Design and fast synthesis of C-terminal duplicated potent C2-symmetric P1/P1'-modified HIV-1 protease inhibitors

Article abstract of DOI:10.1021/jm9910371

An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C₂-symmetric C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds. These were synthesized with the objective to identify sui

Full text of DOI:10.1021/jm9910371

J . Med. Chem. 1999, 42, 3835-3844
3835
Design a n d F a st Syn th esis of C-Ter m in a l Du p lica ted P oten t C2-Sym m etr ic
P 1/P 1′-Mod ified HIV-1 P r otea se In h ibitor s
Mathias Alterman,† Hans O. Andersson,| Neeraj Garg,† Go¨ran Ahlse´n,‡ Seved Lo¨vgren,| Bjo¨rn Classon,
U. Helena Danielson,‡ Ingmar Kvarnstro¨m,∇ Lotta Vrang,§ Torsten Unge,| Bertil Samuelsson, and
Anders Hallberg*,†
Department of Organic Pharmaceutical Chemistry, Uppsala University, BMC, Box 574, SE-751 23 Uppsala, Sweden,
Department of Biochemistry, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden, Medivir AB, Lunastigen 7,
SE-141 44 Huddinge, Sweden, Department of Chemistry, Linko¨ping University, SE-581 83 Linko¨ping, Sweden,
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden, and
Department of Molecular Biology, Uppsala University, BMC, Box 590, SE-751 24 Uppsala, Sweden
Received March 16, 1999
An analysis of the X-ray structure of a complex of HIV-1 protease with a linear C2-symmetric
C-terminal duplicated inhibitor guided the selection of a series of diverse target compounds.
These were synthesized with the objective to identify suitable P1/P1′ substituents to provide
inhibitors with improved antiviral activity. Groups with various physical properties were
attached to the para-positions of the P1/P1′ benzyloxy groups in the parent inhibitor. A
p-bromobenzyloxy compound, prepared in only three steps from commercially available starting
materials, was utilized as a common precursor in all reactions. The subsequent coupling
reactions were completed within a few minutes and relied on palladium catalysis and flash
heating with microwave irradiation. All of the compounds synthesized exhibited good inhibitory
potency in the protease assay, with Ki values ranging from 0.09 to 3.8 nM. A 30-fold
improvement of the antiviral effect in cell culture, compared to the parent compound, was
achieved with four of the inhibitors. The differences in Ki values were not correlated to the
differences in antiviral effect, efficiency against mutant virus, or reduced potency in the presence
of human serum. The poorest enzyme inhibitors in fact belong to the group with the best
antiviral effect. The binding features of two structurally related inhibitors, cocrystallized with
HIV-1 protease, are discussed with special emphasis on the interaction at the enzyme/water
phase.
In tr od u ction
shall exhibit good bioavailability and be easy to syn-
thesize at low cost.
The etiological agent of acquired immune deficiency
syndrome (AIDS), the human immunodeficiency virus
(HIV), is a retrovirus of the lentivirus subfamily.1 The
RNA genome of HIV encodes an essential aspartic pro-
tease, which has become one of the prime targets for
chemotherapeutic intervention in the treatment of
AIDS.2 The C2-symmetric dimeric protease processes the
viral precursor polyproteins gag and gag/ pol into struc-
tural proteins and enzymes. Four HIV protease inhibi-
tors, saquinavir, ritonavir, indinavir, and nelfinavir,
have been approved to date for AIDS therapy, mainly
in combination with reverse transcriptase inhibitors.
This therapy has led to reduction of the viral load, and
an increase in the number of CD4+ lymphocytes has
been demonstrated in HIV-infected individuals.3 How-
ever, side effects4 and the clinical emergence of resistant
mutants5 suggest that there will be a need for an ever-
increasing armament of new protease inhibitors, prefer-
ably with unique resistance profiles. Such inhibitors
We are engaged in a program where derivatized
carbohydrates are employed as C2-symmetric HIV-1
protease inhibitors.6 Application of the concept of C-
terminal duplication combined with suitable selection
of the P2/P2′ substituents provided previously a series
of potent protease inhibitors with P1/P1′ benzyloxy
groups, e.g. 1, which could be prepared in three steps
from commercially available starting materials.6d With
the objective of identifying inhibitors with improved
antiviral activity, the X-ray crystal structure of a
complex of HIV-1 protease with the inhibitor 1 was
examined. Molecular modeling suggested that larger
groups in the para-positions of the P1/P1′ benzyloxy side
chains, located near the exterior surface of the active
site and close to the solvent, could be accommodated
readily. The para-position was anticipated to serve as
a handle for modification aimed at improved antiviral
activity. So far, the effect of modifications to the P1 (or
P1′) side chain of linear nonsymmetrical inhibitors on
enzyme inhibitory potency, antiviral activity, and oral
bioavailability has been the subject of only a relatively
limited number of studies.7 The P1/P1′ structure-
activity relationships were found to differ among the
structural classes of inhibitors investigated, and unfor-
tunately the predictability of the pharmacokinetic be-
havior is often poor.7a,b,i
* Corresponding author.
† Department of Organic Pharmaceutical Chemistry, Uppsala Uni-
versity.
‡ Department of Biochemistry, Uppsala University.
§ Medivir AB.
∇ Linko¨ping University.
Stockholm University.
| Department of Molecular Biology, Uppsala University.
10.1021/jm9910371 CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/03/1999

3836 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Alterman et al.
Sch em e 1
Sch em e 2
We aimed at exploring the P1/P1′ sites of the carbo-
hydrate-derived inhibitor 2, an equipotent valine ana-
Resu lt a n d Discu ssion
Ch em istr y. The synthesis of the bromo precursor 3
was accomplished, as depicted in Scheme 1, following a
similar procedure to that previously reported for the
synthesis of 1 and 2.6d Thus, the L-1,4:3,6-mannarodi-
lactone, prepared in one step from commercially avail-
able L-mannonic-γ-lactone, was alkylated in dioxane
with 4-bromobenzyl imidate 14 to deliver 15, which
precipitated from the reaction medium and was eventu-
ally isolated in 88% yield. Subsequent treatment of 15
with excess valine methylamide in 1,2-dichloroethane
afforded the bromo derivative 3, which precipitated as
a white powder and was isolated in 64% yield.
The target compounds 4-13 (Scheme 2) were pre-
pared by palladium-catalyzed coupling reactions pro-
moted by microwave irradiation8 as shown in Table 1.
We have recently studied flash heating with model
compounds and devised a protocol that allowed the
Suzuki, Stille, and Heck couplings to be completed in
minutes9 instead of hours or days10 which is frequently
required with traditional heating techniques. Here we
demonstrate that additionally the more complex orga-
nopalladium precursor 3, with potential for undesired
water elimination, constitutes a suitable substrate in
all of the reaction types.
