Synthesis and antiviral activity of new anti-HIV amprenavir bioisosteres

Article abstract of DOI:10.1021/jm0208323

Starting from the chemical structure of the recent FDA-approved anti-HIV drug Amprenavir (Agenerase), a potent HIV-protease inhibitor, we have designed new series of Amprenavir bioisoteres in which the methylene group of the benzyl group was replaced by a

Full text of DOI:10.1021/jm0208323

J . Med. Chem. 2002, 45, 3321-3324
3321
Brief Articles
Syn th esis a n d An tivir a l Activity of New An ti-HIV Am p r en a vir Bioisoster es
Luc Rocheblave,†,‡ Fre´de´ric Bihel,†,‡ Ce´line De Michelis,†,‡ Ghislaine Priem,†,‡ J e´roˆme Courcambeck,†,‡
Brice Bonnet,§ J ean-Claude Chermann,‡ and J ean-Louis Kraus*,†,‡
Laboratoire de Chimie Biomole´culaire, Faculte´ des Sciences de Luminy, case 901, Universite´ de la Me´diterrane´e,
70 route Le´on Lachamp, 13288 Marseille Cedex 9, France, INSERM U322, Campus Universitaire de Luminy, BP33,
13273 Marseille Cedex 9, France, and ENSSPICAM, Universite´ Aix-Marseille III, 13397 Marseille Cedex 20, France
Received J anuary 23, 2002
Starting from the chemical structure of the recent FDA-approved anti-HIV drug Amprenavir
(Agenerase), a potent HIV-protease inhibitor, we have designed new series of Amprenavir
bioisoteres in which the methylene group of the benzyl group was replaced by a sulfur atom.
This structural modification has required an original multistep synthesis. Unfortunately,
introduction of the sulfur atom abolished or drastically decreased both inhibitory activity on
recombinant HIV protease and HIV infection protection on MT4 cell cultures.
In tr od u ction
Inhibition of the virally encoded aspartyl protease1-5
of the human immunodeficiency virus (HIV) results in
the production of immature noninfectious virions.6,7 This
discovery has led to new strategies for the treatment of
AIDS, based on the inhibition of this essential viral
F igu r e 1. Structure of the Amprenavir bioisostere 10.
protein. Most successful to date in this area is the use
of structures8 that mimic the transition state9 from the
sponding pure diastereomeric forms (10a 1, 10a 2, 10b1,
10b2). These compounds were evaluated for their ability
to inhibit recombinant HIV-1 protease and for their anti-
HIV activity on infected MT4 cells.
cleavage of the enzyme’s natural substrate. Among the
recently approved anti-HIV protease drugs, Amprenavir
(Agenerase), or VX-478, is of particular interest because
of its reduced size compared to that of Indinavir,
Ritonavir, or Saquinavir.10 Recently Amprenavir mono-
therapy was terminated because of moderate results in
clinical trials.11 Part of this failure was due to the
induction of mutations and associated resistances.
Indeed, most clinically used antiprotease drugs12 have
induced multiple HIV mutations,13-15 bringing to light
the necessity of finding new anti-HIV drugs.
Taking into account these structural and clinical
observations of Amprenavir, we investigated the pos-
sibility of modifying the structure of Amprenavir, using
the concept of bioisosterism.16 Our approach was to use
this bioisosterism concept by replacing the methylene
group of the benzyl substituent in the Amprenavir
derivative by a sulfur atom (Figure 1).
Ch em istr y
The synthesis of this new series of Amprenavir
analogues required the original synthetic route shown
in Scheme 1. Allylamine 1 treated with Boc2O in CH2-
Cl2 at 0 °C led to the corresponding N-allylcarbamate
2, which after oxidation17 with mCPBA gave the corre-
sponding epoxide 3. Selective opening of these epoxides
using isobutylamine in methanol, followed by N-sulfo-
nylation18 using 4-nitrobenzenesulfonyl chloride in ac-
etonitrile, resulted in the formation of the corresponding
N-substituted sulfonamidohydroxypropylene derivative
5. Oxidation of this intermediate in Swern conditions19
led to the corresponding ketone 6 in 88% yield. After
Boc deprotection of compound 6, the resulting free
amine derivative coupled with S(+)-3-hydroxytetrahy-
drofuran using DSC (N,N′-disuccinimidyl carbonate20)
led to carbamate 7, which gave the desired 4-aminoben-
zenesulfonamide ketone 8 after reduction with activated
palladium. By introduction of only one thiophenoxy
group, regioselectively at the R position of the ketone
intermediate was overcome using S-phenylbenzene
thiosulfonate21 as a reagent in the presence of lithium
diisopropylamide (LDA) in dry dichloroethane. The use
of LDA22 appeared to be suitable to allow specifically
the monocarbanion formation at the R position of the
ketone. In contrast, as we have previously reported,23
the use of sodium hydride followed by n-butyllithium
This bioisosteric replacement could induce several
modifications in terms of size, shape, electronic distri-
bution, lipophilicity, chemical reactivity, and hydrogen
bonding capacity. Among these physicochemical modi-
fications, chemical and biochemical aspects could be of
major interest. We describe herein the original synthesis
of Amprenavir bioisostere mixture 10 and the corre-
* To whom correspondence should be addressed. Phone: +33-0-4-
91-82-91-41. Fax: +33-0-4-91-82-94-16. E-mail: kraus@luminy.univ-
mrs.fr.
† Universite´ de la Me´diterrane´e.
‡ Campus Universitaire de Luminy.
§ Universite´ Aix-Marseille III.
10.1021/jm0208323 CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/20/2002

