Synthesis and biological activity of potent HIV-1 protease inhibitors based on Phe-Pro dihydroxyethylene isosteres

Article abstract of DOI:10.1021/jm3001136

Peptidomimetic inhibitors of HIV-1 PR are still a key resource in the fight against AIDS. Here we describe the synthesis and biological activity of HIV-1 PR inhibitors based on four novel dihydroxyethylene isosteres of the Phe-Pro and Pro-Pro dipeptides. The isosteres, containing four stereogenic centers, were synthesized in high yield and excellent stereoselectivity via the cyclization of epoxy amines derived from α-amino acids. The inhibitors were assembled by coupling the isosteres with suitable flanking groups and were screened against recombinant HIV PR showing activities in the subnanomolar to micromolar range. Two Phe-Pro-based inhibitors active at the nanomolar level were further investigated: both inhibitors combine the ability to suppress HIV-1 replication in infected MT-2 cells with low cytotoxicity against the same cells, thereby displaying a high therapeutic index. These results demonstrate the potential of the new Phe-Pro dihydroxyethylene isostere as a core unit of powerful HIV-1 PR inhibitors.

Full text of DOI:10.1021/jm3001136

Article
pubs.acs.org/jmc
Synthesis and Biological Activity of Potent HIV-1 Protease Inhibitors
Based on Phe-Pro Dihydroxyethylene Isosteres
Fabio Benedetti,*,† Federico Berti,*,† Sara Budal,† Pietro Campaner,† Francesca Dinon,†
Alessandro Tossi,‡ Radka Argirova,§ Petia Genova,§ Vasil Atanassov,# and Anton Hinkov⊥
†Department of Chemical and Pharmaceutical Sciences and ‡Department of Life Sciences, University of Trieste, via Giorgieri 1. 34127
Trieste, Italy
§National Center for Infectious and Parasitic Diseases, Department of Virology, Sofia 1233, Bulgaria
⊥
#Laboratory of Bioocordination and Bioanalytical Chemistry and Faculty of Chemistry and Pharmacy, St. Kliment Ohridski Sofia
University, Sofia, Bulgaria
S
* Supporting Information
ABSTRACT: Peptidomimetic inhibitors of HIV-1 PR are still a key resource in the fight against AIDS. Here we describe the
synthesis and biological activity of HIV-1 PR inhibitors based on four novel dihydroxyethylene isosteres of the Phe-Pro and Pro-
Pro dipeptides. The isosteres, containing four stereogenic centers, were synthesized in high yield and excellent stereoselectivity
via the cyclization of epoxy amines derived from α-amino acids. The inhibitors were assembled by coupling the isosteres with
suitable flanking groups and were screened against recombinant HIV PR showing activities in the subnanomolar to micromolar
range. Two Phe-Pro-based inhibitors active at the nanomolar level were further investigated: both inhibitors combine the ability
to suppress HIV-1 replication in infected MT-2 cells with low cytotoxicity against the same cells, thereby displaying a high
therapeutic index. These results demonstrate the potential of the new Phe-Pro dihydroxyethylene isostere as a core unit of
powerful HIV-1 PR inhibitors.
HIV-1 PR is a dimeric aspartyl protease consisting of two
identical monomers of 99 residues.5 The presence of a two-fold
symmetry axis in the homodimer inspired the development
of C2-symmetric inhibitors, and interest has focused on
peptidomimetics based on the Phe-Phe isostere 16 (Figure 1)
INTRODUCTION
■
Since the appearance of saquinavir in clinical practice in 1995,
inhibitors of the protease of human type-1 immune deficiency
virus (HIV-1 PR) have represented the key weapon in the
battle against AIDS pandemics.1 HIV-PR is responsible for the
cleavage of the viral polyprotein precursor to give structural
proteins and three viral enzymes. Inhibition of the protease
blocks viral replication and maturation, resulting in lowering of
the viral load and a general improvement in the clinical state of
AIDS patients. The introduction of first generation inhibitors
ritonavir, nelfinavir, and indinavir has changed the nature of the
AIDS epidemics from a terminal illness to a manageable one.2
More recently, new protocols based on potent reverse
transcriptase (RT) inhibitors have become available;3 never-
theless, the development of potent protease inhibitors, such as
darunavir4 currently used in combination with reverse tran-
scriptase inhibitors, confirms that protease inactivation remains
an essential therapeutic tool. However, the long-term efficacy of
PR-targeted therapies is limited by the onset of drug resistance
due to mutant viral strains; thus, the need still exists for the
development of new, efficient inhibitors.
Figure 1. Phe-Phe and Phe-Pro isosteres.
for which several synthetic approaches have been developed.7,8
Potent inhibitors have been obtained from both (S,S,S,S) and
(S,R,R,S) stereoisomers of 1.6−9
Received: January 26, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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Despite the C2 symmetry of the protease, it has been observed
that nonsymmetrical inhibitors often perform better than symmet-
rical ones. The superior activity of the nonsymmetric inhibitor
may reflect the local lack of symmetry at the catalytic aspartate
residues, which are believed to be in different protonation states
in the active enzyme.10 The diol isostere might then be forced
to establish nonsymmetrical interactions with the two aspartate
residues, thus making symmetry at the core level unimportant.
Accordingly, in the solid state, symmetrical inhibitors are often
found to bind HIV-Pr in a nonsymmetric fashion.11
There are several ways in which the symmetry of the core
unit 1 can be perturbed (Figure 1). Removal of a hydroxy group
leads to the hydroxyethylene isostere 2. Ritonavir and lopinavir
are significant examples of inhibitors containing this Phe-Phe iso-
stere.12 Another possibility is to replace one of the benzyl groups
of 1 with the side chain of a different amino acid. HIV-PR can
recognize Phe-Pro and Tyr-Pro dipeptides as the specific cleavage
site, and this specificity can been exploited in the design of
selective inhibitors.13 Replacement of a benzyl group of 1 with the
side chain of proline thus leads to the Phe-Pro isostere 3.
was used as the nucleophilic partner in a Horner−Emmons
olefination with the orthogonally protected 2-hydroxypyr-
rolidine 7a (Scheme 2), a synthetic equivalent of 4-amino-
butanal. 7a, in turn, was obtained by diisobutylaluminium hydride
(DIBALH) reduction of the corresponding N-Cbz-lactam.15
The Horner−Emmons reaction requires carefully controlled
conditions to avoid aza-Michael cyclization of the product 8a, giv-
ing pyrrolidine 9 as a 1:1 mixture of diastereoisomers (Scheme 2).
This side reaction occurs in ethanol with potassium carbonate
as a base, and in THF with potassium tert-butoxide or 1,8-diaza-
bicycloundec-7-ene (DBU)/lithium chloride. On the contrary,
if the reaction is carried out in acetonitrile and potassium car-
bonate16 and is stopped at 75% conversion, the required enone
8a can be readily isolated and purified. The 15.6 Hz coupling
constant between the vinyl protons confirms the E stereo-
chemistry of the enone. Chelation-controlled reduction of 8a
with lithium tri-tert-butoxyaluminium hydride (LTBAH) in
ethanol17 gives the allylic alcohol 10a in 48% yield from 6. The
synthesis proceeds with peracid epoxidation of 10a, giving the
syn-2,3-epoxy alcohol 11a as a single diastereoisomer. Finally,
removal of the Cbz protecting group by catalytic hydrogenation
gave the corresponding free amine which spontaneously
cyclized onto the epoxide by a 5-exo process, as predicted,14
giving the mono-Boc-protected isostere 3a as a single diastereo-
isomer, in 31% overall yield from the starting phosphonate 6
(Scheme 2). A comparable yield has been obtained when the
synthesis was performed on a 100 g scale.