The aryl- and heteroarylboronic acids were reacted
with 3 in a sealed vessel in the presence of a catalytic
amount of palladium tetrakis(triphenylphosphine) with
sodium carbonate as base.10b Microwave irradiation at
45 W for 4 min in a mixture of dimethoxyethane,
ethanol, and water allowed isolation of the compounds
4-7 in high yields. A microwave induced 9-BBN cou-
logue of 1, and selected a variety of diverse functional
groups to be coupled to the para-position of the P1/P1′
benzyloxy substituents. Herein we report rapid micro-
wave-promoted palladium-catalyzed syntheses of a se-
ries of new P1/P1′-modified derivatives, 4-13, all pre-
pared from one single precursor. In addition to inhibition
analysis on purified enzyme, the antiviral effect in cell
culture using wild type and mutant virus and also the
effect with 50% human serum are presented. Finally,
the characteristic binding features of two of the inhibi-
tors are discussed.

C2-Symmetric HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3837
Ta ble 1. Synthetic Methods for Preparation of C2-Symmetric Carbohydrate-Derived Inhibitors Modified in P1/P1′, Enzyme
Inhibition, and Antiviral Activity in Cell Culture
a
b
Standard error 20%. ED50 for reference substances tested in the same assay: ritonavir (ED50 0.06 µM), indinavir (ED50 0.06
µM), saqinavir (ED50 0.01 µM), nelfinavir (ED50 0.04 µM). c nd, not determined. Compounds tested with 50% human AB+ serum.
d
e Mutations in MT4/HIV-1: V32I, M46I, V82A. f Mutations in MT4/HIV-1: M46I, V82F, V84I. Executed with traditional thermal
g
heating.
pling, which has not been reported previously, was used
for the preparation of 8. Thus, phenylethyl 9-BBN was
synthesized by hydroboration of styrene,11 whereafter
the bromo precursor 3, palladium tetrakis(triphenylphos-
phine), and sodium carbonate were added to the reaction
vessel. Exposure to microwave irradiation (60 W) for 2
min delivered 8, although in moderate yield (38%).
Trimethylheteroaryl tin or tributylheteroaryl tin com-
pounds were employed as coupling partners in the Stille
reactions.10c The reactions were conducted at 60 W for

3838 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Alterman et al.
2 min in the presence of palladium tetrakis(triphen-
ylphosphine) and with dimethylformamide as solvent
to furnish 9-11. In the coupling reactions with the
pyridyl tin compounds, cupric oxide was employed as
additive, but in the corresponding coupling with the
thiazolyl tin derivative, silver(I) oxide was found to be
more suitable.12
Heck reactions with methyl acrylate and with 1,2-
cyclohexanedione13 were conducted with microwave
irradiation for 2 min (60 W) in aqueous dimethylfor-
mamide.10a The isolation of the dione 13 was not
successful by standard chromatography. Starting mate-
rial contaminated the product, and all efforts to achieve
full conversion of 3, e.g. increased power, prolonged
reaction time, or addition of a second portion of catalyst,
failed and led to formation of degradation products.
Therefore the reaction was executed with traditional
heating at 100 °C for 48 h to afford pure 13 in 53% yield.
It should be emphasized that the reaction protocols
described here permit all reaction types to be performed
with an aryl bromide. Thus, it is not necessary to employ
the more reactive aryl iodides, as arylpalladium precur-
sors, although aryl iodides should be useful also in flash
heated reactions.9
HIV P r otea se In h ibition . The HIV-1 protease was
cloned and heterologously expressed in Escherichia coli
and purified as described elsewhere.14 The Ki values for
the synthesized compounds were determined by a
fluorometric assay15 (Table 1).
pronounced antiviral activity (ED50 0.04 µM) and were
similar in activity to the phenyl analogue 4 (ED50 0.04
µM), ritonavir (ED50 0.06 µM), and indinavir (ED50 0.06
µM), evaluated in the same assay system. Derivatives
9 and 10, with electron-deficient weakly basic pyridine
rings, which were expected to increase water solubility,
were approximately equipotent to 2 in the protease
assay but showed no obvious advantages in the cell
system. An increase in antiviral activity was encoun-
tered with the thiazole 11, as compared to the parent
compound 2 and the pyridyl compounds 9 and 10,
corroborating with recent findings by Bold et al.7a
Compound 5, with electron-withdrawing 3-nitrophenyl
groups in the para-positions but lacking basic nitrogens,
was the weakest inhibitor in the series with respect to
the enzyme assay. Despite this, the nitro compound,
unlike the pyridyl compounds 9 and 10, exhibited good
antiviral activity in the cell system. The linear unsatur-
ated ester 12, on the other hand, which was the most
potent enzyme inhibitor in the series is 20-fold less
efficient than the nitro compound 5 in exerting an
antiviral effect in the cell assay.
It was previously reported that a tethered polar
carboxyl group attached to the para-position of a P1′
substituent gives high affinity to the protease but
results in a low antiviral effect. This was anticipated
to be attributed to poor cell penetration.7b,e A cyclic
unsaturated hydroxy ketone, which combined hydrogen
bond-donating and hydrogen bond-accepting capacity
and was designed to be reminiscent of the classical
hydroxyketocoumairn, a bioiostere of a carboxyl group,16
was attached to the P1/ P1′ aryl groups. Although
binding to the protease with good affinity was achieved
with 13, the antiviral effect (ED50 3.7 µM) was still poor.
The most active compounds in the cell assay were
evaluated further in cell culture with mutant virus,
selected by increased concentrations of ritonavir. Three
amino acids were substituted in the protease of the
mutant virus used. All of the compounds 4-7 suffered
a 10-fold decrease in antiviral activity with the mutant
virus, except for compound 5, which retained activity
against one of the mutants. Despite this loss the
inhibitors were in fact 20-fold more active than the lead
compound 2 against one mutant virus. For comparison,
compounds 3, 9, and 11 were also included in this study.
The antiviral activity of compounds 9 and 11 was low
against mutant virus, but the bromo compound 3
exhibited similar antiviral activity in all of the assays
used.
Nonspecific protein binding of the inhibitors was
assessed in a modified cell assay with 50% of human
serum. A dramatic decrease of the antiviral activity was
encountered for most of the tested inhibitors, e.g. more
than a 20-fold drop in antiviral activity for the four most
active compounds (4-7). Surprisingly, the antiviral
activity of the thiazole derivative 11 remained at the
same level in the presence of serum.
