Design, synthesis, and evaluation of conformationally constrained tongs, new inhibitors of HIV-1 protease dimerization

Article abstract of DOI:10.1021/jm9803976

The active form of human immunodeficiency virus type 1 protease (HIV-1 PR) is a homodimeric structure in which two subunits are linked through a two-stranded antiparallel β-sheet consisting of the N- and C-termini of each monomer. To inhibit the dimerizat

Full text of DOI:10.1021/jm9803976

J . Med. Chem. 1999, 42, 957-962
957
Articles
Design , Syn th esis, a n d Eva lu a tion of Con for m a tion a lly Con str a in ed Ton gs, New
In h ibitor s of HIV-1 P r otea se Dim er iza tion
Ahmed Bouras,§ Nicole Boggetto, Zohra Benatalah,§ Eve de Rosny, Sames Sicsic,*,§ and
Miche`le Reboud-Ravaux
BIOCIS, URA-CNRS 1843, Faculte´ de Pharmacie, Universite´ de Paris-sud, 5 rue J . B. Cle´ment,
F-92296 Chaˆtenay-Malabry Cedex, France, and Laboratoire d’Enzymologie Mole´culaire et Fonctionnelle,
De´partement de Biologie Supramole´culaire et Cellulaire, Institut J acques Monod, Universite´s de Paris VI et VII,
2 place J ussieu, F-75251 Paris Cedex 05, France
Received J uly 6, 1998
The active form of human immunodeficiency virus type 1 protease (HIV-1 PR) is a homodimeric
structure in which two subunits are linked through a two-stranded antiparallel â-sheet
consisting of the N- and C-termini of each monomer. To inhibit the dimerization process or
disrupt the dimeric interface leading to inactive enzyme, conformationally constrained
“molecular tongs” have been designed and synthesized to interfere with one monomer end in
a â-sheet fashion. These molecules are based on two peptidic strands attached to an aromatic
scaffold. Inhibitions (submicromolar range) were obtained with molecular tongs containing
tripeptidic or tetrapeptidic arms attached to a pyridinediol- or naphthalenediol-based scaffold
(Kid ) 0.56-4.5 µM at pH 4.7 and 30 °C). Kinetic studies are in agreement with an interface
inhibition mechanism.
In tr od u ction
tor. This type of inhibition was confirmed by Schramm.13
Babe´ et al. designed inhibitors containing two pen-
tapeptide strands mimicking the N- and C-termini of a
HIV-1 PR monomer connected with a three-glycine
spacer.10 Schramm et al. have proposed other inhibitors
containing a five-glycine spacer to connect polypeptides,
which were shown to be competitive site-directed inhibi-
tors at high salt concentrations and mixed antidimer
and competitive inhibitors at low salt concentrations.13
More recently, Zutshi et al. synthesized interfacial
peptides cross-linked with alkyl chains in order to
reproduce the â-sheet structure with one HIV-1 PR
monomer.14 Their best inhibitor (IC50 ) 2 µM) was
constructed with HN-PQITLW-OH and HN-STLNF-OH
polypeptidic chains tethered with a C14 alkyl chain. The
major drawback of these cross-linked inhibitors is
related to their high conformational freedom which
induces an unfavorable entropy term in the interaction
energy of the inhibitor-HIV-1 PR monomer complex.
HIV-1 protease (PR) has been studied and reviewed
extensively, due to its importance as a therapeutic
target to contend with AIDS. It plays a critical role in
the maturation and infectivity of new viral particles.1,2
Nevertheless, viral resistances to protease inhibitors
administered alone or in combination with reverse
transcriptase inhibitors have been described in vivo3
suggesting that there is a need for structurally diverse
antiproteases. All the protease inhibitors used in HIV-1
therapy (Saquinavir, Ritonavir, and Indinavir) interact
with the active site preventing the binding of enzyme
natural substrates. Another binding locus for HIV-1 PR
inhibitors is the extended antiparallel â-sheet formed
by the interdigitation of N-terminal (residues 1-4) and
C-terminal (residues 96-99) â-strands of each monomer
(Figure 1). Indeed, HIV-1 PR consists of two identical
99-residue monomers assembled into a C2-symmetric
structure, forming the active enzyme.4-6 The interac-
tions between the four termini contribute over 50% to
the stabilizing force in the dimer. Blocking the assembly
of the HIV-1 PR homodimer or disrupting the dimeric
interface was suggested to be a novel means of inhibit-
ing protease activity. The strategy of using interface
peptides reproducing C- and N-terminal fragments to
inhibit enzymatic activity has been applied success-
fully.7-14 Zhang et al.8 analyzed the kinetics of dimer-
ization inhibition by the C-terminal tetrapeptide frag-
ment and found it acted as a pure dimerization inhibi-
Our strategy has been directed toward the design of
more rigid “molecular tongs” based on a conformation-
ally constrained scaffold attached to two peptidic strands.
