HIV-1 protease inhibitors with a transition-state mimic comprising a tertiary alcohol: Improved antiviral activity in cells

Article abstract of DOI:10.1021/jm901165g

By a small modification in the core structure of the previously reported series of HIV-1 protease inhibitors that encompasses a tertiary alcohol as part of the transition-state mimicking scaffold, up to 56 times more potent compounds were obtained exhibiting EC₅₀ values down to 3 nM. Three of the inhibitors also displayed excellent activity against selected resistant isolates of HIV-1. The synthesis of 25 new and optically pure HIV-1 protease inhibitors is reported, along with methods for elongation of the inhibitor P1′ side chain using microwave-accelerated, palladium-catalyzed cross-coupling reactions, the biological evaluation, and X-ray data obtained from one of the most potent analogues cocrystallized with both the wild type and the L63P, V82T, I84 V mutant of the HIV-1 protease.

Full text of DOI:10.1021/jm901165g

J. Med. Chem. 2010, 53, 607–615 607
DOI: 10.1021/jm901165g
HIV-1 Protease Inhibitors with a Transition-State Mimic Comprising a Tertiary Alcohol: Improved
Antiviral Activity in Cells†
‡
‡
‡
‡
‡
§
€
A.K. Mahalingam, Linda Axelsson, Jenny K. Ekegren, Johan Wannberg, Jacob Kihlstrom, Torsten Unge,
Hans Wallberg, Bertil Samuelsson, Mats Larhed,*,‡ and Anders Hallberg‡
‡Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden,
§Department of Cell and Molecular Biology, Structural Biology, BMC, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden, and
Medivir AB, P.O. Box 1086, SE-141 44, Huddinge, Sweden
Received August 5, 2009
By a small modification in the core structure of the previously reported series of HIV-1 protease
inhibitors that encompasses a tertiary alcohol as part of the transition-state mimicking scaffold, up to 56
times more potent compounds were obtained exhibiting EC50 values down to 3 nM. Three of the
inhibitors also displayed excellent activity against selected resistant isolates of HIV-1. The synthesis of
25 new and optically pure HIV-1 protease inhibitors is reported, along with methods for elongation of
the inhibitor P10 side chain using microwave-accelerated, palladium-catalyzed cross-coupling reactions,
the biological evaluation, and X-ray data obtained from one of the most potent analogues cocrystallized
with both the wild type and the L63P, V82T, I84 V mutant of the HIV-1 protease.
Introduction
Almost three decades after the discovery of AIDS, the
pandemic is more severe than ever.1 There are, however,
glimpses of light in this otherwise dark period. The HIV-1
protease inhibitors, introduced in 1995, are certainly a bless-
ing for HIV/AIDS infected patients.2-4 In combination with
other anti-HIV-1 drugs, the protease inhibitors provide pro-
longed lifetime and better quality of life by efficiently suppres-
sing the formation of new infectious virus particles in patients.
However, two main factors restrict the clinical benefits of this
class of antiviral agents. First, many of the HIV-1 protease
inhibitors on the market, especially in the first generation,
suffer from poor pharmacokinetic properties due to poor
aqueous solubility, low metabolic stability, high protein bind-
ing, and poor membrane permeability. Because of the phar-
macokinetic problems, high doses of drugs are needed in order
to keep viral plasma levels down. Poor patient compliance is
commonly observed as a consequence of the severe side effects
that the high doses give.3,5 The new contributions to the
available arsenal of HIV-1 protease inhibitors, and the addi-
tion of low-dose ritonavir as a pharmacokinetic booster have
dealt with many of the pharmacokinetic problems, but there is
still room for improvement.6-9 Second, the rate at which the
virus reproduces and the high number of errors made in the
viral replication process create a large amount of mutated
viral strains. Thus, resistance toward the marketed HIV-1
protease inhibitors is a serious threat to efficient HIV-
treatment.6,8,10-14 The development of new HIV-1 pro-
tease inhibitors addressing these issues is therefore of high
importance.
†PDB codes for structure 11 with wild type HIV-1 protease and with
Figure 1. Biological data for compounds A and B and the general
structure of the new series of inhibitors with a shielded hydroxyl
group, C.
mutant strain L63P, V82T, I84 V are 2wkz and 2wl0, respectively.
*To whom correspondence should be addressed. Phone: þ46-18-
4714667. Fax: þ46-18-4714474. E-mail: Mats.Larhed@orgfarm.uu.se.
r
2009 American Chemical Society
Published on Web 12/04/2009
pubs.acs.org/jmc

608 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Scheme 1. Synthesis of Inhibitors 10-18a
Mahalingam et al.
a Reagents and conditions: (a) (1) benzaldehyde, KOtBu, benzene, room temp, (2) H2SO4, 56%; (b) mCPBA, AIBN, DCE, reflux, 60%; (c) Pd/C,
HCOOH, Et3N, EtOAc, 80 °C, 96%; (d) (1S,2R)-1-amino-2-indanol, 2-hydroxypyridine, DCM, sealed tube, 60 °C, 66%; (e) TBDPSCl, imidazole,
DCM, room temp, 80%; (f) (1) 2-methoxypropene, pyridinium p-toluenesulphonic acid, DCM, 0 °C, (2) TBAF, THF, room temp, 69%; (g) (1)
Dess-Martin periodinane, DCM, room temp, (2) 8, Na(OAc)3BH, AcOH, THF, room temp, 40%; (h) 11, 12: R0Sn(nBu)3, Pd(PPh3)2Cl2, CuO, DMF,
MW, 120 °C, 50 min, 40% and 31%; 10, 13-18: R0-boronic acid/ester, Pd(PPh3)2Cl2, Na2CO3 (aq), EtOH, DME, MW, 120 °C, 30 min, 20-62%.
In 2005, we introduced a new class of HIV-1 protease
inhibitors, structurally related to indinavir and atazanavir,
comprising a shielded tertiary alcohol as part of the transi-
tion-state mimicking scaffold, as exemplified by compounds
A15-17 and B18 in Figure 1. A tertiary alcohol unit in the
transition-state mimic in protease inhibitors had previously
been only briefly reported in the literature, and generally, it
resulted in less potent compounds compared to the secondary
alcohol analogues.19-23 The two reported series of HIV-1
protease inhibitors (Figure 1), both encompassing a tertiary
alcohol, differ inthe length of the central carbon tether. Potent
inhibitors were obtained with Ki values down to 2.1 nM, and
several of the compounds exhibited excellent membrane
permeability in a Caco-2 assay.15,16,18 Unfortunately, the
compounds had only moderate cellular antiviral activities,
the best EC50 value was 170 nM. Furthermore, rapid meta-
bolism of several inhibitors in liver microsomes was encoun-
tered, A had an intrinsic clearance of 154 μL/min/mg.