In Vitr o An ti-HIV Activity. The anti-HIV activity
was assayed in MT4 cells according to a previously
published procedure15 using the colorimetric XTT assay
to monitor the cytopathogenic effects. Anti-HIV activity
measured in the presence of human serum (hs) was
performed in MT4 cells with human AB+ serum.
Mutants were obtained by passaging cell-free virus in
the presence of stepwise-increased concentrations of
ritonavir15 (Table 1).
St r u ct u r e-Act ivit y R ela t ion sh ip s a n d X-r a y
Cr ysta llogr a p h ic Da ta . As suggested from modeling,
all target compounds examined (3-13) exhibited good
inhibitory potencies in the protease enzyme assay with
Ki’s in the nanomolar range (Table 1). We first replaced
the P1 and P1′ phenyl groups with biphenyl moieties
and thereby obtained an inhibitor (4) with a slightly
higher Ki than that of 2. More importantly, a 40-fold
increase in antiviral activity was observed. The diphen-
ylethane derivative 8 was prepared to map the region
closer to the exterior of the enzymatic cleft. This
modification resulted in an increased Ki value, and the
antiviral activity dropped 50-fold as compared to 4. This
is contrary to expectations based on previous studies of
related P1/ P1′ benzyloxyphenyl analogues, where the
antiviral activity was often found to be enhanced
strongly by this carbon chain extension.7l We decided
to replace the p-hydrogen atom with various lipophilic,
electron-rich, and electron-deficient heterocycles, with
hydrogen bond-accepting capacities, since Thompson et
al. have demonstrated with several examples that
p-oxygen substituents in P1 or P1′ enhance cell penetra-
tion relative to the related parent compounds.7m Both
the 2- and 3-substituted electron-rich thienyl compounds
6 and 7 were less potent than the parent compound 2.
However, both of these compounds demonstrated a
Compounds 4, 6, 7, and 9 were chosen for evaluation
of oral absorption in rats. At 30-40 mg/kg all of the
compounds failed to attain measurable blood levels
(<0.02 µg/mL). Nelfinavir tested in the same assay gave
1.9 µM at 26 mg/kg.
The 3-thienyl and 3-pyridyl compounds 6 and 9 were
selected for cocrystallization with the HIV-1 protease

C2-Symmetric HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3839
F igu r e 1. Compounds 1, 6, and 9 adopt the same overall conformation. The P1 and P2 arms are in contact with each other
through close-packing interactions. In this figure the inhibitor compounds are rotated such that the central hydroxyl is pointing
toward the reader. This figure was drawn with the program Molscript2.02.18
and crystallographic structure determination. The struc-
tures were determined with 2.1 and 2.0 Å resolutions
for the complexes with the HIV-1 protease and com-
pounds 6 and 9, respectively. As expected, the previously
determined compound 1 and compounds 6 and 9 have
the same overall conformation (Figure 1). The aromatic
side groups of the P1/P1′ arms pack closely to the
backbone and side groups of the P2/P2′ arms. The
inhibitors are chemically C2-symmetrical with two
central hydroxyl groups, which both could be used
alternatively for the binding to the catalytic aspartate
residues. Due to the 3D conformation of the inhibitor,
only one of these hydroxyls can be utilized for binding
to the aspartates. This arrangement leads to an asym-
metric binding of the inhibitor to the protease, which
in turn induces deviations from exact symmetrical
arrangement of the protein monomers. Interestingly,
only one arrangement of the hydroxyls is found in the
crystal structure. The deviation from exact symmetry
is also revealed by the slightly different conformations
of the P1/P1′ arms and the waters and residues sur-
rounding them.
Comparison of the three protease inhibitor structures
shows how the water molecules at the entrances to the
S1/S1′ sites, which are also at the entrance to the S3/
S3′ sites, are displaced by the penetrating thienyl and
pyridyl groups (Figure 2). In the complex of compound
1 with the protease, four water molecules are firmly
bound at the entrance at each side of the protease dimer.
These water molecules are linked to each other in a
hydrogen bond network, which also involves Arg8/108.
The thienyl and pyridyl rings of the extended P1/P1′
arms displace three of these water molecules on both
F igu r e 2. Displacement of water molecules to fill the entrance
to the S1/S1′-S3/S3′ sites. In green are marked amino acids,
which make up the entrance and are in contact with the
sides. In the compound 6 structure (Figure 2c,d), two
new water molecules are bound to the thienyl ring and
Arg8/108. Since the Ki values of compounds 2, 6, and 9
inhibitors. In the structure of compound 1 in complex with
do not differ significantly, it could be concluded that the
net entropy, from release of the waters and binding the
larger compounds 6 and 9, seems to balance the net
enthalpy from loss of the water hydrogen bonds and
creations of the thienyl and pyridyl interactions.17 The
structures also demonstrate that these elongated P1/
P1′ arms fill out the entrances completely (Figure 2).
The electron density for the thiophene ring does not
uniquely define the position for the sulfur atom. This
observation indicates that the thiophene ring may be
bound in two orientations, with approximately equal
occupancy in both orientations. Whether this is also the
situation for the pyridine cannot be excluded. However,
the positions of coordinated water molecules support the
orientation represented in Figures 1 and 2.
the protease, four water molecules (light blue) are bound
through a hydrogen bond network covering the entrance (a,b).
Filling out the entrance by extension of the P1/P1′ inhibitor
arms with thienyl (compound 6, c,d) and pyridyl (compound
9, e,f) results in displacement of water molecules. The view in
panels b, d, and f is 90° away from the view in panels a, c,
and d and from the top of the molecule. This figure was drawn
with the program Molscript2.02.18
of interactions with the elongated P1/P1′ arms. Due to
close-packing interactions between Pro81/181 and the
thienyl and pyridyl groups, the loop aa78-83/178-183
has moved 0.3-0.4 Å toward the P1/P1′ arms. Interac-
tions with backbone atoms of Gly47/147 and the side
chain of Phe53/153 have caused the flaps (aa47-53/
147-153) to move 0.2-0.5 Å. In the compound 6
complex, the side chain of Arg8/108 has moved toward
the thienyl groups and packs at van der Waals distances
The protein backbone and side chains contract slightly
at the entrances to the S1/S1′-S3/S3′ sites as the result

3840 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Alterman et al.
reactions in a multimode domestic microwave oven producing
nonuniform irradiation. Ca u tion ! It is im p or ta n t to n ote
th a t w h en ca r r yin g ou t m icr ow a ve-h ea ted r ea ction s in
closed vessels, qu ite la r ge p r essu r es m a y bu ild u p , a n d
th er efor e it is im p er a tive th a t a n a p p r op r ia te sep tu m
is u tilized a s a p r essu r e r elief d evice. Standard workup:
organic layers were dried with MgSO4 and concentrated in
vacuo.