The objective of this study was to investigate the synthesis of
nonsymmetric HIV-1 PR inhibitors based on the novel diamino
diol Phe-Pro isostere 3 and to compare their biological activities
with those of the corresponding inhibitors containing the C2-
symmetric Phe-Phe isostere 1 described earlier.9 To this end,
we have synthesized three nonsymmetric Phe-Pro isosteres
(3a−c) based on the general structure 3 (Scheme 1). The
The six-membered proline isostere 3b was similarly obtained,
starting from N-Cbz-2-hydroxypiperidine (7b) (Scheme 2).18
Also in this case cyclization was completely regioselective, tak-
ing place exclusively in the 6-exo mode.
Scheme 1. Synthesis of Phe-Pro Isosteres by Epoxy Amine
Cyclization
The S,R,R,R diastereoisomer 3c was synthesized by a
modification of this approach (Scheme 3). The S,S alcohol
10c was obtained by reduction of the enone 8a with L-
Selectride in methanol. It has been demonstrated that, in polar
solvents, L-Selectride reduction of amino ketones gives
preferentially the Felkin−Anh product.19 Although the stereo-
selectivity was only 3:1 in favor of the desired product, the
required alcohol 10c was easily separated from the S,R isomer
by flash chromatography. Epoxidation of 10c and cyclization of
the epoxy amine 11c, under the usual conditions for N-Cbz
deprotection, gave the S,R,R,R isostere 3c.
The symmetrical, monoprotected, Pro-Pro isostere 5 was
synthesized by the approach already described for the Phe-Pro
isosteres 3a,b (Scheme 4). The proline-derived phosphonate 12
was obtained by the reaction of Boc-protected proline methyl
ester with lithiated methyl dimethylphosphonate. Horner−Emmons
reaction of phosphonate 12 was carried out in acetonitrile, to
prevent the unwanted in situ aza-Michael cyclization of the
enone 13, which did not exceed 8% under these conditions
(Scheme 4). Reduction of the carbonyl of 13, however, proved
much more difficult than expected, most likely due to the hin-
drance of the Boc-protected five-membered ring. Addition of
sodium borohydride led to the reduction of the C−C double
bond; addition of cerium chloride restored the desired chemo-
selectivity20 but without any stereoselectivity. After unsuccessful
attempts with lithium borohydride and LTBAH, the alcohol 14
was eventually obtained, as a single stereoisomer, by reduction
with DIBALH in toluene. The S,R stereochemistry was con-
firmed by NMR analysis of the corresponding oxazolidinone
16. The value of 7.3 Hz for the coupling constant between the
vicinal ring protons and 6% NOE measured for the same pro-
tons are both consistent with a syn arrangement of the protons
synthesis of the isosteres relies on the cyclization of
phenylalanine-derived epoxy amines (4) for the generation of
a five- or six-membered proline mimic, as shown in the scheme
for 3a,b.14 The same approach has also been applied to the
synthesis of the monoprotected Pro-Pro isostere 5. A series of
powerful HIV-PR inhibitors with IC50 in the nanomolar and
subnanomolar range has been obtained by combining isosteres
3 and 5 with selected peptide or nonpeptide flanking residues.
We will show that inhibitors containing the nonsymmetric
isostere 3a are superior to the corresponding inhibitors based
on the symmetric isosteres 1 and 5.
RESULTS AND DISCUSSION
■
Synthesis of the Isosteres. In our synthesis of Xaa-Pro
diamino diol isosteres,14 the carbon skeleton of the isosteres was
assembled by the Horner−Emmons reaction between an amino
acid-derived phosphonate and a linear aldehyde containing an
ω-carboxylate group which was later converted into amine by a
Curtius rearrangement. This approach has been modified to
reduce the number of steps and avoid temporary protections
along the pathway. Thus, phenylalanine-derived phosphonate 68
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a
Scheme 2. Synthesis of Isosteres 3a,b
a
Reagents and conditions: (a) K2CO3, CH3CN, 25 °C, 50% (for 8a); (b) DBU, LiCl, CH3CN, 25 °C, 65% (for 8b); (c) LTBAH, EtOH, −78 °C,
94−96%; (d) mCPBA, CH2Cl2, 51−68%; (e) H2, 10% Pd−C, MeOH, 25 °C, 95−96%.
a
Synthesis of the Inhibitors. Both symmetrically and
unsymmetrically substituted peptidomimetic inhibitors can be
obtained from isosteres 3a−c and 5 by appropriate manage-
ment of the unprotected and protected amino groups. To test
the activity of inhibitors based on these new cores, a small set of
10 inhibitors was synthesized by coupling the isosteres with
selected peptide or nonpeptide carboxylic acids (Chart 1). The
peptide residues of inhibitor 17 (Chart 1) correspond to a se-
quence recognized by HIV-PR already exploited for the develop-
ment of potent inhibitors.22 Similarly, the AcTrp-Val dipeptide
present in inhibitors 18 and 19 was shown to be particularly effi-
cient when combined with a Phe-Phe dihydroxyethylene isostere,
as in inhibitor 27.9 In inhibitors 19−26 the dihydroxyethylene
core is combined with derivatives of phenoxyacetic acid, a ligand
specifically targeted at the enzyme’s S2 pocket.23
Scheme 3. Synthesis of Isostere 3c
Unsymmetrically substituted inhibitors 17, 19, 22 were
obtained by acylation of the unprotected secondary amino group of
the isostere 3, followed by deprotection and acylation of the primary
amine (Scheme 5: method A). The symmetrically substituted inhib-
itors 18a, 20, 21, 23a−26 were obtained by Boc deprotection of
the corresponding isostere followed by the simultaneous acylation
of the primary and secondary amino groups (Scheme 5: method B).
a
Reagents and conditions: (a) L-Selectride, MeOH, −78 °C, 61%; (b)
mCPBA, CH2Cl2, 0 °C, 72%; (c) H2, Pd−C 5%, MeOH, 25 °C, 92%.
and therefore with the S,R configuration of 14.21 Epoxidation
and deprotection/cyclization of the epoxy amine 15 finally gave
the S,S,S,S Pro-Pro isostere 5.
a
Scheme 4. Synthesis of Pro-Pro Isostere 5
a
Reagents and conditions: (a) LiCH2P(O)(OCH3)2, THF, −78 °C, 70%; (b) N-Cbz-2-hydroxypyrrolidine (7a), K2CO3, CH3CN, 25 °C, 76%;
(c) DIBALH, toluene, −78 °C, 51%; (d) mCPBA, CH2Cl2, 0 °C, 22%; (e) H2, 5% Pd−C, CH3OH, 25 °C, 95%; (f) NaH, THF, 25 °C
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a
Chart 1. HIV-Pr Inhibitors Based on Phe-Pro and Pro-Pro Dihydroxyethylene Isosteres
a
Phe-Phe-based inhibitors 27 and 28 are included for comparison.
