 1H NMR (CDCl3-CD3OD) δ -0.07 (m,
4H), 0.15 (m, 2H), 0.35 (m, 5H), 0.57 (m, 3H), 1.35 (m, 2H),
1.55 (m, 2H), 1.75 (m, 1H), 2.52 (m, 1H), 3.24 (d, J ) 10.3 Hz,
1H), 3.60 (s, 3H), 5.88 (s, 1H), 6.85 (m, 1H), 7.04 (t, J ) 8.0
Hz, 1H), 7.15 (m, 2H), 7.27 (s, 1H), 7.37 (s, 1H); HRMS (EI)
523.2137 found (calcd 523.2141 for C28H33N3O5S); TLC Rf 0.32
(5% methanol in dichloromethane).
N-[3-[Cyclop r op yl[6-[1-[2-(m eth oxyeth oxy)eth yl]-3-(2-
m eth oxyeth oxy)p r op yl]-4-h yd r oxy-2-oxo-2H-p yr a n -3-yl]-
m et h yl]p h en yl]-1-m et h yl-1H -im id a zole-4-su lfon a m id e
(XVII): 1H NMR (CDCl3) δ 0.07 (m, 1H), 0.29 (m, 1H), 0.46
(m, 1H), 0.61 (m, 1H), 1.7-2.0 (m, 5H), 2.72 (m, 1H), 3.32 (s,
3H), 3.33 (s, 3H), 3.3-3.6 (m, 13H), 3.64 (s, 3H), 6.03 (s, 1H),
6.98 (m, 1H), 7.1-7.3 (m, 4H), 7.51 (s, 1H); HRMS (EI) m/z
619.2578 found (calcd 619.2563 for C30H41N3O9S); TLC Rf 0.21
(5% methanol in dichloromethane). Anal. (C30H41N3O9S) C,
H, N.
0.36 (5% ethyl acetate in dichloromethane). Anal. (C30H33
NO5S) C, H, N.
-
N -[3-[Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclop r op yl-
m et h yl)et h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]-
p h en yl]-4-flu or oben zen esu lfon a m id e (XXII): 1H NMR
(CDCl3) δ -0.08 (m, 4H), 0.1-0.6 (m, 10H), 1.4-1.6 (m, 4H),
1.65 (m, 1H), 2.55 (m, 1H), 3.39 (d, J ) 9.9 Hz, 1H), 6.11 (s,
1H), 6.9-7.3 (m, 7H), 7.72 (m, 2H), 9.7 (br s, 1H); HRMS (EI)
537.1977 found (calcd 537.1985 for C30H32NO5SF); TLC Rf 0.39
(10% ethyl acetate in dichloromethane).
N -[3-[Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclop r op yl-
m et h yl)et h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]-
p h en yl]-4-cya n ob en zen esu lfon a m id e (XIII): 1H NMR
(CDCl3) δ -0.03 (m, 4H), 0.1-0.7 (m, 10H), 1.4 (m, 2H), 1.6
The four individual diastereomers of compound XVIII were
obtained via chiral HPLC preparative work; their physical data
are summarized as follows.
N-[3-[[R or S]-Cyclop r op yl[4h h yd r oxy-2-oxo-6-[1(R)-
(p h en ylm eth yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]-1-
m et h yl-1H -im id a zole-4-su lfon a m id e (diastereomer 1 of

2408 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12
Thaisrivongs et al.
Ta ble 8. Summary of Selected Diffraction Data Collection and
Refinement Statistics of HIV-2 Protease-XVIIIa Complex
(m, 2H), 1.75 (m, 1H), 2.58 (m, 1H), 3.33 (d, J ) 10.2 Hz, 1H),
5.98 (s, 1H), 6.9 (m, 1H), 7.11 (t, J ) 8.1 Hz, 1H), 7.2 (m, 2H),
7.67 (m, 2H), 7.82 (m, 2H); HRMS (EI) 544.2035 found (calcd
544.2032 for C31H32N2O5S); TLC Rf 0.30 (10% ethyl acetate in
dichloromethane).