to the aromatic ring. In the compound 9 complex, Arg8/
108 has moved away from the inhibitor. The structure
studies of these protease inhibitor complexes demon-
strate the flexibility in the protease molecule, which
confirms a previous observation by Nugiel et al. who
studied the same region of the protease.7d
Con clu sion
(4-Br om oben zyl)-2,2,2-tr ich lor oa cetim id a te (14). To a
stirred solution of 4-bromobenzyl alcohol (50.0 g, 0.267 mmol)
in toluene (250 mL) under a nitrogen atmosphere was slowly
added sodium (1.23 g, 0.0535 mmol). The solution was left for
20 min. To this was added 2,2,2-trichloroacetonitrile (26.7 mL,
0.267 mmol). The slight exothermic reaction was stirred for 1
h at room temperature. The orange-colored mixture was
filtered through a glass funnel and concentrated. Drying in
vacuo gave 84.3 g (95%) of (4-bromobenzyl)-2,2,2-trichloroace-
timidate as a slightly grayish solid. The product is 95% pure
(1H NMR) and can be used without further purification: IR
In summary, application of the concept of C-terminal
duplication and concomitant modifications of the P1/
P1′ positions has led to the potent protease inhibitors
4-7 with a 30-fold improvement of the antiviral effect
as compared to the parent compound 2 in vitro. These
compounds were also consistently more potent than 2
in the presence of human serum. With regard to HIV
protease inhibition, it has been demonstrated that a
wide variety of groups are tolerated in P1/P1′ and that
the inhibitory potency in the enzyme assay is largely
retained in all of the compounds synthesized. We
observed no significant alteration in the Ki values as a
result of a possible water displacement from the S1/S1′
sites by thienyl and pyridyl groups. This is consistent
with the view of the para-positions of P1/P1′ being
essentially “neutral” to the thermodynamics of enzyme
binding.7m We conclude that the Ki values did not
always translate to antiviral activity in the cell-based
assay and that the poorest enzyme inhibitors 5 and 6,
although still inhibiting in the nanomolar range, belong
to the group with the best antiviral effect. Furthermore,
we have developed fast and flexible protocols for the
synthesis of the inhibitors, starting from a commercially
available carbohydrate and relying on palladium-
catalyzed coupling reactions promoted by microwave
irradiation as key reactions. These reactions constitute
the first example of the application of palladium-
catalyzed reactions conducted with flash heating to the
area of medicinal chemistry of which we are aware. We
foresee that this heating technique will find many
applications in particular in the context of combinatorial
chemistry.
1
(KBr) ν 3332, 1668 cm-1; H NMR (CDCl3) δ 8.41 (br s, 1H),
7.51 (m, 2H), 7.31 (m, 2H), 5.29 (s, 2H); 13C NMR (CDCl3) δ
162.3, 138.4, 131.7, 129.4, 122.3, 69.9. A small portion was
recrystallized from toluene and submitted for elemental
analysis. Anal. (C9H7Cl3NO‚1/8H2O) C, H, N.
2,5-O-Bis(4-b r om ob e n zyl)-L -m a n n a r o-1,4:3,6-d ila c-
ton e (15). A dried 1-L round flask was charged with L-1,4:
3,6-mannarodilactone (5.00 g, 28.7 mol) and dry dioxane (400
mL) under nitrogen a atmosphere. The mixture was heated
until the dilactone was completely dissolved, and then the oil
bath was removed. To the stirred solution were added a small
portion of triflic acid (0.1 mL) and (4-bromobenzyl)-2,2,2-
trichloroacetimidate (28.6 g, 86.2 mmol). The rest of the triflic
acid (0.6 mL) was added dropwise under vigorous stirring. The
exothermic reaction was stirred for 2 h. The product precipi-
tated from the solution and was collected by filtration. The
white crystals were washed with dioxane and dried in vacuo.
Additional product was collected by filtering the filtrate
through a funnel with 2 cm of NaHCO3 and 2 cm of silica.
The filtrate was concentrated and dried in vacuo overnight.
The remaining solid was washed with diethyl ether (100 mL).
The ether was decanted off, and the washing procedure was
repeated three times. The product was filtered and dried in
vacuo to give totally 12.9 g (88%) of compound 15 as white
crystals: IR (KBr) ν 3050, 2882, 1797 cm-1; [R]D ) -110° (c )
1
1.02, DMSO, 22 °C); H NMR (DMSO-d6) δ 7.60 (d, J ) 8.25
Hz, 4H), 7.36 (d, J ) 8.25 Hz, 4H), 5.25 (d, J ) 3.6 Hz, 2H),
4.89 (d, J ) 3.6 Hz, 2H) 4.75 (m, 4H); 13C NMR (DMSO-d6) δ
Exp er im en ta l Section
171.7, 136.4, 131.6, 130.1, 121.2, 74.9, 74.4, 71.3. Anal. (C20H16
Br2O6) C, H.
-
Ch em istr y. Gen er a l In for m a tion . Melting points were
recorded on an electrothermal melting point apparatus and
are uncorrected. Optical rotations were obtained on a Perkin-
Elmer 241 polarimeter. Specific rotations ([R]D) are reported
in deg/dm, and the concentration (c) is given in g/100 mL in
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis(4-br om oben zyloxy)-3,4-d ih yd r oxy-
h exa n ed ia m id e (3). Compound 15 (6.00 g, 11.7 mmol) was
dissolved in 1,2-dichloroethane (100 mL). To the solution was
added 6 equiv of valine methylamide (9.15 g, 70.3 mmol). The
flask was sealed with a silicon septum and heated to 50 °C
for 48 h. The white product precipitated from the solution and
was collected by filtration. The product was washed with
CHCl3 (30 mL) and dried in vacuo to give 5.79 g (64%) of
compound 3 as off-white crystals: IR (KBr) ν 3290, 3099, 2962,
1642 cm-1; [R]D ) +3.9° (c ) 1.03, DMSO, 22 °C); 1H NMR
(DMSO-d6) δ 7.88 (q, J ) 4.6 Hz, 2H), 7.72 (d, J ) 8.9 Hz,
2H), 7.52 (d, J ) 8.6 Hz, 4H), 7.28 (d, J ) 8.6 Hz, 4H), 4.81 (d,
J ) 7.6, 2H), 4.43 (s, 4H), 4.16 (dd, J ) 8.9, 6.6 Hz, 2H), 3.99
(d, J ) 7.9 Hz, 2H), 3.83 (app d, 2H), 2.59 (d, J ) 4.6 Hz, 6H),
1.97 (m, 2H), 0.85 (d, J ) 6.6 Hz, 6H), 0.82 (d, J ) 6.9 Hz,
6H); 13C NMR (DMSO-d6) δ 171.1, 170.4, 137.5, 131.1, 129.7,
120.6, 79.3, 70.3, 69.7, 57.7, 30.6, 25.5, 19.3, 18.2. A small
portion was recrystallized from CHCl3 to give 3 as off-white
crystals, which were subjected to elemental analysis. Anal.