The advantage of this strategy is that the two peptidic
strands of the tongs could be originally suitably oriented
by the scaffold allowing the formation of an antiparallel
â-sheet with one HIV-1 PR monomer as illustrated in
Figure 2, leading to entropic benefit. Three original rigid
scaffolds have been chosen to link the two interface
peptides. We explored the influence on the enzyme
activity of (i) the nature of the scaffold and (ii) the length
and sequence of the peptidic chains. Kinetic analyses
* To whom correspondence should be addressed. E-mail:
sames.sicsic@cep.u-psud.fr.
§ Universite´ de Paris-sud.
Universite´s de Paris VI et VII.
10.1021/jm9803976 CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/27/1999

958 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Bouras et al.
F igu r e 1. Stereoview of the antiparallel â-sheet formed by interdigitation of N- and C-terminal strands of each monomer of
HIV-1 PR.
Sch em e 1
parallel â-sheet structure and provide a suitable dis-
tance between them, close to the distance between the
NH amide backbone of Pro 1 and Cys 95 of each
monomer of dimeric HIV-1 PR (Figures 1 and 2). Using
molecular modeling based on the crystallographic struc-
ture of HIV-1 PR, three constrained scaffolds were
designed based on resorcinol, 2,6-pyridinediol, and 2,7-
naphthalenediol on which carboxypropyl arms were
bound (Figure 2). A distance of 10 Å between the two
carbons of the carboxyl groups was reachable (Figure
2). The carboxypropyl arms also allowed some flexibility
which may be necessary during the complexation pro-
cess with one monomer of HIV-1 PR. For synthesis
convenience, symmetrical tongs with two identical pep-
tidic arms were synthesized using first the natural
sequences TLNF and QITL (Figure 2).
Our strategy was to simplify these sequences in order
F igu r e 2. (A) Strategy of molecular tongs based on confor-
to decrease the steric hindrance (TLVV versus TLNF)
or the strand length (3 versus 4 amino acids) whereas
the hydrophobic character was maintained (V instead
of F or T). The sequence VLV was the ultimate simpli-
fication of TLV. To appreciate the usefulness of the two
arms, molecules with only one peptidic arm were also
prepared (Scheme 1). The scaffolds 1, 3, and 5 were
synthesized using classical condensation reactions of
ethyl 4-bromobutanoate with resorcinol, 2,6-pyridinediol,
and 2,7-naphthalenediol, respectively. Partial hydrolysis
of diester 5 with methanolic KOH gave rise to acid ester
6, and total hydrolysis of compounds 1, 3, and 5 led to
diacids 2, 4, and 7, respectively. Finally, tongs 8-27
(Table 1) were obtained by condensation of the methyl
esters of di-, tri-, or tetrapeptides on diacids 2, 4, and 7
using isobutyl chloroformate as the condensing agent.
mationally constrained scaffolds. (B) Structures of molecular
tongs.
were performed in order to determine whether the
designed molecules act as competitive or antidimer
inhibitors.
