Microwave-accelerated synthesis of P1′-extended HIV-1 protease inhibitors encompassing a tertiary alcohol in the transition-state mimicking scaffold

Article abstract of DOI:10.1021/jm051239z

Two series of P1′-extended HIV-1 protease inhibitors comprising a tertiary alcohol in the transition-state mimic exhibiting K_i values ranging from 2.1 to 93 nM have been synthesized. Microwave-accelerated palladium-catalyzed cross-couplings were utilized to rapidly optimize the P1′ side chain. High cellular antiviral potencies were encountered when the P1′ benzyl group was elongated with a 3- or 4-pyridyl substituent (EC₅₀ = 0.18-0.22 μM). X-ray crystallographic data were obtained for three inhibitors cocrystallized with the enzyme.

Full text of DOI:10.1021/jm051239z

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J. Med. Chem. 2006, 49, 1828-1832
Brief Articles
Microwave-Accelerated Synthesis of P1′-Extended HIV-1 Protease Inhibitors Encompassing a
Tertiary Alcohol in the Transition-State Mimicking Scaffold
Jenny K. Ekegren,† Nina Ginman,‡ Åsa Johansson,† Hans Wallberg,§ Mats Larhed,† Bertil Samuelsson,§ Torsten Unge,‡ 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, Lunastigen 7, SE-141 44, Huddinge, Sweden
ReceiVed December 13, 2005
Two series of P1′-extended HIV-1 protease inhibitors comprising a tertiary alcohol in the transition-state
mimic exhibiting Ki values ranging from 2.1 to 93 nM have been synthesized. Microwave-accelerated
palladium-catalyzed cross-couplings were utilized to rapidly optimize the P1′ side chain. High cellular antiviral
potencies were encountered when the P1′ benzyl group was elongated with a 3- or 4-pyridyl substituent
(EC50 ) 0.18-0.22 µM). X-ray crystallographic data were obtained for three inhibitors cocrystallized with
the enzyme.
Introduction
Increasing numbers of HIV/AIDS infected patients and related
deaths, along with severe treatment-associated complications,
make the HIV/AIDS pandemic more complex than ever.1,2 The
introduction of HIV protease inhibitors (PIs) in the mid 1990s
dramatically changed the situation for HIV/AIDS patients.3-6
Combination therapy initially including one protease inhibitor
and two nucleoside reverse transcriptase inhibitors, the so-called
highly active antiretroviral therapy (HAART), furnished a sharp
decline in HIV/AIDS related mortality for patients receiving
this therapy.7,8 Seven PIs are available on the market today
(December 2005), and yet another one has been granted
accelerated approval by the FDA (tipranavir, June 2005).9,10
One of the major obstacles to overcome in the development
of HIV protease inhibitors has been the low oral bioavailability
due to insufficient membrane permeation properties, rapid
metabolism, and/or protein binding.11-13 The discovery of PIs
that combine high oral bioavailability with potent anti-HIV
activity remains challenging, although the identification of
atazanavir represented a major step forward (Figure 1).14-17
We recently reported a new class of HIV-1 protease inhibitors
which were structurally related to both atazanavir and indi-
navir18,19 but comprising a tertiary alcohol as part of the
transition-state mimicking unit, the best compound being 1 with
a Ki value of 2.4 nM (Figure 1).20 However, in assays with
HIV-1 infected MT4 cells, the antiviral activities were low (EC50
values at best 1.1 µM). Thus, it was decided to further optimize
inhibitor 1 in order to improve the potency on the cellular level.
In this effort, structure 1 and the corresponding m-bromo
derivative 8 were expected to serve as suitable arylpalladium
precursors for microwave-assisted cross-coupling reactions
(Scheme 1).21-24 We herein report microwave synthesis and
Figure 1. The new class of HIV protease inhibitors exemplified by
the most potent compound from the first generation (1) and the approved
inhibitors atazanavir and indinavir.
biological evaluations of two series of P1′-elongated analogues,
demonstrating that compounds with improved activity in cell
culture rapidly can be identified. X-ray crystallographic data
for three of these new inhibitors bound to the HIV-1 protease
are discussed.
Results
Chemistry. The synthesis of bromo scaffolds 1 and 8
followed the protocol recently published for 120 starting from
4-25 or 3-bromobenzylhydrazine and N-(methoxycarbonyl)-L-
tert-leucine, prepared according to published procedures (Scheme
1).15,26 The hydrazide coupling was performed using 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC),
N-methylmorpholine (NMM), and 1-hydroxybenzotriazole
(HOBT), affording the bromo-substituted hydrazides 5 and 6
(Scheme 1). These hydrazides were then used to regioselectively
ring-open the epoxide 720 in Ti(OiPr)4-catalyzed reactions
yielding key structures 1 and 8, respectively.
