Synthesis, X-ray analysis, and biological evaluation of a new class of stereopure lactam-based HIV-1 protease inhibitors

Article abstract of DOI:10.1021/jm201620t

In an effort to identify a new class of druglike HIV-1 protease inhibitors, four different stereopure β-hydroxy γ-lactam-containing inhibitors have been synthesized, biologically evaluated, and cocrystallized. The impact of the tether length of the central spacer (two or three carbons) was also investigated. A compound with a shorter tether and (3R,4S) absolute configuration exhibited high activity with a K_i of 2.1 nM and an EC₅₀ of 0.64 μM. Further optimization by decoration of the P1′ side chain furnished an even more potent HIV-1 protease inhibitor (K_i = 0.8 nM, EC₅₀ = 0.04 μM). According to X-ray analysis, the new class of inhibitors did not fully succeed in forming two symmetric hydrogen bonds to the catalytic aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-carbon spacer) and the longer inhibitors (three-carbon spacer).

Full text of DOI:10.1021/jm201620t

Article
pubs.acs.org/jmc
Synthesis, X-ray Analysis, and Biological Evaluation of a New Class of
Stereopure Lactam-Based HIV-1 Protease Inhibitors
†
†
†
†
‡
†
̈
Xiongyu Wu, Per Ohrngren, Advait A. Joshi, Alejandro Trejos, Magnus Persson, Riina K. Arvela,
Hans Wallberg,§ Lotta Vrang,§ Åsa Rosenquist,§ Bertil B. Samuelsson,§ Johan Unge,∥ and Mats Larhed*,†
†Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, P.O. Box 574, SE-751 23
Uppsala, Sweden
‡Department of Cell and Molecular Biology, Structural Biology, BMC, Uppsala University, P.O. Box 596, SE-751 24 Uppsala, Sweden
§Medivir AB, P.O. Box 1086, SE-141 22 Huddinge, Sweden
∥MAX IV-Laboratory, Lund University, P.O. Box 118, SE-221 00 Lund, Sweden
S
* Supporting Information
ABSTRACT: In an effort to identify a new class of druglike HIV-1
protease inhibitors, four different stereopure β-hydroxy γ-lactam-
containing inhibitors have been synthesized, biologically evaluated,
and cocrystallized. The impact of the tether length of the central
spacer (two or three carbons) was also investigated. A compound with
a shorter tether and (3R,4S) absolute configuration exhibited high
activity with a Ki of 2.1 nM and an EC50 of 0.64 μM. Further
optimization by decoration of the P1′ side chain furnished an even
more potent HIV-1 protease inhibitor (Ki = 0.8 nM, EC50 = 0.04 μM).
According to X-ray analysis, the new class of inhibitors did not fully
succeed in forming two symmetric hydrogen bonds to the catalytic
aspartates. The crystal structures of the complexes further explain the difference in potency between the shorter inhibitors (two-
carbon spacer) and the longer inhibitors (three-carbon spacer).
lopinavir, atazanavir, tipranavir, and darunavir),15 improving
pharmacokinetic properties and reducing adverse effects are still
INTRODUCTION
■
More than 25 years after the identification of the causative
issues that need to be addressed.16−18 Further, the rapid
replication and the high mutation rate of the HIV-1 virus,19,20
together with the mutation pressure induced by today’s
pharmacotherapies, will lead to an increase in the problems
associated with resistant virus strains. Thus, we cannot expect
the good results currently seen with HAART to continue if new
drugs are not developed and introduced onto the market.17,21
We have been engaged in the development of novel HIV-1
PIs since 1997.22 In our most recent program we developed
novel classes of potent HIV-1 PIs incorporating a shielded
tertiary alcohol as part of the transition state mimic.23−28
Inspired by the structure of the potent inhibitor Atazanavir
(ATZ)29,30 (Figure 1), we used a similar hydrazide moiety in
the prime side31 of our new tert-hydroxy-containing PIs. By
altering the length of the central backbone, using a one-, two-,
or three-carbon spacer (Figure 1, series A,23−25 B,26 and C,27,28
respectively), we focused on optimizing the interaction with the
catalytically active aspartic acid residues of the enzyme.
agent of AIDS,1,2 HIV/AIDS is still a major challenge to
society. The latest WHO/UNAIDS report (2010) states that
the number of people living with HIV has risen to 33.3 million,
with more than 2.6 million new cases annually and almost 5000
AIDS-related deaths per day.3
With the introduction of the first HIV-1 protease inhibitor
(PI) (saquinavir4) in 1995 and the development of highly active
antiviral therapy (HAART)5−7 the clinical outcome of HIV/
AIDS changed from a lethal to a manageable, but chronic,
disease in the developed world.8−10 The early PIs suffered from
poor pharmacokinetic profiles and caused severe side effects
such as hepatic toxicity and lipodystrophy.11,12 For these
reasons and with a frequent daily dosing regimen, they were not
the first-hand choice in HAART. The most common
combinations in early HAART were instead two nucleoside
reverse transcriptase inhibitors (NRTIs) together with a non-
nucleoside reverse transcriptase inhibitor (NNRTI). The
development of NNRTI- and/or NRTI-resistant HIV strains
and the introduction of new PIs, with a once-daily dose regime
and improved effect profiles, have made the combination of a
PI together with two NRTIs a more frequent choice for first
line treatment in HAART.8,13,14
The prepared tert-hydroxy comprising PIs rendered good
affinity and potency.23−28 Class B, with the two-carbon spacer,
yielded the best results, with values of Ki and EC50 as low as 1
Although saquinavir has, to date, been followed by eight
other PIs (ritonavir, indinavir, fosamprenavir, nelfinavir,
Received: November 30, 2011
Published: February 29, 2012
© 2012 American Chemical Society
2724
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
Figure 1. Examples of the previous series of tertiary alcohol-containing HIV-1 PIs. Spacers are indicated in red: A, one-carbon spacer (Ki = 5.5
nM);23−25 B, two-carbon spacer (Ki = 2.3 nM);26 C, three-carbon spacer (Ki = 2.8 nM);27 D, novel lactam-based inhibitors with two-carbon spacer
(Ki = 0.8 nM) and altered stereocenters indicated by asterisks; E, three-carbon spacer (Ki = 4.2 nM). ATZ is included for comparison (Ki = 2.7
nM).37
and 3 nM, respectively.26 In all three series (A−C), inhibitors
CHEMISTRY
■
with high membrane permeability were identified, as well as
Starting from (S)-4-hydroxydihydrofuran-2(3H)-one (1a) or
inhibitors with good metabolic stability,23−28 providing
(R)-4-hydroxydihydrofuran-2(3H)-one (1b), four HIV-1 PR
pharmacokinetic properties well in the range of HIV PIs
inhibitors with a two-carbon spacer and with varied stereo-
already on the market, e.g., ATZ.23
chemistry in the lactam ring were synthesized (Scheme 1).