Although knowing that it is difficult to predict pharmaco-
kinetic outcomes of minor structural modifications, we
decided to address an alternative central scaffold of the
inhibitors. Thus, we became interested in synthesizing a third
series of compounds (C, Figure 1), having a two-carbon tether
rather than one- and three-carbon tethers (A and B, Figure 1)
between the quaternary carbon and the hydrazide β-nitrogen
in the transition-state mimicking scaffold. X-ray structures of
the A and B type of inhibitors combined with computational
modelingledustobelieve thatthe hydroxyfunctionalityofthe
new C-class of inhibitors would be positioned closer to
Asp-25, possibly increasing potency compared to the pre-
viouslysynthesized inhibitors. Optimizationby P10 elongation
was performed in order to improve cell-based antiviral activ-
ities. Furthermore, the indanolamine moiety present in most
of the previously synthesized inhibitors is known to undergo
30-hydroxylation of the indan and to be metabolically
unstable.24,25 Therefore a few examples of inhibitors with
other P2-groups were assessed in the study. Herein, the
synthesis of the new inhibitors, the biological evaluation,
and X-ray data obtained from one of the compounds cocrys-
tallized withboth thewildtype HIV-1protease anda clinically
relevant mutant of the HIV-1 protease are presented.
Results
Chemistry. The bromide (S)-9, serving as an aryl palla-
dium precursor for cross-coupling reactions, was synthesized
from commercially available γ-butyrolactone (1, Scheme 1).
A mixed aldol condensation between this lactone and
benzaldehyde gave alkene 2. mCPBAa epoxidation and
palladium(0)-catalyzed reduction resulted in the racemic
tertiary alcohol-containing compound 4. Ring-opening of
the lactone moiety by (1S,2R)-1-amino-2-indanol in the
presence of a stoichiometric amount of 2-hydroxypyridine
afforded triols (S)-5 and (R)-5, which were separated by flash
column chromatography. Protecting group manipulations
were necessary to enable selective oxidation of the primary
alcohol in (S)-5. This was performed via protection of the
primary hydroxyl group in (S)-5 with TBDPSCl, subsequent
blocking of the secondary alcohol with 2-methoxypropene,
followed by deprotection of the TBDPS-group to give (S)-7.
Diol (S)-7 was then oxidized to the corresponding aldehyde
using Dess-Martin periodinane. Reductive amination with
hydrazide 8,15 under acidic conditions, yielded the 4-bromo-
substituted inhibitor (S)-9. The procedure was repeated with
(R)-5, producing diastereomer (R)-9, which also was evalu-
ated in the enzyme assays. Compound (S)-9 was next used in
microwave-accelerated, palladium(0)-catalyzed cross-
coupling reactions26-30 to afford various para P10-extended
inhibitors, all with retained (S)-configuration at the quater-
nary carbon (Table 1). Stille-couplings were utilized to
a Abbreviations: mCPBA, 3-chloroperoxybenzoic acid; TBDPSCl,
tert-butyldiphenylsilylchloride; KOtBu, potassium tert-butoxide; AIBN,
2,20-azobis(2-methylpropionitrile); DCE, 1,2-dichloroethane; TBAF,
tetrabutylammonium fluoride; HATU, N,N,N0,N0-tetramethyl-O-(7-aza-
benzotriazol-1-yl)uronium hexafluorophosphate; DIEA, diisopropyl-
ethylamine; IBX, 2-Iodoxybenzoic acid; SQV, Saquinavir; RTV, Ritonavir;
IDV, Indinavir; LPV, Lopinavir; ATV, Atazanavir; DRV, Darunavir.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 609
Table 1. Synthesis, Enzyme Inhibition Data, Antiviral Activity, and Cytotoxicity of Compounds 9-18, 20, 22, 25, 27, and 29-34a
a Reaction conditions: see Schemes 1-3. All the inhibitors have the (S) absolute configuration at the tertiary alcohol unless otherwise stated. b The Ki
values for the marketed drugs are from an in house assay. Values in parentheses are literature data. c The EC50 values for the marketed drugs are from an
in house assay. Values in parentheses are literature data. d Caco-2. e Parent compound remaining (%) (PCR). f From (S)-7. g From (R)-7.

610 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Scheme 2. Synthesis of Inhibitors 20, 22, 25, and 27a
Mahalingam et al.
Scheme 3. Synthesis of Inhibitors 29-34a
a Reagents and conditions: (a) (S)-2-amino-N,3,3-trimethylbutan-
amide or (S)-2-amino-N,3-dimethylbutanamide or (S)-2-amino-N-(2-
methoxyethyl)-3-methylbutanamide, 2-hydroxypyridine, DCE, 80 °C,
63-75%; (b) (1) IBX, DCE, 80 °C, (2) 8, DCE, AcOH, Na(OAc)3BH,
room temp, 2-28%; (c) 32: 2-pyridylSn(nBu)3, Pd(PPh3)2Cl2, CuO,
DMF, 120 °C, 50 min, 17%. 33, 34: R0-B(OH)2, Pd(OAc)2, [(t-Bu)3-
PH]BF4, K2CO3, H2O, 1,2-dimethoxyethane, MW, 80 °C, 20 min, 44%
and 10%, respectively.
a Reagents and conditions: (a) (1) Dess-Martin periodinane, DCM,
room temp, (2) 19 or 21, Na(OAc)3BH, AcOH, THF, room temp, 21%
and 28%, respectively; (b) benzylamine or 4-bromobenzylamine, Ti-
(OiPr)4, dry THF, 34-57%; (c) (S)-2-(methoxycarbonylamino)-3,3-
dimethylbutanoic acid, HATU, dry DMF, DIEA, 10-17%. (S)-25
and 27 were isolated as epimeric mixtures 3:1.
(S)-2-amino-N,3-dimethylbutanamide (F, Scheme 3), and
(S)-2-amino-N-(2-methoxyethyl)-3-methylbutanamide (G,
Scheme 3). Only intermediate 28 (R = E) was isolated as the
primary alcohol and the diastereoisomers separated. In the
cases of 30 and 31, separation of the two diastereoisomers
was done after the reductive amination with 8. P10 variations
were again introduced using Suzuki and Stille-cross cou-
plings on (S)-29 to furnish 32-34.
produce pyridines 11 and 12 from the corresponding tribu-
tyltin reagents in the microwave cavity. The products 11 and
12 were isolated after purification of the reaction mixtures by
preparative RPLC-MS (40% and 31% yield, respectively,
Table 1). Compounds 10 and 13-18 were synthesized using
Suzuki cross-couplings with boronic acid or ester reactants
at 120 °C for 30 min of microwave heating followed by
RPLC-MS purification (20-62% yield, Table 1).
Biological Evaluations. Enzyme inhibition data and anti-
viral activities of compounds 9-18, 20, 22, 25, 27, and 29-34
are summarized as Ki and EC50 values in Table 1. Previously
synthesized derivatives A and B (Figure 1) are included in
Table 1 as reference inhibitors. Compounds 9-18, 20, and 22
(excluding (R)-9) were potent enzyme inhibitors, with Ki
values ranging from 1.2 to 12 nM (Table 1). In the cell-based
Similarly, the P10-benzyl analogue of (S)-9, compound 20,
was synthesized from (S)-7 and hydrazide 1915 (Scheme 2),
and derivative 22, with a one-carbon-elongated P10 group,
was produced from hydrazide 21.31 Compounds 25 and 27,
comprising a central amide functionality instead of a hydra-
zide moiety, making the backbone shorter than for previously
synthesized type A compounds, were produced by ring-open-
ing of epoxide 2315 with benzylamine and 4-bromobenzyla-
mine, followed by amide coupling with (S)-2-(methoxy-
carbonylamino)-3,3-dimethylbutanoic acid (Scheme 2).