Acylations were carried out under standard peptide coupling con-
ditions. Assembly of the six-membered inhibitors 18b and 23b
was more difficult, as the piperidine nitrogen of the core unit 3b
(Scheme 5, n = 2) is far less reactive than that of its pyrrolidine
analogues 3a and 3c (n = 1). Thus, inhibitor 18b was obtained
from deprotected 3b with the mixed anhydride method, using
the AcTrpValCOOH peptide and isobutyl chloroformate
(method C); 23b was obtained by acylation of deprotected
3b with 2,6-dimethylphenoxyacetyl chloride (method D).23d
HIV-PR Inhibition and Structure−Activity Relation-
ships. IC50 values (chart 1) were determined at pH 5.5 using a
commercial recombinant wild-type HIV-1 PR and the fluorogenic
substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-NH2
(Abz-NF*-6).
and S3, S3′ subsites (17, 18). Inhibitor 17 is more potent than
its analogue JG-365 based on a hydroxyethylamine Phe-Pro
isostere,22 and the efficacy of the Phe-Pro core 3a is confirmed
by inhibitor 18a, the most powerful compound within this set.
Its IC50 lies below the limit of our test24 but is at least 1 order of
magnitude lower than that of the corresponding inhibitor 27,
based on a Phe-Phe dihydroxyethylene isostere.9 Inhibitors
20−26, containing substituted phenoxyacetic acid residues, can
only engage the enzyme’s S1, S2, S1′, S2′ subsites;23a−c never-
theless, they display activities in the nanomolar to micromolar
range. The effect of substitution on the phenoxyacetyl residue
can be assessed on inhibitors 20−24, based on the same Phe-
Pro core. Comparison of inhibitors 20 and 21 indicates that the
introduction of a single ortho methyl group has no effect on the
inhibitor’s activity. On the contrary, replacement of POA with
2,6-dimethylphenoxyacetic acid (DMPOA), as in 22, results in
a 10-fold increase of activity, and a similar improvement is also
Flanking Residues. Powerful, nanomolar HIV-1 PR
inhibitors are obtained when the S,S,S,S isosteres 3a,b are com-
bined with peptide chains that can occupy the protease’s S2, S2′
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a
Scheme 5. Synthesis of Inhibitors 17−25
Figure 3. Detail of the X-ray crystal structure of the complex of HIV-1
PR with inhibitor 19a,25 showing the interactions of the inhibitor with
the S1 (red) and S1′ (yellow) pockets.
a
Reagents and conditions: method A: (a) RCOOH, THF, EDC,
HOBT, 0−25 °C; (b) i: TFA, DCM, 25 °C; ii: R′COOH, THF, EDC,
HOBT, 0−25 °C; method B: as in step b of method A; method C: i:
TFA, DCM, 25 °C; ii: RCOOH, isobutyl chloroformate, NMM,
EtOAc, 25 °C; method D: i: TFA, DCM, 25 °C; ii: RCOCl, EtOAc,
25 °C.
by the pyrrolidine ring (middle right). Larger groups in this
position should allow further hydrophobic interactions to be
established with the subsite and improve the inhibitor’s binding
properties. However, simple expansion of the pyrrolidine ring
to piperidine, as in 19b, does not improve the inhibitor’s
activity: on the contrary, 19b is less active than 19a. Probably
the small increase in the size of the heterocycle is overcome by
the rigidity of the six-membered ring, preventing optimal fit of
the latter with the S1′ subsite. Introduction of hydrophobic
substituents on selected positions of the more flexible pyrro-
lidine ring may provide a better approach to more efficient
inhibitors.
observed when the second POA group is replaced by DMPOA,
as in 23. A superimposition of the minimum energy confor-
mations of inhibitors 20, 21, and 23, obtained by molecular
mechanics (Figure 2), indicates that the phenoxy rings of 20
Stereochemistry. A comparison between 23a and its
(S,R,R,R) isomer 25 reveals a dramatic drop of activity by 3
orders of magnitude. Changes in the configuration of the
hydroxymethyne carbons in hydroxy- and dihydroxyethylene
inhibitors of HIV-PR do not generally have such a large effect
on the inhibitors’ activity.22,26 The lower activity of 25 is most
likely related to the inversion at the pyrrolidine ring carbon,
suggesting that it is essential that proline’s original config-
uration is preserved in the isostere.
Symmetry. Inhibitors 23a, 26, and 28 (Chart 1) have
identical DMPOA flanking groups and configuration but
different substituents in the dipeptide isostere core. Although
26 (based on a Pro-Pro isostere) and 28 (based on a Phe-Phe
isostere) possess the same C2 symmetry as the target enzyme,
nonsymmetric inhibitor 23a performs better.
It was mentioned earlier that the preference for nonsym-
metric inhibitors may reflect the intrinsic lack of symmetry of
the enzyme−inhibitor complex.10,11 However, a dynamic effect
may also operate. The enzyme’s binding site is closed by two
symmetric loops (flaps) that encompass the ligand upon bind-
ing. The flaps can adopt open,27 semiopen,28 and closed con-
formations, the latter being generally adopted in the complexes
with small peptides and inhibitors.5a Opening of the flaps is
necessary for binding the large viral polyproteins, but it is still
debated whether the binding of small molecules occurs via the
open form and whether it is a single or a multistep process.29
Recently, the binding of a pentapeptide substrate to HIV-Pr has
been studied by molecular dynamics,30 and it has been found
that, in the main binding pathway, the flaps do not open. The
ligand enters the tight catalytic channel with a preferred orien-
tation, and at least two nonsymmetric intermediates are formed
during the slow rearrangement toward the Michaelis complex.
Nonsymmetric species may thus have a kinetic advantage over
symmetric ones, in the steps preceding the formation of the
final complex.
Figure 2. Superimposition of the minimum energy conformations of
inhibitors 20 (green), 21 (pink), and 23 (yellow) obtained by
molecular mechanics with the MMFF force field.
and 21 adopt a similar orientation, while in 23 the rings are
locked by the two methyl groups in an orthogonal
conformation close to the bioactive conformation adopted by
this ligand in complexes with HIV-PR.23a To adopt a similar
conformation, the 2-methylphenoxyacetyl ligand of inhibitor 21
is expected to pay a significant entropic penalty that compen-
sates for the enthalpic advantage resulting from hydrophobic
interactions between the methyl group of the ligand and
residues in the S2/S2′ pockets. The high efficiency of DMPOA
as S2 ligand is also confirmed by nanomolar inhibitor 19 in
which this residue is present on the primary amino group, while
the acetylated Val-Trp dipeptide is linked to the secondary
amino group. On the contrary, introduction of an acetyl group
in the 4-position of the POA ring, as in 24, confirms that
electron-withdrawing substituents in this position depress the
activity, as already observed in a series of inhibitors based on a
Phe-Phe hydroxyethylene core.23b
Ring Size. Inhibitor 19a was cocrystallized with HIV-PR, and
the X-ray crystal structure of the complex was obtained at 1.3 Å
resolution.25 This structure (Figure 3) shows that the
inhibitor’s benzyl side chain (middle left) rests nicely in the
enzyme’s S1 subsite, while the S1′ subsite is only partially filled
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Antiviral Activity and Cell Toxicity. Following the
remarkable activity of several of the tested inhibitors toward
isolated HIV-PR, we further investigated the antiviral activity
Table 2. Cytotoxicity (CC50) and Maximum Nontoxic
Concentrations (MNC) of 18a and 23a in MT-2 Cells for 72
a
and 96 h Cultivation
of the most promising inhibitors 18a and 23a on HIV-1IIIB
infected MT-2 cells.