X-ray analyses of inhibitors in series A−C cocrystallized with
the enzyme revealed binding modes that were not completely
successful in establishing strong symmetric hydrogen bonds
(<3.0 Å) with both the catalytic residues Asp25 and or Asp125,
originating from each monomer of the HIV-1 protease.32−34
Therefore, we decided to further elaborate the central
transition-state mimic by relocating the hydroxyl group one
position away from the backbone. This strategy was
implemented by making use of a β-hydroxy γ-lactam moiety
equipped with a secondary alcohol. It was hypothesized that the
β-hydroxy γ-lactam would provide a better hydrogen bond
arrangement for the catalytic Asp residues and at the same time
reduce the flexibility, providing a more rigid inhibitor.35,36
Modeling studies supported the hypothesis that a hydroxyl
group in the 4-position of the γ-lactam might provide a new
conformationally constrained transition-state-mimicking scaf-
fold for the development of novel HIV-1 PIs. Since both the
(3R,4S) and the (3R,4R) stereoisomers provided good docking
poses, we decided to synthesize and evaluate all four
stereoisomers of the γ-lactam (Figure 1, D). In addition, two
different lengths of the central tether (two or three carbons)
were investigated (Figure 1, D and E). The prime-side31
hydrazide moiety, inspired by ATZ, has been successfully used
in inhibitors in series A−C and was therefore retained in the
new series of lactam-based inhibitors.
Encouraged by previously reported alkylations,38−40 1a and 1b
were chosen as starting substrates for the two-step alkylation
process. Upon treatment with DMPU, LDA, and the first
a
Scheme 1. Dialkylation of Lactones 1a and 1b
a
Reagents and conditions. Path A: (a) DMPU, LDA, allyl bromide, dry
THF, added at −50 °C, stirred at −50 °C for 1 h; (b) LDA, benzyl
bromide added at −40 °C, stirred at −30 °C for 1 h, giving 2a and 2c
in 49% and 33% isolated yield, respectively. Path B: (c) DMPU, LDA,
benzyl bromide, dry THF, added at −50 °C, stirred at −40 °C for 1 h;
(d) LDA, allyl bromide, added at −40 °C, stirred at −30 °C for 1 h,
giving 2b and 2d in 2% and 5% isolated yield, respectively.
Here we present the synthetic protocols and the inhibitory
potency on enzyme level, as well as the activity in a cell-based
assay, of the new inhibitors (D and E). Also included are
stability and permeability studies of selected compounds,
together with X-ray analyses of three of the inhibitors
cocrystallized with the HIV-1 protease.
2725
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
To be able to collect enough material of 2b and 2d, with
their low-yielding synthetic pathway, a method was developed
to alter the stereochemistry at the hydroxyl group in 2a and 2c.
Oxidation of 2a and 2c with Dess−Martin reagent to the
corresponding ketones was followed by reduction using
NaBH4, affording 2d and 2b following paths A and B,
respectively (Scheme 2), with ratios 2d/2a of 5.7:1 and 2b/
2c of 5.9:1. The diastereomers were separated on a silica flash
column.
Scheme 2. Diastereomeric Enrichment of 2b and 2d
Lactamization of the lactones 2a−d with TBS-protected
indanolamine (3) was performed by adopting the methodology
developed by Orrling et al.43 (Scheme 3). Lactams 4a−d were
isolated in good yields using the ionic liquid 1-butyl-3-
methylimidazolium tetrafluoroborate ([bmim]BF4)44 under
microwave irradiation at 180 °C for 35 min, followed by
protection of the alcohol moiety with TBSOTf under basic
conditions.45,46 (Scheme 3). Although the mixture was heated
to 180 °C, these lactamization conditions are relatively mild
compared to those previously reported.47 The use of highly
polar [bmim]BF4 allowed lactamization to proceed smoothly
without the need of Brønstedt acid.43
a
Reagents and conditions: (a) 2a or 2c, Dess−Martin, DCM, rt, 1 h;
(b) NaBH4, 1% methanol in THF, rt 2 h, 2d + 2a (5.9:1) 92%, 2b +
2c (5.7:1) 85%.
a
Scheme 3. Lactamization of Lactones 2a−d
To synthesize the prime-side moiety 5a, hydrazone 7 was
prepared in almost quantitative yield starting from the BOC-
protected hydrazine 6, as previously reported in the literature48
(Scheme 4). Benzylation of 7 using KOH and 4-bromobenzyl
bromide in anhydrous toluene afforded 8 in good yield.
Catalytic quantities of the phase-transfer catalyst tetrabutylam-
monium hydrogen sulfate (TBAHS) were used to improve
solubility and increase the rate of the reaction.49,50
After the initial workup of the alkylation reaction only
compound 8 was generated, but after flash chromatography
purification, compound 9 was also formed (owing to hydrolysis
of the hydrazone). However, purification in this step was
necessary to remove excess quantities of 4-bromobenzyl
bromide, which was foreseen to cause problems in the later
steps. The mixture of 8 and 9 was deprotected with 4 M HCl in
THF to yield the pure hydrochloride salt of 10.
Owing to the photosensitivity of the free nitrogen in the p-
bromobenzylhydrazine 10, the coupling of 10 with 11,
synthesized as previously reported,23 was performed in a
reaction vessel wrapped in aluminum foil. Moreover, 10, 11,
and HOBt were added under a nitrogen atmosphere at 0 °C,
and the mixture was stirred for 30 min. Subsequently, 4-
methylmorpholine (NMM) and N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide hydrochloride (EDC) were added and the
a
Reagents and conditions: (i) [bmim]BF4, 180 °C, 35 min; (ii)
triethylamine, TBSOTf, DCM, 0−25 °C, overnight, giving isolated
yields of 4a 64%, 4b 53%, 4c 72%, and 4d 50%.
alkylating agent (allyl bromide or benzyl bromide) at −50 °C
followed by a second portion of LDA and the addition of the
second alkylating agent (benzyl bromide or allyl bromide) at
−40 °C, the dialkylated β-hydroxy γ-lactams 2a−d were
synthesized in isolated yields of 2−49% (Scheme 1, paths A
and B).
In the first alkylation, the allyl group in 2a and 2c (or the
benzyl group in 2b and 2d) was introduced trans to the
controlling 4-hydroxyl group as expected, showing facial
selectivity, as previously reported by Meyers et al.41 and
others.39,40,42 In the second alkylation, the benzyl group (or the
allyl group in 2b and 2d) was introduced trans to the 4-
hydroxyl functionality. Consequently, the second alkylation
changed the stereochemistry of the first inserted group, forcing
it to end up cis to the 4-hydroxyl group.40
a
Scheme 4. Synthesis of the Prime Side (5a)
a
Reaction conditions: (a) acetone, MgSO4, AcOH (cat.), reflux, 1 h, 98%; (b) (i) KOH, anhydrous toluene, TBAHS, 50 °C, 20 min; (ii) 3, 100 °C, 2
h, 81%; (c) HCl, THF, reflux, 3 h, quantitative yield; (d) EDCI, HOBt, NMM, DCM, 0−25 °C, 15 h, 77% (61% isolated yield over four steps.).
Steps a and c required no purification.
2726
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
Scheme 5. Oxidation and Reductive Amination To Give Two-Carbon-Spacer-Containing HIV-1 PIs 13a−d
a
Reagents and conditions: (a) 4a−d, OsO4, NaIO4, THF/H2O, rt, overnight; (b) 5a, acetic acid, Na(OAc)3BH, dry THF, rt, overnight, provided
12a in 35% and 12d in 54% isolated yield from 4a and 4d, respectively; (c) TBAF, THF, rt, overnight, provided 13a in 38%, 13b in 60%, 13c in 46%,
13d in 34% isolated yield from 4a−d, respectively.
a
Scheme 6. Synthesis of the 2-Pyridyl-Substituted Two-Carbon-Spacer-Containing HIV-1 PIs 13e−f
a
Reagents and conditions: (a) 14a or 14d, Dess−Martin reagent, dry DCM, rt, 1 h; (b) acetic acid, Na(OAc)3BH, dry THF, rt, overnight; (c) TBAF,
THF, rt, overnight, provided isolated yields of 63% 13e and 38% 13h from 14a and 14d, respectively.
a
Scheme 7. Suzuki−Miyaura Decoration of 12a and 12d To Provide the Two-Carbon-Spacer-Containing HIV-1 PIs 13g−j
a
Reagents and conditions: (a) (i) 12a or 12d, Herrmann’s palladacycle, K2CO3, 3- or 4-pyridylboronic acid, [HP(t-Bu)3]BF4, DME, water,
microwave 140 °C, 20 min; (ii) TBAF, THF, rt, overnight, providing isolated yields of 63% 13g, 59% 13h, 74% 13i, and 66% 13j.