(C32H44Br2N4O8) C, H, N.
1
the specified solvent. H and 13C NMR spectra were recorded
on a J eol J NM-EX 270 spectrometer at 270.2 and 67.8 MHz,
respectively. Chemical shifts are given as δ values (ppm)
downfield from tetramethylsilane. Infrared spectra were re-
corded on a Perkin-Elmer 1600 series FTIR instrument.
Elemental analyses were performed by Mikro Kemi AB,
Uppsala, Sweden, or Analytische Laboratorien, Lindlar, Ger-
many, and were within (0.4% of calculated values. Column
chromatography was performed on silica gel 60 (0.04-0.063
mm; E. Merck). Circular chromatography was performed on
Chromatotron 7924T (TC Research) with 1-mm thick silica gel
60 (0.04-0.063 mm; E. Merck) plates and gradient elution.
Thin-layer chromatography was performed on precoated silica
gel F-254 plates (0.25 mm; E. Merck) and visualized with UV
light and H2SO4 in ethanol or ninhydrin. The palladium
tetrakis(triphenylphosphine) was freshly made according to
procedures described by Heck.19 All microwave reactions were
carried out in heavy-walled Pyrex tubes, sealed with screw-
cap-fitted Teflon septa. The microwave treatment was per-
formed with a MicroWell 10 single-mode cavity (Labwell AB,
Uppsala, Sweden). It is not recommended to repeat these
Gen er a l Meth od for th e P r ep a r a tion of Com p ou n d s
4-7. Meth od I. A mixture of 3 (30 mg, 0.039 mmol), arylbo-
ronic acid (0.19 mmol), 2 M aqueous NaCO3 (39 µL, 0.078
mmol), tetrakis(triphenylphosphine)palladium (2.2 mg, 0.0019

C2-Symmetric HIV-1 Protease Inhibitors
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19 3841
mmol), EtOH (60 µL), H2O (80 µL), and 1,2-dimethoxyethane
(240 µL) was placed in a Pyrex tube and degassed under a
nitrogen flow for 5 min. The tube was sealed with a Teflon
septum and irradiated in a microwave reactor for 4 min at 45
W. The reaction mixture was allowed to cool and then diluted
with CHCl3 (50 mL). The organic layer was washed with
saturated aqueous NaHCO3 (3 × 20 mL), dried, filtered, and
concentrated. The crude product was purified by circular
chromatography (CH2Cl2 f CH2Cl2/MeOH (20:1)) to give pure
4-7.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis(4-p h en ylben zyloxy)-3,4-d ih yd r oxy-
h exa n ed ia m id e (4). The title compound was prepared in 93%
yield (28 mg, 0.036 mmol) according to method I, using
phenylboronic acid (24 mg): IR (KBr) ν 3305, 3080, 2957, 1649
cm-1; [R]D ) +1.9° (c ) 0.99, DMSO, 21 °C); 1H NMR (CDCl3)
δ 7.58 (m, 8H), 7.44 (m, 10H), 7.25 (d, J ) 9.2 Hz, 2H), 7.03
(q, J ) 4.3 Hz, 2H), 5.27 (br s, 2H), 4.71 (m, 4H), 4.35 (dd, J
) 9.2, 4.3 Hz, 2H), 4.21 (br s, 2H), 4.15 (br s, 2H), 2.68 (d, J )
4.6 Hz, 6H), 2.49 (m, 2H), 0.94 (d, J ) 6.6 Hz, 6H), 0.83 (d, J
) 6.6 Hz, 6H); 13C NMR (CDCl3) δ 172.5, 170.6, 141.7, 140.5,
135.3, 129.0, 128.7, 127.7, 127.7, 127.2, 82.0, 73.6, 73.3, 58.4,
29.0, 26.2, 19.8, 17.1. Anal. (C44H54N4O8) C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis[4-(3-n itr op h en yl)ben zyloxy]-3,4-d i-
h yd r oxyh exa n ed ia m id e (5). The title compound was pre-
pared in 85% yield (28 mg, 0.033 mmol) according to method
I, using 3-nitrophenylboronic acid (32 mg): IR (KBr) ν 3286,
3083, 2962, 1647 cm-1; [R]D ) -6.5° (c ) 1.00, DMSO, 22 °C);
1H NMR (DMSO-d6) δ 8.41 (m, 2H), 8.21 (dd, J ) 7.9, 1.5 Hz,
2H), 8.13 (d, J ) 8.4 Hz, 2H), 7.91 (q, J ) 4.7 Hz, 2H), 7.8-
7.7 (m, 8H), 7.48 (d, J ) 8.3 Hz, 4H), 4.87 (d, J ) 7.4 Hz, 2H),
4.54 (s, 4H), 4.19 (dd, J ) 6.6, 8.8 Hz, 2H), 4.07 (d, J ) 7.8
Hz, 2H), 3.91 (app t, 2H), 2.60 (d, J ) 4.6 Hz, 6H), 1.92 (m,
2H), 0.86 (d, J ) 6.6 Hz, 6H), 0.84 (d, J ) 6.6 Hz, 6H); 13C
NMR (DMSO-d6) δ 171.1, 170.4, 148.4, 141.5, 138.5, 136.9,
133.2, 130.4, 128.4, 126.8, 122.1, 120.9, 79.6, 70.7, 69.8, 57.6,
30.6, 25.4, 19.2, 18.1. Anal. (C44H52N6O12‚H2O) C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-b is[4-(3-t h ien yl)b en zyloxy]-3,4-d ih y-
d r oxyh exa n ed ia m id e (6). The title compound was prepared
in 96% yield (29 mg, 0.037 mmol) according to method I, using
3-thiopheneboronic acid (25 mg): IR (KBr) ν 3291, 3100, 2962,
1645 cm-1; [R]D ) -5.2° (c ) 1.00, DMSO, 22 °C); 1H NMR
(DMSO-d6) δ 7.89 (q, J ) 4.3 Hz, 2H), 7.84 (dd, J ) 3.0, 1.5
Hz, 2H), 7.72 (d, J ) 9.1 Hz, 2H), 7.68 (d, J ) 7.9 Hz, 4H),
7.62 (dd, J ) 5.0, 3.0 Hz, 2H), 7.54 (dd, J ) 5.1, 1.5 Hz, 2H),
7.36 (d, J ) 7.9 Hz, 4H), 4.86 (d, J ) 7.3 Hz, 2H), 4.48 (s, 4H),
4.19 (dd, J ) 6.4, 9.0 Hz, 2H), 4.04 (d, J ) 7.4 Hz, 2H), 3.90
(app t, 2H), 2.60 (d, J ) 4.3 Hz, 6H), 1.91 (m, 2H), 0.86 (d, J
) 6.3 Hz, 6H), 0.84 (d, J ) 6.3 Hz, 6H); 13C NMR (DMSO-d6)
δ 171.1, 170.4, 141.4, 137.0, 134.4, 128.1, 127.0, 126.1, 125.9,
120.8, 79.6, 71.7, 69.8, 57.3, 30.3, 24.7, 19.2, 18.1. Anal.