Inhibitors 29-31, with variation in the P2-position, were
synthesized by opening of lactone 4 with (S)-2-amino-
N,3,3-trimethylbutanamide (E, Scheme 3), affording 28,
assay,
a
pronounced P10-substituent dependence was
encountered. The 4-aryl-elongated compounds, except the
p-amide 16, displayed excellent antiviral activity, with EC50
values ranging from 3 to 13 nM. The p-bromo and unsub-
stituted benzyl analogues (S)-9, 20, and 22 were less potent
and, surprisingly, the one-carbon-elongated derivative 22
was almost inactive in this assay (EC50 = 6800 nM, Table 1).
Compounds 25 and 27, shortened by exchanging the hydra-
zide core structure with an amide, were also inactive. The
P2-varied inhibitors 29-34 showed good protease inhibitory

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 611
and antiviral activity, with Ki values from 1.0 to 3.2 nM and
EC50 values from 37 to 170 nM (Table 1). The (S)-confi-
guration of the carbon with the tertiary alcohol was assigned
by comparing the potency between inhibitors 29-34 and the
inhibitors with indanolamine in the P2-position and was not
proven by X-ray crystallography. A rough idea of the
inhibitor cytotoxicity is given by the CC50 value, which is
the inhibitor concentration causing a 50% decrease in cell
proliferation. Three of the evaluated compounds displayed
cell toxic properties, with CC50 values below 10 μM, includ-
ing the bromo-, 3-pyridyl, and benzodioxolane derivatives
(S)-9, 12, and 15 (Table 1). On the basis of the favorable Ki/
EC50/CC50 profile inhibitor 11, together with parent inhibi-
tor 9 and related inhibitor 12, were selected for further
studies. The choice of investigating 12 in these assays instead
of 13, with a lower EC50 value, was based on the better
solubility of nonsymmetric pyridine derivates. In a liver micro-
some homogenate, compounds (S)-9 and 12 were extensively
degraded by metabolic enzymes and intrinsic clearance
values above 300 μL/min/mg were encountered (Table 1).
On the other hand, the 2-pyridyl derivative 11, with the
Atazanavir prime side, turned out to be considerably more
stable, with Clint = 20 μL/min/mg. Compounds (S)-9 and 12
had excellent membrane permeability in a Caco-2 assay (Papp
(Caco-2) 42 ꢀ 10-6 cm/s and 26 ꢀ 10-6 cm/s), whereas 11
penetrated more slowly (Papp (Caco-2) = 3.5 ꢀ 10-6 cm/s).
Inhibitors (S)-9, 11, and 12 were further evaluated against
selected protease inhibitor resistant isolates of HIV-1. The
resistant isolates were obtained by passing cell-free virus in
media containing stepwise increased concentrations of
saquinavir (entries 2 and 3, Table 2), a symmetric diol based
inhibitor38,39 (entry 4, Table 2) and ritonavir (entries 5 and 6,
Table 2), all known to induce clinically relevant mutations in
the HIV-1 protease genome. All three inhibitors were equi-
potent or more potent toward the viral isolate with G48V
and L90M mutations in the protease, commonly encoun-
tered in patients treated with saquinavir,12 compared to the
wild-type enzyme (entry 2, Table 2). The V82A and M46I
mutations, located in the S10 sub site and the flap region of
the enzyme, respectively, are known to cause resistance
toward several of the launched HIV-1 protease inhibitors.11,13
Interestingly, very high potencies against one of the isolates
containing these mutations were obtained with compounds
(S)-9, 11 and 12, with 11 being equally potent toward this
isolate as to the wild-type (entry 4, Table 2). Four mutations,
at amino acid 33, 82, 84, and 90, are named universal
protease associated mutations (UPAMs) and are very
commonly seen in patients failing HIV-therapy.12 Com-
pounds (S)-9, 11, and 12 remain relatively potent when only
one UPAM is present (Entries 2, 4, and 5, Table 2), and with
two UPAMs, L90 M and I84 V (entry 3, Table 2), but not
when both residues 82 and 84 are mutated (entry 6, Table 2).
X-ray Crystallographic Data. The arrangement of com-
pound 11 in the active site of the wild type HIV-1 protease
including the most relevant hydrogen bonds, as deduced
from the 1.7 A X-ray structure, with R/Rfree of 0.23/0.25,
(PDB code 2wkz), is illustrated in Figure 2. For comparison,
Table 2. Antiviral Activity Against Selected HIV-1 Protease Inhibitor
Resistant Isolates
EC50 (μM)
entry
mutations in protease
(S)-9
11
12
1
2
3
4
5
6
wild-type (wt)
G48 V, L90M
0.040
0.008
0.044
0.070
0.074
0.24
0.007
0.008
0.007
0.006
0.024
0.13
0.009
0.010
0.019
0.014
0.027
0.60
A71 V, I84 V L90M
V32I, M46I A71 V, V82A
V32I, M46I V82A
M46I, V82F, I84 V
Ki(nM)
7a
L63P, V82T, I84 V
30
16
16
a Ki atazanavir 6.5 nM.
Figure 2. X-ray crystal structures of H, 11, and I cocrystallized with HIV-1 protease.

612 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Mahalingam et al.
Figure 4. Detail of the superimposition of wt/11 (shown in gray)
and mutant L63P, V82T, I84 V/11 (shown in green), describing the
interaction of the P1-part of inhibitor 11 with V182T and I184 V.
Figure 3. Superimposition of the X-ray structures of inhibitor 11
(gray) and H (green).
the previously synthesized H16 with the 1-carbon tether (class
A) and the 2-pyridyl group in the para-benzyl P10 position
(PDB code 2cem) and I18 with the 3-carbon tether (class B)
and the p-bromo benzyl side chain in P10 (PDB code 2uxz)
are also presented. In the solved structure, compound
11 has rotated 180° in the active site as compared to H and
I. Exemplified by Asp25 from one monomer in the
H/I-case is assigned as Asp125 from the other monomer in
the 11-case.40 The crystal structure of 11 confirmed the (S)
absolute configuration of the tertiary alcohol. It also showed
that while I has a distance of 2.9 A from the tertiary alcohol
to Asp25, 11 and H both have a shorter binding distance,
2.7 A to Asp 25/125. Additionally, the tertiary hydroxyl
group in 11 has a hydrogen bond to the backbone carbonyl
of Gly27 (2.6 A), while in H, the tertiary alcohol is situated
too far away (3.3 A) to form the corresponding hydrogen
bond. Compounds H and I have two hydrogen bonds
from the indanolamine hydroxy group to Gly127 and
to Asp129, while the same hydroxyl group in 11 has one
hydrogen bond to the backbone NH-group of Asp29
(3.0 A) and one hydrogen bond via a water molecule (3.1
and 2.8 A). The conserved water molecule that is hydro-
gen bonded to the nonprime side amide carbonyl, the
hydrazide carbonyl, and the enzyme backbone NH-
groups of Ile50 and Ile150, arranges similarly in H, 11,
and I. While structure 11 has seven direct hydrogen
bonds to the protein and five bonds via water molecules,
inhibitors H and I have six/five and five/three, respec-
tively.