N -[3-[Cyclop r op yl[6-[2-cyclop r op yl-1-(cyclop r op yl-
m et h yl)et h yl]-4-h yd r oxy-2-oxo-2H -p yr a n -3-yl]m et h yl]-
p h en yl]-8-qu in olin esu lfon a m id e (XXIV): 1H NMR (CDCl3)
δ -0.07 (m, 4H), 0.18 (m, 1H), 0.3-0.6 (m, 9H), 1.3-1.6 (m,
5H), 2.53 (m, 1H), 3.32 (d, J ) 9.7 Hz, 1H), 5.96 (s, 1H), 6.87
(m, 1H), 7.0 (m, 2H), 7.13 (s, 1H), 7.5 (m, 2H), 7.9 (m, 2H), 8.2
(m, 2H), 9.05 (m, 1H); HRMS (EI) 570.2188 found (calcd
570.2188 for C33H34N2O5S); TLC Rf 0.40 (10% ethyl acetate in
dichloromethane). Anal. (C33H34N2O5S) C, H, N.
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]p r op a n esu lfon -
a m id e (XXV): 1H NMR δ 0.24 (m, 2H), 0.47 (m, 1H), 0.68
(m, 1H), 0.80 (t, J ) 7.4 Hz, 3H), 0.92 (2t, J ) 7.4 Hz, 3H),
1.55 (m, 2H), 1.75 (m, 3H), 2.5 (m, 1H), 2.75 (m, 1H), 2.85 (m,
1H), 3.0 (m, 2H), 3.44 (2d, 1H), 5.92 (s, 1H), 6.9-7.4 (m, 9H),
9.5 (br, 1H); IR (thin film) 3252, 2967, 1661, 1574, 1415, 1285,
1146 cm-1; HRMS (EI) m/z 495.2074 found (calcd 495.2079 for
C28H33NO5S); TLC Rf 0.18 (10% ethyl acetate in dichlo-
romethane).
space group
unit cell: a, Å
b, Å
c, Å
resolution, Å
P212121
33.671
45.671
131.300
2.2
no. of observations
unique reflections
% completeness
R merge
R factor [refinement]
rms deviations: distance, Å
angle, deg
35912
10225
93.6
0.088
0.181
0.034
3.800
12.607
16.112
fixed dihedrals, deg
flexible dihedrals, deg
0.34 (m, 2H), 0.77 (t, J ) 7.3 Hz, 3H), 1.5 (m, 3H), 2.47 (m,
1H), 2.7-2.9 (m, 2H), 3.23 (d, J ) 9.8 Hz, 1H), 5.79 (s, 1H),
6.8-7.2 (m, 10H), 7.41 (t, 1H), 7.5 (m, 1H), 7.89 (d, J ) 8.2
Hz, 1H), 8.2 (m, 2H), 9.01 (br d, 1H); IR (thin film) 3258, 2964,
1659, 1567, 1413, 1166, 1146 cm-1; HRMS (EI) m/z 580.2028
found (calcd 580.2032 for C34H32N2O5S); TLC Rf 0.48 (20%
ethyl acetate in dichloromethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]ben zen esu lfon -
a m id e (XXVI): 1H NMR δ 0.08 (m, 1H), 0.24 (m, 1H), 0.45
(m, 1H), 0.57 (m, 1H), 0.78 (t, J ) 7.2 Hz, 3H), 1.5-1.7 (m,
3H), 2.49 (m, 1H), 2.75 (dd, 1H), 2.85 (dd, 1H), 3.41 (2d, 1H),
5.84 (s, 1H), 6.8-7.5 (m, 12H), 7.7 (m, 1H), 8.9 (br, 1H); IR
Ch ir a l HP LC con d ition s. Compound XVI was separated
into the two individual enantiomers on a 2.0 × 25 cm (R,R)
Whelk-O 1 column, using 30% isopropyl alcohol, 0.1% acetic
acid, and 0.2% water in hexane at 6.0 mL/min flow rate,
monitoring at 290 nm. On the analytical column (0.46 × 25
cm Chiralpak AD) using 30% isopropyl alcohol, 0.1% acetic
acid, and 0.2% water in hexane at 0.5 mL/min flow rate,
monitoring at 290 nm, the two enantiomers exhibited retention
times of 24.1 and 33.4 min, respectively.