(C40H50N4O8S2‚1/2H2O) C, H, N.
nitrogen atmosphere and then cooled on an ice bath. 9-BBN
(0.5 M) in THF (390 µL, 0.19 mmol) was added via a syringe
through a silicon septum. The reaction mixture was allowed
to reach room temperature and stirred for 4 h. To the reaction
mixture were added 3 (30 mg, 0.039 mmol), K2CO3 (21 mg,
0.16 mmol), tetrakis(triphenylphosphine)palladium (2.7 mg,
0.0023 mmol), and DMF (1 mL) under nitrogen atmosphere.
The stir bar was removed from the mixture, and the tube was
sealed with a Teflon septum and irradiated in the microwave
reactor for 2 min at 60 W. After cooling, CHCl3 (50 mL) was
added to the reaction mixture. The organic layer was separated
and subsequently washed with saturated aqueous NaHCO3
(3 × 20 mL), dried, filtered, and concentrated. The crude
product was purified by circular chromatography (CH2Cl2
CH2Cl2/MeOH (20:1)) to give pure 8 (12 mg, 0.015 mmol) in
38% yield: IR (KBr) ν 3306, 3087, 2926, 1650 cm-1; [R]D
f
)
-9.1° (c ) 1.21, CHCl3, 21 °C); 1H NMR (DMSO-d6) δ 7.88 (q,
J ) 4.6 Hz, 2H), 7.68 (d, J ) 9.0 Hz, 2H), 7.29-7.13 (m, 18H),
4.82 (d, J ) 7.2 Hz, 2H), 4.42 (s, 4H), 4.18 (d, J ) 9.0, 6.4 Hz,
2H), 3.99 (d, J ) 7.4 Hz, 2H), 3.85 (app t, 2H), 2.85 (s, 8H),
2.59 (d, J ) 4.6 Hz, 6H), 1.98 (m, 2H), 0.85 (d, J ) 6,5 Hz,
6H), 0.83 (d, J ) 6.5 Hz, 6H); 13C NMR (DMSO-d6) δ 171.0,
170.5, 141.4, 140.8, 135.4, 128.3, 128.2, 127.6, 125.7, 79.4, 71.1,
69.9, 57.5, 37.0, 36.7, 30.5, 25.4, 19.2, 18.1. Anal. (C48H62N4O8)
C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of Com p ou n d s
9-11. Meth od II. A mixture of 3 (30 mg, 0.039 mmol),
aryltrialkyltin (0.19 mmol), tetrakis(triphenylphosphine)-
palladium (2.2 mg, 0.0019 mmol), metal oxide (0.039 mmol),
and DMF (1 mL) was placed in a Pyrex tube and degassed
under a nitrogen flow for 5 min. The tube was sealed with a
Teflon septum and irradiated in a microwave reactor for 2 min
at 60 W. After cooling, CHCl3 (50 mL) was added to the
reaction mixture. The organic layer was separated and sub-
sequently washed with saturated aqueous NaHCO3 (3 × 20
mL), dried, filtered, and concentrated. The crude product was
dissolved in acetonitrile (100 mL), washed with isohexane (3
× 20 mL), concentrated, and then purified by circular chro-
matography (CH2Cl2 f CH2Cl2/MeOH (9:1)) to give pure 9-11.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-b is[4-(3-p yr id yl)b en zyloxy]-3,4-d ih y-
d r oxyh exa n ed ia m id e (9). The title compound was prepared
in 50% yield (15 mg, 0.020 mmol) according to method II, using
trimethyl-3-pyridyltin20 (47 mg) and CuO (3.1 mg): IR (KBr)
ν 3432, 3080, 2921, 1650 cm-1; [R]D ) -5.0° (c ) 1.4, DMSO,
1
21 °C); H NMR (DMSO-d6) δ 8.87 (dd, J ) 0.7, 2.3 Hz, 2H),
8.56 (dd, J ) 1.7, 4.6 Hz, 2H), 8.05 (ddd, J ) 7.9, 2.5, 1.7 Hz,
2H), 7.90 (q, J ) 4.7 Hz, 2H), 7.75 (d, J ) 8.9 Hz, 2H), 7.70 (d,
J ) 8.3 Hz, 3H), 7.48 (m, 2H), 7.46 (d, J ) 8.3 Hz, 2H), 4.85
(d, J ) 7.4 Hz, 2H), 4.52 (s, 4H), 4.18 (dd, J ) 8.9, 6.5 Hz,
2H), 4.06 (d, J ) 7.9 Hz, 2H), 3.89 (app t, 2H), 2.59 (d, J ) 4.5
Hz, 6H), 1.98 (m, 2H), 0.86 (d, J ) 6.7 Hz, 6H), 0.83 (d, J )
6.7 Hz, 6H); 13C NMR (DMSO-d6) δ 171.1, 170.4, 148.4, 147.6,
138.0, 136.2, 135.3, 134.0, 128.3, 126.7, 123.8, 79.4, 70.7, 69.8,
57.6, 30.5, 25.4, 19.2, 18.1. Anal. (C42H52N6O8‚H2O) C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-b is[4-(2-t h ien yl)b en zyloxy]-3,4-d ih y-
d r oxyh exa n ed ia m id e (7). The title compound was prepared
in 86% yield (26 mg, 0.034 mmol) according to method I, using
2-thiopheneboronic acid (25 mg): IR (KBr) ν 3304, 2960, 1647
cm-1; [R]D ) -0.1° (c ) 0.89, DMSO, 21 °C); 1H NMR (DMSO-
d6) δ 7.89 (q, J ) 4.5 Hz, 2H), 7.72 (d, J ) 9.0 Hz, 2H), 7.62 (d,
J ) 8.3 Hz, 4H), 7.53 (dd, J ) 5.1, 1.2 Hz, 2H), 7.49 (dd, J )
3.4, 1.2 Hz, 2H), 7.37 (d, J ) 8.3 Hz, 4H), 7.12 (dd, J ) 3.6,
5.1 Hz, 2H), 4.83 (d, J ) 7.1 Hz, 2H), 4.48 (s, 4H), 4.16 (dd, J
) 8.6, 6.6 Hz, 2H), 4.03 (d, J ) 7.6 Hz, 2H), 3.88 (app t, 2H),
2.59 (d, J ) 4.6 Hz, 6H), 1.97 (m, 2H), 0.85 (d, J ) 6.6 Hz,
6H), 0.83 (d, J ) 6.8 Hz, 6H); 13C NMR (DMSO-d6) δ 171.1,
170.4, 143.1, 137.3, 133.0, 131.1, 129.7, 128.3, 125.2, 123.7,
79.3, 70.7, 69.5, 57.6, 30.6, 25.4, 19.3, 18.1. Anal. (C40H50N4O8S2)
C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-b is[4-(2-p yr id yl)b en zyloxy]-3,4-d ih y-
d r oxyh exa n ed ia m id e (10). The title compound was prepared
in 54% yield (16 mg, 0.021 mmol) according to method II, using