Resu lts a n d Discu ssion
The strategy of antidimer inhibition of HIV-1 PR was
shown to be successful, more particularly by the use of
interface peptides cross-linked with alkyl chains which
were assumed to mimic the antiparallel â-sheet formed
by N- and C-terminal residues of each monomer of
HIV-1 PR.13,14 Our strategy was based on the design of
“molecular tongs” constrained by a rigid scaffold in order
to orientate correctly two peptidic strands in an anti-

Design and Synthesis of Molecular Tongs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 959
Ta ble 1. Inhibition of HIV-1 PR by Molecular Tongs at pH 4.7 and 30 °C
b
molecule
scaffold
X1
X2
analysisa
IC50 (µM)
8
9
10
11
12
Res
Res
Res
Pyr
Pyr
QIOMe
QIOMe
C
38H60N6O12‚2H2Oc
nif
ni
ni
ni
16
VLVOMe
TLVOH
TVOMe
GLLOMe
VLVOMe
TLVOH
TVOMe
GLLOMe
C48H80N6O12
C
C
33H53N5O12‚H2O
43H71N7O12‚H2O
(Kid ) 4.5 µM)
13
Pyr
TIVOMe
TIVOMe
C
C
45H75N7O14‚2H2O
45H75N7O14‚2H2O
4.2
(Kid ) 1.4 µM)
14
15
16
17
18
19
20
Pyr
Pyr
Pyr
Pyr
Pyr
Naph
Naph
TLVOMe
VLVOMe
TLVOH
QITLOMe
TLNFOMe
QIOMe
TLVOMe
VLVOMe
TLVOH
QITLOMe
TLNFOMe
QIOME
Ig
3
C47H79N7O12
34
ni
10
ni
C
C
C
C
57H95N11O18‚H2O
61H87N11O18‚3H2O
42H62N6O12
d
VLVOMe
VLVOMe
52H82N6O12, 2H2O
2
(Kid ) 0.56 µM)
21
22
23
24
25
26
Naph
Naph
Naph
Naph
Naph
Naph
TLVOMe
TLVOBz
TLVOH
TLYOMe
QITLOMe
TLNFOMe
TLVOMe
TLVOBz
TLVOH
TLYOMe
QITLOMe
TLNFOMe
C
C
50H80N6O14
62H86N6O14‚H2O
ni
ni
10
ni
ni
C
C
C
58H78N6O16‚2H2Oe
62H98N10O18‚2H2O
66H90N10O18‚2H2O
4.4
(Kid ) 1 µM)
27
28
29
30
31
32
33
Naph
Naph
Naph
Naph
Naph
Naph
Naph
TLVVOMe
VLVOMe
TLVOMe
TLYOMe
QITLOMe
TLNFOMe
TLVVOMe
TLVVOMe
OMe
OMe
OMe
OMe
C
C
C
60H96N8O16
36H53N3O9
35H51N3O10
ni
ni
Ih
ni
ni
Ii
C39H51N3O11
C
C
41H61N5O12
43H57N5O12‚2H2O
OMe
OMe
C40H58N4O11‚H2O
10
a
b
Analyses (C, H, N) are within the theoretical values ( 0.4% unless otherwise stated. Standard errors are less than 15%. c N: calcd.
10.13; found, 9.35. N: calcd. 9.97; found, 8.38. e H: calcd. 7.18; found, 7.63. f ni, no inhibition at the solubility limit. 30% inhibition at
6.7 µM. 30% inhibition at 4.5 µM. 30% inhibition at 7 µM.
d
g
h
i
Ta ble 2. 1H NMR Chemical Shifts for the Peptides Branched after N-Deprotection to Scaffolds, Leading to Molecular Tongs
1H NMR chemical shifts (NH, HR, Hâ, Hγ)
molecule
AA1
AA2
AA3
AA4
BocQIOMe
5.55, 4.2, 1.95, 2.35
5.63, 4.0, 3.95, 0.95
7.75, 4.18, 4.1, 1.05
6.25, 3.85
7.6, 4.45, 1.85, 1.15
7.2, 4.24, 1.93, 0.68
7.82, 4.50, 2.50, 1.75
7.45, 4.5, 1.55 (â, γ)
6.55, 4.45, 1.55, 1.65
6.97, 4.46, 1.57, 1.62
6.95, 4.35, 1.5 (â, γ)
7.9, 4.25, 1.8, 1.4
BocTVOMe
BocTIVOMe
ZGLLOMe
ZVLVOMe
ZTLVOMe
BocTLYOMe
BocQITLOMe
BocTLNFOMe
BocTLVVOMe
7.82, 4.48, 2.04
7.5, 4.6, 1.55 (â, γ)
6.75, 4.45, 2.08
6.86, 4.51, 2.13
6.85, 4.75, 2.95
7.83, 4.2, 3.9, 1
8.05, 4.5, 2.48/2.36
7.63, 4.2, 1.95
5.52, 4.00, 2.08
5.87, 4.29, 4.25, 1.14
5.55, 4.15, 4.0, 1.08
6.97, 3.9, 1.75/1.65, 2.05
6.3, 3.9, 3.9, 1
7.67, 4.3, 1.3, 1.7
8.02, 4.4, 2.95
8.05, 4.35, 1.95
7.84, 4.3, 1.4, 1.6
7.55, 4.15, 1.55, 1.4
6.4, 3.9, 3.9, 0.95
All the peptides were synthesized (Table 2) using
isobutyl chloroformate, except for the condensation of
XNGlnOH or XNAsnOH with the N-terminal part of
other amino acids or peptides which were realized with
DCC-HOBt method. Molecules 28-33 (Table 1), which
possess only one peptidic arm branched on the naph-
thalenic scaffold, were obtained by the same condensa-
tion step from acid ester 6.
