Potent HIV protease inhibitors containing a novel (hydroxyethyl)amide isostere

Article abstract of DOI:10.1021/jm9606608

A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has been synthesized. These peptidomimetic structures inhibit viral protease activity at low nanomolar concentrations (IC₅₀ < 10 nM for HIV-1 protease). The inhibition constant (K(i)) for inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases, respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays with 50% effective concentrations (EC₅₀) = 3,7-35 nM. This series of inhibitors was found to exhibit poor bioavailability (<10%) in the rat, following oral administration. The synthesis and biological properties of these compounds are discussed. In addition, an X-ray structure of one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding mode of this novel class of HIV protease inhibitors.

Full text of DOI:10.1021/jm9606608

2164
J . Med. Chem. 1997, 40, 2164-2176
P oten t HIV P r otea se In h ibitor s Con ta in in g a Novel (Hyd r oxyeth yl)a m id e
Isoster e
Pierre L. Beaulieu,*,† Dominik Wernic,† Abraham Abraham,† Paul C. Anderson,† Tibor Bogri,† Yves Bousquet,†
Gilbert Croteau,† Ingrid Guse,† Daniel Lamarre,† Francine Liard,† William Paris,† Diane Thibeault,†
Susan Pav,‡ and Liang Tong‡
Bio-Me´ga Research Division, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval (Que´bec), Canada H7S 2G5, and
Boehringer Ingelheim Pharmaceuticals Inc., 175 Briar Ridge Road, Ridgefield, Connecticut 06877
Received September 23, 1996X
A series of HIV protease inhibitors containing a novel (hydroxyethyl)amidosuccinoyl core has
been synthesized. These peptidomimetic structures inhibit viral protease activity at low
nanomolar concentrations (IC50 < 10 nM for HIV-1 protease). The inhibition constant (Ki) for
inhibitor 19 was determined to be 7.5 pM against HIV-1 and 1.2 nM against HIV-2 proteases,
respectively. Several compounds (19-24) inhibited HIV-1 replication in cell culture assays
with 50% effective concentrations (EC50) ) 3.7-35 nM. This series of inhibitors was found to
exhibit poor bioavailability (<10%) in the rat, following oral administration. The synthesis
and biological properties of these compounds are discussed. In addition, an X-ray structure of
one of these inhibitors (23) in complex with HIV-2 protease provides insight into the binding
mode of this novel class of HIV protease inhibitors.
In tr od u ction
incorporating various isosteres that mimic the hydroly-
sis transition state of an amide bond.2,7 Among them,
hydroxyethylamine (HEA)-based structures (Figure 1)
have led to extremely potent inhibitors of the enzyme.9
Originally, the highly peptidic nature of these HIV
protease inhibitors resulted in poor oral bioavailability
and short half-lives in vivo, thus jeopardizing their use
as therapeutic agents.2 However, manipulation of
physicochemical properties (hydrophobicity/hydrophi-
licity, charge), reduction in size, elimination of peptide
bonds, and formulation have led to compounds with
enhanced oral absorption, allowing their advancement
into clinical trials. Recently, HIV protease inhibitors
have been shown to effectively reduce viral load in
infected patients and are providing clinicians with new
tools for AIDS chemotherapy.10
Our laboratory has been actively involved in the
synthesis and evaluation of HIV protease inhibitors.9g,h
Palinavir (Figure 2) is a highly potent and specific
inhibitor of HIV-1 and HIV-2 proteases. It is also
effective against viral replication in cell culture assays
(Table 1).9h,11 The chemical structure of palinavir is
characterized by a hydroxyethylamine transition state
mimic with the (R)-configuration at the carbon atom
bearing the hydroxyl group12 and a novel 4-substituted
pipecolic amide entity.13 While studies on this novel
class of inhibitors were being pursued toward the
selection of a candidate for development, we also desired
a backup series whose structural features would be
significantly different from those of our lead compound.
We describe herein the synthesis and biological evalu-
ation of a novel class of highly potent HIV protease
inhibitors incorporating a (hydroxyethyl)amidosuccinoyl
motif.14
The past decade has seen full realization of the World
Health Organization’s predictions concerning the cata-
strophic dimensions of the epidemic associated with
human immunodeficiency virus (HIV) infections. It has
been estimated that in addition to the 4.5 million adults
which have already died from HIV-associated condi-
tions, 21 million adults worldwide are currently suffer-
ing from HIV/AIDS infection.1 The magnitude of this
tragedy has triggered an unprecedented effort in the
search for a possible cure to the deadly disease.
Studies on the life cycle of the virus have identified
several areas for therapeutic intervention.2 Most widely
studied, nucleoside as well as non-nucleoside reverse
transcriptase (RT) inhibitors offered the first hope for
increasing life expectancy in patients afflicted by the
infection.3 Unfortunately, emergence of viral resistance
coupled with serious side effects associated with the use
of some nucleoside RT inhibitors has resulted in only
marginal success in combating disease progression.4a
More recently, combinations of nucleoside RT inhibitors
as well as combinations of RT inhibitors and nevirapine
(the first approved non-nucleoside RT inhibitor) have
proven superior to monotherapies in delaying advance-
ment of the disease.4b,c
Another essential protein in the virus life cycle is the
HIV protease. It is responsible for the processing of
encoded gag and gag-pol gene products. Cleavage by
the protease of these polyproteins at specific sites
generates the proteins and enzymes required for viral
maturation.5 The discovery that inhibition of the pro-
tease resulted in the production of noninfectious, im-
mature virions6 triggered extensive efforts in the search
for agents capable of interfering with this enzyme,
thereby inhibiting viral replication.7 As a member of
the aspartyl protease class of enzymes,8 HIV protease
is susceptible to inhibition by peptide-like structures
Resu lts a n d Discu ssion
SAR Stu d ies. In our search for a backup to the
pipecolic amide series of inhibitors (as represented by
palinavir, Figure 2), we hoped that modifications which
substantially altered overall physicochemical properties,
while retaining both enzymatic and antiviral inhibitory
† Boehringer Ingelheim (Canada) Ltd.
‡ Boehringer Ingelheim Pharmaceuticals Inc.
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(96)00660-7 CCC: $14.00 © 1997 American Chemical Society

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2165
Ta ble 2
F igu r e 1.
F igu r e 2.
Ta ble 1. Palinavir: Biological Data
viral strain
IC50 (nM)a
Ki (nM)b
EC50 (nM)b
HIV-1 IIIB
HIV-2 ROD
4
10
0.031
0.13
5
25
a
b
HPLC-based peptide substrate cleavage assay. See ref 11.
potencies, would translate into beneficial pharmacoki-
netic effects (oral absorption, sustained duration of
action, longer half-lives).
In recent years, the (R)-hydroxyethylamine dipeptide
insert was incorporated as a key component in highly
potent peptidomimetic inhibitors of HIV protease.
Saquinavir9b and palinavir9h are examples of compounds
which contain a basic nitrogen atom. The (R)-HEA unit
has also appeared in neutral derivatives such as ureas15
and sulfonamides.16 To our knowledge, however, there
are only two isolated reports of inhibitors based on
elaboration of this unit via an amide linkage, forming
a hydroxyethylamide transition state mimic (Figure 1).17
In designing a backup series of inhibitors, we not only
desired structural novelty, but also reduced synthetic
complexity. Pipecolic amide derivative 1 (Table 2),9h
a
trimmed-down version of our lead series (palinavir,
Figure 2), was selected as a starting point for the
development of a novel class of inhibitors. Removal of
the substituent in the 4-position of the pipecolic ring of
palinavir results in a substantial decrease in potency,
and the carbobenzyloxy N-terminus is not optimal for
binding in the S3 subsite of the enzyme.9b Still, com-
pound 1 retained sufficient potency in our enzymatic
assay (IC50 ) 62 nM)18 to be used as a reference point.
We anticipated that replacement of the pipecolic amide
portion of 1 by a 1,2-cyclohexanedicarboxylic acid would
not alter drastically the overall interactions between the
enzyme and inhibitor. Indeed, this succinic acid deriva-
tive 2, prepared as a 1:1 mixture of cis-isomers inhibited
toward a bioactive conformation. Such an effect can be
achieved through conformational rigidification (such as
for compound 2) or by attachment of bulky alkyl groups
at strategic locations along the inhibitor backbone.
Replacement of the 1,2-cyclohexanedicarboxylic moiety
by a tert-butylsuccinic fragment gave compounds 3-6
(Table 2). As can be seen from the data in Table 2, the
ability of the compounds to inhibit HIV protease varies
drastically depending on the position and orientation
of the tert-butyl group. Isomer 3 with the tert-butyl
substituent on the carbon to the tert-butylamide group
activity of HIV-1 protease to a similar extent (IC50
)
140 nM). Novel succinic diamide derivative 2 satisfied
our initial criteria of structural novelty, ease of synthe-
sis, and altered physicochemical properties (the basic
pipecolic ring nitrogen in 1 now exists as a neutral
amide nitrogen). Efforts were then directed at improv-
ing potency by optimizing interactions between the
inhibitor and the enzyme.
((R)-configuration) showed the highest potency (IC50
)
3.0 nM). All other isomers (4-6) were 8-1600 times
less potent. Several other alkyl substituents were
examined in place of the tert-butyl group, using 3 as a
reference point (e.g. isopropyl, phenyl, cyclohexyl, neo-
pentyl; data not shown). None proved as effective as
our original choice. The optimal effect observed in the
case of the bulkier tert-butyl group with a (R)-configu-
ration at the point of attachment can be a consequence
Our past experience in the field of peptidomimetics19
suggested that the role of the cyclohexane ring in 2
might be one of orienting the backbone of the inhibitor

2166 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Beaulieu et al.
Ta ble 3
of its greater ability to direct the inhibitor backbone
toward a bioactive conformation or to a favorable
lipophilic interaction of the tert-butyl group itself with
the enzyme active site (or a combination of both).
Removal of this tert-butyl group (entry 7) resulted in a
dramatic 2750-fold drop in potency. Some insight on
the mode of binding of these inhibitors was gained with
the help of an X-ray crystal structure of a complex with
HIV-2 protease and will be discussed in a separate
section.
Inhibitors have been known to bind to the protease
with either of two possible configurations of the critical
hydroxyl group in the transition state mimic.20 In view
of the structural uniqueness of our succinic series, we
verified that the (R)-configuration at that center was
optimal. The (S)-epimer (compound 8) was prepared
and showed a 17-fold loss in potency relative to 3
(examination of the crystal structure clearly suggests
that this configuration will not position the hydroxyl
group for optimal H-bonding to the catalytic Asp resi-
dues, and hence, there should be a drop in potency).
Our initial SAR studies had quickly identified a novel
(R)-hydroxyethylamidosuccinoyl peptidomimetic, rep-
resented by compound 3, which inhibited the enzymatic
activity of HIV-1 protease with an IC50 of 3.0 nM,
comparable to that of palinavir itself (Table 1). Having
reached the sensitivity limit of our assay,18 in order to
further optimize our inhibitors, we chose to operate in
a less potent series, which would allow a more accurate
comparison of inhibitory potencies. This could be
achieved by removal of the P2 amino acid residue (Val)
from compound 3 to give truncated analog 9 with an
IC50 of 19 nM (Table 3). Compound 9 was used as a
reference point for probing structural requirements at
the C-terminus.
Ta ble 4
As can be seen upon examination of the data pre-
sented in Table 3 (compounds 9-15), a variety of
structural modifications were applied to the C-terminus
amide group. This portion of the molecule proved
sensitive to both size and degree of substitution. tert-
Butylamide 9, isopropylamide 10, 1-ethylpropylamide
11, and anilide 15 gave best results (19, 19, 15, and 14
nM, respectively). However, because of side reactions
occurring during the synthesis of anilide 15, aliphatic
C-termini were selected for further studies. Having
examined structural requirements at the C-terminus of
these shorter inhibitors, the 1-ethylpropyl group was
incorporated in place of the tert-butyl ending in the
longer series. Resulting inhibitor 16 (Table 4) exhibited
potency comparable (IC50 ) 2.9 nM) to that of 3.
entry
-AA-
IC50 (nM)
EC50 (nM)
16a
17
18
Val
Asn
Thr
2.9
3.8
8.2
9.3
127
165
a
Compound 3 (Table 2) with a C-terminal tert-butyl group had
an EC50 of 15 nM.
Next, the ability of 3 and 16 to inhibit viral replication
in cell culture assays was assessed. The antiviral
activity was determined with HIV-1 strain IIIB in
acutely infected C8166 T cells. Both compounds were
potent inhibitors of viral replication in vitro (EC50 ) 15
and 9.3 nM, respectively, Table 4). No apparent cyto-
toxicity was noticed in this cell line at concentrations
up to 1 M (up to 10 M for less active compounds). On
those grounds, slightly more potent 16 was chosen as
the next reference point for further optimization.
16 indicated that a variety of amino acid residues were
tolerated at the P2 position. In particular, valine (entry
16) gave a highly potent inhibitor of HIV-1 protease and
viral replication in vitro. Interestingly, asparagine,
which is found at the P2 position of some natural gag
and pol cleavage sites (e.g., p17/p24, p1/p6, TF/PR, and
PR/RT),5a and found superior with regard to antiviral
activity in other series,9b resulted in an inhibitor with
almost 15-fold reduced potency in cell culture (17, EC50
) 127 nM, Table 4). Threonine-containing analog 18
also showed antiviral properties in this cell assay,
comparable to those of 17. These findings parallel those
made in the pipecolic series of inhibitors where potency
in cell culture also decreased when Val was substituted
for Asn.9h The viral replication assay results presented
Examination of X-ray structures of inhibitors bound
to HIV-18 or HIV-221 protease suggested that the S2
subsite of the enzymes is comprised of uncharged
residues which provide a mostly lipophilic environment
to potential ligands. Optimization of our lead inhibitor