Superimposition of H and 11 reveals a large difference in
the location of the P1 benzylic group (Figure 3). The hydro-
phobic interaction between Val182 and 11 is increased
through a change in the position of one of the methyl groups,
which bend down toward the benzylic group. The P1 aro-
matic ring in 11 is close enough to obtain three hydrophobic
interactions with one of the methyl groups of Val182.41 In H,
only the para position of the P1 aromatic ring is close enough
to have two hydrophobic interactions with Val82. In this
case, the distance is 3.7 A to one of the methyl groups of
Val82 and 3.8 A to the tertiary carbon. The meta position of
the benzylic group in 11 also forms edge on cation-π
interactions42 with the guanidine amino group of Arg108
(4.0 A), which are enhanced by the fact that the amino group
in Arg108 is tilted toward the inhibitor. The corresponding
distance from the benzylic group in H to Arg108 is 7.3 A. On
the other hand, H is situated closer to Ile84 and has three
possible hydrophobic interactions with both the ipso carbon
(3.5 A) and both ortho positions (3.6 and 3.9 A) of the P1
aromatic ring, whereas 11 can only interact with Ile184 via
the benzylic carbon (3.4 A).
The 1.9 A X-ray structure determination of 11 cocrystal-
lized with the mutant protease L63P, V82T, I84V (PDB code
2wl0), shows a loss of hydrophobic interaction between the
benzylic group in P1 and amino acids 182 and 184 after
mutating to the less bulky threonine and valine (Figure 4).
Both the R- and β-methyl groups of Val182 form hydro-
phobic interactions with the P1 benzylic moiety,41,43 but only
the methyl group of the mutant Thr182 forms the corre-
sponding hydrophobic interactions.44 There is also an im-
proved hydrophobic interaction between the P1 benzylic
carbon and the ethyl group of Ile184 (3.4 A) compared to
the methyl group in the mutated ValI84 (4.2 A). The potency
of 11 toward the viral isolate M46I, V82F, I84 V, slightly
different from the cocrystallized species, is decreased (entry
6, Table 2). The Ki-value for 11 against L63P, V82T, I84 V is
increased 10 times (entry 7, Table 2) compared to the wild
type value of 1.7 nM (Table 1).
Discussion
Part of the explanation for the higher inhibiting potency of
the type C inhibitors with the two-carbon-tether, which is
supported by the X-ray crystallography results with inhibitor
11, is that the tertiary alcohol does not only hydrogen bond to
Asp125 but also to the Gly27 carbonyl in the protein back-
bone. The P1 substituent of 11 also fits much better into the S1
and S3 pockets, as exemplified by the improved interactions
with Val182 and Arg108. Importantly, the change of the core
scaffold improved the EC50 value from 1100 nM for the
p-bromo analogue of A16 to 40 nM for (S)-9. It can be
hypothesized that the two-carbon-length scaffold gives this
27-fold improvement in EC50 values due to an improved
masking of the tertiary alcohol. Optimization by arylation
of the benzylic P10-part of (S)-9 furnished good cell-based
activities, with a 10-fold decrease in EC50 values. A compar-
ison between the results obtained from inhibitor 11 with the
corresponding in house assay data for the marketed inhibi-
tors, indinavir and atazanavir, indicates that 11 competes
favorably with these drugs. With a Ki value of 1.7 nM,
compound 11 is only a few times less potent as atazanavir
(Ki = 0.5 nM) and indinavir (Ki = 0.3 nM). Even more
interestingly, in the antiviral cell-based assay 11 (EC50
7 nM) was considerably more potent than indinavir (EC50
=
=
50 nM) and was equally potent as atazanavir (EC50 = 8 nM),
which was the first protease inhibitor to be given on a once a
day basis.45 The EC50 value of 11 is also in the same range as
lopinavir (EC50 = 10 nM). Compared to the most recent

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 613
HIV-1 protease inhibitor on the market, darunavir, 11 is only
two times less potent in cell assays than darunavir (EC50 = 4
nM). HIV-1 protease inhibitors are well-known to be rapidly
degradedbycytochromeP-450 enzymes, contributingtoa low
oral bioavailability of the drugs.7 In this sense, inhibitor 11
delivered a favorable result with a reasonably low intrinsic
clearance of 20μL/min/mgin liver microsomes. Theinhibitors
where the indanolamine had been replaced with N-alkylated
amino acid based P2’s were less potent, but the known
metabolic problems with the indanolamine warrant future
investigations. The Papp (Caco-2)-values and antiviral activ-
ities in the cell assay give somewhat contradictory results,
suggesting that the membranes are different in Caco-2 and in
HIV-infected MT4 cells (or indicate an activity of membrane
transport proteins). Compound 11 has an excellent profile
in enzyme assays with HIV-1 protease resistant viral iso-
lates, with the exception of the combined mutations at resi-
dues 82 and 84. The reason for that, as can be seen from
the cocrystallized X-ray structures, is probably a loss of
hydrophobic interactions. Taking all the above-mentioned
features into account, inhibitor 11 is a highly promising new
lead structure in the development of novel HIV-1 protease
inhibitors.
Plus instruments; 1H at 399.9 MHz and 13C at 100.6 MHz or 1H
at 399.8 MHz and 13C at 100.5 MHz. 3-[1-Phenyl-meth-(E)-
ylidene]-dihydro-furan-2-one (2),46-48 2-phenyl-1,5-dioxa-
spiro[2.4]heptan-4-one (3),48,49 and 3-benzyl-3-hydroxy-dihydro-
furan-2-one (4)46,50 were previously reported. All products were
>95% pure according to LC-MS (ELSD).
Materials. All the reagents were purchased from commercial
suppliers and used without further purification. Dichloro-
methane (DCM) and tetrahydrofuran (THF) were distilled
under nitrogen immediately before use. For DCM, calcium
hydride was used as a drying agent, and for THF, sodium/
benzophenone ketyl were used.
3-[1-Phenyl-meth-(E)-ylidene]-dihydro-furan-2-one (2). To a
cooled (0 °C) solution of γ-butyrolactone (5.0 g, 58 mmol) and
benzaldehyde (5.85 g, 55.1 mmol) in dry benzene (75 mL) was
added KOtBu (7.82 g, 69.7 mmol) portionwise. After the addi-
tion, the thick orange solution was stirred at room temp for 6 h.
The mixture was acidified with dilute H2SO4 (aq) and extracted
with Et2O (3 ꢀ 30 mL). The organic layer was dried (MgSO4)
and evaporated under reduced pressure. The residue was pur-
ified on flash column chromatography (silica gel, EtOAc/pet-
1
roleum ether, 1:5) to yield 2 (5.4 g, 56%) as a white solid. H
NMR (CDCl3, 400 MHz): δ 7.94 (s, 1H), 7.49-7.34 (m, 5H),
3.91 (m, 2H), 2.85 (m, 2H). 13C NMR (CDCl3, 100 MHz):
δ 172.8, 136.9, 134.9, 130.3, 130.1, 129.2, 123.8, 65.8, 27.7.