Table 1 indicates the results of inhibition of HIV-1
PR by the synthesized molecules within their limit of
solubility in test medium. No inhibition was observed
with the resorcinol-based tongs 8-10, whereas inhibi-
tions were obtained with pyridinediol- and naphtha-
lenediol-based tongs. These results allowed to estimate
the influence of the scaffolds and of the length and
sequence of the attached peptidic strands on the inhibi-
tion potency. Comparing the inhibitions of the py-
ridinediol-based tongs 15, 16, and 18 bearing VLV-OMe
(IC50 ) 3 µM), TLV-OH (IC50 ) 34 µM), and TLNF-OMe
(IC50 ) 10 µM), respectively, with the equivalent naph-
thalenediol-based tongs 20 (IC50 ) 2 µM), 23 (IC50 ) 10
µM), and 26 (IC50 ) 4.4 µM), it can be deduced that the
naphthalenediol-based scaffold was slightly better than
the pyridinediol-based scaffold. In both series of tongs,
the preferred sequences for inhibition could be classified
as follows: VLV-OMe (15) ∼ TIV-OMe (13) > GLL-OMe
(12) ∼ TLV-OMe (14) for the tripeptidic strands and
VLV-OMe (15, 20) > TLNF-OMe (18, 26) if tripeptidic
and tetrapeptidic strands are compared. Tongs with the
peptidic sequences QI-OMe (19), TV-OMe (11), TLY-
OMe (24), TLVV-OMe (27), and QITL-OMe (17, 25) did
not behave as inhibitors within their solubility limits.
Generally, the inhibition was favored by the presence
of two peptidic strands as exemplified by the comparison
of 20 versus 28 and 26 versus 32. The replacement of
the ester end by the corresponding acid (14 versus 16)
altered physicochemical properties, especially the solu-
bility in buffer which was increased by a factor of 10,
while a comparable enzymatic inhibitory potency was
retained. Finally, it should be noted that pyridinediol-
and naphthalenediol-based scaffolds without attached
peptidic strands (3 and 5) were devoid of inhibitory

960 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Bouras et al.
F igu r e 4. Schematic structure of the complex HIV-1 PR
monomer/tong 13 showing potential ionic interaction between
Phe-O- 99 of the protease and the protonated pyridine of
compound 13.
was obtained for compound 20 (Kid ) 0.56 µM) which
has two VLV-OMe strands attached to the naphtha-
lenediol-based scaffold. It was verified that in the
experimental conditions, the competitive inhibitor
acetylpepstatin led to straight lines with the same
intercept on the y axis.
F igu r e 3. Plot of [E]0/ v versus v for hydrolysis of the
x
x
i
fluorogenic substrate by HIV-1 PR at pHi 4.7 and 30 °C in the
absence (b) and presence of (A) 3 µM (2), 6 µM (9), compound
13; (B) 1 µM (9), 2 µM (2) compound 20. The enzyme
concentrations were 2.37-9.47 nM, and the substrate concen-
tration was 5.2 µM. The straight lines are curves calculated
by regression fit to the data.