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2167
Ta ble 5
IC50 (nM)
EC50 (nM)
HIV-1
entry
19
20
21
22
23
24
palinavir
-AA-
X
HIV-1
HIV-2
Val
Val
Asn
Asn
Thr
Thr
CH
N
CH
N
CH
N
2.5
2.2
2.2
1.9
3.6
3.0
4.0
42
38
3.7
5.8
11
35
7.0
13
6.7
6.3
185
135
10
5.0
in Table 4 indicate that several of our novel peptidomi-
metic structures are capable of inhibiting the protease
and disrupting viral replication in vitro.
F igu r e 3. Stereodiagram showing the overlap of the bound
conformation of 23 (thick lines) and palinavir (thin lines). The
bound water molecules (W301 and W302) are shown as crosses
and are labeled (W302 is present only in the palinavir
complex).
Incorporation of a 2-quinolinecarboxyl group at the
P3 position of HIV protease inhibitors generally results
in improved antiviral properties.9b,h Similar observa-
tions were made in the present series of inhibitors as
shown in Table 5 (entries 19, 21, and 23). The effect
was most pronounced in the case of 21 and 23, incor-
porating an Asn or Thr residue in the P2 position, when
a significant 10- and 20-fold improvement was observed
in their respective antiviral potencies. We also exam-
ined the use of the 2-quinoxaline group (entries 20, 22,
and 24) in an attempt to alter physicochemical proper-
ties and improve aqueous solubility of our inhibitors.
The latter turned out to be slightly less potent than their
quinoline counterparts, but nevertheless, all compounds
in Table 5 are potent inhibitors of HIV-1 protease,
leading to antiviral activity in vitro comparable to that
of palinavir (with the exception of 22).
Compounds 19-24 were also tested in an HIV-2
protease assay (see the Experimental Section) and
exhibited somewhat reduced potency against this en-
zyme (Table 5). The observed specificity in favor of
HIV-1 is probably a consequence of less favorable
interactions between the inhibitors and HIV-2 in both
the S2 and S1′-S2′ regions of the active site. Unfortu-
nately, since an X-ray structure of 23 in complex with
HIV-1 protease is not available for comparison, further
rationalization as well as the significance of these
observations relative to the resistance potential of this
class of inhibitors is not possible at this time.
inhibitor itself to enter cells. Similar observations were
made in the case of palinavir.11
As mentioned previously, some of our compounds
display some specificity toward HIV-1 versus HIV-2
proteases. In the case of compound 19 (Ki for HIV-2 )
1.2 nM), there is a 160-fold difference between the
measured Ki’s. Most of our inhibitors were also tested
for inhibition of human plasma renin, another aspartyl
protease enzyme, and were found inactive at concentra-
tions up to 5 M.
Bioa va ila bility.24 Inhibitors 19, 20, 21, and 23 were
evaluated for oral bioavailability in rats (n ) 3) at a po
dose of 20 mg/kg (formulated in methyl cellulose:Tween-
80, 0.5:0.01% in water) and iv dose of 1 mg/kg (formu-
lated in DMA/NaH2PO4 0.04 M in 10% dextrose/water:
30/50/20). Compounds 20 and 21 gave very low plasma
levels after oral administration (Cmax’s <150 and <25
nM were measured in plasma for 20 and 21, respec-
tively). Inhibitors 19 and 23 gave slightly better
responses after po administration with average Cmax’s
of 170 and 530 nM, respectively. In the case of 23,
comparison of the area under the curves (AUC) between
the oral and intravenous routes translated into an
absolute oral bioavailability of 8.6%.
Ki Deter m in a tion s a n d Sp ecificity. The inhibition
constant (Ki) was measured for compound 19 (HIV-1 and
HIV-2 proteases) using the method for tight binding
inhibitors of Morrison and Walsh11,22 (see the Experi-
mental Section for details). As expected, compound 19
is an extremely potent inhibitor of HIV-1 protease with
an observed Ki of 7.5 pM.23 Examination of Tables 4
and 5 reveals that, in many instances, measured IC50’s
and EC50’s are of the same order of magnitude, our
compounds effectively inhibiting viral replication at low
nanomolar concentrations. In the case of compound 19,
there is a 500-fold difference between the affinity of the
compound for the protease (Ki) and the EC50 measured
in cell culture. This could result from a number of
factors including protein binding of the inhibitor and
metabolic stability in cell, as well as the ability of the
X-r a y Stu d ies. The crystal structure of HIV-2 pro-
tease in complex with 23 was determined at 2.5 Å
resolution. The final R factor was 17.9%, and the model
had small deviations from ideal geometric parameters
(see Supporting Information). As observed with other
peptidomimetic inhibitors, 23 binds in an extended
conformation which, in this particular case, spans the
S3-S2′ pockets of the enzyme (Figure 3). The amide
groups of the inhibitor are hydrogen-bonded to the
protease dimer either directly or through water mol-
ecules. The critical hydroxyl group of the transition
state mimic ((R)-configuration at the carbinol atom) is
located in close proximity to the catalytic Asp25 and
Asp25′ residues. The P3 to P1 groups of the inhibitor are
similar to those for the pipecolic series, and their
binding modes and interactions with the protease are

2168 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Beaulieu et al.
F igu r e 4. Stereoplot showing the S1′-S2′ interactions between inhibitor 23 and HIV-2 protease. Residues from HIV-2 protease
are shown (carbon atoms in white) and labeled. Inhibitor 23 is shown in green for carbon atoms. The corresponding fragment
for palinavir is shown in cyan. The water molecules that are associated with inhibitor binding are depicted as green and cyan
spheres for the inhibitor 23 and palinavir complexes, respectively.
similar.21 The binding mode of the right-hand side of
the inhibitor differs in many respect to that of the
pipecolic series (Figure 4). The carbonyl oxygen atom
of the P1′ residue in 23 is about 1 Å away from the
corresponding atom in the other series21 due to reversal
of the peptide direction. As a result, the position of the
conserved water molecule W301 has moved by about 0.5
Å to maintain hydrogen-bond contact with the P1′
carbonyl. However, the conformation of the flap resi-
dues on the enzyme is not affected. The carbonyl oxygen
atom of the P2′ residue is hydrogen-bonded to the amido
nitrogen of residue 29, replacing the water molecule
W302 that was observed in earlier structures.21 The
amide group of the P2′ residue is hydrogen-bonded to
the carbonyl group of residue 48. The tert-butyl group
is inserted into the S1′ pocket, and the 3-pentyl group
is located between the S2′ and S4′ pockets. One branch
of the pentyl group is in a similar position in the S2′
pocket as the tert-butyl group was in the pipecolic
inhibitors.21 The other half is extended toward the S4′
pocket and is partly exposed to solvent. It is difficult
to rationalize the potency differences that are observed
between all four possible isomers of the tert-butylsuc-
cinic fragment (compounds 3-6) based on this crystal
structure alone, since some conformational changes in
the inhibitors are very likely to occur to compensate for
the modifications. Additionally, this region of the
protease is known to be somewhat flexible.21 The
dramatically larger potency loss for compound 5 could
be due to a steric hindrance between the tert-butyl group
and the main chain of residue 48 in the flap of the
protease (based on an examination of the structure of
the complex with 23). However, again there are likely
to be conformational readjustments in compound 5 upon
binding to the protease. Also apparent from this crystal
structure is the fact that the S3′ pocket of the enzyme
remains largely unoccupied.
In h ibitor Syn th esis. As mentioned earlier, an
important element in the design of our backup series
of inhibitors was to minimize synthetic complexity. This
was achieved by means of a highly convergent approach
in which simple building blocks were prepared and
assembled in the late stages of the synthesis. This
strategy allowed rapid preparation of a large number
of inhibitors for SAR studies. The right-hand side of
our inhibitors was readily constructed by condensation
of succinic anhydrides with amines. cis-Succinamic
derivative 2625 was prepared in racemic form from 25,
as shown in eq 1, and used as such. Homochiral tert-
(1)
butylsuccinic amides 28-34 were prepared from the
corresponding anhydrides (R)- and (S)-27 as depicted
in Scheme 1 for the (R)-enantiomers. Starting (R)- and
(S)-tert-butylsuccinic anhydrides 27 were prepared
through resolution of racemic tert-butylsuccinic acid
with chiral R-methylbenzylamines, following a literature
procedure.26 Opening of anhydride 27 with amines was
found to be quite regioselective (ca. 9:1) when conducted
at low temperature, and resulting amides 28-34 could
be purified to high homogeneity by simple recrystalli-
zation. Succinamic derivative 38 was prepared via
monobenzyl ester 35 obtained by reaction of 27 with
benzyl alcohol in pyridine (regioisomer 36 was also
obtained in appreciable amounts, but could be removed
by flash chromatography). Coupling to tert-butylamine
using benzotriazol-1-yl-1,1,3,3-tetramethyluronium tet-
rafluoroborate (TBTU)27 in the presence of N-methyl-
morpholine gave 37, which was hydrogenolyzed to 38
under standard conditions. tert-Butylamide intermedi-
ate 40 was prepared from 39 as shown in eq 2.28
The differences in potency toward HIV-1 and HIV-2
proteases from some of the inhibitors listed in Table 5
is difficult to rationalize based on this X-ray structure
alone. Several possibilities could be invoked such as the
presence of a Thr residue in the P2 position, exposure
of the hydrophobic pentyl group to solvent, and the
displacement of W302 from the structure. However,
similar observations could be made for a complex of 23
with HIV-1 protease, and detailed structural analysis
will have to await future studies.
(2)
The central portion of our inhibitors, which consti-
tutes the transition state mimic, was readily obtained

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2169
C8 column (3.9 × 150 mm), 10 f 80% CH3CN in 50 mM NaH2-
PO4 (pH 4.4) in 25 min then 5 min isocratic, 1.0 mL/min flow
rate, UV detection at 215 nm. System B: Waters (Milford MA)
Deltapack C18 column (2 × 150 mm), 0 f 100% CH3CN (0.1%
TFA) in 0.1% TFA in 25 min then 5 min isocratic, 0.5 mL/min
flow rate, UV detection at 215 nm.
Sch em e 1
Flash chromatography was performed on Merck silica gel
60 (0.040-0.063 mm). Analytical thin layer chromatography
(TLC) was carried out on precoated (0.25 mm) Merck silica
gel F-254 plates.
Ma ter ia ls. The decapeptide substrate VSFNFPQITL-NH2
was synthesized using standard solid phase methodology. The
fluorogenic substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-
Gln-Arg-NH2, was purchased from Bachem. Protease-free
BSA, MES, and EDTA were purchased from Sigma. Common
N-protected amino acids, reagents, and solvents were from
commercial sources and used as received without purification.
Benzotriazol-1-yl-1,1,3,3-tetramethyluronium tetrafluorobo-
rate (TBTU)27 was purchased from Richelieu Biotechnologies
Inc. (Montre´al, Canada).
IC50 Det er m in a t ion s Usin g a n HIV P r ot ea se HP LC-
Ba sed Assa y.30 Recombinant HIV-1 and HIV-2 proteases
were expressed from Escherichia coli and purified as previ-
ously described. Enzymatic assays were performed in 100 mM
MES, 300 mM KCl, 5 mM EDTA, 1 mg/mL BSA, pH 5.5.
DMSO (final concentration was brought to 4.5%) was used to
aid solubilization. To 20 L of a 400 M solution of VSFNF-
PQITL-NH2 substrate was added 10 L of various concentra-
tions of the inhibitor tested. The enzymatic reaction was
initiated by the addition of 12 nM of enzyme in 10 L for a
final concentration of 3 nM. The assay mixture was then
incubated for 45 (HIV-2 protease assay) or 60 (HIV-1 protease
assay) min at 37 °C and the reaction terminated by addition
of 200 L of 1%TFA/H2O. Cleavage products and substrate
were separated by reversed-phase HPLC on a Perkin-Elmer
3 × 3CR C8 column as previously described.30a A nonlinear
curve fit using the Hill model was applied to the percent
inhibition-concentration data, and 50% effective concentra-
tions (IC50) were calculated through the use of SAS (Statistical
Software System, SAS Institute Inc., Cary, NC).
Extr a cellu la r p 24 Assa y. In h ibition of Vir a l Rep lica -
tion . The extracellular core protein level, determined by
ELISA with the HIV-1 p24 antigen (Ag) Coulter kits, was used
to monitor the replication of the HIV-1 IIIB strain. C8166 cells
were infected at a MOI of 0.001 in complete medium and were
incubated in the presence of serially diluted inhibitors for 3
days. The percentage of inhibition was obtained by determi-
nation of the core protein level from pools of supernatants
(eight replicas). A nonlinear curve fit using the Hill model
was applied to the percent inhibition-concentration data, and
EC50’s were obtained through the use of the SAS software. No
apparent cytotoxicity was noticed in this cell line at concentra-
tions up to 1 M (up to 10 M for less active compounds).
Ki Deter m in a tion s.11,22 For classical competitive inhibi-
tors, the velocity of the inhibited reaction was measured at
several fixed concentrations, while varying substrate concen-
trations. Enzyme activity was monitored by the fluorescence
change associated with the cleavage of the fluorogenic sub-
strate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-NH2
(10 M) in presence of HIV-1 protease (1 nM) or HIV-2
protease (20 nM) as previously described.11 The assay was
performed in a final volume of 2 mL of 100 mM sodium acetate,
4 mM EDTA, 1.5 M sodium chloride, 0.025% sodium azide, 1
mg/mL BSA, pH 5.5. The final concentration of DMSO was
brought to 1%. The high-salt concentration improved sub-
strate binding affinity, therefore reducing the contribution of
dimer dissociation to the data. The reaction was run at 23 °C
and initiated by the addition of the enzyme. The fluorescence
change associated with the substrate cleavage was monitored
using a Perkin-Elmer LS-50B fluorometer equipped with a
high-speed 4-place magnetic stirrer. The excitation wave-
length was 320 nm and the emission wavelength 417 nm. Data
were collected for a maximum of 400 s, corresponding to a
substrate conversion to product of no greater than 25%. All
data was analyzed using SAS software. The quadratic equa-
tion describing tight-binding inhibition31 was used for the
through aminolysis29 of (2S,3S)-N-Boc-epoxide 41
(Scheme 2). Aminoalkyl epoxide 41 is a key intermedi-
ate in the synthesis of palinavir and other HEA-based
HIV protease inhibitors. We recently described a
practical synthesis of this versatile building block that
is amenable to the preparation of large quantities of
material.12 Coupling of 41 to succinamic acids using
TBTU/N-methylmorpholine gave key intermediate 43.
Deprotection (4 N HCl in dioxane) followed by capping
with benzyl chloroformate gave inhibitors 9-15. Depro-
tection of 43 as above, followed by coupling to N-Cbz-
protected amino acids, gave compounds 2-7 and 16-
18. Hydrogenolysis of the benzyloxycarbonyl group of
16-18, followed by coupling (TBTU/N-methylmorpho-
line) to quinoline-2-carboxylic acid, gave inhibitors 19,
21, and 23. Coupling to quinoxaline-2-carboxylic acid
gave 20, 22, and 24. Compound 8 with the (S)-
configuration at the carbon atom bearing the hydoxyl
group was prepared in the same way as 3, except that
the (2R,3S)-isomer of 41 was used.29
Con clu sion . We have discovered a highly potent (Ki
for HIV-1 protease in the low picomolar range for 19)
series of HIV protease inhibitors which contain a novel
(R)-(hydroxyethyl)amide isostere. The compounds in-
hibit viral replication at low nanomolar concentrations.
The synthesis of these inhibitors is accomplished in a
convergent manner from readily available building
blocks. Unfortunately, oral absorption of these com-
pounds was found to be poor, and further development
of this series was discontinued.
Exp er im en ta l Section
Gen er a l. NMR spectra were recorded on Bruker AC200
or AMX400 spectrometers in deuterated solvents and were
referenced to TMS ( scale). Mass spectral data was obtained
in FAB mode on a MF 50 TATC instrument operating at 6 kV
and 1 mA using thioglycerol or nitrobenzyl alcohol (NBA) as
a matrix support.
HPLC homogeneities were determined under reversed-
phase conditions. System A: Waters (Milford MA) Symmetry