trimethyl-2-pyridyltin20 (47 mg) and CuO (3.1 mg): IR (KBr)
ν 3304, 3085, 2959, 1650 cm-1; [R]D ) -4.3° (c ) 0.47, MeOH,
1
21 °C); H NMR (DMSO-d6) δ 8.65 (ddd, J ) 4.8, 1.2, 0.8 Hz,
2H), 8.05 (d, J ) 8.3 Hz, 4H), 7.96-7.82 (m, 6H), 7.76 (d, J )
8.9 Hz, 2H), 7.45 (d, J ) 8.3 Hz, 4H), 7.34 (ddd, J ) 7.2, 4.8,
1.3 Hz, 2H), 4.87 (d, J ) 7.3 Hz, 2H), 4.53 (s, 4H), 4.19 (dd, J
) 8.8, 6.8 Hz, 2H), 4.06 (d, J ) 7.6 Hz, 2H), 3.93 (app t, 2H),
2.60 (d, J ) 4.5 Hz, 6H), 1.98 (m, 2H), 0.86 (d, J ) 6.7 Hz,
6H), 0.83 (d, J ) 6.6 Hz, 6H); 13C NMR (DMSO-d6) δ 171.1,
170.4, 155.7, 149.5, 138.9, 137.9, 137.2, 127.8, 126.3, 122.5,
120.1, 79.5, 70.8, 69.7, 57.6, 30.5, 25.4, 19.2, 18.1. Anal.
(C42H52N6O8‚1/2H2O) C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis[4-(2-p h en yleth yl)ben zyloxy]-3,4-d i-
h yd r oxyh exa n ed ia m id e (8). A dried Pyrex tube was charged
with styrene (22 µL, 0.19 mmol) and THF (20 µL) under a
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis[4-(2-th ia zolyl)ben zyloxy]-3,4-d ih y-
d r oxyh exa n ed ia m id e (11). The title compound was prepared

3842 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 19
Alterman et al.
in 53% yield (16 mg, 0.021 mmol) according to method II, using
tributyl-2-thiazolyltin (73 mg) and Ag2O (9.0 mg): IR (KBr) ν
3363, 3081, 2960, 1650 cm-1; [R]D ) -2.3° (c ) 0.47, DMSO,
were diluted in human AB+ serum (Sigma) and added to the
cells after 1 h of virus adsorption. The final medium contained
50% human serum and 10% fetal calf serum. The viral
replication was analyzed after 6 days by determining the
amount of HIV p24 antigen.
Mutant HIV was achieved by passaging cell-free virus in
the presence of stepwise increased concentrations of ritonavir.
When virus was grown in the highest possible nontoxic
concentration, it was passaged once in medium without
compound and the supernatant was frozen in aliquots at -70
°C. The cellular DNA was analyzed with respect to the
nucleotide sequence of the HIV protease gene.
1
21 °C); H NMR (CD3OD-CDCl3, 3:1) δ 7.90 (d, J ) 8.3 Hz,
4H), 7.82 (d, J ) 3.3 Hz, 2H), 7.49 (d, J ) 3.3 Hz, 2H), 7.48 (d,
J ) 8.3 Hz, 4H), 4.68 (s, 4H), 4.24 (q, J ) 5.9 Hz, 2H), 4.18
(m, 2H), 4.10 (m, 2H), 2.72 (d, J ) 4.6 Hz, 6H), 2.19 (m, 2H),
0.92 (d, J ) 6.9 Hz, 6H), 0.88 (d, J ) 6.6 Hz, 6H); 13C NMR
(CD3OD-CDCl3, 3:1) δ 173.3, 172.7, 170.0, 144.0, 140.4, 133.8,
129.4, 127.5, 120.4, 81.1, 72.8, 72.2, 59.3, 31.1, 26.5, 19.9, 18.1.
Anal. (C38H48N6O8S2) C, H, N.
N1,N6-Bis[(1S)-2-m eth yl-1-(m eth ylca r ba m oyl)p r op yl]-
(2R,3R,4R,5R)-2,5-bis{4-[(E)-2-m eth oxyca r bon yleth en yl]-
ben zyloxy}-3,4-d ih yd r oxyh exa n ed ia m id e (12). A mixture
of 3 (30 mg, 0.039 mmol), methyl acrylate (35 µg, 0.39 mmol),
diisopropylethylamine (27 µL, 0.16 mmol), Pd(OAc)2 (0.87 mg,
0.0039 mmol), PPh3 (2.6 mg, 0.093 mmol), H2O (0.15 mL), and
DMF (0.85 mL) was added to a Pyrex tube and degassed under
a nitrogen flow for 5 min. The tube was sealed with a Teflon
septum and irradiated in a microwave reactor for 2 min at 60
W. After cooling, CHCl3 (50 mL) was added to the reaction
mixture. The organic layer was separated and subsequently
washed with saturated aqueous NaHCO3 (3 × 20 mL), dried,
filtered, and concentrated. The crude product was purified by
circular chromatography (CH2Cl2 f CH2Cl2/MeOH (20:1)) to
give pure 12 (23 mg, 0.029 mmol) in 76% yield: IR (KBr) ν
3307, 3098, 2960, 1650 cm-1; [R]D ) -6.7° (c ) 1.00, DMSO,
Or a l Absor p tion . Male Sprague-Dawley rats (weight:
approximately 200 g) from B&K Universal AB, Sollentuna,
Sweden, were used for this study. The rats were fasted 18 h
before administration of the compound. The test compounds
were dissolved in propylene glycol in an administration volume
of 4 mL/kg. Two rats were used for each compound in the
screening of plasma levels after oral administration of 4 (40
mg/kg), 6 (40 mg/kg), 7 (40 mg/kg), 9 (30 mg/kg), and nelfinavir
(26 mg/kg). The rats were dosed by gavage at time 0, and blood
samples were collected in heparin (20 IE/mL) from the tail vein
at 0.5, 1, 2, and 4 h. Blood samples were immediately
centrifuged at 3000-3500g for 10 min, and the plasma was
stored at -18 °C until assayed. After protein precipitation,
the plasma samples were directly injected onto the HPLC
system. Drug concentrations in plasma were determined by
reversed-phase liquid chromatography and UV detection.