* Corresponding author. Tel: +46-18-4714284. Fax: +46-18-
4714474. E-mail: Anders.Hallberg@orgfarm.uu.se.
† Department of Medicinal Chemistry, Uppsala University.
‡ Department of Cell and Molecular Biology, Uppsala University.
§ Medivir AB.
10.1021/jm051239z CCC: $33.50 © 2006 American Chemical Society
Published on Web 02/11/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1829
Scheme 1a
conducted with 5-10 mol % dichlorobis(triphenylphosphine)-
palladium (Pd(PPh3)2Cl2), 5 equiv of the appropriate organobo-
ronic acid, and aqueous Na2CO3 as base in a DME/EtOH
mixture.31 Microwave heating at 120 °C for 30 min rendered
compounds 9, 12, 13, 17, 19-21, 24, and 25 (Tables 1 and 2) in
low to good isolated yields (26-62%), with the exception of
sluggish pyridine-4-boronic acid which yielded para derivative
12 in only 17% yield. Stille coupling reactions were applied to
afford compounds 10, 11, 18, 22, and 23 (Tables 1 and 2) using
the corresponding tributyltin reagents and microwave irradiation
for 20 min at 130 °C with 1 equiv of CuO32 and 5 mol %
Pd(PPh3)2Cl2 in DMF. This generally furnished the target
products in lower isolated yields (17-27%) compared to the
Suzuki reactions, a trend which has been observed previously
for symmetric HIV-1 PIs.33 Alkynes 14-16 and 26-28 (Tables
1 and 2) were prepared using the Sonogashira reaction from
phenylacetylene or ethynylpyridines in the presence of 5-8 mol
% Pd(PPh3)2Cl2 and 7-10 mol % CuI. Initially, a protocol
including diethylamine as base and microwave processing for
30-40 min at 140 °C was applied for the Sonogashira reactions.
However, this procedure promoted decomposition of the bromo
precursors 1 and 8. The conditions were therefore altered to
include the less nucleophilic triethylamine as base at a lower
temp (130 °C) for 60 min. This slightly milder method generally
provided cleaner reactions and somewhat higher isolated yields
of the acetylenic products, 22-45% (Table 1), compared to the
19-27% achieved with diethylamine as base (Table 2). All final
compounds were purified by RPLC-MS with acetonitrile in
0.05% aqueous HCOOH as mobile phase to provide the pure
products. Some of the analogues were found to partly form salts
a Reagents: (a) EDC, HOBT, NMM, EtOAc, room temp, 54-76%; (b)
Ti(OiPr)4, THF, 40 °C, 46-55%.
The P1′-elongated analogues 9-28 (Tables 1 and 2) were
prepared by a series of sequential Suzuki,27 Stille,28 or Sono-
gashira29 reactions performed under air with 20-60 min of
controlled microwave irradiation in septum-sealed reaction
vessels.22,23,30 To enable comparison of the meta and para
inhibitor sets, cross-coupling reactions yielding the phenyl, 2-,
3-, and 4-pyridyl, two-carbon elongated 2-phenylalkenyl, and
phenyl- and 2- and 3-pyridylalkynyl-substituted analogues were
performed with both 1 and 8. For the para series, four additional
heterocyclic and bicyclic compounds were prepared by pal-
ladium-catalyzed couplings (Table 1). Suzuki reactions were
Table 1. Synthesis and Antiviral Activity of P1′ Para-Substituted Inhibitors 9-20a
a Microwave conditions: Suzuki reactions: Pd(PPh3)2Cl2, Na2CO3 (aq), EtOH, DME, 120 °C, 30 min; Stille reactions: Pd(PPh3)2Cl2, CuO, DMF, 130
°C, 20 min; Sonogashira reactions: Pd(PPh3)2Cl2, Et3N, CuI, DMF, 130 °C, 60 min. b Indinavir: Ki ) 0.52 nM,19 atazanavir: Ki ) 2.66 nM.16 c Indinavir:
d
EC50 ) 0.041 µM,6,36 atazanavir: EC50 ) 0.0039 µM.37 Papp (Caco-2) ) 33 × 10-6 cm/s. e Clint ) 154 µL/min/mg.