2170 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Beaulieu et al.
Sch em e 2
determination of Ki(app) and the enzyme concentration. Previ-
ous determination of the Km under similar assay conditions,11
in presence of 0.25-10 M substrate, allowed calculation of
the Ki assuming a competitive mode of inhibition.
and charged with magnesium turnings (126.0 g, 5.2 mol) and
reagent grade THF (5 L). 2-Chloro-2-methylpropane (480.4
g, 5.2 mol) was added slowly over 5 h and the gray slurry
stirred for an additional 48 h at room temperature. The
suspension was cooled to -75 °C (dry ice/acetone bath), and
diethyl maleate (447.0 g, 2.60 mol) in THF (1 L) was added
dropwise over 4 h, maintaining an internal reaction temper-
ature close to -75 °C. After completion, stirring was continued
for an additional 2 h at -75 °C and the reaction quenched at
that temperature by dropwise addition (20 min) of saturated
aqueous ammonium chloride (500 mL). The reaction mixture
was acidified to pH 7 by slow addition of 6 N HCl (ca. 500
mL), and precipitated white solids were removed by filtration
using THF for washings. The filtrate was concentrated under
reduced pressure to a volume of 2.5 L and extracted with
hexanes (5 × 200 mL). The extracts were combined, washed
successively with water (3 × 100 mL) and brine (2 × 100 mL),
and dried over MgSO4/Norit decolorizing charcoal. Removal
of solvents under reduced pressure and distillation of the
residue under high vacuum gave racemic 2-tert-butylsuccinic
acid diethyl ester as a yellow oil (323.2 g, 54 % yield): bp 105-
107 °C (1-0.2 Torr).
The diester (321.6 g, 1.40 mol) and concentrated HCl (1.5
L) were refluxed overnight, and the partially hydrolyzed
reaction mixture was concentrated under vacuum to a yellow
oil. The material was again refluxed with fresh concentrated
HCl (1.5 L) for 24 h. After the mixture was cooled to room
temperature, the desired racemic 2-tert-butylsuccinic acid
separated as white crystals. After being cooled to 0 °C, the
product was collected by filtration and rinsed with hexanes (3
× 200 mL). The white solid was dissolved in ether (1.4 L),
and the solution was washed with brine (3 × 50 mL) and dried
over MgSO4. After concentration under reduced pressure to
a volume of 250 mL and addition of hexanes (500 mL), the
product separated as white crystals that were collected and
dried in vacuo (210.8 g, 87% yield): mp 127-130 °C (lit.34 mp
133 °C). Anal. Calcd for C8H14O4: C, 55.16; H, 8.10. Found:
C, 54.93; H, 8.19.
Cr ysta l Str u ctu r e Deter m in a tion . Crystals of HIV-2
protease in complex with inhibitor 23 were prepared by the
hanging drop vapor diffusion method. The reservoir solution
contained 0.3-0.5 M NaCl, 0.1 M NaOAc (pH 5.4), and 0.02%
NaN3.21 The crystals belong to space group P43212 with cell
dimensions of a ) b ) 62.6 Å and c ) 115.4 Å, isomorphous
with those reported earlier.21 Diffraction data to 2.5 Å
resolution were collected at 4 °C with an R-Axis imaging plate
system. The data processing statistics are summarized in the
Supporting Information. The structure of HIV-2 protease
reported earlier21 was used as the initial model for the
structure refinement with the X-Plor program,32 using reflec-
tion data between 6 and 2.5 Å resolution. The inhibitor
molecule was clearly visible in the 2Fo - Fc electron density
map after one cycle of simulated annealing refinement with
the protease atoms. Water molecules were located from
difference electron density maps in the later stages of the
refinement. The final atomic model was obtained after a cycle
of refinement with the TNT program.33 The X-ray coordinates
for the complex of 23 with HIV-2 protease have been deposited
in the Brookhaven Protein Data Bank (filename 1J LD).
Syn th esis of In h ibitor s. (()-cis-2-(ter t-Bu tylca r ba m -
oyl)cycloh exa n eca r boxylic Acid (26): prepared from cis-
1,2-cyclohexanedicarboxylic anhydride and tert-butylamine;25
mp 130-132 °C (lit.25 mp 128.5-130 °C); IR (KBr) 3360,
3200-2400, 1711, 1625, 1551 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 11.25 (broad s, 1 H), 7.06 (broad s, 1 H), 2.64
(broad m, 1 H), 2.44 (dt, J ) 8.7, 4.3 Hz, 1 H), 2.07-1.96 (m,
1 H), 1.90-1.82 (m, 1 H), 1.65-1.15 (m, 6 H), 1.22 (s, 9 H);
13C NMR (50 MHz, DMSO-d6) 175.0, 173.3, 49.7, 42.4, 42.0,
28.5, 27.9, 25.5, 23.7, 22.7; MS (FAB) m/z 228 (MH+). Anal.
Calcd for C12H21NO3: C, 63.41; H, 9.31; N, 6.16. Found: C,
63.63; H, 9.26; N, 6.15.
(R/S)-2-ter t-Bu tylsu ccin ic Acid . This compound was
prepared by a modified literature procedure:34 A 12 L three-
necked flask, equipped with a mechanical stirrer, reflux
condenser, and a 1 L addition funnel, was purged with argon
(R)-2-ter t-Bu tylsu ccin ic Acid . The (R)-enantiomer was
obtained by resolution of the racemate using (S)-R-methyl-
benzylamine as described in the literature:26 mp 126-128 °C