3322 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Brief Articles
Sch em e 1a
a
Reagents: (i) Boc2O, CH2Cl2; (ii) mCPBA, CH2Cl2; (iii) iBuNH2, MeOH; (iv) 4-nitrobenzenesulfonyl chloride, K2CO3, CH3CN; (v) TFAA,
DMSO, Et3N, CH2Cl2; (vi) (1) TFA, CH2Cl2, (2) TEA, DSC, (S)-3-hydroxytetrahydrofuran, CH2Cl2; (vii) H2, Pd/C, EtOH; (viii) PhSSO2Ph,
LDA, ClCH2CH2Cl; (ix) NaBH4, MeOH.
Ta ble 1. Amprenavir Analogues and Associated Biological
allowed the formation of the 1,3-dithiophenoxy ketone
Activities
intermediate. The last step of the synthesis required
reduction of the obtained R-sulfanyl ketone intermediate
9. Compound 10 was obtained from 9 by reduction using
NaBH4 as reducing reagent. Compound 10 was a
racemic mixture of four diastereoisomers (10a 1, 10a 2,
10b1, 10b2). Since separation by HPLC could not be
achieved directly, separation of diastereoisomers was
achieved as follows. First, the mixture of the R-thiophe-
noxy ketone diastereoisomers 9 was separated on a
chiralpack AS column into isomers 9a and 9b. It should
be emphasized that the different fractions containing
9a and 9b had to be kept cold (-30 °C) in order to avoid
racemization through the enolization process. These
fractions were directly reduced into their corresponding
R-thiophenoxy alcohols using NaBH4 as reagent. Reduc-
tion of ketones 9a and 9b led respectively to diastere-
oisomeric mixtures (10a 1 + 10a 2) and (10b1 + 10b2).
configuration
b
c
d
IC50 EC50 CC50
(µM) (µM) (µM)
SIe
a
compd
*C1 *C2 *C3
[R]D
10
racemic mixture 0°
nd
1.4
50
>50 >50 nd
>50 >1
10a 1
10a 2
10b1
10b2
S
S
S
S
S
S
R
+6.8°
c ) 2.92
-0.5°
c ) 1.46
S
S
11.6 >50 >50 nd
R
R
S
S
-24.8° 12.5 >50 >50 nd
c ) 3.23
R
R
-3.6°
c ) 1.92
nd
16.7 >50 >50 nd
Amprenavir
0.001 0.01 >50 >5000
a
[R]D: optical rotation was measured at 20 °C in c (g/100 mL)
Separation of the diastereoisomeric mixtures (10a 1
+
b
concentration. IC50
HIV-1 protease cleavage by 50%. c EC50
required to inhibit syncytia formation by 50% on MT4 cells. CC50
:
concentration in µM required to inhibit
10a 2) and (10b1 + 10b2) was achieved using the same
HPLC protocole (R-thiophenoxy alcohols were less sen-
sitive to racemization than their corresponding ketone
precursors). The four diastereoisomers (10a 1, 10a 2,
10b1, 10b2) were isolated, characterized (1H and 13C
NMR, mass spectroscopy, elemental analyses), and
screened for antiviral evaluation.
:
concentration in µM
d
:
concentration in µM required to cause 50% death of uninfected
MT4 cells. e SI: selective index ) CC50/EC50 (nd) not determi-
nated).
Under our assay conditions, the racemic mixture
tested elicited anti-HIV activity with an EC50 value up
to 50 µM. Under the same conditions, Amprenavir
inhibited syncytium formation with EC50 ) 0.01 µM. The
results obtained led us to separate the four constitutive
isomers (10a 1, 10a 2, 10b1, 10b2) from the diastereoiso-
meric mixture (compound 10) and to screen each of them
under the same assay conditions.The results show that
none of the four separated isomers were found active
at concentrations below 50 µM. Compound 10a 1 (S, S,
R), in which the configurations of the three asymmetric
Resu lts a n d Discu ssion
Compounds 10 obtained as mixture of diastereoiso-
mers were first screened for their inhibitory effects on
HIV replication in MT4 cell cultures. Anti-HIV activities
were determined according to a procedure described by
Rey et al.24,25 by the observation of the fusogenic effect
(syncytium formation assay). Antiviral potencies (EC50)
and cytotoxicities (CC50) are reported in Table 1.