These results may be discussed as follows: (i) The
naphthalenediol- and pyridinediol-based scaffolds ap-
pear efficient to induce peptidic interface inhibition on
HIV-1 PR, while the resorcinol-based scaffold appears
inefficient. Indeed, compounds 9 and 10 were not
inhibitors at concentrations for which equivalent naph-
thalenediol-based tongs 20 and 23 and pyridinediol-
based tongs 15 and 16 inhibited HIV-1 PR. From a steric
point of view, the necessary distance of 10 Å between
the two peptidic strands of the tongs could be more
easily reached by the naphthalenediol-based tongs than
by the two other types of tongs. On the other hand, from
an electronic point of view, only the pyridinediol-based
tongs could form a favorable ionic interaction between
the positively charged pyridine in the medium test and
the negatively charged Phe-O- 99 of the C-end of HIV-1
PR monomer15 (Figure 4). This argument is supported
by the fact that scaffold 3‚HCl had a pKA ) 6.15 as
measured potentiometrically. The assumption that ster-
ic effect and/or electronic effect is favorable for the tongs
to induce inhibitory activity could explain why the
resorcinol-based tongs were devoid of activity. (ii) The
peptidic sequence TLNF-OMe reproducing the internal
C-terminal strands of the antiparallel â-sheet of HIV-1
PR led to the active tongs 18 and 26, while the peptidic
sequence QITL-OMe reproducing the external N-termi-
nus â-sheet strand led to the inactive tongs 17 and 25.
These results are consistent with those described in
the literature where the best inhibitions were observed
with interfacial peptides reproducing the C-terminal
strand.7-9,13 (iii) Surprisingly, the short peptidic se-
quence VLV-OMe appeared as the best sequence for
inducing inhibitory potency (compounds 15 and 20),
although it did not reproduce the sequences involved
in the natural antiparallel â-sheet of HIV-1 PR.
activity, as peptides alone, not attached to a rigid
scaffold, excepted for TLV-OMe and TLVV-OMe which
behaved as weak inhibitors (IC50 > 30 µM).
To determine the type of inhibition which occurred
with tongs, kinetic analyses of the inhibition process
according to Zhang et al.8 were performed with com-
pounds 12, 13, 20, and 26. Plots of [E]0/ v versus
v
x
x
i
i
constructed for the four compounds studied gave straight
lines with the same slope as without inhibitor, whatever
the inhibitor concentration (Figure 3). These patterns
were consistent with a dissociative inhibition described
by the minimal kinetic scheme of eq 1, where D is the
dimer, M is the monomer, S is the substrate, P is the
product, and I is the inhibitor with Kid ) [M][I]/[MI],
Kd ) [M]2/[D], and Km and kcat are the kinetic param-
eters for the enzyme catalysis of substrate S hydrolysis.
In our conditions the constant Kd was equal to 1.5 (
0.25 nM. The values of Kid (equal to [I]b0/(b - b0)) were
calculated from the intercepts in the presence (b) and
absence (b0) of inhibitor according to eq 2.
These Kid values are indicated in Table 1; the best value

Design and Synthesis of Molecular Tongs
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6 961
monitored over a period of 5 min at 30 °C. For the more potent
inhibitors the inhibition pathway was characterized using the
kinetic analysis of Zhang et al.8 The kinetic measurements
were carried out with constant initial substrate concentration
[S]0 , Km (Km ) 103 µM)17 using at least five different
concentrations of enzyme (2.37-11.8 nM) for various concen-
trations of inhibitor.
Molecu la r Mod elin g. Molecular modeling was performed
using a SGI Iris Indigo system (XS 4000) and the Sybyl 6.3
program from Tripos. Energy calculations were done using
Tripos force field, Gesteiger-Marsilii charges, and a dielectric
constant of 1. Crystallographic protease structure 8HVP
downloaded from the Protein Data Bank was used. One
monomer and the TLNF and QITL strands of the other
monomer were retained. The tongs were built on the TLNF
and QITL fragments in such a way that the minimization
procedure of the obtained complexes converged easily (termi-
nation gradient of 0.5 kcal/mol‚Å).