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2171
(lit.26 mp 122-123 °C); [R]21D -28.3 ° (c 5, acetone) [lit.26 [R]20
over MgSO4, volatiles were removed under reduced pressure
and the residue was purified by flash chromatography using
9:1 hexanes/EtOAc as eluant. (S)-2-tert-Butylsuccinic N1-tert-
butylamide 4-benzyl ester (S)-37 was obtained as a white solid
(1.02 g, 85% yield): mp 55-57 °C; Rf 0.47 (3:7 hexane/EtOAc);
D
-26.5° (c 5, acetone)]; IR (KBr) 3400-2400, 1708 cm-1; H
NMR (400 MHz, CDCl3) 11.40 (broad s, 1 H), 2.80 (dd, J )
16.2, 12.1 Hz, 1 H), 2.65 (dd, J ) 12.1, 2.5 Hz, 1 H), 2.52 (dd,
J ) 16.1, 2.5 Hz, 1 H), 1.02 (s, 9H); 13C NMR (50 MHz, DMSO-
d6) 175.2, 173.9, 51.1, 32.7, 31.9, 27.9; MS (FAB) m/z 175
(MH+). Determination of the enantiomeric purity: an aliquot
of the resolved acid was derivatized by heating (30 min) to
120 °C in a sealed tube with (S)-2-octanol saturated with HCl
gas. The material was analyzed by GC (Supelcowax 10, 210
°C/5 min, 2 °C/min f 235 °C, 20 °C/min f 280 °C/5 min; 0.6
mL/min flow, FID detector): tR 11.68 min (99.3%, (R)-isomer,
98.5% ee); tR 12.72 min (0.7%, (S)-isomer).
1
[R]23 +31.3° (c 1, CHCl3); IR (KBr) 3344, 2962, 1738, 1646,
D
1544 cm-1; H NMR (400 MHz, DMSO-d6) 7.39-7.30 (m, 6
1
H), 5.07 (d, J ) 12.9 Hz, 1 H, part of AB), 5.02 (d, J ) 12.6
Hz, 1 H, part of AB), 2.63 (dt, J ) 14.4, 4.5 Hz, 1 H), 2.47-
2.37 (m, 2 H), 1.19 (s, 9 H), 0.90 (s, 9 H); 13C NMR (100 MHz,
DMSO-d6) 172.2, 171.9, 136.2, 128.3, 127.8, 127.7, 65.2, 50.7,
49.9, 32.5, 28.3, 27.6; MS (FAB) m/z 320 (MH+). Anal. Calcd
for C19H29NO3: C, 71.44; H, 9.15; N, 4.38. Found: C, 71.48;
H, 8.95; N, 4.42.
(S)-2-ter t-Bu tylsu ccin ic Acid . This compound was ob-
tained from the mother liquors of the above resolution: after
acidification, the free acid (enriched in the (S)-enantiomer) was
isolated and resolved as above in the presence of (R)-R-
The benzyl ester from above (0.886 g, 2.77 mmol) was added
to a suspension of 20% palladium hydroxide on charcoal (50
mg) in MeOH (20 mL), and the mixture was stirred under 1
atm of hydrogen gas of 2 h. Following removal of the catalyst
by filtration and removal of volatiles under reduced pressure,
the residue was triturated with hexanes, collected by filtration,
and dried under vacuum. The title succinamic acid derivative
(S)-38 was obtained as a white solid (0.562 g, 88% yield): mp
185-189 °C. [R]25D +35.0° (c 1, MeOH); IR (KBr) 3377, 1722,
1640, 1551 cm-1; 1H NMR (400 MHz, DMSO-d6) 11.28 (broad
s, 1 H), 7.31 (broad s, 1 H), 2.49 (dd, J ) 15.6, 11.1 Hz, 1 H),
2.37 (dd, J ) 11.1, 2.7 Hz, 1 H), 2.23 (dd, J ) 15.6, 2.7 Hz, 1
H), 1.22 (s, 9 H), 0.89 (s, 9 H); 13C NMR (50 MHz, DMSO-d6)
174.0, 172.4, 50.8, 49.9, 32.6, 32.1, 28.5, 27.7; MS (FAB) m/z
230 (MH+). Anal. Calcd for C12H23NO3: C, 62.85; H, 10.11;
N, 6.11. Found: C, 62.71; H, 10.14; N, 6.12.
methylbenzylamine: mp 123-126 °C; [R]21 +28.1° (c 5,
D
acetone); GC (after derivatization of an aliquot as described
for the (R)-enantiomer) tR 12.46 min (1.9%, (R)-isomer), tR
13.78 min (98.1%, (S)-isomer, 96.17% ee). IR, 1H NMR, 13C
NMR, and MS (FAB) data were identical to that of the (R)-
isomer.
(R)-2-ter t-Bu tylsu ccin ic An h yd r id e [(R)-27].26 (R)-2-
tert-Butylsuccinic acid (100.0 g, 0.574 mol) was dissolved in
acetic anhydride (1 L) and the solution stirred overnight at
room temperature. The reaction mixture was then stirred for
another 5 h at 100 °C. Volatiles were removed under reduced
pressure at 40 °C, and the residue was distilled under high
vacuum to give anhydride (R)-27 as a clear oil which solidified
on cooling (85.0 g, 95% yield): bp 100-105 °C (0.25-0.5 Torr);
mp 62-64 °C (lit.26 mp 63-64 °C); [R]23D -12.8° (c 4, benzene)
[lit.26 [R]20D -11.4° (c 4, benzene)]; 1H NMR (400 MHz, CDCl3)
3.03-2.90 (m, 2 H), 2.80 (dd, J ) 17.2, 4.1 Hz, 1 H), 1.09 (s,
9 H); 13C NMR (100 MHz, DMSO-d6) 173.1, 171.4, 50.7, 32.8,
31.3, 26.7; MS (CI-isobutane) m/z 157 (MH+); GC (derivatiza-
tion and GC conditions as described for (R)-2-tert-butylsuccinic
acid) tR 11.32 min (96.6%, (R)-isomer, 93.2% ee), tR 12.36 min
(3.4%, (S)-isomer).
(R)-2-ter t-Bu tylsu ccin ic N1-ter t-Bu tyla m id e [(R)-38]:
prepared in the same manner as the (S)-enantiomer, starting
from (R)-2-tert-butylsuccinic anhydride; mp 185-189 °C; [R]23
D
-39.2° (c 0.5, MeOH). IR, H NMR, 13C NMR, and MS data
1
were identical to that of the (S)-enantiomer. Anal. Calcd for
C12H23NO3: C, 62.85; H, 10.11; N, 6.11. Found: C, 63.03; H,
9.88; N, 6.01.
(R)- a n d (S)-2-ter t-Bu t ylsu ccin ic N4-Am id es 28-34.
Gen er a l P r oced u r e. The (R)- or (S)-2-tert-butylsuccinic
anhydride 27 (1 equiv) was dissolved in pyridine and the
solution cooled to -40 °C (dry ice/acetone). The amine (1.2
equiv) in pyridine was added dropwise and the mixture stirred
for 10 min. The cooling bath was removed and the solution
stirred overnight at room temperature. Pyridine and excess
amine were evaporated under vacuum, and the oily residue
was dissolved in EtOAc. The solution was washed successively
with 20% aqueous citric acid (4×) and brine (2×) and then
dried over MgSO4. Removal of volatiles under reduced pres-
sure and purification by crystallization or flash chromatogra-
phy gave desired amides (28-34) as white solids.
(S)-2-ter t-Bu tylsu ccin ic An h yd r id e [(S)-27]: obtained as
above starting from (S)-2-tert-butylsuccinic acid; mp 62-66 °C;
1
[R]21 +9.5° (c 4, benzene); H NMR and 13C NMR data were
D
identical to that of (R)-isomer; MS (FAB) m/z 157 (MH+); GC
(after derivatization of an aliquot as above) tR 12.52 min (1.7%,
(R)-isomer), tR 13.86 min (98.3%, (S)-isomer, 96.6% ee).
(S)-2-ter t-Bu tylsu ccin ic N1-ter t-Bu tyla m id e [(S)-38].
(S)-2-tert-Butylsuccinic anhydride (5.00 g, 32 mmol) was
dissolved in pyridine (30 mL) and the solution cooled in ice-
water. Benzyl alcohol (3.46 g, 32 mmol) and 4-(dimethylami-
no)pyridine (0.40 g, 3.3 mmol) were added, and the mixture
was stirred for 4 days at room temperature. Volatiles were
removed under vacuum, and the residue was dissolved in
EtOAc. The solution was washed with 20% aqueous citric acid
and brine and then dried over MgSO4. Concentration under
reduced pressure gave an oil that was purified by flash
chromatography on silica gel using 15:80:5 EtOAc/hexanes/
MeOH. Pure benzyl (S)-2-tert-butylmonosuccinate (S)-35 was
obtained as a colorless oil (1.33 g, 15 % yield): Rf 0.38 (30:
(S)-2-ter t-Bu t ylsu ccin ic N4-ter t-b u t yla m id e [(S)-28]:
crystallized from EtOAc (74% yield); mp 167-168 °C; [R]23
D
-2.5° (c 1, MeOH); IR (KBr) 3337, 1716, 1635, 1561 cm-1
;
1H NMR (400 MHz, CDCl3) 11.1-10.2 (broad s, 1 H), 5.72
(broad s, 1 H), 2.66 (dd, J ) 11.1, 3.4 Hz, 1 H), 2.53-2.38 (m,
1 H), 2.29 (dd, J ) 14.3, 3.5 Hz, 1 H), 1.31 (s, 9 H), 0.99 (s, 9
H); 13C NMR (50 MHz, DMSO-d6) 175.4, 170.7, 51.0, 49.8,
34.4, 31.9, 28.6, 27.9; MS (FAB) m/z 230 (MH+). Anal. Calcd
for C12H23NO3: C, 62.85; H, 10.11; N, 6.11. Found: C, 62.53;
H, 10.27; N, 6.04.
65:5 EtOAc/hexane/MeOH); [R]23D +13.0° (c 1, CHCl3); [R]23
365
+55.8° (c 1, CHCl3); IR (neat) 1750, 1715 cm-1; 1H NMR (400
MHz, CDCl3) 7.38-7.29 (m, 5 H), 5.13 (d, J ) 12.4 Hz, 1 H,
part of AB), 5.09 (d, J ) 12.4 Hz, 1 H, part of AB), 2.83 (dd, J
) 16.7, 11.6 Hz, 1 H), 2.70 (dd, J ) 11.6, 3.0 Hz, 1 H), 2.54
(dd, J ) 16.7, 3.0 Hz, 1 H), 1.01 (s, 9 H); 13C NMR (50 MHz,
DMSO-d6) 174.9, 172.2, 136.1, 128.4, 128.0, 127.9, 65.5, 50.9,
32.5, 31.9, 27.7; MS (FAB) m/z 265 (MH+). Anal. Calcd for
C15H20O4: C, 68.16; H, 7.63. Found: C, 68.51; H, 7.45.
The ester from above (1.00 g, 3.78 mmol), TBTU (1.40 g,
4.36 mmol), and N-methylmorpholine (1.90 g, 18.78 mmol)
were dissolved in dry DMF (20 mL). tert-Butylamine (1.60 g,
21.87 mmol) was added and the mixture stirred overnight at
room temperature. The reaction mixture was concentrated
under vacuum to one-fourth of the original volume and diluted
with EtOAc (100 mL) and the solution washed successively
with 2 N NaOH (3 × 20 mL), 1 N HCl (3 × 20 mL), water (2
× 20 mL), and brine (2 × 20 mL). After the mixture was dried
(R)-2-ter t-Bu tylsu ccin ic N4-ter t-bu tyla m id e [(R)-28]:
crystallized from EtOAc (68% yield); mp 166-167 °C; [R]23
D
+2.6° (c 1, MeOH). IR, 1H NMR, 13C NMR, and MS data were
identical to that of the (S)-enantiomer. Anal. Calcd for
C12H23NO3: C, 62.85; H, 10.11; N, 6.11. Found: C, 62.79; H,
10.32; N, 6.10.
(R)-2-ter t-Bu tylsu ccin ic N4-isop r op yla m id e [(R)-29]:
crystallized from EtOAc (48% yield); mp 137-141 °C; [R]23
365
-24.3° (c 1, MeOH); IR (neat) 3336, 1706, 1631, 1560 cm-1
;
1H NMR (200 MHz, CDCl3) 11.8-9.8 (broad s, 1 H), 5.92
(broad d, J ) 7.1 Hz, 1 H), 4.14-3.88 (m, 1 H), 2.67 (dd, J )
11.2, 3.5 Hz, 1 H), 2.57-2.41 (m, 1 H), 2.32 (dd, J ) 14.1, 3.3
Hz, 1 H), 1.12 (d, J ) 6.6 Hz, 3 H), 1.10 (d, J ) 6.6 Hz, 3 H),
1.00 (s, 9 H); 13C NMR (50 MHz, DMSO-d6) 175.3, 170.0,
51.1, 40.2, 33.8, 31.9, 27.8, 22.5, 22.4; MS (FAB) m/z 216