The four diastereomers of compound 4 were obtained on a
1.0 × 25 cm (R,R) Whelk-O 1 column, using 25% isopropyl
alcohol and 0.05% acetic acid in hexane at 9.0 mL/min flow
rate, monitoring at 310 nm. On the analytical column (0.46
× 25 cm (R,R) Whelk-O 1 column) using 25% isopropyl alcohol
and 0.05% acetic acid in hexane at 1.0 mL/min flow rate,
monitoring at 290 nm, the four diastereomers exhibited
retention times of 19.5, 26.5, 40.7, and 42.5 min, respectively.
These four individual diastereomers were separately carried
through the reaction sequence to produce the four individual
diastereomers of compound XVIII.
Cr ysta llogr a p h y: Cr ysta lliza tion . Preparation and pu-
rification of the recombinant HIV-2 protease has been de-
scribed previously.33 Protein preparation of HIV-2 protease
contains a Lys57/Leu mutation but has been found to be
indistinguishable in activity and specificity from the wild type
enzyme. Crystals of HIV-2 protease complexed with diaste-
reomer 1 of compound XVIII were obtained by co-crystalliza-
tion experiments in which 2 µL of the inhibitor solution (0.1
mg/µL concentration) in DMSO was added to 130 µL of the
freshly thawed, ice-cold protease solution (ca. 6 mg/mL con-
centration) and the mixture equilibrated on ice for 60 min.
The undissolved inhibitor that precipitates upon mixing was
removed by centrifugation. Crystals were grown at room
temperature in 10-µL hanging drops (equal volumes of protein
and precipitant) by vapor diffusion against a precipitant of 30-
35% (w/v) PEG 4000 at pH 6.6-7.0 (0.1 M imidazole) and at
pH 7.2-7.6 (0.1 M HEPES buffer).
Da ta Collection . A single crystal of the inhibitor/protease
complex with approximate dimensions of 0.3 × 0.2 × 0.2 mm
was used for data collection. Diffraction data were collected
using a Siemens area detector, with X-rays generated by a
Siemens rotating anode source operating at 45 kV, 96 mA.
Measurements were made as a series of 0.25° frames, with
an exposure time of 180 s per frame. Data sets were processed
using XENGEN data reduction software.34 Table 8 sum-
marizes the data collection along with statistics from the
refinement of the models. The effective resolution was taken
as the maximum resolution for which the mean I/σ was greater
than 2.0. Data beyond this maximum were discarded.
Str u ctu r e Refin em en t. Since the space group of the
protease/inhibitor complex was the same as one previously
refined in our laboratory,33 refinement of the protease model
(thin film) 3253, 2964, 1661, 1572, 1414, 1284, 1158 cm-1
;
HRMS (EI) m/z 529.1927 found (calcd 529.1923 for C31H31
-
NO5S); TLC Rf 0.23 (10% ethyl acetate in dichloromethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H-p yr a n -3-yl]m eth yl]p h en yl]-4-m eth oxyben -
zen esu lfon a m id e (XXVII): 1H NMR δ 0.11 (m, 1H), 0.24 (m,
1H), 0.44 (m, 1H), 0.58 (m, 1H), 0.78 (t, J ) 7.5 Hz, 3H), 1.6