2727
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
Scheme 8. Synthesis of Three-Carbon-Spacer-Containing HIV-1 PIs 19a−e
a
Reagents and conditions: (a) (i) 9-BBN, dry THF, 80 °C, 6 h; (ii) 2 M NaOH, 30% H2O2 in H2O, ethanol, rt, 2 h, 78%; (b) Et3N, 50% SO3Py in
DMSO, dry DCM, 0−20 °C, 3 h; (c) 5a, acetic acid, Na(OAc)3BH, dry THF, 35 °C, 3 h, 35%; (d) TBAF, THF, rt, overnight, 19a 61%; (e) (i)
Herrmann’s palladacycle, K2CO3, arylboronic acid, [HP(t-Bu)3]BF4, 105 °C, 1.5 h; (ii) TBAF, THF, rt, overnight, 19b 45%, 19c 35%, and 19d 30%;
(f) (i) 2-(tributylstannyl)pyridine, Pd(PPh3)2Cl2, CuO, DMF, 105 °C, 2 h; (ii) TBAF, THF, rt, overnight, 19e 16%.
reaction mixture was gradually heated to 25 °C and stirred
under a nitrogen atmosphere for 15 h, giving 5a in good
isolated yield (77%, 61% overall isolated yield starting from 38
mmol of 6).
(Scheme 6) was synthesized starting from the 4-(2-pyridinyl)-
benzaldehyde, as previously described.27
The alcohols 14a and 14d were isolated as side products in
reductive amination reactions to produce 12a and 12d,
respectively. Dess−Martin reagent was used to oxidize 14a
and 14d to the corresponding aldehyde intermediates (Scheme
5, v and viii, respectively), followed by reductive amination
with 5b using acetic acid and Na(OAc)3BH in dry THF and
subsequent TBAF-mediated deprotection to give useful yields
of the inhibitors 13e and 13f (Scheme 6).
The TBS-protected inhibitors 12a and 12d were decorated
using the corresponding phenyl- or pyridylboronic acids in
Suzuki−Miyaura cross-coupling in which Herrmann’s pallada-
cycle (0.1 equiv) was used as a palladium precatalyst together
with K2CO3 (3.3 equiv) and [HP(t-Bu)3]BF4 (0.2 equiv) in
DME/water. The reaction mixtures were heated to 140 °C for
20 min under focused microwave irradiation in sealed reaction
vessels.55−57 Cross-coupling was followed by deprotection of
the hydroxyl groups using TBAF in THF at room temperature,
giving inhibitors 13g−j in good isolated yields (Scheme 7 and
Table 2).
Next the allylic double bonds in lactams 4a−d were
oxidatively cleaved to give the corresponding aldehydes v−
viii using osmium tetraoxide and sodium periodate in THF/
water (3:1) at room temperature36,51 (Scheme 5). Note that
the nomenclature for the absolute configuration for the lactam
carbon in position 3 changes when comparing the lactams 4a−
d, the intermediates v−viii, and 12 and 13 because of changes
in the assigned priority according to the sequence rule.52,53
The aldehydes were quickly flushed through a short silica
column. Reductive amination between the crude aldehydes and
the prime side (5a) was performed in dry THF using acetic
acid, followed by treatment with Na(OAc)3BH, to afford the
crude TBS-protected products. The TBS protecting groups
were removed using TBAF, and the inhibitors 13a−d, carrying
a two-carbon tether, were isolated in good yields (Scheme 5).
The TBS-protected inhibitors 12a and 12d (but not 12b and
12c) were isolated, purified, and fully characterized before the
final deprotection.
To be able to incorporate the new lactam scaffold into
inhibitors with the three-carbon spacer, corresponding to the
previously published C series (Figure 1), the allylic compound
4a was refluxed in THF at 80 °C with 9-BBN for 6 h. After
addition of NaOH, H2O2, and ethanol at room temperature and
another 2 h stirring, the primary alcohol 16 was isolated in
good yield (Scheme 8).58 The alcohol 16 was oxidized to the
To evaluate the effect of different P1′ side chains, a small
series of P1′ p-phenyl- and p-pyridyl-substituted inhibitors was
produced. The known problem of rapid protodeboronation of
2-pyridylboronic acid54 prevented us from conducting
functionalization of 12a and 12d directly via Suzuki−Miyaura
cross-coupling. Thus, to introduce the 2-pyridyl as a para-
substituent in P1′, the 2-pyridine-substituted hydrazide 5b
2728
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
Table 1. Isolated Yields, Enzyme Inhibition Data, and Antiviral Activity of 12a, 12d, and 13a−d
a
b
For preparation of inhibitors, see Scheme 5. The asterisk (∗) indicates not determined. Isolated yields in the final reductive amination (12a, 12d)
or reductive amination−deprotection step (13a−d). ATZ: Ki = 2.7 nM.37 ATZ: EC50 = 0.0039 mM.37 ATZ Clint = 90 μL min−1 mg−123 [140 μL
c
d
e
min−1 mg−164]. ATZ Papp(Caco-2) = 5.3 × 10−6 cm/s.
f
corresponding aldehyde (17) using 50% SO3Py in DMSO
together with triethylamine in DCM at 0−25 °C.59,60
enzyme, all four stereoisomers were synthesized and evaluated
regarding binding and in a cell-based assay, giving the results
summarized in Table 1. Comparisons with previous series of
tertiary-alcohol-based HIV-1 PIs (A−C) could easily be
conducted by using the indanolamide in the P2 position and
the p-bromophenyl as the P1′ side chain.
The aldehyde was thereafter used in a reductive amination
reaction with 5a using Na(OAc)3BH as reducing agent at 35 °C
to give 18 in moderate isolated yield (Scheme 8). The
nomenclature for the absolute configuration of the lactam
carbon in position 4 changes when comparing the 13a−j and
19a−e series because of changes in the assigned priority
according to the sequence rule in IUPAC’s guidelines.52,53
Deprotection of 18 using TBAF in THF gave inhibitor 19a
in a good yield. Inhibitor 18 was also used as starting material
in Suzuki−Miyaura cross-coupling with phenyl- and pyridylbor-
onic acids together with Herrmann’s palladacycle, K2CO3, and
[HP(tBu3)]BF4, heated by microwave irradiation to 105 °C for
1.5 h. Deprotection of the TBS groups using TBAF in THF
gave 19b−d in 35−40% isolated yields. By use of 2-
(tributylstannyl)pyridine, compound 18 was subjected to Stille
type coupling in DMF under microwave irradiation (105 °C, 2
h) using CuO and with Pd(PPh3)2Cl2 as precatalyst.61−63 The
Stille coupling was followed by TBAF-mediated deprotection
giving inhibitor 19e in moderate isolated yield.