2172 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Beaulieu et al.
(MH+). Anal. Calcd for C11H21NO3: C, 61.37; H, 9.83; N, 6.51.
Found: C, 61.34; H, 9.95; N, 6.46.
(400 MHz, DMSO-d6) 7.28-7.13 (m, 5 H), 6.38 (d, J ) 9.3
Hz, 1 H), 3.72 (m, 1 H), 3.33 (m, 1 H), 2.78 (dd, J ) 13.8, 5.7
Hz, 1 H), 2.65 (dd, J ) 13.6, 9.2 Hz, 1 H), 2.55-2.41 (m, 2 H),
1.31 (s, 9 H); 13C NMR (100 MHz, DMSO-d6) 155.5, 139.5,
129.1, 128.0, 125.7, 77.5, 72.6, 53.8, 44.3, 36.8, 28.2; MS (FAB)
m/z 281 (MH+). Anal. Calcd for C15H24N2O3: C, 64.26; H, 8.63;
N, 9.99. Found: C, 63.91; H, 8.66; N, 9.80.
P r epar ation of In h ibitor s. Gen er al P r ocedu r e (Sch em e
2). (2R,3S)- or (2S,3S)-N3-Boc-1,3-diamino-2-hydroxy-4-phe-
nylbutanes 42 were coupled to equimolar amounts of succinic
acid monoamides using TBTU (1.2 equiv) as coupling agent,
N-methylmorpholine (5-6 equiv) as base, and dry DMF as
solvent. Coupling reactions were allowed to proceed until
complete as judged by TLC or reversed-phase HPLC (C-18
column, 0.06% TFA/0.06% TFA in acetonitrile gradients)
analysis. After removal of DMF under vacuum, the residue
was diluted with EtOAc and the solution washed successively
with 2 N NaOH, 1 N HCl, water, and brine. After the mixture
(R)-2-ter t-Bu tylsu ccin ic N4-(3-a m in op en tyl)a m id e [(R)-
30]: crystallized from ether/hexanes (75% yield); mp 124-126
°C; [R]23 -8.0° (c 1, CHCl3); [R]23 -33.3° (c 1, CHCl3); IR
D
365
(KBr) 3274, 1728, 1637, 1565 cm-1 1H NMR (400 MHz,
;
CDCl3) 5.41 (broad d, J ) 8.9 Hz, 1 H), 3.8-3.7 (m, 1 H),
2.79 (dd, J ) 11.4, 3.5 Hz, 1 H), 2.58-2.50 (m, 1 H), 2.41 (dd,
J ) 14.3, 3.5 Hz, 1 H), 1.58-1.45 (m, 1 H), 1.41-1.27 (m, 1
H), 1.02 (s, 9 H), 0.88 (t, J ) 7.6 Hz, 3 H), 0.86 (t, J ) 7.6 hz,
3 H); 13C NMR (50 MHz, DMSO-d6) 175.4, 170.9, 51.4, 51.2,
34.0, 32.0, 27.9, 27.1, 27.0; MS (FAB) m/z 244 (MH+). Anal.
Calcd for C13H25NO3: C, 64.97; H, 10.36; N, 5.76. Found: C,
63.99; H, 10.54; N, 5.70.
(R)-2-ter t-Bu tylsu ccin ic N4-(4-a m in oh ep tyl)a m id e [(R)-
31]: crystallized from ether/hexanes (67% yield); mp 129-130
°C; [R]23 -9.2° (c 1, CHCl3); [R]23 -38.0° (c 1, CHCl3); IR
D
365
(KBr) 3295, 1714, 1705, 1654, 1609, 1549 cm-1; 1H NMR (200
MHz, CDCl3) 11.5-11.0 (broad s, 1 H), 5.44 (broad d, J )
9.0 Hz, 1 H), 3.90 (broad m, 1 H), 2.79 (dd, J ) 10.8, 3.7 Hz,
1 H), 2.61-2.45 (m, 1 H), 2.39 (dd, J ) 14.3, 3.8 Hz, 1 H),
1.5-1.2 (m, 8 H), 1.01 (s, 9 H), 0.93-0.83 (m, 6 H); 13C NMR
(50 MHz, DMSO-d6) 175.3, 170.5, 51.2, 47.3, 37.0, 33.9, 31.9,
27.8, 18.7, 18.6, 14.0, 13.9; MS (FAB) m/z 272 (MH+). Anal.
Calcd for C15H29NO3: C, 66.38; H, 10.77; N, 5.16. Found: C,
66.02; H, 10.87; N, 5.07.
was dried over MgSO4
and the solvent removed under reduced
pressure, intermediate N-Boc derivatives 43 were obtained in
good yield as white solids. When necessary, purification was
carried out by flash chromatography on silica gel. The
compounds had 1H NMR and MS data compatible with their
assigned structures.
The above N-Boc intermediates were deprotected by stirring
with 4 N hydrogen chloride in dioxane, until complete as
shown by TLC or reversed-phase HLC analysis. Removal of
volatiles under vacuum followed by trituration with ether gave
the desired hydrochloride salts as white solids which were used
without further purification.
(R)-2-ter t-Bu tylsu ccin ic N4-cycloh exyla m id e [(R)-32]:
crystallized from EtOAc (68% yield); mp 188-190 °C; [R]23
D
-1.7° (c 1, MeOH); [R]23
-16.7° (c 1, MeOH); IR (neat)
365
3343, 1702, 1619, 1562 cm-1; 1H NMR (200 MHz, DMSO-d6)
11.90 (s, 1 H), 7.70 (d, J ) 8.0 Hz, 1 H), 3.45 (broad m, 1 H),
2.48-2.27 (m, 2 H), 2.20 (dd, J ) 13.7, 2.7 Hz, 1 H), 1.75-
1.45 (m, 6 H), 1.37-0.94 (m, 4 H), 0.90 (s, 9 H); 13C NMR (50
MHz, DMSO-d6) 175.3, 170.0, 51.1, 47.4, 33.8, 32.6, 32.5,
31.9, 27.8, 25.3, 24.6; MS (FAB) m/z 256 (MH+). Anal. Calcd
for C14H25NO3: C, 65.85; H, 9.87; N, 5.49. Found: C, 65.55;
H, 9.97; N, 5.53.
In h ibitor s 1-8, 16-18. Crude hydrochloride salts from
above were coupled with N-Cbz-protected amino acids (1.1
equiv) using TBTU (1.1 equiv) as coupling agent, N-methyl-
morpholine (6 equiv) as base, and dry DMF as solvent.
Couplings were allowed to proceed until completion as judged
by TLC or reversed-phase HPLC analysis. After usual work-
up, inhibitors were purified by flash chromatography on silica
gel using EtOAc/hexanes or EtOAc/MeOH mixtures as eluant.
In h ibitor s 9-15. Crude hydrochloride salts derived from
43 were dissolved in a mixture of THF and triethylamine (4-5
equiv). Benzyl chloroformate (1.1-1.2 equiv) was added and
the mixture stirred at room temperature until completion as
shown by TLC or reversed-phase HPLC analysis. Following
the usual workup, the inhibitors were purified by flash
chromatography on silica gel using EtOAc/hexanes mixtures
as eluant.
In h ib it or s 19-24. N-Cbz-protected derivatives (16-18)
were hydrogenolyzed (1 atm of H2 gas) over 20% palladium
hydroxide on charcoal (5-10% w/w catalyst/substrate) in
MeOH as solvent. Deprotection was usually complete in a few
hours as shown by TLC or reversed-phase HPLC analysis.
After removal of the catalyst by filtration and concentration
in vacuo, free amines were obtained as white solids. The latter
were coupled to 2-quinolinecarboxylic acid or 2-quinoxalin-
ecarboxylic acid using TBTU and N-methylmorpholine in DMF
in the usual way. Inhibitors were purified to >90% homoge-
neity (as determined by reversed-phase HPLC in two solvent
systems) by flash chromatography on silica gel using EtOAc/
MeOH mixtures as eluant.
(R)-2-ter t-Bu tylsu ccin ic N4-(3-am in o-2,4-dim eth ylh exyl)-
a m id e [(R)-33]: purified by flash chromatography on silica
gel using 95:5 chloroform/MeOH as eluant (76% yield); mp
130-134 °C; [R]23 -8.3° (c 1, CHCl3); [R]23
-36.8° (c 1,
D
365
CHCl3); IR (KBr) 3288, 1706, 1629, 1553 cm-1; 1H NMR (400
MHz, DMSO-d6) 11.32 (broad s, 1 H), 7.28 (d, J ) 9.9 Hz, 1
H), 3.35 (dt, J ) 9.9, 6.7 Hz, 1 H), 2.25 (m, 2 H), 2.31-2.22
(m, 1 H), 1.70 (m, J ) 6.7 Hz, 2 H), 0.93 (s, 9 H), 0.79 (d, J )
6.7 Hz, 3 H), 0.78 (d, J ) 6.7 Hz, 3 H), 0.77 (d, J ) 6.7 Hz, 3
H), 0.76 (d, J ) 6.7 Hz, 3 H); 13C NMR (50 MHz, DMSO-d6)
175.3, 171.2, 58.3, 51.3, 33.8, 31.9, 28.9, 28.8, 27.9, 20.3, 20.2,
17.8, 17.7; MS (FAB) m/z 272 (MH+). Anal. Calcd for C15H29
-
NO3: C, 66.38; H, 10.71; N, 5.16. Found: C, 66.70; H, 10.87;
N, 5.16.
(R)-2-ter t-Bu tylsu ccin ic N4-p h en yla m id e [(R)-34]: crys-
tallized from EtOAc (71% yield); mp 153-156 °C; [R]23 -1.0°
D
(c 1, MeOH); [R]23
-10.5° (c 1, MeOH); IR (neat) 3297,
365
1715, 1687, 1634, 1600, 1544 cm-1; 1H NMR (200 MHz, DMSO-
d6) 12.04 (s, 1 H), 9.97 (s, 1 H), 7.57 (d, J ) 8.0 Hz, 2 H),
7.27 (t, J ) 7.9 Hz, 2 H), 7.00 (t, J ) 7.5 Hz, 1 H), 2.8-2.44
(m, 3 H), 0.97 (s, 9 H); 13C NMR (50 MHz, DMSO-d6) 175.3,
170.3, 139.3, 128.7, 122.9, 118.9, 50.9, 34.9, 31.9, 27.9; MS
(FAB) m/z 250 (MH+). Anal. Calcd for C14H19NO3: C, 67.45;
H, 7.68; N, 5.62. Found: C, 67.26; H, 7.74; N, 5.71.
N-ter t-Bu tylsu ccin a m ic a cid (40): prepared from succinic
anhydride and tert-butylamine;28 mp 130-131.5 °C (lit.28a mp
134-135 °C); IR (KBr) 3358, 3200-2300, 1705, 1613, 1561
cm-1; 1H NMR (400 MHz, DMSO-d6) 11.2 (broad s, 1 H), 7.36
(broad s, 1 H), 2.36 (m, 2 H), 2.26 (m, 2 H), 1.23 (s, 9 H); 13C
NMR (50 MHz, DMSO-d6) 173.9, 170.3, 49.8, 30.6, 29.2, 28.5;
MS (CI-NH3) m/z 174 (MH+).
In h ibitor 2: 1:1 mixture of isomers; mp 132-136 °C; Rf 0.27
and 0.31 (7:3:0.5 EtOAc/hexane/MeOH); IR (KBr) 3298, 1652,
1
1537 cm-1; H NMR (400 MHz, DMSO-d6) 7.72 (broad d, J
) 2.4 Hz, 0.5 H), 7.70 (broad d, J ) 2.4 Hz, 0.5 H), 7.52 (t, J
) 5.7 Hz, 0.5 H), 7.48 (t, J ) 5.7 Hz, 0.5 H), 7.4-7.28 (m, 5
H), 7.24-7.06 (m, 6 H), 7.04 (broad d, J ) 3.6 Hz, 0.5 H), 7.01
(broad d, J ) 3.9 Hz, 0.5 H), 5.07-4.99 (m, 2 H), 4.92 (d, J )
5.4 Hz, 0.5 H), 4.89 (d, J ) 5.1 Hz, 0.5 H), 3.90 (m, 1 H), 3.75
(m, 1 H), 3.51-3.31 (m, 2 H), 3.00 (broad d, J ) 2.7 Hz, 0.5
H), 2.97 (broad d, J ) 2.7 Hz, 0.5 H), 2.95-2.79 (m, 1 H), 2.60
(dd, J ) 13.8, 10.5 Hz, 1 H), 2.56-2.5 (m, 1 H), 2.49-2.42 (m,
1 H), 2.06-1.90 (m, 2 H), 1.81 (m, 1 H), 1.70 (m, 1 H), 1.64-
1.41 (m, 3 H), 1.35-1.21 (m, 2 H), 1.21 (s, 9 H), 0.74-0.68 (m,
6 H); 13C NMR (50 MHz, DMSO-d6) 174.2, 173.4, 170.7,
170.6, 155.9, 139.2, 139.1, 137.1, 129.2, 128.3, 127.8, 127.6,
125.6, 72.1, 71.7, 65.4, 60.6, 53.0, 49.8, 43.8, 43.4, 43.2, 42.4,
42.1, 35.2, 34.9, 30.3, 28.5, 27.2, 27.1, 26.5, 23.2, 19.2, 18.1;
(2R ,3S )-N 3-Boc-1,3-d ia m in o-2-h yd r oxy-4-p h e n ylb u -
ta n e [(2R,3S)-42]: prepared by aminolysis of (2S,3S)-N-Boc-
3-amino-1,2-epoxy-4-phenylbutane (2S,3S)-4112 following the
literature procedure.29
(2S ,3S )-N 3-Boc-1,3-d ia m in o-2-h yd r oxy-4-p h e n ylb u -
ta n e [(2S,3S)-42]: prepared according to literature proce-
dure;29 mp 75-78 °C; Rf 0.46 (7:2:1 CHCl3/MeOH/Et3N); [R]23
D
-40.7° (c 1, CHCl3); IR (KBr) 3358. 1691, 1517 cm-1; 1H NMR