-
MNC (μM)
72 h
0.01
0.0001
CC50 (μM)
inhibitor
96 h
72 h
96 h
The anti-HIV effect of the two inhibitors has been evaluated
by a rapid and sensitive in vitro microtiter infection assay based
on cytolysis quantitation by vital dye 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake as an
end point for infection.31 The effect of inhibitors on
endogenous reverse transcriptase activity of HIV-1IIIB-infected
MT-2 supernatants was considered as an additional marker
for their ability to block HIV-1 replication. MT-2 cells were
infected and incubated with 18a and 23a for 72−96 h, and then
reverse transcriptase activity was measured in the supernatants.
To protect the infected cells from cytolysis directly induced
from the virus, a multiplicity of infection (MOI) of 0,1 was
applied for the test.31 Results are in Table 1.
18a
23a
0.0003
0.0001
0.5
2500
1000
300
a
Mean values of six to eight parallels per experiment with standard
deviation ≤10%; each experiment was run in triplicate (see Supporting
Information).
The two inhibitors express a different degree of cytotoxicity in
the tested cells, and this phenomenon is dose-dependent. After a
prolonged exposure (96 h), the cytotoxicity of 18a measured by
MNC increases up to 33 times and about 5000 times by CC50,
while no significant differences in MNC are observed with 23a at
different incubation times.
CONCLUSIONS
■
Table 1. Antiviral Activity of Inhibitors 18a and 23a
We recently introduced epoxy amine cyclization as a versatile
approach for the synthesis of diamino diol Xaa-Pro dipeptide
isosteres.14 This approach has now been optimized by using
2-hydroxypyrrolidine and 2-hydroxypiperidine as synthetic
equivalents of the corresponding ω-amino aldehydes in the
initial Horner−Emmons olefination. Epoxy amines can thus be
obtained in a more direct way and with higher yields. The
improved methodology has been applied to the synthesis of
three Phe-Pro isosteres (3a−c) and a C2-symmetric Pro-Pro
isostere (5) containing a pyrrolidine or piperidine ring as a pro-
line mimic. All isosteres were obtained in good yields and
excellent stereoselectivity.
A set of 12 peptidomimetic HIV-PR inhibitors was syn-
thesized by coupling the isosteres with peptide residues, chosen
for their known affinity for the target enzyme, and nonpeptide
residues derived from phenoxyacetic acid; several inhibitors dis-
play high activity against HIV-1 PR, with IC50 in the low nano-
molar range. Comparison of inhibitors with identical DMPOA
flanking residues grafted onto S,S,S,S diamino diol Phe-Pro,
Pro-Pro, and Phe-Phe isosteres reveals that the nonsymmetric
Phe-Pro-based inhibitor 23a performs better than the C2
symmetric partners 26 and 28, despite the latter two inhibitors
having the same symmetry of the target enzyme. The efficiency
of nonsymmetric inhibitors based on the Phe-Pro isostere 3a
is confirmed by the higher activity of inhibitor 18a (Chart 1)
with respect to the symmetric Phe-Phe inhibitor 27.9 We
propose that nonsymmetric binding of the inhibitors with HIV-
PR, regardless of the intrinsic symmetry of the inhibitor, is at
the origin of this behavior, either through a thermodynamic effect
or through a kinetic effect due to the presence of nonsymmetric
intermediates along the path to the enzyme−inhibitor complex.
Inhibitors 18a and 23a, based on a five-membered proline
mimic, were further studied for their ability to inhibit HIV-1
replication. Both inhibitors display high antiviral activity, in the
nanomolar to picomolar range, combined with low cytotoxicity,
resulting in a high therapeutic index in the 103 and 104 range,
respectively.
concentration infection assay MTT-A540
RT activity in
b
a
inhibitor
(nM)
(% cell survival)
supernatants (%)
18a
10
5
>90
>90
78
24
50
24
25
26
29
28
26
23a
0.1
0.001
80
0.0001
87
0.00001
0.000001
0.0000001
90
>90
>90
a
Percent cell survival of virus-infected cells at different inhibitor
b
concentrations (see Experimental Section). From duplicate experi-
ments. RT activity (%) is the ratio between RT activity in super-
natants of inhibitor-treated HIV-1-infected cultures and that in
untreated HIV-1-infected MT-2 cells (100%). Abacavir (see ref 32)
was used as control (>90% cell survival and >90% inhibition of RT
activity).
Data in Table 1 indicate that both 18a and 23a protect virus-
infected cells for over 90% at nanomolar (18a) and sub-
picomolar (23a) concentrations and significantly depress RT
activity. Although the virus-induced cytolytic effect is not com-
pletely suppressed, it is much lower than that observed when
MOI was ≥1 (data not shown), this being a clear indication
that the inhibitors had modified de novo-synthesized HIV
virions rendering them noninfectious.31 The extent of
protection does not correlate with RT activity (Table 1), and
a control experiment with recombinant RT confirms that this
enzyme is not a target for the inhibitors. On the contrary, native
viral protease obtained by lysis of virions from tissue culture
fluids of infected H9/HTLV IIIB cells is efficiently inhibited by
18a and 23a with IC50 values of 5 1 nM and 1.5 0.9 pM,
respectively.33 The inversion of the inhibitors’ activities in this
assay with respect to the assay with recombinant HIV-PR
(Chart 1) is likely due to the different conditions for the two
tests (enzyme concentration, substrate, pH, buffer and ionic
strength, presence of albumin; see Experimental Section).
The cytotoxicity of 18a and 23a was evaluated on MT-2 cell
lines. MNC was evaluated as the maximal concentration which
altered neither the morphology of MT-2 cell monolayers nor
the cell survival rate, while CC50 was evaluated by % cell
survival. In vitro cytotoxicities are summarized in Table 2.
In conclusion, we have demonstrated that isostere 3a, avail-
able from phenylalanine in only five steps and excellent yield, is
a precursor of efficient HIV-PR inhibitors, when coupled with
suitable peptide or nonpeptide residues. The potential of this
new peptidomimetic core is well illustrated by inhibitor
23a that combines low molecular weight with subpicomolar
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N-Cbz-2-hydroxypiperidine 7b18 (800 mg, 3.4 mmol) and DBU
(440 mg, 2.8 mmol) were then added, and the mixture was stirred
at 25 °C for 16 h, neutralized with 10% HCl, and extracted with ethyl
acetate. Workup as described for 8a, gave the enone (1.02 g, 65%), mp
antiproliferative activity and low cytotoxicity, thus appearing
particularly promising for further development. The synthesis
and evaluation of second generation, rationally designed inhib-
itors based on Phe-Pro isostere 3a that have high predicted
inhibitory potencies, promising ADME profiles, and the
potential to avoid drug resistance due to favorable interactions
with the PR backbone are currently underway.34
25
72−73 °C (isopropyl ether). [α]D −0.62 (c 0.73). IR (nujol): 3362,
1743, 1691, 1620 cm−1. 1H NMR (CDCl3) δ: 1.40 (m, 13H), 2.16 (m,
2H), 3.05 (m, 4H), 4.77 (m, 2H), 5.08 (s, 2H), 5.25 (d, 1H, J =
69 Hz), 6.11 (d, 1H, J = 15.7 Hz), 6.85 (m, 1H), 7.29 (m, 10H) ppm.
13C NMR (CDCl3) δ: 25.1, 28.4, 29.6, 32.2, 38.6, 40.7, 58.4, 66.7, 79.8,
126.9, 127.2, 127.9, 128.3, 128.4, 128.5, 128.8, 129.4, 129.6, 136.3,
136.6, 149.0, 155.2, 156.5, 197.5 ppm. ES-MS m/z 481.2 [MH]+.