In accordance with the initial docking studies, inhibitors 13a
(3R,4S) and 13d (3R,4R) exhibited good activity in the enzyme
assay (Ki of 2.1 and 6.4 nM, respectively) as well as in the cell-
based evaluation (EC50 of 0.64 and 0.35 μM, respectively). The
stereoisomers 13b (3S,4S) and 13c (3S,4R) did not show any
activity and, as expected, neither did the TBS-protected
inhibitors 12a and 12d. The metabolic stability and
permeability of the two active inhibitors were investigated.
High metabolic clearance was observed for both 13a and 13d.
However, 13d also showed good results in the Caco-2
permeability study. Because of its low solubility, inhibitor 13a
was not tested in the permeability assay. Compound 13b
exhibited slight cell toxic properties, with a CC50 of 15 μM.
The lactam scaffold inhibitors 13a and 13d ((3R,4S) and
(3R,4R), respectively) yielded the most potent inhibitors and
were therefore selected for further optimization.
When the P1′ position is optimized by replacing the p-bromo
substituent of the P1′ phenyl group in 13a and 13d with
heteroaromatic moieties, the inhibitors showed improved
protease inhibitor potency and, most importantly, increased
antiviral activity (Table 2, 13e−j).
RESULTS
■
Since the preliminary docking studies suggested that two of the
stereoisomers in the lactam moiety ((3R,4S) and (3R,4R)), in
the two-carbon-tethered inhibitors would fit well in the
2729
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
Table 2. Isolated Yields, Enzyme Inhibition Data, and Antiviral Activity of Compounds 13g−j
a
b
For preparation of inhibitors, see Schemes 6 and 7. The asterisk (∗) indicates not determined. Isolated yields of 13e−f from 14a or 14d (Scheme
6) or in the coupling deprotection step of 13g−j (Scheme 7). ATZ: Ki = 2.7 nM.37 ATZ: EC50 = 0.0039 mM.37 ATZ Clint = 90 μL min−1 mg−1
c
d
e
23
[140 μL min−1 mg−1 64]. ATZ Papp(Caco-2) = 5.3 × 10−6 cm/s.
f
The best inhibitors, having (3R,4S) configuration and 3- or
4-pyridylbenzyl as the P1′ moiety (13g and 13i), exhibited 10
times higher potency than 13a in the cell-based antiviral activity
assay, the best EC50 values being 40 nM (Table 2). The 2-
pyridyl-substituted inhibitor (13e) showed lower activity than
the 3- and 4-pyridyl-substituted analogues (13g and 13j,
respectively). The improved EC50 upon decorations with 2-, 3-,
and 4-pyridyls has previously been demonstrated showing the
same trend.24
The (3R,4R) compounds showed less improvements, but all
inhibitors decorated with pyridine functionalized in P1′ were
observed to have higher potency than the precursor bromo
compound 13d. The position of the nitrogen in the
heteroaromatic P1′ group showed the same general trend as
in the (3R,4S) inhibitors, with the meta- and para-positions
providing the best potency (Table 2).
When the backbone spacer was elongated from two to three
carbons, as in 19a−e, inhibitors with lower potency than the 13
series were obtained (Table 3). This is in accordance with
results previously reported for the linear series of tertiary
alcohol inhibitors, e.g., comparing the B26 and C27 series
(Figure 1). However, with the p-phenyl or p-4-pyridyl groups in
the P1′ position, submicromolar values of EC50 were observed
in the antiviral cell based assay (19b and 19d).
As mentioned above, permeability (Caco-2) and stability
(Clint) studies were performed on some of the inhibitors
prepared (13a, 13d, 13g, 13h, 19a, 19c, and 19e). Compound
19a showed high permeability (>20 × 10−6 cm/s), while all
other inhibitors investigated showed moderate permeability
((3−20) × 10−6 cm/s). The value of Clint varied from 120 to
>300 μL min−1 mg−1 (Tables 1−3). These results are in the
same range as those previously reported for ATZ (Papp = 5.3 ×
23
Heteroaromatic functionalization of P1′ provided inhibitors
with increased stability compared to 13a and 13d. Compound
13h gave the best result (Clint of 120 μL min−1 mg−1). Both
13g and 13h were observed to possess moderate permeability
in the Caco-2 studies, with Papp of 3.8 × 10−6 and 5.1 × 10−6
cm/s, respectively.
10−6 cm/s, Clint = 90 μL min−1 mg−1 [140 μL min−1
mg−1 64]). There was no major difference between the 13
and the 19 series with respect to Clint and Papp, and the
rigidification of the backbone seemed to be well tolerated
compared to the linear inhibitors.23,27,28 The metabolic stability
was improved when the bromo group in 13a and 19a was
2730
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
a
Table 3. Isolated Yields, Enzyme Inhibition Data, and Antiviral Activity for Compounds 19a−e
a
b
For conditions, see Scheme 8. The asterisk (∗) indicates not determined. Isolated yields of 19a for the deprotection step, and isolated yields of
19b−e for the coupling−deprotection step. ATZ: Ki = 2.7 nM.37 ATZ: EC50 = 0.0039 mM.37 ATZ Clint = 90 μL min−1 mg−1 [140 μL min−1
c
d
e
23
mg−1 64]. ATZ Papp(Caco-2) = 5.3 × 10−6 cm/s.
f
substituted by the heteroaromatic pyridyls, although the
permeability was unfortunately reduced at the same time.
X-ray Structure Analysis. A drug-resistant strain of the
HIV-1 protease (Leu63Pro, Val82Thr, Ile84Val)65 was
cocrystallized with the active PIs 13i, 19b, and 19d for X-ray
crystallographic studies of the complexes. Data were obtained
for all complexes, and the structures were refined to high
resolution (for refinement statistics, see Supporting Informa-
tion). The resulting electron density maps allowed unambig-
uous modeling of the inhibitors within the binding site.
Overviews of the binding patterns are presented in Figure 2.
Previously published structures of HIV-1 PIs 20,27 21,28 and
ATZ29 are included for comparison (Figure 3).
In the two-carbon linker compound 13i this β-hydroxy group
forms hydrogen bond interactions to the two catalytic aspartic
acids with 2.7 and 3.0 Å. The β-hydroxy group in the three-
carbon inhibitors 19b and 19d only form hydrogen bonds to
Asp25, with 2.7 and 2.6 Å, respectively (Figure 2). This loss of a
hydrogen bond for the 19 series compounds is due to the
different spatial conformation of the lactam ring, apparently as a
result of the longer central backbone (Figure 4). As the only
difference between the structures of 13i and 19d is the length
of the backbone tether, this is a likely explanation of the lower
antiviral potency of compound 19d compared with 13i.
None of the cocrystallized PIs in these novel series formed a
symmetrical binding pattern with the catalytic aspartic acids
(Asp25 and Asp125) such as that seen in ATZ. Together with
the hydrazide carbonyl oxygen, the carbonyl oxygen in the
lactam ring in both 13i and 19d creates hydrogen bonds to the
structural water bridging the inhibitors and the Ile50 and Ile150
in the flap region with hydrogen bond lengths of 2.7−3.3 Å
(Figure 2).
The position of the P2−P3 indanolamide in 13i, 19b, and
19d is not markedly affected by the introduction of the β-
hydroxy γ-lactam, absent in 20. While in 20 the indanolhy-
droxyl group was close enough to form a hydrogen bond to
Arg108 and for the Arg108 to make an edge-on cation−π
interaction with the P1 phenyl group, the distance to the
indanol group in 13i seems to prevent this bond from forming
(Figure 5).