Ch em istr y. Gen er a l Syn th esis of Sca ffold s. Exa m p le
of Eth yl 4-((7-(4-Eth oxy-4-oxobu toxy)-2-n a p h th yl)oxy)-
bu ta n oa te (5). To a stirred solution of 2,7-dihydroxynaph-
thalene (10 g, 0.062 mol) and potassium carbonate (21.4 g,
0.155 mol) in DMF (200 mL) was added slowly ethyl 4-bro-
mobutanoate (36.3 g, 0.186 mol). After 3 h solvent and excess
reagent were evaporated, and the residue was dissolved in
CH2Cl2 (100 mL). This solution was washed with water (2 ×
50 mL), dried (MgSO4), and evaporated. The resulting residue
was chromatographed (silica gel, CH2Cl2) giving rise to 5 as
an oily product (18.4 g, 75% yield): 1H NMR (CDCl3) δ 7.5 (d,
2H, J ) 8 Hz), 6.94 (s, 2H), 6.90 (d, 2H, J ) 8 Hz), 4.07 (q,
2H, J ) 7 Hz), 4.06 (q, 2H, J ) 7 Hz), 3.9 (t, 4H, J ) 7 Hz),
2.5 (t, 4H, J ) 7 Hz), 2.0 (m, 4H), 1.16 (t, 3H, J ) 7 Hz), 1.15
(t, 3H, J ) 7 Hz); 13C NMR (CDCl3) δ 173.6, 157.3, 135.8, 129,
124.3, 116.1, 106, 66.6, 60.4, 51.6, 30.8, 24.6. Anal. (C22H28O6).
Our results could be compared with those of Zutshi
et al.14 who synthesized interfacial peptides cross-linked
with polyalkyl chains. An IC50 value of 57 µM was
obtained for the molecule displaying two identical
interfacial peptides STLNF-OH cross-linked by the
flexible spacer -(CO)-(CH2)12-(CO)-. With a rigid spacer
(naphthalene) and a shorter peptidic sequence (TLNF-
OMe, compound 26), a better inhibitory potency was
obtained (IC50 ) 4.4 µM, Kid ) 1 µM). The more potent
inhibitor described by Zutshi et al. (IC50 ) 2µM) was
obtained with two different peptidic sequences repro-
ducing the N- and C-termini of HIV-1 PR monomer
(PQILW-OH and STLNF-OH, respectively, cross-
linked with a 14-methylene spacer). With two identical
and shorter peptidic strands (VLV-OMe, compound 20),
a comparable inhibitory efficiency was obtained (IC50
) 2 µM, Kid ) 0.56 µM). The advantage of our tongs
may be explained by the structural difference of the two
types of spacers: aromatic-based spacers introduce a
steric constraint which likely provides a positive en-
tropic effect in contrast with the highly flexible spacers
reported by Zutshi et al. A similar strategy based on a
rigid scaffold was described by LaBrenz et al. in order
to understand the energetics of â-sheet-based molecular
recognition between a constrained peptide host and a
linear peptide guest.15 Finally, another advantage of
constrained tongs resides in their symmetrical character
allowing easy syntheses.
Con clu sion
E t h yl
4-((3-(4-et h oxy-4-oxob u t oxy)p h en yl)oxy)b u -
In this work, we have demonstrated the validity of
the concept of constrained tongs built as two peptidic
strands attached to a rigid naphthalenediol- or py-
ridinediol-based scaffold for inducing efficient antidimer
inhibitions of the HIV-1 PR. This approach also shows
that molecules with only tripeptidic strands which did
not reproduce the N- and C-termini of HIV-1 PR
monomer led to efficient inhibition. The best inhibitor
is structurally simple and very easy to synthesize.
ta n oa te (1): 85% yield; 1H NMR (CDCl3) δ 7.11 (t, 1H, J ) 7
Hz), 6.43 (m, 3H), 4.11 (q, 4H, J ) 7 Hz), 3.94 (t, 4H, J ) 7
Hz), 2.41 (t, 4H, J ) 7 Hz), 2.06 (m, 4H), 1.23 (t, 6H, J ) 7
Hz); 13C NMR (CDCl3) δ 173, 159.9, 129.6, 106.6, 101.2, 66.5,
60.2, 30.6, 24.4, 14. Anal. (C18H26O6).
Eth yl 4-((6-(4-eth oxy-4-oxobu toxy)-2-p yr id yl)oxy)bu -
ta n oa te (3): 59% yield; 1H NMR (CDCl3) δ 7.29 (t, 1H, J ) 8
Hz), 6.1 (d, 2H, J ) 8 Hz), 4.14 (t, 4H, J ) 6 Hz), 3.97 (q, 4H,
J ) 7 Hz), 2.32 (t, 4H, J ) 7.5 Hz), 2.0-1.86 (m, 4H), 1.09 (t,
6H, J ) 7 Hz); 13C NMR (CDCl3) δ 173.4, 162.6, 140.9, 101.4,
64.7, 60.3, 31.0, 24.6, 14.2. Anal. (C17H25O6‚1/4H2O).