1830 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5
Brief Articles
Table 2. Synthesis and Antiviral Activity of P1′ Meta-Substituted Inhibitors 21-28a
a Microwave conditions: Suzuki reactions: Pd(PPh3)2Cl2, Na2CO3 (aq), EtOH, DME, 120 °C, 30 min; Stille reactions: Pd(PPh3)2Cl2, CuO, DMF, 130
b
°C, 20 min; Sonogashira reactions Pd(PPh3)2Cl2, Et2NH, CuI, DMF, 140 °C, 30-40 min. Papp (Caco-2) ) 11 × 10-6 cm/s. c Clint ) 190 µL/min/mg.
Figure 2. X-ray arrangement of inhibitor 1, 10, and 11 in the active site of the HIV-1 protease. The most relevant hydrogen bonding interactions
between the inhibitors and the enzyme amino acid residues are represented by green dashed lines. The P1′ pyridyl groups of 10 and 11 are angled
away from Arg108 via hydrogen bonding through a water molecule. The electronegative property of the bromine in 1 enables the P1′ group to close
pack against Arg108.
with the formic acid. For these compounds, additional HRMS
analyses were performed, and data presenting the actual mass
within 5 ppm of the theoretical masses are included in the
Supporting Information. The somewhat disappointing isolated
yields can partly be explained by the rigorous purification
procedure and partly by the complexity of the structures. Please
note that all cross-couplings were performed without protection
of the hydroxyl groups34 and that no byproducts from water
elimination were detected.
Biological Evaluations. Antiviral activities for compounds
8-28 are summarized as Ki and EC50 values in Tables 1 and 2.
The previously investigated p-bromo compound 1 is included
as a reference. Within the para collection (1, 9-20, Table 1),
all structures seemed to be well accommodated in the active
site of the HIV-1 protease, and Ki values varying between 2.1
and 20 nM were encountered (Table 1). More interestingly,
higher P1′ substituent dependence as deduced from the EC50
values was observed in the assay with HIV-1 infected MT4 cells.
All compounds were active at concentrations up to 10 µM with
EC50 values ranging from 0.18 to 1.1 µM except analogues 13,
14, and 20 with hydrophobic phenyl groups attached at a two-
carbon distance from the parent P1′ benzyl group. The para 3-
and 4-pyridyl derivatives 11 and 12 exhibited notably higher
cellular activities (EC50 ) 0.18 and 0.22 µM, respectively) than
1 (EC50 ) 1.1 µM). In the meta library, (8, 21-28, Table 2)
slightly less potent enzyme inhibitors compared to the corre-
sponding para analogues were obtained with Ki values ranging
from 12 to 93 nM. Surprisingly, only two of the meta
compounds exhibited antiviral activity on the cellular level, the
best one being the 4-pyridine analogue 24 with an EC50 value
of 2.0 µM (Table 2). Four compounds exhibited cell toxicity
with CC50 values below 10 µM (15, 18, 22, and 23, Tables 1
and 2). The 3-pyridyl analogues 11 and 23 (Ki ) 5.0 and 12
nM, respectively) were further examined in a Caco-2 cell assay
and for stability in liver microsome homogenate (Tables 1 and
2). Compound 11 exhibited excellent permeability (Papp ) 33
× 10-6 cm/s) whereas the corresponding meta analogue 23 was
more slowly transported across the Caco-2 cell membrane (Papp
) 11 × 10-6 cm/s). The observed fast intrinsic clearance of
these compounds in liver microsomes (Clint ) 154 and 190 µL/
min/mg, respectively) could be attributed to rapid oxidative
metabolism of the P2 aminoindanol group.35
X-ray Crystallographic Data. The arrangements of com-
pound 1, 10, and 11 in the active site of the HIV-1 protease
including the most relevant hydrogen bonds, as deduced from
X-ray crystallography, are presented in Figure 2 (PDB codes

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 5 1831
2cej, 2cem, and 2cen, respesctively). The tertiary hydroxyl group
in 1, 10, and 11 form a hydrogen bond to one of the catalytic
aspartic acid residues (Asp25) in the enzyme and the hydrazide
R- and â-nitrogens are hydrogen bonded to Gly27. Arg108 plays
a crucial role for the positioning of the extended P1′ side chains,
and in the case of 1, the electronegative bromine can favorably
close pack against the positively charged Arg side chain.
Furthermore, in the complexes with compounds 10 and 11, a
water molecule is correctly positioned for hydrogen bonding
between the pyridyl nitrogen atom and the Arg side chain. This
arrangement forces the P1′ groups in 10 and 11 to be lifted up
and away from Arg108. As a result, the position of the enzyme
Pro181 residue differs (1.5 Å) between the three inhibitor/HIV-1
complexes. The P1′ groups are embedded in the enzyme S1′-
S3′ cavity formed by amino acid residues 179-183.