(E,2S,3R)-8-Benzyloxycarbonylamino-2-tert-butoxycarbony-
lamino-3-hydroxy-1-phenyloct-4-ene (10a). Enone 8a (5.0 g,
10.4 mmol), in 40 mL of dry ethanol, was added, under inert atmo-
sphere, to a stirred suspension of LTBAH (6.62 g, 26 mmol) in dry
ethanol (30 mL), while keeping the temperature between −78 and
−60 °C. When all the enone was reduced, the mixture was quenched
with 16 mL of 10% aqueous citric acid. The mixture was diluted with
water (50 mL) and extracted with ethyl acetate. The organic phases
were extracted with brine and dried over anhydrous sodium sulfate.
The solvent was removed, and the residue was purified over silica,
giving 4.7 g (96%) of a white solid, mp 113−114 °C (isopropyl ether).
EXPERIMENTAL SECTION
■
Melting points are not corrected. 1H NMR and 13C NMR spectra were
recorded on a Varian 500 MHz spectrometer or a Jeol EX 400 MHz in
CDCl3 (unless otherwise stated), with tetramethylsilane as internal
standard. Electrospray mass spectra were obtained with a Bruker
Daltonics Esquire 4000 spectrometer. Optical activities were measured
with a Perkin-Elmer 261 polarimeter, in methanol unless otherwise
stated. IR spectra were recorded on a Thermo-Nicolet AVATAR 320
FT-IR instrument. Enzyme kinetics were followed with a fluorogenic
substrate on a Perkin-Elmer LS50B spectrofluorimeter. Flash chro-
matography was performed on silica gel 60 (Merck, 230−400 mesh).
THF was distilled, dried over KOH, and then redistilled from sodium−
benzophenone ketyl. Dichloromethane was dried over CaCl2 and
distilled. Dry acetonitrile and dimethylformamide were purchased from
Sigma-Aldrich. Methanol and ethanol were dried over CaH2 and distilled.
All inhibitors were >95% pure, as determined by HPLC analysis (C4,
water−acetonitrile).
25
1
[α]D −20.23 (c 0.44). IR (KBr): 3451, 3341, 1713 cm−1. H NMR
(CDCl3) δ: 1.35 (s, 9H), 1.60 (m, 2H), 2.12 (m, 2H), 2.79 (m, 2H),
3.12 (broad, 1H), 3.22 (m, 2H), 3.79 (m, 1H), 4.12 (m, 1H), 4.78
(broad, 1H), 4.93 (broad, 1H), 5.10 (s, 2H), 5.51 (dd, 1H, J = 15.4,
6.6 Hz); 5.67 (m, 1H), 7.34 (m, 10H) ppm . 13C NMR (CDCl3) δ:
28.4, 29.1, 29.2, 36.5, 40.1, 56.8, 66.8, 74.7, 79.7, 126.4, 128.2, 128.3
128.5, 128.6, 129.3, 129.4, 129,5, 130.0, 132.4, 136.7, 138.2, 156.5,
156.6 ppm. ES-MS m/z 469 [MH]+.
Dimethyl (S)-3-tert-Butoxycarbonylamino-2-oxo-4-phenyl-
butylphosphonate (6). A 2.5 M solution of n-butyllithium in
hexanes (86 mL, 214 mmol) was added to a solution of methyl
dimethylphosphonate (23.0 mL, 214 mmol) in dry THF (200 mL) at
−78 °C, under argon atmosphere. The mixture was stirred for 15 min,
and then N-Boc-L-phenylalanine methyl ester (10 g, 35.8 mmol) in
THF (70 mL) was added. The resulting solution was stirred at −78 °C
for 2 h and then at −30 °C for 1 h and finally neutralized with 20%
aqueous citric acid. The aqueous phase was extracted with ethyl acetate
(3 × 75 mL), and the organic phases were extracted with sat. NaHCO3
and brine and then dried over anhydrous sodium sulfate. The solvent
was removed, and the crude product was purified over silica to give the
phosphonate 6 (12.4 g, 89%) as a white solid, mp 65−67 °C
(E,2S,3R)-9-Benzyloxycarbonylamino-2-tert-butoxycarbony-
lamino-3-hydroxy-1-phenylnon-4-ene (10b). Enone 8b (1.62 g,
3.48 mmol) and LTBAH (2.65 g, 10.2 mmol), as described for the
synthesis of 10a, gave the alcohol 10b (1.52 g, 94%), mp 76−78 °C
25
(isopropyl ether). [α]D −24.3 (c 0.41). IR: 3348, 1688, 1620 cm−1.
1H NMR (CDCl3) δ: 1.26−1.51 (m, 13H), 2.09 (m, 2H), 2.80 (m,
2H), 3.16 (m, 2H), 3.94 (broad, 1H), 4.13 (m, 1H), 4.78 (broad, 1H),
4.86 (broad, 1H), 5.07 (s, 2H), 5.53 (dd, 1H, J = 15.16, 6.2), 5.67 (m,
1H), 7.19−7.33 (m, 10H) ppm. 13C NMR (CDCl3) δ: 28.2, 29.2, 31.7,
36.1, 40.7, 56.6, 66.6, 76.3, 79.5, 126.4, 128.0, 128.2, 128.5, 128.6,
129.0, 129.3, 129.5, 133.2, 136.6, 138.1, 156.4, 156.4 ppm. ES-MS m/z
483.2 [MH]+.
25
(isopropyl ether). [α]D −52 (c 0.4). IR (KBr): 3280, 1723, 1713,
1
1225 cm−1. H NMR (CDCl3) δ: 1.39 (s, 9H), 2.93 (dd, 1H, J = 8.3,
14.2 Hz), 3.18 (dd, 1H, J = 6.1, 14.2 Hz), 3.10 (dd, 1H, J = 14.2, 22.0
Hz), 3.26 (dd, 1H, J = 14.2, 22.0 Hz), 3.75 (d, 3H, J = 11.5 Hz), 3.77
(d, 3H, J = 11.5 Hz), 4.55 (m, 1H), 5.43 (d, 1H, J = 7.8 Hz), 7.24 (m,
5H) ppm. 13C NMR (CDCl3) δ: 28.3, 37.0, 38.3 (d, J = 130 Hz), 53.2,
61.3, 80.1, 127.0, 127.8, 128.7, 136. 6, 155.3, 201.0 (d, J = 5.5 Hz)
ppm. ES-MS m/z 372 [MH]+.
(E,2S,3S)-8-Benzyloxycarbonylamino-2-tert-butoxycarbony-
lamino-3-hydroxy-1-phenyloct-4-ene (10c). L-Selectride (6.4 mL
of a 1.0 M solution in THF, 6.4 mmol) was added at −78 °C and
under an inert atmosphere to a solution of 8a (1.0 g, 2.14 mmol) in
30 mL of dry methanol. After 3 h, the solution was acidified to pH 5 with
1 N HCl, and the solvent was evaporated. The residue was partitioned
between ethyl acetate and sat. NaHCO3. The aqueous phase was extra-
cted with ethyl acetate, and the combined organic phases were extracted
with brine and dried over anhydrous sodium sulfate. The solvent was
removed, and the residue was purified over silica, giving the alcohol 10c
(E,S)-10-Benzyloxycarbonylamino-2-tert-butoxycarbonylami-
no-3-oxo-1-phenyloct-4-ene (8a). Phosphonate 6 (3.87 g, 1 mmol)
was added to a suspension of dried K2CO3 (4.14 g, 3 mmol) in
anhydrous acetonitrile (10 mL), and the suspension was stirred at
25 °C for 15 min. N-Cbz-2-hydroxypyrrolidine 7a15 (2.3 g, 1 mmol) in
10 mL of dry acetonitrile was added dropwise, and the mixture was
stirred under argon for 48 h, neutralized with 10% (w/v) citric acid,
and extracted with ethyl acetate. The organic phase was extracted with
water and brine and dried over anhydrous sodium sulfate. The solvent
was evaporated, and the residue was purified over silica giving the
enone 8a (2.3 g, 50%), mp 75−77 °C (isopropyl ether). [α]D25 −0.59
25
(610 mg, 61%), mp 87−89 °C (isopropyl ether). [α]D −36.8 (c 0.5).