A complicating factor for the comparisons of the inhibitor
complexes was the fact that compounds 20 and ATZ were
rotated 180° compared to compounds 13i, 19b, 19d, and
21.66−68
The amino acids are labeled according to the novel
inhibitor−enzyme complexes presented herein (13i, 19b, and
19d). The overall binding configurations for 13i, 19b, and 19d
to the protease are, as expected, in good accordance with those
of previously published linear inhibitors 20,27 21,28 as well as
with ATZ, despite the novel β-hydroxy γ-lactam moiety.
Lactam Moiety. On the basis of the modeling studies, it
was postulated that the β-hydroxyl group of the lactam moieties
forms hydrogen bonds with the catalytic aspartic acids (Asp25
and Asp125).
2731
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
Figure 2. Comparison of the overall X-ray conformations and binding patterns of compounds 13i (top left, PDB code 2uxz), 19b (bottom left, PDB
code 4a6c), and 19d (bottom right, PDB code 4a6b) in the active site of HIV-1 protease. Compound 13i forms five direct hydrogen bonds to the
protease and five more via water molecules. The corresponding binding interactions for 19b and 19d are four direct bonds and six more through
water bridges. In all three complexes, two of the interactions via water are due to the structural water coordinating Ile50 and Ile150 in the protein
flaps.
P1′ Site. In accordance with the previously observed results
for 21,28 the P1′ outer phenyl group in both 13i and 19d
interacts through a hydrophobic interaction with Pro81 (3.3−
3.8 Å) and an edge−face π−π interaction to Phe153 (3.7−3.8
Å). The differences in length of the central motif as well as in
the length of the extension of 13i, 19d, 20, 21, and ATZ in the
P1′ site are nicely accommodated through corresponding shifts
in the positions of Phe153 and Pro81 (Figure 5).28
These interactions are likely to improve the binding constant
and is the most likely explanation of the better binding of
compound 13i than 13d, differing only in the length of the
extension in the P1′ site. In a previously examined complex with
compound 20, the interaction with Phe153 was not possible, as
the corresponding moiety only reached far enough for a van der
Waal interaction with Pro81. Neither is the interaction with
Phe153 observed in the complex with ATZ. Since the binding
modes of 19b and 19d are very similar (Figure 2), only 19d was
included in the analysis, as the structure of the complex could
be interpreted at higher resolution.
DISCUSSION
■
Chemistry. The introduction of the β-hydroxy γ-lactams as
new scaffolds was intended to provide more rigid PIs and to
relocate the hydroxyl group from the backbone to enable more
symmetric binding to the catalytically active Asp25 and Asp125
of the HIV-1 protease. The outcome of the dialkylation
reactions performed to obtain 2a−d was in accordance with the
results described by Amat et al. in 2007, although they observed
a larger substrate-dependent variability.69 When introducing the
benzyl moiety in the first alkylation, as in the cases of 2b and
2d, the yields were lower (2% and 5%, respectively) than when
the allyl group was introduced before the benzyl moiety (as in
2a and 2c, with yields of 49% and 33%, respectively). The same
trend has been reported by Johnson et al. with 4-substitued
lactams40 but was not observed in the 5-substitued examples
presented by Meyers et al., in which the order of addition did
not affect the yields.41 Probable reasons for the lower yields
observed by Johnson et al. were steric and/or electrostatic
2732
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
Figure 3. Previously published PIs for comparison: ATZ (PDB code 3EL9), 20 (PDB code 2uxz),27 and 21 (PDB code 2xye).28
interactions between the 4-hydroxy group and the bulkier 3-
benzyl moiety present after the first alkylation, compared to the
smaller allyl group. These findings followed the reasoning
presented by Huang et al.,70 who proposed stereoelectronic
factors to be the major explanation in this class of stereo-
selective two-step alkylation reactions.
In the present work, the diastereoselectivity controlled by the
stereochemistry of the 4-hydroxy group was strong enough to
allow highly enantiomerically enriched isomers to be obtained
in all cases.
There was an urgent need for a robust method for the
synthesis of the prime side hydrazide moiety (5a). The
procedures used previously were cumbersome and low yielding
because of the use of toxic and environmentally hazardous
hydrazine hydrate and/or tedious purification protocols.
Previously used synthetic procedures were not satisfactory,
since the quantities of prime side were not sufficient to support
our lead optimization program throughout. The synthetic route
to the prime side hydrazide moiety 5a presented here provided
an efficient way of producing sufficient amounts and constitutes
an improvement in yield as well as a reduction in work
compared to previous methods.27,71 With this convenient
method, there was no need to use hazardous hydrazine hydrate,
and the purification protocol resulted in a good yield.
Biological Evaluation and X-ray Structure Analysis.
The biological results obtained from the novel lactam-
containing inhibitors are summarized in Tables 1−3. Evaluation
of the four stereoisomers (13a−d) gave two active and two
nonactive PIs (Table 1). The (3R,4R) and (3R,4S) stereo-
isomers in the lactam ring showed the best results, with 13a and
13d being the most potent compounds (Ki < 10 nM and EC50
< 1 μM).
The most important structure−activity feature appears to be
the direction of the benzyl in the P1 position. With R-
stereochemistry at the α-carbon (13a and 13d), the direction of
the β-hydroxy substituent (position 4) appears to be of less
importance for inhibition with 13a and 13d being almost
equipotent. With S-stereochemistry at the α-carbon, 13b and
13c showed almost no inhibiting effect on the enzyme or in the
cell-based antiviral activity assay (Table 1) and, as expected, the
TBS-protected inhibitors 12a and 12d did not show any
inhibitory potency.
Figure 4. The position of the β-hydroxy group of the lactame ring,
involved in hydrogen binding to both Asp25 and Asp125 in 13i (gold),
is different in 19d (purple) and 19b (not shown) exhibiting the three-
carbon linker. This leads to a loss of a hydrogen bond to one of the
catalytic aspartates. The position of the β-hydroxy group involved in
hydrogen binding in 19d is 2.1 Å from the position observed in 13i.
2733
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
Figure 5. Comparison of the positioning of the cocrystallized inhibitors in the S2−S3 pocket and interaction with Pro81 and Phe153 in the S1′
pocket. The effect on the S2−S3 site is visualized at residues Asp29, Asp30, Arg108, and Pro181. (a) Superimposition of 13i (gold) and 19d
(purple). As a result of an additional CH2 group in 19d, the indanol group of 19d dislocates Asp30 compared to 13i. (b) Superimposition of 19d
and 20 (blue). The lactam group present in the new series of compounds as in 19d mimics the conformation of 20, also exhibiting the three-carbon
linker, very well. With the lactam ring present, the position of the indanol ring, and therefore also Asp30/130, is more similar to the situation in 13i
comprising the two-carbon linker. (c) Superimposition of 13i and ATZ (black). Despite the differences of functional elements between 13i and ATZ
in the S2−S3 site, the common ribbon of the compounds overlap well. In the P1′ site 13i and 19d extend further than ATZ, inducing spatial
rearrangement of Phe153 and Pro81. Compound 19d induced side chain and main chain atom displacements in Phe153 and Pro81 up to 2.5 and 1.7
Å, respectively, compared to ATZ complex positions. Corresponding displacements with 13i as substrate are 0.9 and 0.5 Å. None of the new
compounds induced a shift in the position of Arg108, as was seen in compound 20.27
When the P1′ side chain was decorated with heteroaromatic
moieties (Table 2), at best a 10-fold improvement in inhibitory
potency was observed (13g and 13i, EC50 = 0.04 μM).