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2173
MS (FAB) m/z 623 (MH+); homogeneity 96.93% (system A, sum
of two isomers), 98.72% (system B, sum of two isomers).
31.1, 30.2, 28.4, 19.1, 18.0; MS (FAB) m/z 569 (MH+); homo-
geneity 91.41% (system A), 94.07% (system B).
In h ibitor 8: mp 240-243 °C; Rf 0.40 (3:7 hexane/EtOAc);
[R]23D -15.2° (c 1, MeOH); [R]23365 -50.1° (c 1, MeOH); IR (KBr)
3346, 3263, 2962, 1708, 1648, 1532 cm-1; 1H NMR (400 MHz,
DMSO-d6) 7.66 (d, J ) 9.0 Hz, 1 H), 7.43 (t, J ) 5.7 Hz, 1
H), 7.40-7.28 (m, 5 H), 7.24 (s, 1 H), 7.22-7.11 (m, 6 H), 5.08
(d, J ) 4.5 Hz, 1 H), 5.04 (broad s, 2 H), 3.99 (broad q, J ) 7.8
Hz, 1 H), 3.88 (dd, J ) 8.4, 7.2 Hz, 1 H), 3.50 (m, 1 H), 3.18
(m, 1 H), 2.84-2.67 (m, 3 H), 2.37-2.28 (m, 2 H), 2.09 (m, 1
H), 1.91 (m, 1 H), 1.17 (s, 9 H), 0.84 (s, 9 H), 0.78 (d, J ) 7.2
Hz, 3 H), 0.76 (d, J ) 7.2 Hz, 3 H); 13C NMR (50 MHz, DMSO-
d6) 173.6, 171.4, 170.9, 156.0, 138.9, 137.1, 129.1, 128.3,
128.0, 127.7, 127.6, 125.9, 69.3, 65.4, 60.5, 52.4, 51.9, 49.8, 37.0,
34.7, 32.2, 30.3, 28.4, 27.7, 19.3, 18.0; MS (FAB) m/z 625
(MH+); homogeneity 96.60% (system A), 98.23% (system B).
In h ibitor 9: mp 70-74 °C; Rf 0.35 (3:7 hexane/EtOAc);
In h ibitor 3: mp 220-223 °C; Rf 0.25 (3:7 hexane/EtOAc);
[R]23D, -11.1° (c 1, MeOH); [R]23
-34.5° (c 1, MeOH); IR
365
(KBr) 3437, 3348, 3263, 2961, 1710, 1646, 1541 cm-1
.
1H
NMR (400 MHz, DMSO-d6) 7.74 (d, J ) 9.0 Hz, 1 H), 7.64
(t, J ) 5.5 Hz, 1 H), 7.4-7.28 (m, 5 H), 7.28 (s, 1 H), 7.23-
7.06 (m, 5 H), 7.02 (d, J ) 9.3 Hz, 1 H), 5.07 (d, J ) 12.9 Hz,
1 H, part of AB), 5.01 (d, J ) 12.6 Hz, 1 H, part of AB), 4.93
(d, J ) 4.5 Hz, 1 H), 3.91 (m, 1 H), 3.79 (dd, J ) 8.7, 7.5 Hz,
1 H), 3.50 (m, 1 H), 3.44-3.36 (m, 1 H), 2.65 (dd, J ) 13.6,
10.3 Hz, 1 H), 2.46-2.37 (m, 2 H), 2.14 (dt, J ) 10.5, 9.3 Hz,
1 H), 1.86 (m, 1 H), 1.21 (s, 9 H), 0.91 (s, 9 H), 0.74 (d, J ) 6.7
Hz, 3 H), 0.72 (d, J ) 6.9 Hz, 3 H); 13C NMR (50 MHz, DMSO-
d6) 174.0, 171.2, 170.6, 155.9, 139.3, 137.1, 129.2, 128.3,
127.8, 127.6, 125.6, 72.5, 65.4, 60.4, 53.3, 51.8, 49.8, 42.4, 35.0,
34.7, 32.3, 30.4, 28.5, 27.9, 19.3, 18.0; MS (FAB) m/z 625
(MH+); homogeneity 91.72% (system A), 91.64% (system B).
[R]23 +2.0° (c 1, MeOH); IR (KBr) 3314, 2962, 1705, 1654,
D
1538 cm-1; H NMR (400 MHz, DMSO-d6) 7.63 (t, J ) 5.7
1
In h ibitor 4: mp 213-215 °C; Rf 0.25 (3:7 hexane/EtOAc);
[R]23D +1.2° (c 1, MeOH); [R]23365 +16.9° (c 1, MeOH); IR (KBr)
Hz, 1 H), 7.34-7.12 (m, 11 H), 4.96 (d, J ) 12.9 Hz, 1 H, part
of AB), 4.92 (d, J ) 4.5 Hz, 1 H), 4.88 (d, J ) 12.9 Hz, 1 H,
part of AB), 3.68-3.56 (m, 1 H), 3.52-3.40 (m, 2 H), 3.06 (dd,
J ) 13.8, 3.0 Hz, 1 H), 2.90 (m, 1 H), 2.57 (dd, J ) 13.6, 11.0
Hz, 1 H), 2.46-2.35 (m, 2 H), 2.14 (dt, J ) 10.8, 8.7 Hz, 1 H),
1.21 (s, 9 H), 0.91 (s, 9 H); 13C NMR (50 MHz, DMSO-d6)
173.9, 171.1, 155.8, 139.6, 137.4, 129.2, 128.2, 127.9, 127.5,
127.0, 125.7 , 72.8, 64.7, 55.6, 51.7, 49.8, 42.5, 35.4, 34.8, 32.3,
28.5, 27.9; MS (FAB) m/z 526 (MH+); homogeneity 96.38%
(system A), 96.51% (system B).
3310, 2963, 1699, 1652, 1537 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 7.73-7.67 (m 2 H), 7.4-7.3 (m, 6 H), 7.23-7.06
(m, 5 H), 7.02 (d, J ) 9.0 Hz, 1 H), 5.09-5.00 (m, 2 H), 4.84
(d, J ) 5.4 Hz, 1 H), 3.93 (m, 1 H), 3.76 (dd, J ) 8.7, 7.8 Hz,
1 H), 3.52 (m, 1 H), 3.31 (m, 1 H), 3.00 (dd, J ) 13.9, 2.9 Hz,
1 H), 2.90 (m, 1 H), 2.61 (dd, J ) 13.9, 10.3 Hz, 1 H), 2.44-
2.36 (m, 2 H), 1.22 (s, 9 H), 0.90 (s, 9 H), 0.72 (d, J ) 6.6 Hz,
3 H), 0.69 (d, J ) 6.9 Hz, 3 H); 13C NMR (50 MHz, DMSO-d6)
174.1, 171.3, 170.6, 155.9, 139.2, 137.1, 129.3, 128.3, 127.8,
127.6, 125.6, 71.5, 65.4, 60.6, 53.1, 51.7, 49.8, 42.5, 35.1, 34.6,
32.2, 30.3, 28.5, 27.8, 19.2, 18.1; MS (FAB) m/z 625 (MH+);
homogeneity 92.35% (system A), 92.65% (system B).
In h ibitor 10: mp 62-66 °C; Rf 0.23 (3:7 hexane/EtOAc);
[R]23 -6.0° (c 1, MeOH); IR (KBr) 3306, 2964, 1702, 1647,
D
1540 cm-1; H NMR (400 MHz, DMSO-d6) 7.64 (t, J ) 5.4
1
Hz, 1 H), 7.58 (d, J ) 7.8 Hz, 1 H), 7.33-7.11 (m, 10 H), 4.95
(d, J ) 13.2 Hz, 1 H, part of AB), 4.90 (m, 1 H), 4.87 (d, J )
13.2 Hz, 1 H, part of AB), 3.78 (m, J ) 6.9 Hz, 1 H), 3.60 (m,
1 H), 3.52-3.38 (m, 2 H), 3.05 (dd, J ) 13.8, 2.7 Hz, 1 H), 2.89
(m, 1 H), 2.56 (dd, J ) 13.6, 11.2 Hz, 1 H), 2.45-2.35 (m, 2
H), 2.21-2.11 (m, 1 H), 1.01 (d, J ) 6.3 Hz, 3 H), 0.99 (d, J )
6.3 Hz, 3 H), 0.91 (s, 9 H); 13C NMR (50 MHz, DMSO-d6)
173.8, 170.5, 155.8, 139.6, 137.4, 129.2, 128.2, 127.9, 127.5,
127.0, 125.7, 72.8, 64.7, 55.6, 51.5, 42.5, 35.4, 34.1, 32.3, 27.8,
22.4, 22.3; MS (FAB) m/z 512 (MH+); homogeneity 98.25%
(system A), 99.47% (system B).
In h ibitor 5: mp 165-168 °C; Rf 0.22 (3:7 hexane/EtOAc);
[R]23 -6.7° (c 1, MeOH); IR (KBr) 3297, 2963, 1698, 1652,
D
1534 cm-1; H NMR (400 MHz, DMSO-d6) 7.68 (d, J ) 9.0
1
Hz, 1 H), 7.62 (t, J ) 5.4 Hz, 1 H), 7.4-7.28 (m, 5 H), 7.24 (s,
1 H), 7.22-7.05 (m, 5 H), 7.02 (d, J ) 9.3 Hz, 1 H), 5.05 (d, J
) 12.6 Hz, 1 H, part of AB), 5.01 (d, J ) 12.6 Hz, 1 H, part of
AB), 4.95 (d, J ) 5.4 Hz, 1 H), 3.90 (m, 1 H), 3.74 (dd, J ) 9.0,
7.5 Hz, 1 H), 3.43 (m, 1 H), 3.23 (dt, J ) 13.2, 5.1 Hz, 1 H),
3.00-2.90 (m, 2 H), 2.59 (dd, J ) 13.8, 10.2 Hz, 1 H), 2.47-
2.37 (m, 2 H), 2.13 (dt, J ) 10.5, 9.0 Hz, 1 H), 1.80 (m, 1 H),
1.21 (s, 9 H), 0.90 (s, 9 H), 0.72 (d, J ) 6.6 Hz, 3 H), 0.69 (d,
J ) 6.9 Hz, 3 H); 13C NMR (50 MHz, DMSO-d6) 172.7, 172.0,
170.5, 155.9, 139.1, 137.1, 129.3, 128.3, 127.8, 127.6, 125.7,
71.7, 65.4, 60.5, 53.1, 51.3, 49.8, 42.7, 34.9, 34.3, 32.3, 30.4,
28.5, 27.7, 19.2, 18.1; MS (FAB) m/z 625 (MH+); homogeneity
98.49% (system A), 99.66% (system B).
In h ibitor 11: mp 121-125 °C; Rf 0.33 (3:7 hexane/EtOAc);
[R]23 +0.8° (c 1, MeOH); [R]23 -5.1° (c 1, MeOH); IR (neat)
D
365
3328, 2963, 1702, 1642, 1534 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 7.63 (t, J ) 5.6 Hz, 1 H), 7.41 (d, J ) 9.0 Hz, 1
H), 7.33-7.11 (m, 10 H), 4.95 (d, J ) 13.2 Hz, 1 H, part of
AB), 4.91 (d, J ) 4.5 Hz, 1 H), 4.87 (d, J ) 13.2 Hz, 1 H, part
of AB), 3.60 (m, 1 H), 3.53-3.42 (m, 3 H), 3.05 (dd, J ) 13.5,
3.0 Hz, 1 H), 2.80 (m, 1 H), 2.55 (dd, J ) 13.6, 11.2 Hz, 1 H),
2.49-2.42 (m, 2 H), 2.20 (dt, J ) 10.8, 8.4 Hz, 1 H), 1.45-1.32
(m, 2 H), 1.32-1.18 (m, 2 H), 0.92 (s, 9 H), 0.78 (t, J ) 7.5 Hz,
3 H), 0.75 (t, J ) 7.5 Hz, 3 H); 13C NMR (50 MHz, DMSO-d6)
173.7, 171.1, 155.8, 139.6, 137.4, 129.2, 128.2, 127.9, 127.5,
127.1, 125.7, 72.8, 64.7, 55.6, 51.8, 51.0, 42.5, 35.4, 34.3, 32.3,
27.9, 26.9, 26.8, 10.4, 10.2; MS (FAB) m/z 540 (MH+); homo-
geneity 96.95% (system A), 99.89% (system B).
In h ibitor 6: mp 263-264 °C; Rf 0.36 (95:5 CHCl3/MeOH);
[R]23 -0.2° (c 1, MeOH); IR (KBr) 3280, 2962, 1702, 1653,
D
1541 cm-1; H NMR (400 MHz, DMSO-d6) 7.71 (d, J ) 9.0
1
Hz, 1 H), 7.56 (t, J ) 6.6 Hz, 1 H), 7.40-7.28 (m, 5 H), 7.25 (s,
1 H), 7.22-7.07 (m, 5 H), 7.01 (d, J ) 9.3 Hz, 1 H), 5.04 (m, 2
H), 5.00 (d, J ) 4.8 Hz, 1 H), 3.88 (m, 1 H), 3.77 (dd, J ) 8.4,
7.8 Hz, 1 H), 3.48-3.37 (m, 2 H), 2.96 (dd, J ) 13.8, 3.0 Hz, 1
H), 2.80-2.71 (m, 1 H), 2.59 (dd, J ) 13.6, 10.3 Hz, 1 H), 2.46-
2.37 (m, 2 H), 2.13 (q, J ) 9.9 Hz, 1 H), 1.82 (m, 1 H), 1.20 (s,
9 H), 0.90 (s, 9 H), 0.73 (d, J ) 6.6 Hz, 3 H), 0.71 (d, J ) 6.0
Hz, 3 H); 13C NMR (50 MHz, DMSO-d6) 172.7, 172.0, 170.6,
155.9, 139.1, 137.1, 129.3, 128.3, 127.8, 127.7, 127.6, 125.6,
72.2, 65.3, 60.5, 53.2, 51.4, 49.8, 42.7, 35.1, 34.4, 32.3, 30.3,
28.5, 27.7, 19.2, 18.1; MS (FAB) m/z 625 (MH+); homogeneity
98.15% (system A), 99.11% (system B).
In h ibitor 12: mp 143-147 °C; Rf 0.36 (3:7 hexane/EtOAc);
[R]23 +0.4° (c 1, MeOH); [R]23 -7.0° (c 1, MeOH); IR (neat)
D
365
3317, 2955, 1696, 1642, 1538 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 7.63 (t, J ) 5.5 Hz, 1 H), 7.41 (d, J ) 8.7 Hz, 1
H), 7.34-7.13 (m, 10 H), 4.95 (d, J ) 12.9 Hz, 1 H), part of
AB), 4.92 (d, J ) 4.2 Hz, 1 H), 4.87 (d, J ) 12.9 Hz, 1 H, part
of AB), 3.74-3.43 (m, 4 H), 3.07 (dd, J ) 13.6, 3.1 Hz, 1 H),
2.80 (m, 1 H), 2.56 (dd, J ) 13.8, 11.1 Hz, 1 H), 2.49-2.40 (m,
2 H), 2.17 (dt, J ) 10.8, 10.2 Hz, 1 H), 1.32-1.12 (m, 8 H),
0.93 (s, 9 H), 0.83 (t, J ) 6.9 Hz, 3 H), 0.81 (t, J ) 6.6 Hz, 3
H); 13C NMR (50 MHz, DMSO-d6) 173.6, 170.8, 155.8, 139.6,
137.4, 129.2, 128.2, 127.9, 127.5, 127.0, 125.7, 72.8, 64.7, 55.7,
51.8, 47.2, 42.5, 36.9, 35.4, 34.3, 32.3, 27.9, 18.8, 18.5, 13.9;
MS (FAB) m/z 568 (MH+); homogeneity 99.58% (system A),
99.56% (system B).
In h ibitor 7: mp 216-218 °C; Rf 0.24 (95:5 CHCl3/MeOH);
[R]25D -11.0° (c 0.5, MeOH); IR (KBr) 3299, 1692, 1645, 1536
1
cm-1; H NMR (400 MHz, DMSO-d6) 7.71 (t, J ) 5.4 Hz, 1
H), 7.66 (d, J ) 8.9 Hz, 1 H), 7.39-7.27 (m, 6 H), 7.22-7.05
(m, 5 H), 6.96 (d, J ) 9.3 Hz, 1 H), 5.03 (m, 2 H), 4.97 (d, J )
5.4 Hz, 1 H), 3.91 (m, 1 H), 3.75 (broad t, J ) 8.2 Hz, 1 H),
3.46 (m, 1 H), 3.37 (m, 1 H), 2.97 (dd, J ) 13.8, 3.0 Hz, 1 H),
2.89 (m, 1 H), 2.60 (dd, J ) 13.8, 10.5 Hz, 1 H), 2.29 (m, 4 H),
1.82 (m, J ) 6.9 Hz, 1 H), 1.23 (s, 9 H), 0.72 (d, J ) 6.6 Hz, 3
H), 0.69 (d, J ) 6.6 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6)
171.8, 170.9, 170.5, 155.8, 139.0, 136.9, 129.2, 128.1, 127.7,
127.6, 127.5, 125.5, 71.7, 65.3, 60.5, 53.0, 49.7, 42.4, 34.9, 31.7,
In h ibitor 13: mp 121-124 °C; Rf 0.44 (1:1 hexane/EtOAc);
[R]23D -0.9° (c 1, MeOH); [R]23365 -10.0° (c 1, MeOH); IR (neat)
3329, 2933, 1699, 1648, 1538 cm-1 1H NMR (400 MHz,
;