IR (Nujol): 3452, 3341, 1713 cm−1. 1H NMR (CD3CN, 50 °C) δ: 1.32
(s, 9H), 1.56 (quint, 2H, J = 6.9 Hz), 2.05 (q, 2H, J = 6.9 Hz), 2.67 (dd,
1H, J = 9.2, 13.7 Hz), 2.89 (dd, 1H, J = 5.8, 13.7 Hz), 3.12 (q, 2H, J =
6.6 Hz), 3.70 (m, 1H), 4.01 (s, 1H), 5.06 (m, 3H), 5.39 (s, 1H), 5.49
(dd, 1H, J = 6.2 Hz, 15.4 Hz), 5.63 (m, 1H), 7.34 (m, 10H) ppm. 13C
NMR (CD3CN, 50 °C) δ: 28.0, 29.0, 29.2, 37.4, 40.2, 56.8, 66.0, 72.7,
78.5, 126.1, 127.8, 127.9, 128.3, 128.6, 131.1, 131.5, 137.5, 139.4, 156.0,
156.5 ppm. ES-MS m/z 469 [MH]+.
1
(c 0.73). H NMR (CDCl3) δ: 1.35 (s, 9H), 1.59 (t, 2H, J = 6.9 Hz),
2.18 (q, 2H, J = 6.9 Hz), 2.96 (dd, 1H, J = 5.9, 13.7 Hz), 3.05 (dd, 1H,
J = 6.8, 13.7 Hz), 3.15 (q, 2H, J = 6.2 Hz); 4.77 (m, 1H), 4.99 (broad,
1H), 5.12 (s, 2H), 5.31 (d, 1H, J = 7.7 Hz), 6.09 (d, 1H, J = 15.7 Hz),
6.85 (m, 1H), 7.10−7.34 (m, 10H) ppm. 13C NMR (CDCl3) δ: 28.4
(two carbons), 29.8, 38.5, 40.5, 58.5, 66.7, 79.8, 126.9, 127.9, 128.2,
128.4, 128.6, 128.8, 129.5, 136.3, 136.6, 148.4, 155.2, 156.5, 197.5
ppm. ES-MS m/z 465.1 [MH]+.
(E,S)-10-Benzyloxycarbonylamino-2-tert-butoxycarbony-
lamino-3-oxo-1-phenylnon-4-ene (8b). LiCl (1.44 g, 3.4 mmol),
dried at 220 °C for 4 h, was added to a stirred solution of phos-
phonate 6 (1.26 g, 3.4 mmol) in 65 mL of dry acetonitrile.
(2S,3S,4R,5R)-8-Benzyloxycarbonylamino-2-tert-butoxycar-
bonylamino-4,5-epoxy-3-hydroxy-1-phenyloctane (11a).
mCPBA (314 mg, 1.1 mmol) in DCM (5 mL) was added at 0 °C to
a solution of allylic alcohol 10a (426 mg, 0.91 mmol) in DCM (10 mL).
The mixture was stirred at room temperature for 16 h, diluted with
15 mL DCM, extracted with 10% aqueous sodium metabisulfite (2 ×
25 mL), sat. NaHCO3 (2 × 25 mL), and brine, and dried over anhydrous
sodium sulfate. The solvent was evaporated, and the residue was purified
over silica to give the epoxide 11a (300 mg, 68%), mp 99−101 °C
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Journal of Medicinal Chemistry
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(isopropyl ether). [α]D25 +8.33 (c 0.3). H NMR (CDCl3) δ: 1.35 (m,
11H), 1.61 (m, 2H), 2.63 (d, 1H, J = 6.2 Hz), 2.86 (m, 1H), 2.96 (m,
3H), 3.23 (m, 2H), 3.55 (m, 1H), 3.99 (m, 1H), 4.71 (m, 1H), 4.88
(broad, 1H), 5.08 (s, 2H), 7.32 (m, 10H) ppm. 13C NMR (CDCl3) δ:
26.5, 28.3, 28.7, 36.6, 40.6, 55.7, 58.4, 59.0, 66.8, 71.7, 79.7, 126.6, 128.2
(two signals), 128.6 (two signals), 129.5 (two signals), 136.6, 137.6, 156.5
(two signals) ppm. ES-MS m/z 485 [MH]+, 429 [MH − C4H8]+.
(2S,3S,4R,5R)-9-Benzyloxycarbonylamino-2-tert-butoxycar-
bonylamino-4,5-epoxy-3-hydroxy-1-phenylnonane (11b). The
product (2.10 g, 51%) was obtained from 15b (4.00 g, 8.3 mmol) and
mCPBA (2.90 g, 9.95 mmol), as described for 11a. Mp 103−105 °C
d6, 90 °C) δ: 1.39 (s, 9H), 1.78 (m, 2H), 1.98 (m, 1H), 2.13 (m, 1H),
3.26 (dd, 2H, J = 3.5, 21.2 Hz), 3.36 (m, 2H), 3.69 (d, 6H, J = 10.6
Hz), 4.36 (dd, 1H, J = 4.8, 8.8 Hz) ppm. 13C NMR (DMSO-d6 90 °C)
δ: 23.8, 28.6 (two carbons), 36.6 (d, J = 132.7 Hz), 47.1, 53.0, 66.1 (d,
J = 3.8 Hz), 79.6, 154.7, 201.6 (d, J = 6.9 Hz) ppm. ES-MS m/z 322
[MH]+.
(E,S)-6-Benzyloxycarbonylamino-1-(N-tert-butoxycarbonyl-
pyrrolidin-2-yl)hex-2-en-1-one (13). The enone (1.8 g, 76%, oil)
was obtained from phosphonate 12 (1.82 g, 5.69 mmol), N-Cbz-
2-hydroxypyrrolidine 7a15 (334 mg, 1.51 mmol), and K2CO3 (2.36 g,
17 mmol), as described for the synthesis of 8a. [α]D25 −29.6 (c 0.58).
1H NMR (CDCl3, pair of rotamers) δ: 1.31−1.40 (s, 9H), 1.61−1.80
(m, 5H), 2.21 (m, 1H), 2.22 (q, 2H, J = 7.3 Hz), 3.17 (m, 2H), 3.48
(m, 2H), 4.31−4.51 (m, 1H), 5.04 (m, 3H), 6.20 (d, 1H, J = 15.7 Hz),
6.90 (m, 1H) ppm. 13C NMR (CDCl3, pair of rotamers) δ: 23.8−24.3,
28.3−28.5, 29.3, 29.8−30.4, 40.5, 46.8−46.9, 63.4−64.3, 66.7, 79.7−
80.0, 126.0, 127.0, 128.1−128.6, 136.6, 147.3, 154.0−154.1, 156.5,
198.5, 198.8 ppm. ES-MS m/z 417.2 [MH]+.