Compared to the 2-pyridyl inhibitor 13e (EC50 = 0.190 μM),
the 3- and 4-pyridyl-substituted inhibitors (13g and 13i,
respectively) with (3R,4S) stereochemistry afforded 5 times
higher potency, with EC50 of 40 nM. Despite the fact that ATZ
contains a 2-pyridinyl in position P1′,37 our previous series with
one- or three-carbon spacers showed better potency for the 3-
and 4-pyridinyl-substituted inhibitors.24,27,28 With the linear
two-carbon spacer the 2-, 3-, and 4-pyridinyls gave equipotent
inhibitors.26 This result was also obtained with the lactam-
containing inhibitors with the three-carbon extended PIs in the
19 series.
Comparing p-bromide functionalized inhibitors 13a and 19a,
a 5-fold loss of potency within measured Ki and EC50 values
were observed. However, the same trend is present in both
series (13 and 19, Table 3) as seen with the shorter inhibitors.
The pyridyls (19c−e) showed slightly better inhibition
compared to the p-bromo compound 19a. The p-phenyl
substituted 19b was among the most potent inhibitors,
concurring with recent reports.26,28
with two- and three-carbon spacers was in good agreement with
the observed variation in enzyme binding activity.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details of syntheses and reaction conditions, X-ray statistics,
and description of biological assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +46 18-471 4667. Fax: +46 18 471 4374. E-mail: mats.
larhed@orgfarm.uu.se.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Swedish Research Council (VR) and the Swedish
Foundation for Strategic Research (SSF) for financial support,
and we thank Prof. Torsten Unge for help with preparation of
the crystals.
REFERENCES
■
CONCLUSIONS
■
(1) Barre-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.;
Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Vezinet-Brun, F.;
Rouzioux, C.; Rozenbaum, W.; Montagnier, L. Isolation of a T-
Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune
Deficiency Syndrome (AIDS). Science 1983, 220, 868−871.
(2) Gallo, R. C.; Sarin, P. S.; Gelmann, E. P.; Robert-Guroff, M.;
Richardson, E.; Kalyanaraman, V. S.; Mann, D.; Sidhu, G. D.; Stahl, R.
E.; Zolla-Pazner, S.; Leibowitch, J.; Popovic, M. Isolation of Human T-
Cell Leukemia Virus in Acquired Immune Deficiency Syndrome
(AIDS). Science 1983, 220, 865−867.
(3) Global Report: UNAIDS Reoport on the Global AIDS Epidemic
2010; UNAIDS: Geneva, 2010; 364 pp.
(4) Roberts, N. A.; Martin, J. A.; Kinchington, D.; Broadhurst, A. V.;
Craig, J. C.; Duncan, I. B.; Galpin, S. A.; Handa, B. K.; Kay, J.; Krohn,
A.; Lambert, R. W.; Merrett, J. H.; Mills, J. S.; Parkes, K. E. B.;
We have successfully introduced β-hydroxy γ-lactams providing
a rigid backbone moiety and replaced the previously used tert-
hydroxy group with a sec-hydroxy group. All four stereoisomers
were synthesized, incorporated into the full inhibitor and
evaluated. In addition, the length of the central spacer was
varied (two or three carbons). Functionalization of the two
most potent stereoisomers (3R,4S) (13a) and (3R,4R) (13d)
with heteroaromatic moieties in the p-benzyl P1′ position
improved the potency, rendering Ki values down to 0.7 nM and
EC50 values down to 0.04 μM. Three inhibitors were
cocrystallized with the HIV-1 protease enzyme providing
information about the binding of the hydroxy lactams to the
enzyme. The change in binding pattern between the inhibitors
2734
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
Redshaw, S.; Ritchie, A. J.; Taylor, D. L.; Thomas, G. J.; Machin, P. J.
Rational Design of Peptide-Based HIV Proteinase-Inhibitors. Science
1990, 248, 358−361.
(5) Hammer, S. M.; Squires, K. E.; Hughes, M. D.; Grimes, J. M.;
Demeter, L. M.; Currier, J. S.; Eron, J. J. Jr.; Feinberg, J. E.; Balfour, H.
H. Jr.; Deyton, L. R.; Chodakewitz, J. A.; Fischl, M. A. A Controlled
Trial of Two Nucleoside Analogues plus Indinavir in Persons with
Human Immunodeficiency Virus Infection and CD4 Cell Counts of
200 per Cubic Millimeter or Less. AIDS Clinical Trials Group 320
Study Team. N. Engl. J. Med. 1997, 337, 725−733.
(6) Palella, F. J. Jr.; Delaney, K. M.; Moorman, A. C.; Loveless, M.
O.; Fuhrer, J.; Satten, G. A.; Aschman, D. J.; Holmberg, S. D.
Declining Morbidity and Mortality among Patients with Advanced
Human Immunodeficiency Virus Infection. HIV Outpatient Study
Investigators. N. Engl. J. Med. 1998, 338, 853−860.
(7) Mocroft, A.; Vella, S.; Benfield, T. L.; Chiesi, A.; Miller, V.;
Gargalianos, P.; d’Arminio Monforte, A.; Yust, I.; Bruun, J. N.; Phillips,
A. N.; Lundgren, J. D. Changing Patterns of Mortality across Europe in
Patients Infected with HIV-1. Lancet 1998, 352, 1725−1730.
(8) Yeni, P. Update on HAART in HIV. J. Hepatol. 2006, 44, S100−
S103.
(9) Pokorna, J.; Machala, L.; Rezacova, P.; Konvalinka, J. Current and
Novel Inhibitors of HIV Protease. Viruses 2009, 1, 1209−1239.
(10) Ghosh, R. K.; Ghosh, S. M.; Chawla, S. Recent Advances in
Antiretroviral Drugs. Expert Opin. Pharmacol. 2011, 12, 31−46.
(11) Carr, A. Toxicity of Antiretroviral Therapy and Implications for
Drug Development. Nat. Rev. Drug Discovery 2003, 2, 624−634.
(12) Hawkins, T. Understanding and Managing the Adverse Effects
of Antiretroviral Therapy. Antiviral Res. 2010, 85, 201−209.
(13) Walmsley, S. Protease Inhibitor-Based Regimens for HIV
Therapy: Safety and Efficacy. JAIDS, J. Acquired Immune Defic. Syndr.
2007, 45, S5−S13.
(14) Mehellou, Y.; De Clercq, E. Twenty-Six Years of Anti-HIV Drug
Discovery: Where Do We Stand and Where Do We Go? J. Med. Chem.
2010, 53, 521−538.
(15) Wensing, A. M. J.; van Maarseveen, N. M.; Nijhuis, M. Fifteen
Years of HIV Protease Inhibitors: Raising the Barrier to Resistance.
Antiviral Res. 2010, 85, 59−74.
(16) Randolph, J. T.; DeGoey, D. A. Peptidomimetic Inhibitors of
HIV Protease. Curr. Top. Med. Chem. 2004, 4, 1079−1095.
(17) Anderson, J.; Schiffer, C.; Lee, S.-K.; Swanstrom, R. Viral
Protease Inhibitors. Handb. Exp. Pharmacol. 2009, 189, 85−110.
(18) Este, J. A.; Cihlar, T. Current Status and Challenges of
Antiretroviral Research and Therapy. Antiviral Res. 2010, 85, 25−33.
(19) Roberts, J. D.; Bebenek, K.; Kunkel, T. A. The Accuracy of
Reverse-Transcriptase from HIV-1. Science 1988, 242, 1171−1173.