3-pyridine analogue 11 which gave seven times higher cellular
antiviral potency than parent compound 1, showed no indications
of cell toxicity and exhibited excellent membrane permeability
properties.
Experimental Section
General Procedures for the Palladium-Catalyzed Reactions.
Method A. Aryl bromide 1 or 8 (1 equiv), boronic acid (5 equiv),
Pd(PPh3)2Cl2 (0.05-0.1 equiv), 2 M Na2CO3 (aq, 3 equiv), EtOH
(0.6 mL), and DME (2.4 mL) were stirred in a Smith process vial
sealed under air at 120 °C for 30 min in the microwave cavity.
Five drops of formic acid were added to the mixture, and then the
solvent was evaporated followed by purification on RPLC-MS.
Method B. Aryl bromide 1 or 8 (1 equiv), tin reagent (4 equiv),
Pd(PPh3)2Cl2 (0.05 equiv), CuO (1-1.1 equiv), and DMF (2 mL)
were stirred in a Smith process vial sealed under air at 130 °C for
20 min in the microwave cavity. CH2Cl2 was added to the mixture
followed by washing with saturated NaHCO3 (aq). The organic
phase was dried (Na2SO4) and evaporated, and then the residue
was redissolved in CH3CN and washed with isohexane. The CH3-
CN phase was evaporated, and the crude product was purified using
RPLC-MS.
Discussion
Generally, the para P1′ elongated structures exhibited higher
enzyme affinity compared to the corresponding meta com-
pounds, which could be due to an unfavorable angle of the P1′
side chains formed in the meta analogues for approach into the
S1′-S3′ pocket. Not more than two inhibitors in the meta series
were active on the cellular level, the best compound being 24
(EC50 ) 2.0 µM, Table 2). Thus, structure 24 was considerably
Method C. Aryl bromide 1 (1 equiv), alkyne (2 equiv), Et3N
(10 equiv), Pd(PPh3)2Cl2 (0.05 equiv), CuI (0.1 equiv), and DMF
(2.1 mL) were stirred in a Smith process vial sealed under air at
130 °C microwave heating for 60 min. Evaporation of most of the
solvent was followed by purification using RPLC-MS.
less potent than the corresponding para derivative 12 (EC50
)
Method D. Aryl bromide 8 (1 equiv), alkyne (1.2-1.3 equiv),
Et2NH (8.7 equiv), Pd(PPh3)2Cl2 (0.08 equiv), CuI (0.07-0.09
equiv), and DMF (2 mL) were stirred in a Smith process vial sealed
under air at 140 °C microwave heating for 30-40 min. Workup
was performed by extracting the mixture with CH2Cl2 and H2O.
The organic phase was evaporated, and then the product was
purified by RPLC-MS.
0.22 µM, Table 1), although it should be taken into account
that 24 also is a rather weak enzyme inhibitor (Ki ) 93 nM,
Table 2). The low cellular potencies of the meta analogues are
not easy to explain, although a somewhat slower membrane
transport, as deduced from the Caco-2 assay, is observed with
the meta compound 23 (Papp ) 11 × 10-6 cm/s, Table 2) as
compared to the corresponding para derivative 11 (Papp ) 33
× 10-6 cm/s, Table 1). By comparing the membrane permeation
properties of compound 11 and 23 with data obtained from
atazanavir and indinavir in the same assay the high Papp values
of both 11 and 23 are exceptional.20 This is probably due to
intramolecular hydrogen bonding with the tertiary alcohol, which
should prevent solvation of the hydroxyl group and result in a
lower desolvation energy for entering the membrane phase.38-40
In addition, conformational changes of these flexible molecules
to attain a transient, more permeable structure could contribute
to the fast membrane transport.38 However, considering the
favorable Papp values and in particular the Ki value of 12 nM,
we can at present not explain why compound 23 was devoid of
activity in the cell assay. Furthermore, it is notable that the
2-pyridyl derivative 10, that is structurally most similar to
atazanavir, is less potent than both the 3- and 4-pyridyl
derivatives 11 and 12.
Acknowledgment. We gratefully acknowledge the Swedish
Research Council (VR) and the Swedish Foundation for
Strategic Research (SSF) for financial support.
Supporting Information Available: Experimental details and
spectroscopic data for compounds 6 and 8-28, elemental analysis
data, X-ray structure determination details and statistics, procedures
for enzyme, cytotoxicity and Caco-2 assays, and liver microsome
stability evaluation. This material is available free of charge via
the Internet at http://pubs.acs.org.
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