2-Phenyl-1,5-dioxa-spiro[2.4]heptan-4-one (3). To a solution
of 2 (4.0 g, 22.9 mmol) and 3-chloroperoxybenzoic acid (6.18 g,
27.6 mmol) in DCE (70 mL) was added AIBN (50 mg) at 80 °C,
and the mixture was refluxed in the dark for 6 h. The resulting
solution was cooled and filtered, the solvent was removed under
reduced pressure, and the residue dissolved in DCM. The
organic phase was washed successively with saturated, aqueous
solutions of NaHCO3 (20 mL), KI (20 mL), Na2S2O3 (20 mL),
and NaHCO3 (20 mL) and then dried (MgSO4) and evaporated
under reduced pressure. Purification by flash column chromato-
graphy (silica gel, EtOAc/petroleum ether, 1:4) gave 3 (2.62 g,
60%) as a white solid. A similar reaction without AIBN gave 3 in
46% yield. The material had spectroscopic properties identical
with those reported in the literature.49
Conclusion
In summary, by modifying the core structure of a series of
tertiary alcohol containing HIV-1 protease inhibitors, and by
varying the P10 side chains of the structure, as well as the P2
part, compounds exhibiting excellent antiviral activities were
obtained (EC50 values down to 3 nM). One of the best
inhibitors within the series of 25 compounds, 11, was also
potent toward several resistant isolates of HIV-1 and was only
slowly degraded by metabolic enzymes. In addition, no signs
of cytotoxicity were recorded for this inhibitor. X-ray crystal-
lography data showed a better fit of the P1 moiety of 11 in the
S1and S3 pockets. Alsoanadditionalhydrogenbondbetween
the tertiary alcohol in 11 and the HIV-1 protease enzyme was
detected, which could explain the higher potencies compared
to the previous series.
3-Benzyl-3-hydroxy-dihydro-furan-2-one (4). To a mixture of
3 (1.90 g, 10 mmol), Pd/C (Degussa type E101 NE/W, 0.53 g, 2.5
mol % Pd), and 20 mL of EtOAc in a reaction tube was added
formic acid (604 μL, 16 mmol) and triethylamine (2.09 mL, 15
mmol). The tube was sealed with a screw cap and heated at 80 °C
for 3 h. The reaction mixture was allowed to cool to room
temperature, the catalyst was filtered off, and volatiles were
evaporated under reduced pressure. The residue was purified by
silica flash column chromatography (i-hexane/EtOAc 1:1) to
give the alcohol as a colorless solid (1.85 g, 9.63 mmol, 96%). 1H
NMR (CDCl3, 400 MHz): δ 7.34-7.23 (m, 5H), 4.26 (m, 1H),
Experimental Section
General Information. Chemistry. The microwave reactions
were performed in a Smith synthesizer producing controlled
irradiation at 2450 MHz with a power of 0-300 W. The reaction
temperature was determined using the built-in online IR-sensor.
Flash column chromatography was performed on Merck silica
gel 60 (40-63 μm). Analytical thin layer chromatography was
3.75 (m, 1H), 3.05 (s, 2H), 3.04 (s, 1H), 2.39-2.24 (m, 2H). 13
C
NMR (CDCl3, 100 MHz): δ 178.9, 134.2, 130.1, 128.7, 127.5,
75.5, 65.3, 43.4, 34.0.
done using aluminum sheets precoated with silica gel 60 F254
.
UV light visualized components. Analytical RPLC-MS was
performed on a Gilson HPLC system with a Finnigan AQA
quadropole mass spectrometer with detection by UV (DAD)
using an Onyx Monolithic C18 column (50 mm ꢀ 4.6 mm) or a
Gilson HPLC system with a Finnigan Thermoquest MSQ
quadropole mass spectrometer with detection by a SEDERE
ELSD (Sedex 55) and MS (ESIþ) using an Onyx Monolithic C18
column (50 mm ꢀ 4.6 mm). For both systems with CH3CN in
0.05% aqueous HCOOH as mobile phase at a flow rate of 4 mL/
min. Preparative RPLC-MS was performed on a Gilson HPLC
system with a Finnigan AQA quadropole mass spectrometer
using a Zorbax SB-C8, 5 μm 21.2 mm ꢀ 150 mm (Agilent Tech-
nologies) column, with CH3CN in 0.05% aqueous HCOOH as
mobile phase at a flow rate of 15 mL/min. The IMAC support
used for metal ion removal was Chelating Superose (32 μmol/
mL Zn2þ capacity) prepacked in a 1.5 cm ꢀ 1 cm I.D. column.
1H and 13C NMR spectra were recorded on Varian Mercury
(S)-2-Benzyl-2,4-dihydroxy-N-((1S,2R)-2-hydroxy-indan-1-
yl)-butyramide ((S)-5). To compound 4 (0.5 g, 2.6 mmol) and
2-hydroxypyridine (0.27 g, 2.8 mmol) in dry DCM (15 mL) was
added (1S,2R)-1-amino-2-indanol (0.43 g, 2.8 mmol). The reac-
tion mixture was stirred at 60 °C for 24 h in a sealed tube and
then evaporated. The residue was dissolved in EtOAc (80 mL)
and washed with 1 M HCl (20 mL), followed by saturated
aqueous NaHCO3 (20 mL) and thereafter dried (MgSO4),
filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel, acetone/petroleum ether,
1:3) yielding the two diastereomeric triols; (S)-5 (0.26 g) and (R)-
5 (0.33 g) as white solids in a total yield of 66%. 1H NMR
(CD3OD, 400 MHz): δ 7.29-7.15 (m, 9H), 5.11 (d, J = 4.8 Hz,
1H), 4.19 (m, 1H), 3.82 (m, 2H), 3.13-3.05 (m, 2H), 2.96-2.80
(m, 2H), 2.30 (m, 1H), 1.93 (m, 1H). 13C NMR (CD3OD, 100
MHz): δ 176.1, 141.3, 140.3, 136.5, 130.4, 127.7, 127.6, 126.6,
126.4, 125.0, 124.0, 78.3, 72.7, 58.4, 57.1, 45.7, 40.3, 39.7.

614 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2
Mahalingam et al.
(S)-2-Benzyl-4-(tert-butyl-diphenyl-silanyloxy)-2-hydroxy-
N-((1S,2R)-2-hydroxy-indan-1-yl)-butyramide ((S)-6). To a stir-
red solution of (S)-5 (0.245 g, 0.72 mmol) and imidazole (0.073 g,
1.08 mmol) in dry DCM (25 mL) was added TBDPS-Cl (0.2 g,
0.75 mmol) and left overnight with stirring. The reaction
mixture was diluted, washed with water, dried (MgSO4),
evaporated, and purified by flash column chromatography to
yield (S)-6 (0.334 g, 80%). 1H NMR (CDCl3, 400 MHz): δ
7.71-7.66 (m, 4H), 7.48-7.36 (m, 8H), 7.34-7.24 (m, 4H),
7.22-7.18 (m, 2H), 7.12-7.04 (m, 2H), 5.37 (s, 1H), 5.24 (m,
1H), 4.16-3.95 (m, 3H), 3.10 (d, J = 13.6 Hz, 1H), 3.05 (m, 2H),
2.81 (d, J = 16.4 Hz, 1H), 2.40 (m, 1H), 2.15 (m, 1H), 1.09 (s,
9H). 13C NMR (CDCl3, 100 MHz): δ 174.8, 140.7, 137.1, 135.7,
135.6, 132.3, 131.0, 130.4, 128.3, 128.2, 128.1, 127.1, 127.0,
125.3, 124.0, 80.9, 73.4, 63.3, 57.4, 46.4, 39.1, 38.9, 27.9, 19.2.