(20) Mastrolorenzo, A.; Rusconi, S.; Scozzafava, A.; Barbaro, G.;
Supuran, C. T. Inhibitors of HIV-1 Protease: Current State of the Art
10 Years after Their Introduction. From Antiretroviral Drugs to
Antifungal, Antibacterial and Antitumor Agents Based on Aspartic
Protease Inhibitors. Curr. Med. Chem. 2007, 14, 2734−2748.
(21) Clavel, F.; Hance, A. J. HIV drug resistance. N. Engl. J. Med.
2004, 350, 1023−1035.
(22) Hulten, J.; Bonham, N. M.; Nillroth, U.; Hansson, T.;
Zuccarello, G.; Bouzide, A.; Aqvist, J.; Classon, B.; Danielson, U. H.;
Karlen, A.; Kvarnstrom, I.; Samuelsson, B.; Hallberg, A. Cyclic HIV-1
Protease Inhibitors Derived from Mannitol: Synthesis, Inhibitory
Potencies, and Computational Predictions of Binding Affinities. J. Med.
Chem. 1997, 40, 885−897.
(23) Ekegren, J. K.; Unge, T.; Safa, M. Z.; Wallberg, H.; Samuelsson,
B.; Hallberg, A. A New Class of HIV-1 Protease Inhibitors Containing
a Tertiary Alcohol in the Transition-State Mimicking Scaffold. J. Med.
Chem. 2005, 48, 8098−8102.
(24) Ekegren, J. K.; Ginman, N.; Johansson, A.; Wallberg, H.; Larhed,
M.; Samuelsson, B.; Unge, T.; Hallberg, A. Microwave-Accelerated
Synthesis of P1′-Extended HIV-1 Protease Inhibitors Encompassing a
Tertiary Alcohol in the Transition-State Mimicking Scaffold. J. Med.
Chem. 2006, 49, 1828−1832.
(25) Ekegren, J. K.; Gising, J.; Wallberg, H.; Larhed, M.; Samuelsson,
B.; Hallberg, A. Variations of the P2 Group in HIV-1 Protease
Inhibitors Containing a Tertiary Alcohol in the Transition-State
Mimicking Scaffold. Org. Biomol. Chem. 2006, 4, 3040−3043.
(26) Mahalingam, A. K.; Axelsson, L.; Ekegren, J. K.; Wannberg, J.;
Kihlstrom, J.; Unge, T.; Wallberg, H.; Samuelsson, B.; Larhed, M.;
Hallberg, A. HIV-1 Protease Inhibitors with a Transition-State Mimic
Comprising a Tertiary Alcohol: Improved Antiviral Activity in Cells. J.
Med. Chem. 2010, 53, 607−615.
̈
(27) Wu, X.; Ohrngren, P.; Ekegren, J. K.; Unge, J.; Unge, T.;
Wallberg, H.; Samuelsson, B.; Hallberg, A.; Larhed, M. Two-Carbon-
Elongated HIV-1 Protease Inhibitors with a Tertiary-Alcohol-
Containing Transition-State Mimic. J. Med. Chem. 2008, 51, 1053−
1057.
̈
(28) Ohrngren, P.; Wu, X.; Persson, M.; Ekegren, J. K.; Wallberg, H.;
Vrang, L.; Rosenquist, A.; Samuelsson, B.; Unge, T.; Larhed, M. HIV-1
Protease Inhibitors with a Tertiary Alcohol Containing Transition-
State Mimic and Various P2 and P1′ Substituents. MedChemComm
2011, 2, 701−709.
(29) Bold, G.; Fassler, A.; Capraro, H.-G.; Cozens, R.; Klimkait, T.;
̈
Lazdins, J.; Mestan, J.; Poncioni, B.; Rosel, J.; Stover, D.; Tintelnot-
̈
Blomley, M.; Acemoglu, F.; Beck, W.; Boss, E.; Eschbach, M.;
Hurlimann, T.; Masso, E.; Roussel, S.; Ucci-Stoll, K.; Wyss, D.; Lang,
M. New Aza-Dipeptide Analogues as Potent and Orally Absorbed
HIV-1 Protease Inhibitors: Candidates for Clinical Development. J.
Med. Chem. 1998, 41, 3387−3401.
(30) Piliero, P. J. Atazanavir: A Novel HIV-1 Protease Inhibitor.
Expert Opin. Invest. Drugs 2002, 11, 1295−1301.
(31) Schechter, I.; Berger, A. On the Size of the Active Site in
Proteases. I. Papain. Biochem. Biophys. Res. Commun. 1967, 27, 157−
162.
(32) Miller, M.; Schneider, J.; Sathyanarayana, B. K.; Toth, M. V.;
Marshall, G. R.; Clawson, L.; Selk, L.; Kent, S. B. H.; Wlodawer, A.
Structure of Complex of Synthetic HIV-1 Protease with a Substrate-
Based Inhibitor at 2.3 Å Resolution. Science 1989, 246, 1149−1152.
(33) Wlodawer, A.; Miller, M.; Jaskolski, M.; Sathyanarayana, B. K.;
Baldwin, E.; Weber, I. T.; Selk, L. M.; Clawson, L.; Schneider, J.; Kent,
S. B. H. Conserved Folding in Retroviral Proteases: Crystal Structure
of a Synthetic HIV-1 Protease. Science 1989, 245, 616−621.
(34) Brik, A.; Wong, C.-H. HIV-1 Protease: Mechanism and Drug
Discovery. Org. Biomol. Chem. 2003, 1, 5−14.
(35) Freidinger, R. M.; Veber, D. F.; Perlow, D. S.; Brooks, J. R.;
Saperstein, R. Bioactive Conformation of Luteinizing-Hormone-
Releasing Hormone: Evidence from a Conformationally Constrained
Analog. Science 1980, 210, 656−658.
(36) Thaisrivongs, S.; Pals, D. T.; Turner, S. R.; Kroll, L. T.
Conformationally Constrained Renin Inhibitory Peptides: γ-Lactam-
Bridged Dipeptide Isostere as Conformational Restriction. J. Med.
Chem. 1988, 31, 1369−1376.
(37) Robinson, B. S.; Riccardi, K. A.; Gong, Y.-F.; Guo, Q.; Stock, D.
A.; Blair, W. S.; Terry, B. J.; Deminie, C. A.; Djang, F.; Colonno, R. J.;
Lin, P.-F. BMS-232632, a Highly Potent Human Immunodeficiency
Virus Protease Inhibitor That Can Be Used in Combination with
Other Available Antiretroviral Agents. Antimicrob. Agents Chemother.
2000, 44, 2093−2099.
(38) Herrmann, J. L.; Schlessinger, R. H. Method for Alkylating
Lactones. J. Chem. Soc., Chem. Commun. 1973, 711−712.
(39) Maldaner, A. O.; Pilli, R. A. Stereoselective Alkylation of N-Boc-
2-pyrrolidinones and N-Boc-2-piperidinones. Synthesis and Character-
ization of Disubstituted Lactams. Tetrahedron 1999, 55, 13321−13332.
(40) Johnson, T. A.; Jang, D. O.; Slafer, B. W.; Curtis, M. D.; Beak, P.
Asymmetric Carbon−Carbon Bond Formations in Conjugate
Additions of Lithiated N-Boc Allylic and Benzylic Amines to
Nitroalkenes: Enantioselective Synthesis of Substituted Piperidines,
Pyrrolidines, and Pyrimidinones. J. Am. Chem. Soc. 2002, 124, 11689−
11698.
(41) Meyers, A. I.; Seefeld, M. A.; Lefker, B. A.; Blake, J. F. Origin of
Stereochemistry in Simple Pyrrolidinone Enolate Alkylations. J. Am.