1
25
(isopropyl ether). [α]D −8.7 (c 0.32). IR (nujol): 3341, 1686, 1620
cm−1. 1H NMR (CDCl3) δ: 1.34 (m, 13H), 1.49 (m, 2H), 1.97 (d, 1H,
J = 4.04 Hz), 2.84 (m, 3H), 2.96 (m, 1H), 3.16 (m, 2H), 3.53 (m, 1H),
3.98 (m, 1H), 4.81 (m, 1H), 4.94 (m, 1H), 5.06 (s, 2H), 7.20−7.33
(m, 10H) ppm. 13C NMR (CDCl3) δ: 23.1, 28.4, 29.7, 31.0, 36.3, 40.9,
55.0, 56.1, 59.1, 66.7, 72.0, 79.6, 126.5, 128.5, 128.6 (multiple
overlapping signals), 129.5, 136.7, 137.8, 155.8, 156.5 ppm. ES-MS
m/z 499.2 [MH]+.
(R,E)-6-Benzyloxycarbonylamino-1-((S)-N-tert-butoxycarbo-
nylpyrrolidin-2-yl)hex-2-en-1-ol (14). DIBALH in toluene (5.2
mmol) was added at −78 °C to a stirred solution of enone 13 (2.16 g,
5.18 mmol) in 50 mL of toluene. The reaction was monitored by TLC
and neutralized with acetic acid when all the reagent was consumed.
The solvent was evaporated, and the residue was partitioned between
ethyl acetate and sat. NaHCO3. The aqueous phase was extracted
with ethyl acetate, and the combined organic phases were extracted
with brine and dried over anhydrous sodium sulfate. The solvent was
evaporated, and the residue was purified over silica to give a colorless
(2S,3R,4S,5S)-8-Benzyloxycarbonylamino-2-tert-butoxycar-
bonylamino-4,5-epoxy-3-hydroxy-1-phenyloctane (11c). Epox-
idation of 10c (770 mg, 1.64 mmol) with mCPBA (567 mg, 1.98 mmol),
as described for 11a, gave the product 11c (571 mg, 72%) as a colorless
oil. [α]D −14.4 (c 0.25). H NMR (CD3CN, 70 °C) δ: 1.33 (s, 9H),
1.59 (m, 4H), 2.72−2.91 (m, 4H), 3.14 (m, 2H), 3.25 (broad, 1H), 3.32 -
3.44 (2s, 1H, rotamers), 3.88 (m, 1H), 5.06 (s, 2H), 5.15 (broad, 1H),
5.53 (m, 1H), 7.37 (m, 10H) ppm. 13C NMR (CD3CN, 70 °C) δ: 26.1,
27.7, 28.6, 37.7, 40.6, 55.4 and 55.7 (rotamers), 58.6, 59.9, 65.9, 71.1 and
72.6 (rotamers), 78.7, 127.7, 127.9, 128.3 (2 overlapping signals), 128.4,
129.3, 137.7, 139.1, 155.8 and 156.5 (rotamers) ppm. ES-MS m/z 485
[MH]+.
(1S,2S,3S)-3-tert-Butoxycarbonylamino-4-phenyl-1-((S)-pyr-
rolidin-2-yl)butane-1,2-diol (3a). The Cbz-protected epoxy alcohol
11a (100 mg, 0.206 mmol) in methanol (5 mL) was stirred for 16 h
over 5% Pd−C under a hydrogen atmosphere. The mixture was
filtered over Celite, and the solvent was evaporated to give the crude
product (69 mg, 95%), mp 167 °C (toluene−hexane). [α]D25 −38.8 (c
0.35). 1H NMR (CDCl3) δ: 1.33 (s, 9H), 1.57 (m, 1H), 1.76 (m, 2H),
1.91 (m, 1H), 2.86 (m, 1H), 2.92 (m, 1H), 2.98 (m, 1H), 3.13 (d, 1H,
J = 13.9 Hz), 3.53−3.66 (m, 3H), 3.91 (m, 1H), 4.62 (broad, 3H),
7.34 (m, 5H) ppm. 13C NMR (CDCl3) δ: 25.2, 27.3, 28.3, 36.5, 45.9,
52.8, 61.9, 69.6, 73.1, 80.2, 126.4, 128.4, 129.5, 137.9, 157.1 ppm. ES-
MS m/z 351 [MH]+.
(1S,2S,3S)-3-tert-Butoxycarbonylamino-4-phenyl-1-((S)-pi-
peridin-2-yl)butane-1,2-diol (3b). The product (350 mg, 96%) was
obtained from epoxy alcohol 11b (500 mg, 1 mmol) with the pro-
cedure described for the synthesis of 6a. Mp 120−123 °C (toluene−
hexane). [α]D25 −39.3 (c 0.4). IR (nujol): 3385, 3190 cm−1. 1H NMR
(CDCl3) δ: 1.33 (s, 9H), 1.43−1.81 (m, 6H), 2.48 (td, 2H, J = 9.16,
2.56 Hz), 2.85 (m, 1H), 3.05 (m, 1H), 3.14 (m, 1H), 3.40 (m, 1H),
3.46 (m, 1H), 3.95 (m, 1H), 4.40 (m, 1H), 7.26 (m, 5H) ppm. 13C
NMR (CDCl3) δ: 24.6, 25.9, 28.1, 28.3, 36.4, 46.5, 51.7, 60.3, 70.6,
74.1, 80.5, 126.5, 128.5, 129.6, 137.7, 157.5 ppm. ES-MS m/z 365.3
[MH]+.
25
1
25
1
oil (1.1 g, 51%). [α]D −43.8 (c 0.5). H NMR (CDCl3, pair of
rotamers) δ: 1.43 (s, 9H), 1.57−1.85 (m, 5H), 1.99−2.04 (m, 3H),
3.13 (m, 2H), 3.16 (broad, 1H), 3.41 (m, 1H), 3.76−3.87 (m, 1H),
5.04 (m, 3H), 5.39 (dd, 1H, J = 5.8, 15.7 Hz), 5.62 (m, 1H) ppm. 13C
NMR (CDCl3, pair of rotamers) δ: 24.0, 25.4, 28.3−28.5, 29.4, 29.4−
29.5, 40.6−41.1, 47.5−48.2, 62.7, 66.6, 75.6, 80.4−80.5, 127.9−131.3,
132.4, 156.5, 157.9 ppm. ESI-MS m/z 419.2 [MH]+.
(1S,2R,3R)-6-Benzyloxycarbonylamino-1-((S)-N-tert-butoxy-
carbonylpyrrolidin-2-yl)-2,3-epoxyhexanol (15). The colorless
oil (223 mg, 53%) was obtained from 14 (407 mg, 0.97 mmol) and
mCPBA (336 mg, 1.17 mmol), as described for 11a. [α]D25 9.7 (c 0.3).
1H NMR (CDCl3) δ: 1.42 (s, 9H), 1.61−1.98 (m, 5H), 2.77 (m, 1H),
2.89 (m, 1H), 3.20 (m, 2H), 3.42 (broad, 1H), 3.62 (m, 1H), 3.91 (m,
1H), 5.08 (m, 3H), 7.18−7.32 (m, 10H) ppm. 13C NMR (CDCl3) δ:
24.3, 25.7, 28.5, 28.9, 29.8, 40.6, 47.6, 55.2, 58.8, 61.0, 66.6, 72.4, 80.0,
128.0−128.6, 136.7, 156.2, 156.5 ppm. ESI-MS m/z 434.2 [MH]+.