4-((7-(4-Meth oxy-4-oxobu toxy)-2-n a ph th yl)oxy)bu ta n o-
ic Acid (6). A solution of diester 5 (1 g, 0.26 mmol) and NaOH
(0.08 g, 2 mmol) in methanol (100 mL) was heated (50 °C) for
12 h and then evaporated and the residue put in water (50
mL). The aqueous solution was extracted with CH2Cl2 (2 ×
50 mL) in order to remove the nonhydrolyzed diester, acidified
(HCl, 10%), and extracted with CH2Cl2 (2 × 50 mL). This
organic solution was washed with water, dried (MgSO4), and
evaporated giving 6 as a white cristalline product (0.3 g, 30%
yield): mp 85 °C (petroleum ether-ether); 1H NMR (CDCl3) δ
7.63 (d, 2H, J ) 7 Hz), 7.05-6.9 (m, 4H), 4.12 (t, 2H, J ) 7
Hz), 4.08 (t, 2H, J ) 7 Hz), 3.7 (s, 3H), 2.63 (t, 2H, J ) 7 Hz),
2.55 (t, 2H, J ) 7 Hz), 2.25-2.08 (m, 4H); 13C NMR (CDCl3) δ
179.5, 173.8, 157.3, 135.8, 129.1, 124.3, 116.2, 106.0, 66.6, 66.4,
51.7, 30.6, 24.6, 24.3. Anal. (C19H22O6).
Exp er im en ta l Section
HIV-1 protease was kindly supplied by H. J . Schramm, Max-
Planck Institut fu¨r Biochemie (Martinsried, Germany). It was
expressed using the plasmid pET9c-PR, and the isolation and
purification procedure is described by Billich et al.16 The stock
solution (34 µM protease in 50 mM MES, pH 6.0, 1 mM EDTA,
1 mM DTT, 0.5 M NaCl, 5% v/v glycerol) was stored in small
aliquots at -80 °C. The fluorogenic substrate DABCYL-S-Q-
N-Y-P-I-V-Q-EDANS was purchased from Bachem. Reagents
and solvents were from commercial sources. The fluorescence
measurements were performed using a J obin Yvon spectro-
fluorometer. NMR spectra were done on AC 200 Bruker and
ARX 400 Bruker spectrometers. Elemental analyses were
performed by the Service de microanalyse de la Faculte´ de
Pharmacie. The pKA measurements were performed in water
using a Metrohm 632 pH apparatus equipped with a 614
impulsomat.
En zym a tic Assa ys. Enzymatic assays were performed in
100 mM sodium acetate, 1 mM EDTA, 1 M NaCl, pH 4.7, 3%
DMSO (v/v). Inhibitors and the substrate were dissolved in
DMSO before addition to the buffer. For the determination of
IC50 values, 0.52 µL of a 3 mM solution of substrate (final
concentration, 5.2 µM) was added to 8.5 µL of 4-10 different
concentrations of inhibitors (final volume, 300 µL). The
enzymatic reaction was initiated by the addition of enzyme
(prediluted in buffer containing 1 mg/mL bovine serum
albumin). The final enzyme concentration was 7.5 nM. The
increase in fluorescence at 490 nm (λexc ) 340 nm) was
4-((7-(3-Car boxypr opoxy)-2-n aph th yl)oxy)bu tan oic Acid
(7). A solution of ethanol (50 mL) containing 5 (6 g, 0.016
mmol) and 10% KOH (100 mL) was stirred at reflux for 4 h.
Then the solution was poured in ice and extracted with CH2-
Cl2 (2 × 50 mL). The aqueous layer was acidified (HCl, 10%),
and the precipitate was filtered and washed with water giving
rise to a crystalline product. (5.84 g, 97% yield): mp 144 °C;
1H NMR (CDCl3) δ 7.75 (d, 2H, J ) 7 Hz), 7.25 (d, 2H, J ) 1.3
Hz), 7 (dd, 2H, J ) 7 Hz, J ) 1.3 Hz), 4.1 (t, 4H, J ) 7 Hz), 2.5
(t, 4H, J ) 7 Hz), 2 (m, 4H); 13C NMR (CDCl3) δ 174.1, 157,
136, 129, 123.8, 116, 106.2, 66.4, 30.2, 24.2. Anal. (C18H20O6).