(S)-2-Benzyl-1-((3aS,8aR)-2,2-dimethyl-8,8a-dihydro-3aH-
indeno[1,2-d]oxazol-3-yl)-2,4-dihydroxy-butan-1-one ((S)-7).
To a cooled (0 °C) solution of (S)-6 (0.325 g, 0.56 mmol)
and pyridinium p-toluenesulphonic acid (15 mg, 0.05 mmol)
in dry DCM (20 mL), 2-methoxypropene (0.4 g, 5.6 mmol)
was added and stirred for 6 h at the same temperature. The
solution was washed with saturated NaHCO3 (aq) and brine,
dried (MgSO4), and evaporated under reduced pressure. The
crude product [0.33 g, MS (ESIþ): m/z 620 (MþHþ)] was
dissolved in THF (20 mL), TBAF (0.278 g, 1.06 mmol, 1 M in
THF) was added at room temp, and the mixture was stirred
for 3 h. The solvent was evaporated and the residue dissolved
in DCM, washed with water and brine, dried (MgSO4), and
evaporated. The product was purified by flash column
chromatography (silica gel, acetone/petroleum ether, 1:4)
yielding (S)-7 (0.145 g, 69%) as a white solid. 1H NMR
(CDCl3, 400 MHz): δ 7.34-7.25 (m, 4H), 7.20-7.09 (m,
5H), 5.25 (m, 1H), 4.23 (m, 1H), 4.10-4.00 (m, 2H), 3.15 (d,
J = 12.8 Hz, 1H), 3.06 (dd, J = 5.6, 16.4 Hz, 1H), 2.96 (d, J =
13.2 Hz, 1H), 2.83 (d, J = 16.8 Hz, 1H), 2.40 (m, 1H), 2.16 (s,
3H), 2.10 (m, 1H), 1.05 (s, 3H). 13C NMR (CDCl3, 100 MHz):
δ 175.9, 140.7, 140.5, 136.7, 130.9, 128.3, 127.3, 127.2, 125.4,
124.0, 102.5, 80.3, 73.3, 61.2, 57.6, 46.2, 39.3, 39.2, 31.2, 29.5.
((S)-1-{N0-(4-Bromo-benzyl)-N0-[(S)-3-hydroxy-3-((1S,2R)-
2-hydroxy-indan-1-ylcarbamoyl)-4-phenyl-butyl]-hydrazinocar-
bonyl}-2,2-dimethyl-propyl)-carbamic Acid Methyl Ester ((S)-9).
A solution of (S)-7 (0.13 g, 0.34 mmol) in dry DCM (5 mL) was
added over 1 min to a stirred solution of Dess-Martin period-
inane (0.16 g, 0.37 mmol) in dry DCM (10 mL). After
30 min, the homogeneous mixture was diluted with ether and
poured into cold saturated NaHCO3 (aq, 10 mL) containing
Na2S2O3 (2.2 g). The organic layer was washed with saturated
NaHCO3 (aq) and brine, dried (MgSO4), and evaporated below
20 °C to yield the crude aldehyde [0.082 g, MS (ESIþ): m/z
380 (MþHþ)]. To the aldehyde (0.082 g, 0.21 mmol) and
{(S)-1-[N0-(4-bromo-benzyl)-hydrazinocarbonyl]-2,2-dimethyl-
propyl}-carbamic acid methyl ester 8 (0.086 g, 0.23 mmol) in dry
THF (10 mL) was added acetic acid (0.025 g, 0.42 mmol), and
the mixture was stirred for 10 min and then Na(OAc)3BH (0.133
g, 0.63 mmol) was added and stirring was continued overnight.
The reaction mixture was quenched with water and evaporated.
The residue was dissolved in DCM (20 mL) and washed with
water and brine, and then trifluoroacetic acid (1 mL) was added
and the organic layer was stirred for 30 min. The mixture was
evaporated and washed successively with saturated NaHCO3
(aq), water, and brine and dried (MgSO4). The product was
purified on flash column chromatography (silica gel, acetone/
{(S)-1-[N0-[(S)-3-Hydroxy-3-((1S,2R)-2-hydroxy-indan-1-ylcar-
bamoyl)-4-phenyl-butyl]-N0-(4-pyridin-2-yl-benzyl)-hydrazinocar-
bonyl]-2,2-dimethyl-propyl}-carbamic Acid Methyl Ester (11).
Pd(PPh3)2Cl2 (4.61 mg, 0.0065 mmol) was added to a solution
of (S)-9 (90 mg, 0.129 mmol), 2-(1,1,1-trin-butylstan-
nyl)pyridine (0.191 g, 0.51 mmol), and CuO (10.3 mg, 0.129
mmol) in DMF (2.0 mL) and stirred in a sealed, heavy-walled
Smith process vial at 120 °C for 50 min in the microwave cavity.
The mixture was diluted with DCM (25 mL) and washed with
saturated NaHCO3 (aq, 3 ꢀ 15 mL). The organic layer was dried
(MgSO4) and evaporated. The residue was redissolved in
CH3CN (60 mL) and washed with isohexane (3 ꢀ 20 mL). The
acetonitrile phase was evaporated, and the crude product was
purified using RPLC-MS (45 min gradient of 15-70% CH3CN
in 0.05% aq formic acid), followed by a passage through an
IMAC column in order to remove metal ions, yielding 11 (36.2
mg, 40%) as a white solid. 1H NMR (CD3OD 400 MHz): δ 8.56
(m, 1H), 7.82 (m, 1H), 7.72-7.60 (m, 4H), 7.54 (m, 1H), 7.44 (m,
1H), 7.34-7.16 (m, 6H), 7.06-7.00 (m, 3H), 6.96 (m, 1H), 4.96
(m, 1H), 4.16 (m, 1H), 3.82 (m, 2H), 3.70 (m, 1H), 3.60 (s, 3H),
3.08-2.78 (m, 6H), 2.10 (m, 1H), 1.94 (m, 1H), 0.78 (s, 9H). 13
C
NMR (CD3OD, 100 MHz): δ 176.9, 171.5, 158.2, 157.7, 149.1,
141.5, 140.5, 138.6, 138.1, 138.0, 137.2, 132.9, 132.3, 132.2,
130.8, 129.9, 129.2, 129.1, 127.9, 127.3, 126.9, 126.6, 125.0,
124.5, 122.8, 121.7, 79.3, 73.1, 62.3, 57.7, 53.6, 51.9, 46.6, 39.8,
34.5, 33.9, 26.1.
Acknowledgment. We acknowledge the financial support
from the Swedish Research Council (VR), Knut and Alice
Wallenberg’s Foundation and Medivir AB. We thank Dr.