Cr ysta llogr a p h y. The details of the crystallization and the
structure determination will be published elsewhere. Briefly,
complexes of HIV-1 PR with the compounds 1, 6, and 9 were
crystallized in the space group P21212. The cell parameters
were (compound 1) a ) 59.0, b ) 86.8, c ) 47.0 Å, (compound
6) a ) 58.1, b ) 86.1, c ) 46.1 Å, and (compound 9) a ) 58.5,
b ) 86.7, c ) 46.5 Å. X-ray diffraction data to 1.8, 2.1, and 2.0
Å resolution, respectively, were collected at the synchrotron
stations D41 at LURE, Paris, France, and I711 at MAX-lab,
Lund, Sweden. Data were processed with DENZO21 and scaled
with SCALEPACK.22 The completeness of data was 82.6% with
1
21 °C); H NMR (DMSO-d6) δ 7.88 (q, J ) 4.7 Hz, 2H), 7.74
(d, J ) 8.9 Hz, 2H), 7.68 (d, J ) 8.1 Hz, 4H), 7.65 (d, J ) 15.7
Hz, 2H), 7.37 (d, J ) 8.1 Hz, 4H), 6.62 (d, J ) 16.2 Hz, 2H),
4.84 (d, J ) 7.4 Hz, 2H), 4.49 (s, 4H), 4.17 (dd, J ) 6.4, 9.0
Hz, 2H), 4.02 (d, J ) 7.6 Hz, 2H), 3.86 (app t, 2H), 2.59 (d, J
) 4.6 Hz, 6H), 1.97 (m, 2H), 0.84 (d, J ) 6.4 Hz, 6H), 0.82 (d,
J ) 6.6 Hz, 6H); 13C NMR (DMSO-d6) δ 171.1, 170.3, 166.6,
144.2, 140.6, 133.2, 128.2, 127.9, 117.6, 79.4, 70.6, 69.7, 57.6,
51.4, 30.5, 25.4, 19.2, 18.1. Anal. (C40H54N4O12‚H2O) C, H, N.
N 1,N 6-Bis[(1S )-2-m e t h yl-1-(m e t h ylca r b a m oyl)p r o-
p yl](2R,3R,4R,5R)-2,5-b is[4-(3-cycloh exa n e-1,2-d ion yl)-
ben zyloxy]-3,4-d ih yd r oxyh exa n ed ia m id e (13). A mixture
of 3 (30 mg, 0.039 mmol), 1,2-cyclohexanedione (87 mg, 0.78
mmol), diisopropylethylamine (27 µL, 0.16 mmol), Pd(OAc)2
(0.87 mg, 0.0039 mmol), P(o-tol)3 (2.8 mg, 0.093 mmol), H2O
(0.15 mL), and DMF (0.85 mL) was added to a Pyrex tube with
a magnetic stir bar and degassed under a nitrogen flow for 5
min and sealed. The reaction was heated to 100 °C and stirred
for 48 h. After cooling, CHCl3 (50 mL) was added to the
reaction mixture. The organic phase was separated and
subsequently washed with saturated aqueous NaHCO3 (3 ×
20 mL), dried, filtered, and concentrated. The crude product
was purified on column chromatography (CHCl3/MeOH (20:
1)) to give pure 13 (17 mg, 0.020 mmol) in 52% yield: IR (KBr)
ν 3314, 3106, 2960, 1652 cm-1; [R]D ) -7.3° (c ) 1.01, CHCl3,
19 °C); 1H NMR (DMSO-d6) δ 8.39 (s, 2H), 7.89 (q, J ) 4.6 Hz,
2H), 7.72 (d, J ) 9.0 Hz, 2H), 7.67 (d, J ) 8.3 Hz, 4H), 7.34 (d,
J ) 8.3 Hz, 4H), 4.83 (d, J ) 7.3 Hz, 2H), 4.48 (s, 4H), 4.18
(dd, J ) 8.9, 6.4 Hz, 2H), 3.88 (app t, 2H), 2.70 (m, 2H), 2.60
(d, J ) 4.5 Hz, 6H), 2.52 (m, 2H), 1.98 (m, 4H), 0.85 (d, J )
6.3 Hz, 6H), 0.83 (d, J ) 5.6 Hz, 6H); 13C NMR (DMSO-d6) δ
195.0, 171.1, 170.4,144.1, 137.4, 137.1, 128.1, 127.5, 127.0,
79.5, 70.9, 69.9, 57.6, 36.4, 30.5, 28.4, 25.4, 22.3, 19.3, 18.1.
Anal. (C44H58N4O12‚2H2O) C, H, N.
23
an Rsym of 6.4% for the PR/1 complex, for the PR/6 complex
the corresponding values were 93.1% and 7.2%, and for the
PR/9 complex the corresponding values were 93.2% and 12.8%.
The protein model coordinates from 1AJ V were used for
molecular replacement calculations. Refinements were done
using the program package XPLOR.24 The structures were
refined to an Rcrystal (Rfree) factor25 of 19.1% (22.8%) using 1.8
Å data for the PR/1 complex, 19.3% (26.2%) using 2.1 Å data
for the PR/6 complex, and 18.1% (24.1%) using 2.0 Å data for
the PR/9 complex. Model building were made using the
program O.26
Ack n ow led gm en t. We gratefully acknowledge sup-
port from the Swedish Foundation for Strategic Re-
search (SSF), the Swedish Research Council for Engi-
neering Sciences (TFR), Medivir AB, Huddinge, Sweden,
and the Swedish Medical Research Council (MFR).
Refer en ces
(1) (a) Morrow, C. D.; Park, J .; Wakefield, J . K. Viral Gene Products
and Replication of the Human Immunodeficiency Type 1 Virus.
Am. J . Physiol. 1994, 266, C1135-1156. (b) Clavel, F.; Gue´tard,
D.; Brun-Ve´zinet, F.; Chamaret, S.; Rey, M.; Santos-Ferreira,
M. O.; Laurent, A. G.; Dauguet, C.; Katlama, C.; Rouzioux, C.;
Klatzmann, D.; Champalimaud, J . L.; Montagnier, L. Isolation
of a New Human Retrovirus from West African Patients with
AIDS. Science 1986, 233, 343-346. (c) Gallo, R. C.; Salahuddin,
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