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3323
tion assay. This material is available free of charge via the
Internet at http://pubs.acs.org.
carbons are identical to those of Amprenavir (S, S, R),
was inactive. The four diastereoisomers (10a 1, 10a 2,
10b1, 10b2) were then tested as recombinant HIV
protease inhibitors according to a classical procedure.26
IC50 values are given in Table 1. It can be seen that the
four diastereoisomeric resolved Amprenavir bioisosteres
(10a 1, 10a 2, 10b1, 10b2) were only weak inhibitors of
recombinant HIV protease (IC50 values were 1.4, 11.6,
12.5, and 16.7 µM, respectively). One explanation for
the lack of HIV-protease inhibitory properties of these
thiophenoxy bioisosteres could be their high sensitivity
to hydrolysis. We have seen through the recombinant
anti-HIV-1-protease inhibition studies that the new bio-
isosteres are not protease substrates. The HPLC profile
showed that the peak corresponding to the bioisostere
remained unchanged. Since the bioisosteres were not
degraded by the recombinant HIV-1 protease, we have
also studied their stabilities in normal human serum.
The half-lives (t1/2) of some representative compounds
were determined at 37 °C by the HPLC method. The
half-life value found for compound 10 was 10 min, while
Amprenavir was recovered unchanged after 1440 min.
These results were inconsistent with molecular mod-
eling studies (data not shown) that supported the
hypothesis that the replacement of the methylene group
in the phenylalanine moiety of Amprenavir did not
affect drastically the binding energy of the resulting
analogue within the active site of the HIV-1 protease.
Introduction of a thiophenoxy moiety abolished both
protease inhibition activities and viral cytopathogenicity
protection in HIV-infected MT4 cells, indicating that the
presence of a sulfur atom disturbed the capabilities of
the thiophenoxy drug to form a complex within the HIV-
protease active site.
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