Lotta Vrang and Dr. Asa Rosenquist for discussions and
testing of the mutant isolates. We also thank Mr. Oskar
€
Enstrom for help with crystal production.
Supporting Information Available: Experimental details and
spectroscopic data for compounds 2-7, 9-18, 20, 22, 24-34,
elemental analysis data, X-ray crystal structures and determina-
tion details, procedures for enzyme, antiviral activity and
Caco-2 assays, and liver microsome stability evaluation. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Joint United Nations Programme on HIV/AIDSAIDS Epidemic
Update: December 2007; UNAIDS/WHO: Geneva, 2007.
(2) Brik, A.; Wong, C.-H. HIV-1 protease: mechanism and drug
discovery. Org. Biomol. Chem. 2003, 1, 5–14.
(3) Randolph, J. T.; DeGoey, D. A. Peptidomimetic inhibitors of HIV
protease. Curr. Top. Med. Chem. 2004, 4, 1079–1095.
(4) Abdel-Rahman, H. M.; Al-Karamany, G. S.; El-Koussi, N. A.;
Youssef, A. F.; Kiso, Y. HIV protease inhibitors: peptidomimetic
drugs and future perspectives. Curr. Med. Chem. 2002, 9, 1905–
1922.
(5) Rodrıguez-Barrios, F.; Gago, F. HIV protease inhibition: limited
´
recent progress and advances in understanding current pitfalls.
Curr. Top. Med. Chem. 2004, 4, 991–1007.
(6) Aruksakunwong, O.; Promsri, S.; Wittayanarakul, K.; Nimmanpipug,
P.; Lee, V. S.; Wijitkosoom, A.; Sompornpisut, P.; Hannongbua,
S. Current development on HIV-1 protease inhibitors. Curr. Comput.-
Aided Drug Des. 2007, 3, 201–213.
(7) Armbruster, C. HIV-1 infection: recent developments in treat-
ment and current management strategies. Anti-Infect. Agents
Med. Chem. 2008, 7, 201–214.
(8) Anderson, J.; Schiffer, C.; Lee, S.-K.; Swanstrom, R. Viral protease
1
petroleum ether, 1:3) to yield (S)-9 (0.58 g, 40%). H NMR
inhibitors. Handb. Exp. Pharmacol. 2009, 189, 85–110.
(CDCl3, 400 MHz): δ 7.40-7.09 (m, 13H), 5.07 (d, J = 5.2 Hz,
1H), 4.27 (m, 1H), 3.70 (m, 6H), 3.10-2.78 (m, 6H), 2.20 (m,
1H), 1.92 (m, 1H), 0.69 (s, 9H). 13C NMR (CD3OD, 100 MHz):
δ 177.8, 172.4, 159.1, 142.6, 141.7, 138.1, 137.7, 132.4, 131.8,
129.1, 128.9, 127.9, 127.6, 126.3, 125.5, 122.4, 79.9, 73.8, 63.2,
62.4, 58.4, 55.3, 52.9, 47.2, 40.9, 35.7, 34.7, 26.9.
(9) 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.
(10) Ghosh, A. K.; Sridhar, P. R.; Leshchenko, S.; Hussain, A. K.;
Li, J.; Kovalevsky, A. Y.; Walters, D. E.; Wedekind, J. E.;

Article
Grum-Tokars, V.; Das, D.; Koh, Y.; Maeda, K.; Gatanaga, H.;
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 2 615
R. J.; Lin, P.-F. BMS-232632, a highly potent human immunode-
ficiency virus protease inhibitor that can be used in combination
with other available antiretroviral agents. Antimicrob. Agents Che-
mother. 2000, 44, 2093–2099.
Weber, I. T.; Mitsuya, H. Structure-Based Design of Novel HIV-1
Protease Inhibitors To Combat Drug Resistance. J. Med. Chem.
2006, 49, 5252–5261.
(11) Clavel, F.; Hance, A. J. HIV drug resistance. N. Engl. J. Med. 2004,
350, 1023–1035.
(33) Dorsey, B. D.; Levin, R. B.; McDaniel, S. L.; Vacca, J. P.; Guare, J.
P.; Darke, P. L.; Zugay, J. A.; Emini, E. A.; Schleif, W. A.;
Quintero, J. C.; Lin, J. H.; Chen, I.-W.; Holloway, M. K.;
Fitzgerald, P. M. D.; Axel, M. G.; Ostovic, D.; Anderson, P. S.;
Huff, J. R. L-735,524: the design of a potent and orally bioavailable
HIV protease inhibitor. J. Med. Chem. 1994, 37, 3443–3451.
(34) Molla, A.; Vasavanonda, S.; Kumar, G.; Sham, H. L.; Johnson,
M.; Grabowski, B.; Denissen, J. F.; Kohlbrenner, W.; Plattner,
J. J.; Leonard, J. M.; Norbeck, D. W.; Kempf, D. J. Human serum
attenuates the activity of protease inhibitors toward wild-type
and mutant human immunodeficiency virus. Virology 1998, 250,
255–262.
(35) Sham, H. L.; Kempf, D. J.; Molla, A.; Marsh, K. C.; Kumar, G. N.;
Chen, C. M.; Kati, W.; Stewart, K.; Lal, R.; Hsu, A.; Betebenner,
D.; Korneyeva, M.; Vasavanonda, S.; McDonald, E.; Saldivar, A.;
Wideburg, N.; Chen, X.; Niu, P.; Park, C.; Jayanti, V.; Grabowski,
B.; Granneman, G. R.; Sun, E.; Japour, A. J.; Leonard, J. M.;
Plattner, J. J.; Norbeck, D. W. ABT-378, a highly potent inhibitor
of the human immunodeficiency virus protease. Antimicrob. Agents
Chemother. 1998, 42, 3218–3224.
(36) Ghosh Arun, K.; Dawson Zachary, L.; Mitsuya, H. Darunavir, a
conceptually new HIV-1 protease inhibitor for the treatment of
drug-resistant HIV. Bioorg. Med. Chem. 2007, 15, 7576–7580.
(37) De Meyer, S.; Azijn, H.; Surleraux, D.; Jochmans, D.; Tahri, A.;
Pauwels, R.; Wigerinck, P.; de Bethune, M.-P. TMC114, a novel
human immunodeficiency virus type 1 protease inhibitor active
against protease inhibitor-resistant viruses, including a broad
range of clinical isolates. Antimicrob. Agents Chemother. 2005,
49, 2314–2321.
(12) de Mendoza, C.; Soriano, V. Resistance to HIV protease inhibitors:
mechanisms and clinical consequences. Curr. Drug Metab. 2004, 5,
321–328.
ꢀ
(13) Menendez-Arias, L. Targeting HIV: antiretroviral therapy and
development of drug resistance. Trends Pharmacol. Sci. 2002, 23,
381–388.
(14) Hertogs, K.; Bloor, S.; Kemp, S. D.; Van den Eynde, C.; Alcorn,
T. M.; Pauwels, R.; Van Houtte, M.; Staszewski, S.; Miller, V.;
Larder, B. A. Phenotypic and genotypic analysis of clinical HIV-1
isolates reveals extensive protease inhibitor cross-resistance: a
survey of over 6000 samples. AIDS (London, England) 2000, 14,
1203–1210.