Chem. Soc. 1997, 119, 4565−4566.
2735
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736

Journal of Medicinal Chemistry
Article
(42) Sprules, T. J.; Lavallee, J. F. Unexpected Contrasteric Alkylation
Leading to a Model for 5-Membered Ring Enolate Alkylation: Short
Stereoselective Synthesis of ( )-Acetomycin. J. Org. Chem. 1995, 60,
5041−5047.
(43) Orrling, K. M.; Wu, X. Y.; Russo, F.; Larhed, M. Fast, Acid-Free,
and Selective Lactamization of Lactones in Ionic Liquids. J. Org. Chem.
2008, 73, 8627−8630.
(44) Welton, T.; Hallett, J. P. Room-Temperature Ionic Liquids:
Solvents for Synthesis and Catalysis. 2. Chem. Rev. 2011, 111, 3508−
3576.
(45) Rassu, G.; Zanardi, F.; Battistini, L.; Gaetani, E.; Casiraghi, G.
Expeditious Syntheses of Sugar-Modified Nucleosides and Collections
Thereof Exploiting Furan-, Pyrrole-, and Thiophene-Based Siloxy
Dienes. J. Med. Chem. 1997, 40, 168−180.
(46) Boger, D. L.; Schule, G. Synthesis of Acyclic Precursors to
(3S,4S)-4-Hydroxy-2,3,4,5-tetrahydro-pyridazine-3-carboxylic acid and
Incorporation into a Luzopeptin/Quinoxapeptin Dipeptide. J. Org.
Chem. 1998, 63, 6421−6424.
(47) Zienty, F. B.; Steahly, G. W. N-Substituted 2-Pyrrolidones. J.
Am. Chem. Soc. 1947, 69, 715−716.
(65) Weber, I. T.; Agniswamy, J. HIV-1 Protease: Structural
Perspectives on Drug Resistance. Viruses 2009, 1, 1110−1136.
(66) Navia, M. A.; Fitzgerald, P. M. D.; Mckeever, B. M.; Leu, C. T.;
Heimbach, J. C.; Herber, W. K.; Sigal, I. S.; Darke, P. L.; Springer, J. P.
Three-Dimensional Structure of Aspartyl Protease from Human
Immunodeficiency Virus HIV-1. Nature 1989, 337, 615−620.
(67) Wlodawer, A.; Erickson, J. W. Structure-Based Inhibitors of
HIV-1 Protease. Annu. Rev. Biochem. 1993, 62, 543−585.
(68) Bagossi, P.; Cheng, Y. S. E.; Oroszlan, S.; Tozser, J. Comparison
of the Specificity of Homo- and Heterodimeric Linked HIV-1 and
HIV-2 Proteinase Dimers. Protein Eng. 1998, 11, 439−445.
(69) Amat, M.; Lozano, O.; Escolano, C.; Molins, E.; Bosch, J.
Enantioselective Synthesis of 3,3-Disubstituted Piperidine Derivatives
by Enolate Dialkylation of Phenylglycinol-Derived Oxazolopiperidone
Lactams. J. Org. Chem. 2007, 72, 4431−4439.
(70) Huang, A.; Kodanko, J. J.; Overman, L. E. Asymmetric Synthesis
of Pyrrolidinoindolines. Application for the Practical Total Synthesis of
(−)-Phenserine. J. Am. Chem. Soc. 2004, 126, 14043−14053.
(71) Otteneder, M.; Plastaras, J. P.; Marnett, L. J. Reaction of
Malondialdehyde−DNA Adducts with Hydrazines: Development of a
Facile Assay for Quantification of Malondialdehyde Equivalents in
DNA. Chem. Res. Toxicol. 2002, 15, 312−318.
(48) Zawadzki, S.; Zwierzak, A. Simple Approach to 1-Alkyl-2-
isopropylhydrazines. Pol. J. Chem. 2003, 77, 315−319.
(49) Freedman, H. H. Industrial Applications of Phase-Transfer
Catalysis (Ptc): Past, Present and Future. Pure Appl. Chem. 1986, 58,
857−868.
(50) Wang, M. L.; Tseng, Y. H. Analysis of Phase-Transfer-Catalyzed
O-Alkylation Reaction Using Fractional Factorial Design Method.
Chem. Eng. Commun. 2005, 192, 557−574.
(51) Pappo, R.; Allen, D. S.; Lemieux, R. U.; Johnson, W. S. Osmium
Tetroxide-Catalyzed Periodate Oxidation of Olefinic Bonds. J. Org.
Chem. 1956, 21, 478−479.
(52) Panico, R.; Powell, W. H.; Richer, J. C. A Guide to IUPAC
Nomenclature of Organic Compounds: Recommendations 1993, 1st ed.;
Blackwell Scientific Publications: Oxford, U.K., 1993; 208 pp.
(53) See Supporting Information for illustrations where the
stereochemistry is indicated for each molecule.
(54) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling
Reactions of Organoboron Compounds. Chem. Rev. 1995, 95, 2457−
2483.
(55) Larhed, M.; Hallberg, A. Microwave-Promoted Palladium-
Catalyzed Coupling Reactions. J. Org. Chem. 1996, 61, 9582−9584.
(56) Larhed, M.; Moberg, C.; Hallberg, A. Microwave-Accelerated
Homogeneous Catalysis in Organic Chemistry. Acc. Chem. Res. 2002,
35, 717−727.
(57) Kappe, C. O.; Dallinger, D. The Impact of Microwave Synthesis
on Drug Discovery. Nat. Rev. Drug Discovery 2006, 5, 51−63.
(58) Brown, H. C.; Knights, E. F.; Scouten, C. G. Hydroboration
XXXVI. Direct Route to 9-Borabicyclo[3.3.1]nonane via Cyclic
Hydroboration of 1,5-Cyclooctadiene. 9-Borabicyclo[3.3.1]nonane as
a Uniquely Selective Reagent for Hydroboration of Olefins. J. Am.
Chem. Soc. 1974, 96, 7765−7770.
(59) Parikh, J. R.; Doering, W. V. E. Sulfur Trioxide in Oxidation of
Alcohols by Dimethyl Sulfoxide. J. Am. Chem. Soc. 1967, 89, 5505−
5507.
(60) Zhang, Q.; Jin, H.-X.; Wu, Y. A Facile Access to Bridged 1,2,4-
Trioxanes. Tetrahedron 2006, 62, 11627−11634.
(61) Gronowitz, S.; Bjork, P.; Malm, J.; Hornfeldt, A. B. The Effect of
Some Additives on the Stille Pd0-Catalyzed Cross-Coupling Reaction.
J. Organomet. Chem. 1993, 460, 127−129.
(62) Floistrup, E.; Goede, P.; Stromberg, R.; Malm, J. Synthesis of
̈
̈
estradiol backbone mimics via the Stille reaction using copper(II)
oxide as co-reagent. Tetrahedron Lett. 2011, 52, 209−211.
(63) Farina, V.; Kapadia, S.; Krishnan, B.; Wang, C. J.; Liebeskind, L.
S. On the Nature of the Copper Effect in the Stille Cross-Coupling. J.
Org. Chem. 1994, 59, 5905−5911.
(64) Wempe, M. F.; Anderson, P. L. Atazanavir Metabolism
According to CYP3A5 Status: An in Vitro−in Vivo Assessment.
Drug Metab. Dispos. 2011, 39, 522−527.
2736
dx.doi.org/10.1021/jm201620t | J. Med. Chem. 2012, 55, 2724−2736