2174 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14
Beaulieu et al.
DMSO-d6) 7.63 (t, J ) 5.4 Hz, 1 H), 7.59 (d, J ) 8.1 Hz, 1
H), 7.34-7.12 (m, 10 H), 4.96 (d, J ) 12.9 Hz, 1 H, part of
AB), 4.92 (d, J ) 4.8 Hz, 1 H), 4.88 (d, J ) 12.9 Hz, 1 H, part
of AB), 3.62 (m, 1 H), 3.54-3.40 (m, 3 H), 3.06 (dd, J ) 13.8,
3.0 Hz, 1 H), 2.90 (m, 1 H), 2.58 (dd, J ) 13.5, 11.1 Hz, 1 H),
2.48-2.38 (m, 2 H), 2.18 (dt, J ) 10.5, 8.9 Hz, 1 H), 1.30-1.02
(m, 5 H), 0.92 (s, 9 H); 13C NMR (50 MHz, DMSO-d6) 173.8,
170.4, 155.8, 139.6, 137.4, 129.2, 128.2, 127.9, 127.4, 127.0,
125.7, 72.8, 64.7, 55.6, 51.5, 47.3, 42.5, 35.4, 34.1, 32.4, 32.3,
32.2, 27.8, 25.2, 24.6; MS (FAB) m/z 552 (MH+); homogeneity
100% (system A), 99.33% (system B).
In h ibitor 18: mp 158-161 °C; Rf 0.37 (95:5 EtOAc/MeOH);
[R]23D -10.5° (c 1, MeOH); [R]23365 -37.0° (c 1, MeOH); IR (neat)
3304, 2963, 1685, 1646, 1539 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 7.70 (d, J ) 9.0 Hz, 1 H), 7.60 (t, J ) 5.5 Hz, 1
H), 7.43 (d, J ) 8.7 Hz, 1 H), 7.40-7.28 (m, 4 H), 7.23-7.08
(m, 4 H), 6.74 (d, J ) 8.7 Hz, 1 H), 5.06 (d, J ) 12.3 Hz, 1 H,
part of AB), 5.01 (d, J ) 12.6 Hz, 1 H, part of AB), 4.91 (d, J
) 5.7 Hz, 1 H), 4.71 (d, J ) 5.7 Hz, 1 H), 3.90-3.80 (m, 2 H),
3.77 (m, 1 H), 3.53-3.37 (m, 3 H), 3.00 (dd, J ) 13.9, 3.1 Hz,
1 H), 2.88 (dt, J ) 13.5, 6.3 Hz, 1 H), 2.62 (dd, J ) 13.6, 10.0
Hz, 1 H), 2.48-2.40 (m, 2 H), 2.20 (m, 1 H), 1.45-1.32 (m, 2
H), 1.32-1.18 (m, J ) 7.5 Hz, 2 H), 0.92 (s, 9 H), 0.90 (d, J )
10.2 Hz, 3 H), 0.78 (t, J ) 7.5 Hz, 3 H), 0.75 (t, J ) 7.5 Hz, 3
H); 13C NMR (50 MHz, DMSO-d6) 173.9, 171.2, 169.7, 155.8,
139.3, 137.0, 129.3, 128.3, 127.9, 127.8, 127.7, 125.7, 72.3, 66.9,
65.5, 60.8, 53.3, 51.8, 51.0, 42.4, 35.0, 34.2, 32.3, 27.9, 26.9,
26.8, 19.9, 10.5, 10.3; MS (FAB) m/z 641 (MH+); homogeneity
99.82% (system A), 99.67% (system B).
In h ibitor 19: mp 88-92 °C; Rf 0.55 (95:5 EtOAc/MeOH);
[R]23D +4.8° (c 1, MeOH); [R]23365 +114.8° (c 1, MeOH); IR (neat)
3310, 2963, 1652, 1532 cm-1; 1H NMR (400 MHz, DMSO-d6)
8.60 (d, J ) 8.4 Hz, 1 H), 8.49 (d, J ) 9.6 Hz, 1 H), 8.20 (d,
J ) 8.7 Hz, 1 H), 8.16 (d, J ) 8.4 Hz, 1 H), 8.10 (d, J ) 8.4 Hz,
2 H), 7.89 (dt, J ) 7.6, 1.2 Hz, 1 H), 7.75 (dt, J ) 7.5, 1.0 Hz,
1 H), 7.62 (t, J ) 6.3 Hz, 1 H), 7.42 (d, J ) 8.7 Hz, 1 H), 7.20
(d, J ) 7.5 Hz, 2 H), 7.04 (t, J ) 7.5 Hz, 2 H), 6.88 (t, J ) 7.3
Hz, 1 H), 4.40 (dd, J ) 9.4, 6.4 Hz, 1 H), 3.92 (m, 1 H), 3.50
(m, 3 H), 3.05 (dd, J ) 13.8, 3.0 Hz, 1 H), 2.84 (m, 1 H), 2.60
(dd, J ) 13.5, 10.8 Hz, 1 H), 2.49-2.40 (m, 2 H), 2.20 (m, 1
H), 2.10 (m, 1 H), 1.44-1.16 (m, 4 H), 0.92 (s, 9 H), 0.86 (d, J
) 6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 3 H), 0.78 (t, J ) 7.5 Hz,
3 H), 0.76 (t, J ) 7.5 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6)
173.6, 171.1, 170.0, 163.1, 149.5, 145.8, 139.2, 138.0, 130.6,
129.3, 129.1, 128.9, 128.1, 128.0, 127.7, 125.5, 118.5, 72.4, 57.7,
53.5, 51.8, 51.0, 42.4, 35.2, 34.2, 32.2, 31.3, 27.8, 26.9, 26.8,
19.4, 17.8, 10.4, 10.1; MS (FAB) m/z 660 (MH+); homogeneity
99.74% (system A), 99.40% (system B).
In h ibitor 14: mp 98-100 °C; Rf 0.40 (3:7 hexane/EtOAc);
[R]23 +3.4° (c 1, MeOH); IR (neat) 3316, 2961, 1704, 1650,
D
1538 cm-1; H NMR (400 MHz, DMSO-d6) 7.62 (t, J ) 6.3
1
Hz, 1 H), 7.34-7.11 (m, 11 H), 4.95 (d, J ) 12.9 Hz, 1 H, part
of AB), 4.91 (d, J ) 4.5 Hz, 1 H), 4.87 (d, J ) 12.9 Hz, 1 H,
part of AB), 3.66-3.43 (m, 3 H), 3.36 (dt, J ) 9.9, 6.6 Hz, 1
H), 3.06 (dd, J ) 13.6, 3.1 Hz, 1 H), 2.81-2.73 (m, 1 H), 2.56
(dd, J ) 13.8, 10.8 Hz, 1 H), 2.47 (dd, J ) 10.8, 2.1 Hz, 1 H),
2.22 (dd, J ) 11.7, 2.0 Hz, 1 H), 1.75-1.63 (m, 2 H), 0.94 (s, 9
H), 0.80 (d, J ) 6.6 Hz, 3 H), 0.77 (d, J ) 6.6 Hz, 6 H), 0.73 (d,
J ) 6.9 Hz, 3 H); 13C NMR (50 MHz, DMSO-d6) 173.7, 171.6,
155.7, 139.6, 137.4, 129.2, 128.2, 127.9, 127.5, 127.1, 125.7,
72.8, 64.7, 58.2, 55.6, 51.9, 42.6, 35.3, 34.1, 32.3, 28.8, 28.7,
27.9, 20.2, 20.1, 17.8, 17.5; MS (FAB) m/z 568 (MH+); homo-
geneity 97.34% (system A), 98.69% (system B).
In h ibitor 15: mp 80-84 °C; Rf 0.39 (3:7 hexane/EtOAc);
[R]23 -1.0° (c 1, MeOH); [R]23 +7.0° (c 1, MeOH); IR (neat)
D
365
3309, 2959, 1696, 1661, 1603, 1539 cm-1; 1H NMR (400 MHz,
DMSO-d6) 9.89 (s, 1 H), 7.72 (broad t, J ) 5.4 Hz, 1 H), 7.57
(d, J ) 8.1 Hz, 1 H), 7.32-7.10 (m, 12 H), 7.00 (t, J ) 7.3 Hz,
1 H), 4.95 (d, J ) 12.9 Hz, 1 H, part of AB), 4.88 (d, J ) 6.6
Hz, 1 H), 4.87 (d, J ) 12.9 Hz, 1 H, part of AB), 3.67-3.56 (m,
1 H), 3.56-3.43 (m, 2 H), 3.04 (dd, J ) 11.4, 3.0 Hz, 1 H),
2.90-2.80 (m, 1 H), 2.73 (dd, J ) 14.7, 10.5 Hz, 1 H), 2.60-
2.40 (m, 3 H), 0.97 (s, 9 H); 13C NMR (50 MHz, DMSO-d6)
173.5, 170.6, 155.8, 139.6, 139.3, 137.4, 129.2, 128.6, 128.3,
127.9, 127.4, 127.0, 125.7, 122.9, 119.0, 72.7, 64.7, 55.7, 51.0,
42.5, 35.4, 34.9, 32.3, 28.0; MS (FAB) m/z 546 (MH+); homo-
geneity 99.34% (system A), 99.48% (system B).
In h ibitor 20: mp 134-137 °C; Rf 0.48 (95:5 EtOAc/MeOH);
[R]23 -5.4° (c 1, MeOH); IR (neat) 3313, 2963, 1648, 1528
D
cm-1; 1H NMR (400 MHz, DMSO-d6) 9.51 (s, 1 H), 8.40 (d, J
) 9.3 Hz, 1 H), 8.24 (m, 2 H), 8.08 (d, J ) 9.0 Hz, 1 H), 8.00
(m, 2 H), 7.62 (t, J ) 5.4 Hz, 1 H), 7.43 (d, J ) 8.7 Hz, 1 H),
7.20 (d, J ) 7.5 Hz, 2 H), 7.06 (t, J ) 7.6 Hz, 2 H), 6.87 (t, J
) 7.3 Hz, 1 H), 4.93 (broad s, 1 H), 4.39 (dd, J ) 9.3, 6.9 Hz,
1 H), 4.13 (m, 1 H), 3.55-3.43 (m, 2 H), 3.04 (dd, J ) 13.8, 3.3
Hz, 1 H), 2.84 (m, 1 H), 2.58 (dd, J ) 13.6, 10.6 Hz, 1 H), 2.48-
2.39 (m, 2 H), 2.17 (q, J ) 10.2 Hz, 1 H), 2.09 (m, 1 H), 1.44-
1.17 (m, 4 H), 0.90 (s, 9 H), 0.85 (d, J ) 6.6 Hz, 3 H), 0.83 (d,
J ) 6.6 Hz, 3 H), 0.78 (t, J ) 7.5 Hz, 3 H), 0.74 (t, J ) 7.2 Hz,
3 H); 13C NMR (100 MHz, DMSO-d6) 173.5, 171.1, 169.8,
162.2, 143.6, 143.1, 139.6, 139.2, 132.0, 131.4, 129.5, 129.1,
129.0, 127.7, 125.5, 72.4, 58.0, 53.5, 51.8, 51.0, 42.4, 35.2, 34.2,
32.2, 31.1, 27.8, 26.8, 26.7, 19.3, 18.0, 10.4, 10.1; MS (FAB)
m/z 661 (MH+); homogeneity 95.82% (system A), 97.34%
(system B).
In h ibitor 16: mp 193-196 °C; Rf 0.58 (95:5 EtOAc/MeOH);
[R]23D -14.4° (c 1, MeOH); [R]23365 -45.5° (c 1, MeOH); IR (neat)
3301, 2963, 1699, 1649, 1538 cm-1 1H NMR (400 MHz,
;
DMSO-d6) 7.70 (d, J ) 9.0 Hz, 1 H), 7.60 (t, J ) 6.0 Hz, 1
H), 7.42 (d, J ) 8.7 Hz, 1 H), 7.39-7.28 (m, 5 H), 7.22-7.06
(m, 5 H), 7.00 (d, J ) 9.3 Hz, 1 H), 5.06 (d, J ) 12.6 Hz, 1 H,
part of AB), 5.01 (d, J ) 12.6 Hz, 1 H, part of AB), 4.89 (d, J
) 4.2 Hz, 1 H), 3.89 (m, 1 H), 3.77 (dd, J ) 8.7, 7.5 Hz, 1 H),
3.54-3.40 (m, 3 H), 3.00 (dd, J ) 13.9, 3.1 Hz, 1 H), 2.81 (m,
1 H), 2.59 (dd, J ) 13.8, 10.2 Hz, 1 H), 2.48-2.41 (m, 2 H),
2.18 (q, J ) 10.2 Hz, 1 H), 1.83 (m, 1 H), 1.44-1.16 (m, 4 H),
0.92 (s, 9 H), 0.78 (t, J ) 7.5 Hz, 3 H), 0.75 (t, J ) 7.5 Hz, 3
H), 0.73 (d, J ) 6.3 Hz, 3 H), 0.71 (d, J ) 6.0 Hz, 3 H); 13C
NMR (50 MHz, DMSO-d6) 174.0, 171.2, 170.6, 155.9, 139.3,
137.1, 129.3, 128.3, 127.8, 127.6, 125.6, 72.4, 65.4, 60.4 53.2,
51.9, 51.0, 42.3, 35.0, 34.3, 32.3, 30.4, 27.9, 26.9, 26.8, 19.3,
18.0, 10.5, 10.2; MS (FAB) m/z 639 (MH+); homogeneity 95.61%
(system A), 99.96% (system B).
In h ibitor 21: mp 142-145 °C; Rf 0.24 (95:5 EtOAc/MeOH);
[R]23 -3.4° (c 0.5, MeOH); [R]23 +76.8° (c 0.5, MeOH); IR
D
365
(neat) 3317, 2955, 1696, 1642, 1538 cm-1; 1H NMR (400 MHz,
DMSO-d6) 8.86 (d, J ) 8.4 Hz, 1 H), 8.60 (d, J ) 8.4 Hz, 1
H), 8.20 (d, J ) 8.7 Hz, 1 H), 8.15 (d, J ) 8.4 Hz, 1 H), 8.11 (d,
J ) 8.1 Hz, 1 H), 7.97 (d, J ) 9.0 Hz, 1 H), 7.90 (dt, J ) 7.8,
1.2 Hz, 1 H), 7.74 (dt, J ) 7.5, 0.9 Hz, 1 H), 7.66 (t, J ) 5.7
Hz, 1 H), 7.46 (d, J ) 8.7 Hz, 2 H), 7.20 (d, J ) 7.2 Hz, 2 H),
7.04 (t, J ) 7.5 Hz, 2 H), 6.98-6.92 (m, 2 H), 4.99 (d, J ) 4.8
Hz, 1 H), 4.80 (m, 1 H), 3.89 (m, 1 H), 3.55-3.45 (m, 2 H),
3.43-3.35 (m, 1 H), 3.02 (dd, J ) 13.5, 3.3 Hz, 1 H), 2.96 (dt,
J ) 13.8, 6.4 Hz, 1 H), 2.71-2.56 (m, 3 H), 2.49-2.43 (m, 2
H), 2.21 (q, J ) 10.5 Hz, 1 H), 1.45-1.18 (m, 4 H), 0.93 (s, 9
H), 0.79 (t, J ) 7.5 Hz, 3 H), 0.76 (t, J ) 7.5 Hz, 3 H); 13C
NMR (100 MHz, DMSO-d6) 173.9, 171.7, 171.2, 170.0, 163.2,
149.5, 145.9, 139.1, 137.9, 130.6, 129.2, 129.1, 128.9, 128.2,
128.1, 127.7, 125.5, 118.5, 71.9, 53.6, 51.8, 51.0, 50.1, 42.5, 37.4,
35.2, 34.2, 32.2, 27.9, 26.9, 26.8, 10.4, 10.2; MS (FAB) m/z 675
(MH+); homogeneity 99.94% (system A), 99.82% (system B).
In h ibitor 22: mp 172-175 °C; Rf 0.18 (95:5 EtOAc/MeOH);
In h ibitor 17: mp 193-197 °C; Rf 0.27 (95:5 EtOAc/MeOH);
[R]23D -22.8° (c 1, MeOH); [R]23365 -79.0° (c 1, MeOH); IR (neat)
3300, 2962, 1652, 1538 cm-1; 1H NMR (400 MHz, DMSO-d6)
7.73 (d, J ) 9.0 Hz, 1 H), 7.62 (t, J ) 5.5 Hz, 1 H), 7.46 (d,
J ) 8.7 Hz, 1 H), 7.39-7.28 (m, 4 H), 7.25 (s, 1 H), 7.24-7.08
(m, 6 H), 6.87 (s, 1 H), 5.05 (d, J ) 12.6 Hz, 1 H, part of AB).
5.01 (d, J ) 12.6 Hz, 1 H, part of AB), 4.96 (d, J ) 4.8 Hz, 1
H), 4.30 (m, 1 H), 3.81 (m, 1 H), 3.56-3.42 (m, 2 H), 3.40-
3.13 (m, 1 H), 3.00 (dd, J ) 13.6, 3.1 Hz, 1 H), 2.89 (dt, J )
13.5, 6.9 Hz, 1 H), 2.65 (dd, J ) 13.6, 10.1, Hz, 1 H), 2.49-
2.41 (m, 2 H), 2.40-2.16 (m, 2 H), 1.38 (m, 2 H), 1.28 (m, J )
7.2 Hz, 2 H), 0.92 (s, 9 H), 0.78 (t, J ) 7.5 Hz, 3 H), 0.76 (t, J
) 7.5 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6) 173.9, 171.4,
171.2, 170.8, 155.5, 139.3, 136.9, 129.3, 128.3, 127.8, 127.7,
125.6, 72.1, 65.5, 53.4, 51.8, 51.0, 42.4, 37.5, 35.0, 34.2, 32.3,
27.9, 26.9, 26.8, 10.4, 10.2; MS (FAB) m/z 654 (MH+); homo-
geneity 100% (system A), 99.52% (system B).
[R]23 -8.2° (c 1, MeOH); IR (neat) 3304, 2962, 1656, 1534
D