(15) Ekegren, J. K.; Unge, T.; Safa, M. Z.; Wallberg, H.; Samuelsson,
B.; Hallberg, A. A new class of HIV-1 protease inhibitors contain-
ing a tertiary alcohol in the transition-state mimicking scaffold.
J. Med. Chem. 2005, 48, 8098–8102.
(16) Ekegren, J. K.; Ginman, N.; Johansson, A.; Wallberg, H.; Larhed,
M.; Samuelsson, B.; Unge, T.; Hallberg, A. Microwave accelerated
synthesis of P10-extended HIV-1 protease inhibitors encompassing
a tertiary alcohol in the transition-state mimicking scaffold.
J. Med. Chem. 2006, 49, 1828–1832.
(17) 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.
€
(18) Wu, X.; Ohrngren, P.; Ekegren, J. K.; Unge, J.; Unge, T.; Wallberg,
H.; Samuelsson, B.; Hallberg, A.; Larhed, M. Two-Carbon-Elon-
gated HIV-1 Protease Inhibitors with a Tertiary-Alcohol-Contain-
ing Transition-State Mimic. J. Med. Chem. 2008, 51, 1053–1057.
(19) Rich, D. H.; Bernatowicz, M. S.; Agarwal, N. S.; Kawai, M.;
Salituro, F. G.; Schmidt, P. G. Inhibition of aspartic proteases by
pepstatin and 3-methylstatine derivatives of pepstatin. Evidence
for collected-substrate enzyme inhibition. Biochemistry 1985, 24,
3165–3173.
(20) Rich, D. H. Pepstatin-derived inhibitors of aspartic proteinases. A
close look at an apparent transition-state analog inhibitor. J. Med.
Chem. 1985, 28, 263–273.
(21) Agarwal, N. S.; Rich, D. H. Inhibition of cathepsin D by substrate
analogs containing statine and by analogs of pepstatin. J. Med.
Chem. 1986, 29, 2519–2524.
(22) Godfrey, J. D., Jr.; Gordon, E. M.; Von Langen, D. J. Synthesis of
peptide-derived amino alcohols II. Synthetic methodology for the
preparation of tertiary alcohols. Tetrahedron Lett. 1987, 28, 1603–1606.
(23) Kim, B. M.; Guare, J. P.; Hanifin, C. M.; Arford-Bickerstaff, D. J.;
Vacca, J. P.; Ball, R. G. A convergent synthesis of novel confor-
mationally restricted HIV-1 protease inhibitors. Tetrahedron Lett.
1994, 35, 5153–5156.
(38) “N1,N6-Di[(1S)-2-methyl-1-(methylcarbamoyl)propyl]-(2R,3R,4R,-
5R)-2,5-di(benzyloxy)-3,4-dihydroxyhexanediamide.
€
(39) Alterman, M.; Bjorsne, M.; Muehlman, A.; Classon, B.;
Kvarnstrom, I.; Danielson, H.; Markgren, P.-O.; Nillroth, U.;
€
Unge, T.; Hallberg, A.; Samuelsson, B. Design and synthesis of
new potent C2-symmetric HIV-1 protease inhibitors. Use of
L-mannaric acid as a peptidomimetic scaffold. J. Med. Chem.
1998, 41, 3782–3792.
(40) 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.
(41) The alpha-methyl group of Val 182 (Figure 4) has a distance of
3.8 A to one of the ortho, 3.8 A to the meta and 3.9 A to the para
carbon of the inhibitor benzyl group.
(42) Gallivan, J. P.; Dougherty, D. A. Cation-pi interactions in
structural biology. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9459–
9464.
(43) The beta-methyl group of Val182 (Figure 4) has a distance of 5.1 A
to the ortho, 4.3 and 4.4 A to the meta positions, and 3.9 A to the
para position of the inhibitor benzyl group.
(24) Balani, S. K.; Arison, B. H.; Mathai, L.; Kauffman, L. R.; Miller,
R. R.; Stearns, R. A.; Chen, I. W.; Lin, J. H. Metabolites of
L-735,524, a potent HIV-1 protease inhibitor, in human urine.
Drug Metab. Dispos. 1995, 23, 266–270.
(25) Lin, J. H. Role of pharmacokinetics in the discovery and develop-
ment of indinavir. Adv. Drug Delivery Rev. 1999, 39, 33–49.
(26) Larhed, M.; Moberg, C.; Hallberg, A. Microwave-accelerated
homogeneous catalysis in organic chemistry. Acc. Chem. Res.
2002, 35, 717–727.
(27) Ersmark, K.; Larhed, M.; Wannberg, J. Microwave-enhanced
medicinal chemistry: A high-speed opportunity for convenient
preparation of protease inhibitors. Curr. Opin. Drug Discovery
Dev. 2004, 7, 417–427.
(44) The methyl group of mutant Thr182 (Figure 4) has a distance of 4.0
A to the ipso, 3.8 A to the ortho, 3.7 A to the meta, and 3.8 A to the
para carbon. The distance to the hydroxy oxygen is 5.7 A for
the ortho, 5.1 A for the meta, and 5.0 A for the para carbon of the
inhibitor benzyl group.
(45) Wang, F.; Ross, J. Atazanavir: a novel azapeptide inhibitor of
HIV-1 protease. Formulary 2003, 38, 691–702.
(46) Ekegren, J.; Hallberg, A.; Wallberg, H.; Samuelsson, B.; Mahalingam,
K. Preparation of amino acid hydrazide derivatives and related
compounds as HIV protease inhibitors. Patent WO 2006084688, 2006.
(47) Moiese, J.; Arseniyadis, S.; Cossy, J. Cross-Metathesis between
alpha-Methylene-gamma-butyrolactone and Olefins: A Dramatic
Additive Effect. Org. Lett. 2007, 9, 1695–1698.
(28) Kappe, C. O.; Dallinger, D. The impact of microwave synthesis on
drug discovery. Nat. Rev. Drug Discovery 2006, 5, 51–63.
(29) Caddick, S.; Fitzmaurice, R. Microwave enhanced synthesis. Tet-
rahedron 2009, 65, 3325–3355.
(30) Larhed, M.; Hallberg, A. Microwave-Promoted Palladium-Cata-
lyzed Coupling Reactions. J. Org. Chem. 1996, 61, 9582–9584.
(31) Hydrazides 19 and 21 were kindly supplied by Dr. Kristina Orrling,
Department of Medicinal Chemistry, Uppsala University, Sweden.
(32) 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,
(48) Murray, A. W.; Reid, R. G. Convenient synthesis of alpha-
epoxylactones (4-oxo-1,5-dioxaspiro[2.4]heptanes and -[2.5]octanes).
Synthesis 1985, 35–38.
(49) Shibata, I.; Yamasaki, H.; Baba, A.; Matsuda, H. A Novel Darzens
type reaction promoted by tributylstannylcarbamate. J. Org.
Chem. 1992, 57, 6909–6914.
(50) Curran, D. P.; Ko, S.-B. Synthesis of Optically Active alpha-
Hydroxy Lactones by Sharpless Asymmetric Dihydroxylations of
Ketene Acetals, Enol Ethers, and Ene Lactones. J. Org. Chem.
1994, 59, 6139–6141.