(m, 3H), 2.49 (m, 1H), 2.74 (dd, 1H), 2.86 (dd, 1H), 3.40 (2d,
1H), 3.73 (s, 3H), 5.85 (s, 1H), 6.7-7.3 (m, 12H), 7.65 (m, 2H),
9.0 (br, 1H); IR (thin film) 3253, 2965, 1661, 1579, 1415, 1262,
1155 cm-1; HRMS (EI) m/z 559.2019 found (calcd 559.2028 for
C32H33NO6S); TLC Rf 0.20 (10% ethyl acetate in dichlo-
romethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H -p yr a n -3-yl]m et h yl]p h en yl]-4-ch lor ob en -
zen esu lfon a m id e (XXVIII): 1H NMR δ 0.11 (m, 1H), 0.22
(m, 1H), 0.44 (m, 1H), 0.58 (m, 1H), 0.78 (t, 3H), 1.5-1.7 (m,
3H), 2.49 (m, 1H), 2.7 (m, 1H), 2.85 (m, 1H), 3.38 (d, J ) 9.8
Hz, 1H), 5.91 (s, 1H), 6.8-7.3 (m, 12H), 7.62 (m, 2H), 9.5 (br,
1H); IR (thin film) 3255, 2965, 1660, 1574, 1414, 1281, 1163,
1094 cm-1; HRMS (EI) m/z 563.1528 found (calcd 563.1533 for
C31H30NO5ClS); TLC Rf 0.30 (10% ethyl acetate in dichlo-
romethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H -p yr a n -3-yl]m et h yl]p h en yl]-4-cya n ob en -
zen esu lfon a m id e (XXIX): 1H NMR δ 0.12 (m, 1H), 0.23 (m,
1H), 0.45 (m, 1H), 0.59 (m, 1H), 0.80 (t, 3H), 1.6 (m, 3H), 2.5
(m, 1H), 2.7-2.9 (m, 2H), 3.38 (d, J ) 9.8 Hz, 1H), 5.87 (s,
1H), 6.8-7.2 (m, 9H), 7.45 (br, 1H), 7.57 (m, 2H), 7.78 (m, 2H),
9.27 (br, 1H); IR (thin film) 3253, 2965, 1661, 1573, 1415, 1282,
1165 cm-1; HRMS (EI) m/z 554.1878 found (calcd 554.1875 for
C32H30N2O5S); TLC Rf 0.23 (10% ethyl acetate in dichlo-
romethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H -p yr a n -3-yl]m e t h yl]p h e n yl]-3-p yr id in e -
su lfon a m id e (XXX): 1H NMR δ 0.11 (m, 1H), 0.22 (m, 1H),
0.45 (m, 1H), 0.59 (m, 1H), 0.84 (t, J ) 7.4 Hz, 3H), 1.5-1.8
(m, 3H), 2.51 (m, 1H), 2.81 (dd, J ) 7.0 Hz, J ) 13.5 Hz, 1H),
2.92 (2dd, J ) 7.9 Hz, J ) 13.5 Hz, 1H), 3.28 (2d, J ) 10.3 Hz,
1H), 5.69 (s, 1H), 6.9-7.4 (m, 10H), 8.05 (m, 1H), 8.60 (m, 1H),
8.83 (s, 1H); HRMS (EI) m/z 530.1879 found (calcd 530.1875
for C30H30N2O5S); TLC Rf 0.25 (4% methanol in dichlo-
romethane).
N-[3-[Cyclop r op yl[4-h yd r oxy-2-oxo-6-[1-(p h en ylm eth -
yl)p r op yl]-2H -p yr a n -3-yl]m et h yl]p h en yl]-8-q u in olin e-
su lfon a m id e (XXXI): 1H NMR δ -0.13 (m, 1H), 0.13 (m, 1H),

Design of Novel HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 12 2409
could be initiated without resolving the position of the molecule
in the cell. Structural refinement was carried out using
CEDAR,39 with periodic manual rebuilding using the interac-
tive graphics program CHAIN,37 based on 2|Fo - Fc| and |Fo
- Fc| electron density maps. Electron density maps were
calculated using the XTAL package of crystallographic pro-
grams.38 Inhibitor and solvent molecules were added during
later stages of the refinements. Atomic coordinates of this
structure are being deposited with the Protein Data Bank.40
HIV-1 a n d HIV-2 P r otea se In h ibitor y Assa ys a n d Ki
Va lu e Deter m in a tion . HIV-1 protease41 and HIV-2 pro-
tease33 were purified and refolded from E. coli inclusion bodies.