Potent HIV Protease Inhibitors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 14 2175
cm-1; 1H NMR (400 MHz, DMSO-d6) 9.50 (s, 1 H), 8.89 (d, J
) 8.4 Hz, 1 H), 8.23 (m, 2 H), 8.01 (m, 2 H), 7.93 (d, J ) 9.0
Hz, 1 H), 7.66 (t, J ) 5.7 Hz, 1 H), 7.45 (d, J ) 8.7 Hz, 2 H),
7.20 (d, J ) 7.2 Hz, 2 H), 7.07 (t, J ) 7.6 Hz, 2 H), 6.94 (m, 2
H), 4.97 (d, J ) 4.8 Hz, 1 H), 4.81 (m, 1 H), 3.87 (m, 1 H),
3.54-3.43 (m, 2 H), 3.42-3.35 (m, 1 H), 3.02 (dd, J ) 13.5,
3.3 Hz, 1 H), 2.95 (dt, J ) 13.5, 6.3 Hz, 1 H), 2.72-2.56 (m, 3
H), 2.50-2.40 (m, 3 H), 1.98 (dt, J ) 11.1, 8.7 Hz, 1 H), 1.44-
1.32 (m, 2 H), 1.25 (m, J ) 7.5 Hz, 2 H), 0.91 (s, 9 H), 0.77 (t,
J ) 7.5 Hz, 3 H), 0.75 (t, J ) 7.5 Hz, 3 H); 13C NMR (100
MHz, DMSO-d6) 173.8, 171.7, 171.1, 169.8, 162.4, 143.6,
143.5, 143.0, 139.6, 139.2, 132.0, 131.4, 129.4, 129.2, 129.1,
127.7, 125.5, 71.9, 53.6, 51.7, 51.0, 50.1, 42.5, 37.1, 35.1, 34.2,
32.2, 27.8, 26.8, 26.7, 10.4, 10.2; MS (FAB) m/z 676 (MH+);
homogeneity 99.05% (system A), 97.73% (system B).
(3) Romero, D. L. Advances in the development of HIV reverse
transcriptase inhibitors. Annu. Rep. Med. Chem. 1994, 29, 123-
132.
(4) (a) Sarin, P. S.; Goldstein, A. L. Treatment of AIDS with drugs
targeted to inhibit different stages of the HIV life cycle. Im-
munopharmacol. Immunotoxicol. 1995, 17, 217-245. (b) Rich-
man, D. D. HIV Therapeutics. Science 1996, 272, 1886. (c)
Daquila, R. T.; Hughes, M. D.; J ohnson, V. A.; Fischl, M. A.;
Sommadossi, J . P.; Liou, S. H.; Timpone, J .; Myers, M.; Basgoz,
N.; Niu, M.; Hirsch, M. S.; Costanzo, L.; Ruben, S.; Berzins, B.;
Martinez, A.; Fishman, I.; Kazial, K.; Cort, S. N.; Robinson, P.;
Hall, D.; Macy, H.; Mclaren, C.; Rooney, J .; Warwick, J .;
Cavaillecoll, M.; et al. Nevirapine, zidovudine, and didanosine
compared with zidovudine and didanosine in patients with
HIV-1 infection. A randomized, double-blind, placebo-controlled
trial. Ann. Intern. Med. 1996, 124, 1019-1030.
(5) (a) Darke, P. A.; Huff, J . R. HIV protease as an inhibitor target
for the treatment of AIDS. Adv. Pharmacol. 1994, 25, 399-
454. (b) Darke, P. A.; Nutt, R. F.; Brady, S. F.; Garsky, V. M.;
Ciccarone, T. M.; Leu, C.-T.; Lumma, P. K.; Freidinger, R. M.;
Veber, D. F.; Sigal, I. S. HIV-1 Protease specificity of peptide
cleavage is sufficient for processing of gag and pol polyproteins.
Biochem. Biophys. Res. Commun. 1988, 156, 297-303.
(6) (a) Khol, N. E.; Emini, E. A.; Schleif, W. A.; Davis, L. J .;
Heimbach, J . C.; Dixon, R. A. F.; Scolnick, E. M.; Sigal, I. S.
Active HIV-1 protease is required for viral infectivity. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 4686-4690. (b) Kramer, R. A.;
Schaber, M. D.; Skalka, A. M.; Ganguly, K.; Wong-Staal, F.;
Reddy, E. P. HTLV-111 gag Protein is processed in yeast cells
by the virus pol-protease. Science 1986, 231, 1580-1584.
(7) (a) Boehme, R. E.; Borthwick, A. D.; Wyatt, P. G. Antiviral
agents. Ann. Rep. Med. Chem. 1995, 30, 139-149. (b) Thais-
rivongs, S. HIV protease inhibitors. Annu. Rep. Med. Chem.
In h ibitor 23: mp 105-109 °C; Rf 0.46 (95:5 EtOAc/MeOH);
[R]23 +19.7° (c 1, MeOH); IR (neat) 3318, 2964, 1654, 1532
D
1
cm-1; H NMR (400 MHz, DMSO-d6) 8.67 (d, J ) 8.7 Hz, 1
H), 8.60 (d, J ) 8.4 Hz, 1 H), 8.21 (d, J ) 8.4 Hz, 1 H), 8.17 (d,
J ) 8.4 Hz, 1 H), 8.11 (d, J ) 7.8 Hz, 1 H), 7.97 (d, J ) 9.0 Hz,
1 H), 7.89 (dt, J ) 7.6, 1.2 Hz, 1 H), 7.74 (dt, J ) 7.6, 0.9 Hz,
1 H), 7.68 (t, J ) 5.4 Hz, 1 H), 7.47 (d, J ) 8.7 Hz, 1 H), 7.23
(d, J ) 7.2 Hz, 2 H), 7.14 (t, J ) 7.6 Hz, 2 H), 7.03 (t, J ) 7.3
Hz, 1 H), 5.10 (d, J ) 5.4 Hz, 1 H), 4.97 (d, J ) 4.5 Hz, 1 H),
4.41 (dd, J ) 8.7, 4.2 Hz, 1 H), 4.13 (m, 1 H), 3.93 (m, 1 H),
3.57-3.43 (m, 3 H), 3.06 (dd, J ) 13.8, 3.3 Hz, 1 H), 2.94 (m,
1 H), 2.65 (dd, J ) 13.8, 10.2 Hz, 1 H), 2.47 (m, 2 H), 2.22 (dt,
J ) 10.8, 5.4 Hz, 1 H), 1.45-1.18 (m, 4 H), 1.05 (d, J ) 6.3
Hz, 3 H), 0.93 (s, 9 H), 0.78 (t, J ) 7.5 Hz, 3 H), 0.76 (t, J )
7.5 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6) 173.9, 171.1,
169.3, 163.4, 150.0, 145.9, 139.2, 138.0, 130.6, 129.2, 128.9,
128.2, 128.1, 127.8, 125.6, 118.5, 72.4, 66.9, 58.2, 53.4, 51.7,
51.0, 42.5, 35.1, 34.2, 32.3, 27.8, 26.8, 26.7, 20.1, 10.4, 10.2;
MS (FAB) m/z 662 (MH+); homogeneity 96.89% (system A),
96.86% (system B).
1994, 29, 133-144. (c) Huff, J . R. HIV Protease:
a novel
chemotherapeutic target for AIDS. J . Med. Chem. 1991, 34,
2305-2314. (d) Kempf, D. J .; Sham, H. L. HIV protease
inhibitors. Curr. Pharm. Des. 1996, 2, 225-246.
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HIV-1 protease. Annu. Rev. Biochem. 1993, 62, 543-585.
(9) (a) Rich, D. H.; Green, J .; Toth, M. V.; Marshall, E. G.; Kent, S.
B. Hydroxyethylamine analogs of the p17/p24 substrate cleavage
site are tight binding inhibitors of HIV protease. J . Med. Chem.
1990, 33, 1285-1288. (b) Roberts, N. A.; Martin, J . A.; Kirch-
ington, D.; Broadhurst, A. V.; Craig, J . C.; Duncan, I. B.; Galpin,
S. A.; Handa, B. K.; Kay, J .; Krohn, A.; Lambert, R. W.; Merrett,
J . H.; Mills, J . S.; Parkes, K. E. B.; Redshaw, S.; Ritchie, A. J .;
Taylor, D. L.; Thomas, G. J .; Machin, P. S. Rational design of
peptide-based HIV proteinase inhibitors. Science 1990, 248,
358-361. (c) Tucker, T. J .; Lumma, W. C., J r.; Payne, L. S.;
Wai, J . M.; de Solms, S. J .; Giuliani, E. A.; Darke, P. A.;
Heimbach, J . C.; Zugay, J . A.; Schleif, W. A.; Quintero, J . C.;
Emini, E. A.; Huff, J . R.; Anderson, P. S. A series of potent HIV-1
protease inhibitors containing a hydroxyethyl secondary amine
transition state isostere: synthesis, enzyme inhibition, and
antiviral activity. J . Med. Chem. 1992, 35, 2525-2533. (d)
Ghosh, A. K.; Lee, H. Y.; Thompson, W. J .; Culberson, C.;
Holloway, M. K.; McKee, S. P.; Munson, P. M.; Duong, T. T.;
Smith, A. M.; Darke, P. L.; Zugay, J . A.; Emini, E. A.; Schleif,
W. A.; Huff, J . R.; Anderson, P. S. The development of cyclic
sulfolanes as novel and high-affinity P2 ligands for HIV-1
protease inhibitors. J . Med. Chem. 1994, 37, 1177-1188 and
references cited therein. (e) Ghosh, A. K.; Thompson, W. J .;
Fitzgerald, P. M. D.; Culberson, J . C.; Axel, M. G.; McKee, S.
P.; Huff, J . R.; Anderson, P. S. Structure-based design of HIV-1
In h ibitor 24: mp 109-113 °C; Rf 0.40 (95:5 EtOAc/MeOH);
[R]23 +14.9° (c 1, MeOH); IR (neat) 3317, 2964, 1654, 1527
D
cm-1; 1H NMR (400 MHz, DMSO-d6) 9.50 (s, 1 H), 8.52 (d, J
) 8.7 Hz, 1 H), 8.21 (m, 2 H), 7.99 (m, 3 H), 7.67 (t, J ) 6.0
Hz, 1 H), 7.44 (d, J ) 8.7 Hz, 1 H), 7.21 (d, J ) 7.2 Hz, 1 H),
7.13 (t, J ) 7.5 Hz, 2 H), 6.99 (t, J ) 7.3 Hz, 1 H), 5.09 (d, J
) 5.4 Hz, 1 H), 4.95 (d, J ) 4.5 Hz, 1 H), 4.41 (dd, J ) 8.7, 4.5
Hz, 1 H), 4.10 (m, 1 H), 3.91 (m, 1 H), 3.53-3.40 (m, 3 H),
3.05 (dd, J ) 13.6, 3.0 Hz, 1 H), 2.92 (m, 1 H), 2.63 (dd, J )
13.8, 10.2 Hz, 1 H), 2.45 (m, 2 H), 2.20 (dt, J ) 10.8, 8.4 Hz,
1 H), 1.44-1.31 (m, 2 H), 1.31-1.16 (m, J ) 7.5 Hz, 2 H), 1.03
(d, J ) 6.3 Hz, 3 H), 0.90 (s, 9 H), 0.77 (t, J ) 7.5 Hz, 3 H),
0.75 (t, J ) 7.5 Hz, 3 H); 13C NMR (100 MHz, DMSO-d6)
173.9, 171.1, 169.0, 162.4, 143.5, 143.1, 139.6, 139.2, 132.0,
131.4, 129.4, 129.2, 129.1, 127.8, 125.6, 72.4, 66.9, 58.4, 53.4,
51.7, 51.0, 42.5, 35.1, 34.2, 32.3, 27.8, 26.8, 26.7, 20.1, 10.4,
10.2; MS (FAB) m/z 663 (MH+); homogeneity 98.76% (system
A), 96.51% (system B).
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Ack n ow led gm en t. We are grateful to Elizabeth
Wardrop and Chantal Clouette for technical assistance
in running assays. We would also like to thank Sylvain
Bordeleau, Colette Boucher and Serge Valois for ana-
lytical support and Dr. J orge J aramillo, Dr. Sopone
Nawoot and Elise Ghiro for their involvement in bio-
availability measurements.
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any current masthead page.
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