(S)-1-tert-Butoxycarbonyl-2-((1S,2S)-1,2-dihydroxy-2-((S)-
pyrrolidin-2-yl)ethyl)pyrrolidine (5). Deprotection of N-Cbz
epoxy amine 15 (73 mg, 0.17 mmol), as described for 3a, gave 47.9
mg (95%) of a colorless oil. [α]D25 −18.8 (c 0.35). 1H NMR (CDCl3)
δ: 1.42 (s, 9H), 1.86−2.19 (m, 8H), 3.35 (m, 4H), 3.64 (m, 1H), 3.78
(m, 1H), 3.87 (m, 1H), 3.91 (m, 1H) ppm. 13C NMR (CDCl3) δ:
23.3, 24.2, 27.1, 27.4, 28.4, 45.2, 47.2, 58.6, 62.0, 68.5, 71.1, 80.6,
157.0 ppm. ES-MS m/z 301 [MH]+.
General Procedure for the Preparation of Inhibitors 17, 19,
and 22 (method A). The acid (peptide or phenoxyacetic acid
derivative) (1.1 equiv) was dissolved in the minimum amount of
anhydrous THF; HOBT (1.1 equiv), diisopropylethylamine (1.1
equiv), and isostere (1 equiv) were added. The solution was cooled to
0 °C, and EDC (1.2 equiv) was added. The mixture was stirred for 1 h
at 0 °C and for a further 16 h at 25 °C. The solvent was removed, and
the residue was dissolved in ethyl acetate, extracted with 10% aqueous
citric acid, sat. NaHCO3, and brine and dried over anhydrous Na2SO4.
The solvent was removed in vacuo, and the crude product was purified
on silica with a 9: 1 mixture of DCM−methanol. The monoacylated
product was Boc-deprotected immediately prior to use by treatment
with 40% TFA in DCM for 1 h at 25 °C, followed by coevaporation of
TFA with ethyl ether. Acylation of the primary amine was carried out
as previously, and the product was purified by flash chromatography
and/or preparative HPLC. Details on the synthesis and character-
ization of the inhibitors are in the Supporting Information.
(1R,2R,3S)-3-tert-Butoxycarbonylamino-4-phenyl-1-((R)-pyr-
rolidin-2-yl)butane-1,2-diol (3c). The oily product (65.4 mg, 92%)
was obtained from epoxy alcohol 11c (98 mg, 0.20 mmol) with the
procedure described for the synthesis of 3a. [α]D −17.1 (c 0.3). H
NMR (CDCl3) δ: 1.23−1.37 (m, 11H), 1.64 (m, 2H), 2.85−2.89 (m,
4H), 3.29 (m, 1H), 3.47 (m, 1H), 3.58 (m, 1H), 3.82 (m, 1H), 3.95
(broad, 2H), 5.22 (d, 1H, J = 8.4 Hz); 7.12−7.25 (m, 5H) ppm. 13C
NMR (CDCl3) δ: 24.8, 25.7, 28.4, 39.1, 46.1, 53.3, 60.3, 71.1, 72.4,
79.6, 126.4, 128.4, 128.4, 129.5, 138.4, 156.1 ppm. ES-MS m/z 351
[MH]+.
25
1
Dimethyl 2-((S)-1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)-2-ox-
oethylphosphonate (12). The phosphonate (6.85 g, 70%, yellow
oil) was obtained from N-Boc-L-proline methyl ester (7.0 g, 30.5
mmol), methyl dimethylphosphonate (9.8 mL, 91.7 mmol), and
n-butyllithium (36.6 mL, 91.7 mmol) as described previously for 6.
25
1
[α]D −54.2 (c 0.5). IR (film): 1693, 1255 cm−1. H NMR (DMSO-
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General Procedure for the Preparation of Inhibitors 18, 20,
21, and 23−25 (method B). The isostere (3a or 3c) was Boc-
deprotected and the crude diamine was bis-acylated with 2 equiv of the
acid and purified, as described in method A. Details on the synthesis and
characterization of the inhibitors are in the Supporting Information.
Inhibitor 18b (method C). Isobutyl chloroformate (84 μL,
0.65 mmol) was added, at 0 °C, to a solution of dipeptide N-Ac-
TrpVal-COOH (224 mg, 0.65 mmol) and NMM (183 μL, 1.3 mmol)
in 5 mL of ethyl acetate; the solution was allowed to reach 25 °C, and
Boc-deprotected diol 3b (118.5 mg, 0.32 mmol) in 5 mL of ethyl
acetate was added dropwise. The solution was kept at 25 °C for 2 h,
and aqueous workup gave the solid product, 146 mg (49%). ES-MS
m/z 832 [MH]+.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ministry of University and Research (MIUR Rome-
Italy) for financial support (PRIN project) and the University
of Trieste for partially supporting P.C. and F.D. (Ph.D.
fellowships). The MT-2 cell line was a gift from Dr. Sylvie Liu,
Imperial College, Faculty of Medicine, London, UK. The H9/
HTLV III B line was a gift from Dr. R. Gallo (NIH, Bethesda,
MD).
Inhibitor 23b (method D). DMPOA (65.3 mg, 0.36 mmol) was
dissolved in thionyl chloride, and the solution was heated at 50 °C for
5 h. This solution was then added to a solution of Boc-deprotected
diol 3b (55.2 mg, 0.15 mmol) in 1:1 ethyl acetate and aqueous sodium
hydrogen carbonate (0.6 mmol). After 2 h, the organic layer was washed
with 5% aqueous sodium hydrogen carbonate and brine. The solution
was dried over anhydrous sodium sulfate, the solvent was removed, and
the crude product was crystallized from acetone−hexane. White solid;
41 mg (46%). ES-MS m/z 590 [MH]+.
ABBREVIATIONS USED
■
DMPOA, 2,6-dimethylphenoxyacetic acid; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBT, hydroxybenzotria-
zole; LTBAH, lithium tri-tert-butoxyaluminium hydride; MNC,
maximum nontoxic concentration; MOI, multiplicity of
infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide; NMM, N-methylmorpholine; PR, protease;
RT, reverse transcriptase
Inhibition of Recombinant HIV-Pr. IC50 values were determined
at pH 5.5 using recombinant wild-type HIV-1 PR from Bachem and
the fluorogenic substrate Abz-Thr-Ile-Nle-Phe(p-NO2)-Gln-Arg-NH2
REFERENCES
■
(Abz-NF*-6; Bachem AG, Bubendorf, CH). Atazanavir (IC50
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ASSOCIATED CONTENT
* Supporting Information
Synthesis and characterization of the inhibitors. Description of
the recombinant HIV-PR inhibition assay. Average IC50 values
and standard errors for inhibitors 17−26. Detailed description
of the antiviral activity and cytotoxicity assays. Description of
the native protease inhibition assay. This material is available
free of charge via the Internet at http://pubs.acs.org
■
S
AUTHOR INFORMATION
Corresponding Author
*(F. Benedetti) Phone: +39 040 5583919; fax: +39 040
5582402; e-mail: benedett@units.it. (F. Berti) Phone: +39 040
5583920; fax: +39 040 5582402; e-mail: fberti@units.it.
■
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