The substrate used spans the p17-p24 processing site (R-V-
S-Q-N-Y-P-I-V-Q-N-K) and was derivatized with biotin and
fluorescein isothiocyanate at the amino and carboxy termini,
respectively. The reaction was performed in assay buffer at
substrate concentrations below Km concentrations for 90 min
at room temperature in the dark. The assay buffer consists
of 0.1 M sodium acetate, 1.0 M NaCl, 0.05% NP40. Enzyme
and substrate concentrations were 10 and 50 nM, respectively.
Inhibitors were dissolved in dimethyl sulfoxide, and the
amount in the assay was 2%. After incubation the reaction
was stopped by addition of fluoricon avidin beads at 0.5% (w/
v). The residual bound fluorescence was obtained by process-
ing on an IDEXX Screen Machine from which percent inhibi-
tion values were calculated. A range of inhibitor concentrations
was used. Determination of Ki values with this assay required
analysis under conditions in which substrate concentration
resided substantially below Km and the inhibitor concentra-
tions greatly exceeded the Ki value and the enzyme concentra-
tion. One can solve the remaining substrate after prolonged
incubation times as a quadratic, exponential function in terms
of inhibitor concentrations and time to generate calculated
values for the kinetic constants Km, kcat, and Ki as well as the
enzyme concentration.
MTT Cytotoxicity Assa y.42 Into appropriate wells of a
96-well microtiter plate was added 125 µL of 2× drug dissolved
in RPMI Complete medium, followed by 125 µL of suspension
of MT4 cells containing 2 × 104 cells, and mixed gently.
Various drug concentrations were tested in triplicate. Positive
controls contained cells and RPMI Complete without drug,
while negative controls contained medium only. Each micro-
plate was incubated for 4-7 days at 37 °C in 5% CO2. After
completion of the incubation period, 100 µL of supernatant was
removed, with minimal disturbance of the cells at the bottom
of each well. To each well was added 10 µL of a freshly
prepared MTT solution (5 mg/mL in PBS, Sigma). The plate
was incubated for 4 h at 37 °C in 5% CO2. An additional 50
µL was removed from all wells, and the cells were mixed
thoroughly by vigorously pipetting up and down to resuspend
the formazan dye precipitate. To each well was added 100 µL
of 0.04 N HCl/2-propanol solution with extensive mixing. The
plate was read within 15 min on a dual-wavelength microplate
reader using a test wavelength of 570 nm and a reference
wavelength of 650 nm. The cytotoxic activity of a test
compound was calculated by dividing the mean OD at each
drug concentration by the mean OD of the positive controls,
subtracting the result from 1, and multiplying by 100. The
CCTD50 (cell culture toxicity dose50) value was determined to
be the calculated compound concentration required to inhibit
50% of cell metabolism when OD’s between compound-exposed
cells and compound-free control cells were compared. The
CCTD50 value was calculated by linear regression or by
straight-line extrapolation.
0.2% DMSO without compound. Final DMSO concentration
in each culture was 0.1%. Plates were incubated for 7 days
at 37 °C in 5% CO2. At the end of the 7-day incubation period,
100 µL of supernatant was removed from each test well and
live virus inactivated by the addition of lysis buffer (Coulter
Diagnostics, Hialeah, FL) containing 5% Triton X-100. The
amount of HIV p24 core antigen was quantified with an ELISA
procedure by following the manufacturer’s direction. The IC50
(inhibitory concentration50), the amount of drug necessary to
reduce the concentration of p24 in drug-containing cultures
by 50% when compared to drug-free controls, was calculated
by comparing the quantity of p24 produced in drug-containing
supernatants with that of DMSO-containing drug-free super-
natants. This was accomplished by using the linear portion
of the plot of log10 drug concentration versus percent p24
inhibition and utilizing linear regression or straight line
extrapolation to calculate the drug concentration necessary to
inhibit 50% of non-drug-treated p24 antigen.
Ack n ow led gm en t. We gratefully acknowledge Che-
Shen C. Tomich and Kimberly A. Curry for cloning and
expressing and Alfredo G. Tomasselli and Donna J .
Rothrock for purifying the HIV proteases. We are also
grateful to Carol A. Bannow and Linda L. Maggiora for
the preparation of the biotinylated and fluorescent-
labeled substrate for the enzyme inhibitory assay.
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