4-((3-(3-Car boxypr opoxy)ph en yl)oxy)bu tan oic acid (2):
1
95% yield; mp 170 °C; H NMR (DMSO) δ 7.2-7.1 (m, 1H),

962 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 6
Bouras et al.
(6) Lapatto, R.; Blundel, T.; Hemmings, A.; Overington, J .; Wilder-
spin, A.; Wood, S.; Merson, J . R.; Whittle, P. J .; Danley, D. E.;
Geoghegan, K. F.; Hawrylik, S. J .; Lee, S. E.; Scheld, K. G.;
Hobart, P. M. X-ray Analysis of HIV-1 Proteinase at 2.7 Å
Resolution Confirms Structural Homology among Retroviral
Enzymes. Nature 1989, 342, 299-302.
(7) Babe´, L. M.; Pichuantes, S.; Craik, C. S. Inhibition of HIV
Protease Activity by Heterodimer Formation. Biochemistry 1991,
30, 106-111.
(8) Zhang, Z.-Y.; Poorman, R. A.; Maggiora, L. L.; Heinrickson, R.
L.; Kedzy, F. J . Dissociative Inhibition of Dimeric Enzymes.
Kinetic Characterization of the Inhibition of HIV-1 Protease by
its COOH-Terminal Tetrapeptide. J . Biol. Chem. 1991, 266,
15591-15594.
(9) Schramm, H. J .; Breipohl, G.; Hansen, J .; Henke, S.; J aeger,
E.; Meichsner, C.; Riess, G.; Ruppert, D.; Ru¨cknagel, K.-P.;
Scha¨fer, W.; Schramm, W. Inhibition of HIV-1 Protease by Short
Peptides Derived from the Terminal Segments of the Protease.
Biochem. Biophys. Res. Commun. 1992, 184, 980-985.
(10) Babe´, L. M.; Rose´, J .; Craik, C. S. Synthetic Interface Peptides
alter Dimeric Assembly of the HIV 1 and 2 Proteases. Protein
Sci. 1992, 1, 1244-1253.
6.49-6.45 (m, 3H), 3.93 (t, 4H, J ) 6 Hz), 2.34 (t, 4H, J ) 7
Hz), 1.96-1.83 (m, 4H); 13C MR (CDCl3) δ 183.6. 169.2, 139.4,
116.2, 110.6, 76.0, 39.6, 33.8.
4-((6-(3-Ca r boxyp r op oxy)-2-p yr id yl)oxy)bu ta n oic a cid
(4): 88% yield; mp 92 °C (CH2Cl2-EtOH); 1H NMR (CDCl3) δ
7.45 (t, 1H, J ) 8 Hz), 6.25 (d, 2H, J ) 8 Hz), 4.33 (t, 4H, J )
6 Hz), 2.53 (t, 4H, J ) 7. Hz), 2.15-2.02 (m, 4H); 13C NMR
(CDCl3) δ 175.5, 163.9, 143, 102.5, 66.3, 31.6, 25.8. Anal.
(C13H17NO6‚0.5H2O).
Gen er a l Syn th esis of Ton gs. Exa m p le of 13. In a
solution of anhydrous DMF (20 mL) containing 4 (0.23 g, 0.83
mmol) and N-methylmorpholine (0.17 g, 1.6 mmol) maintained
at -15 °C was added slowly isobutyl chloroformate (0.23 g,
1.6 mmol). The mixture was stirred for 2 h at -15 °C, and
NH2Thr-Ile-Val-OMe‚HCl (0.8 g, 1.6 mmol) and N-methylmor-
pholine (0.17 g, 1.6 mmol) were added. The mixture was stirred
for 15 h at room temperature. The resulting mixture was
filtered, and the filtrate was evaporated to dryness. The crude
solid was washed successively with warm water and methanol
to give 0.7 g of 13 (90% yield).
(11) Franciskovich, J .; Houseman, K.; Mueller, R.; Chmielewski, J .
A Systematic Evaluation of the Inhibition of HIV-1 Protease by
its C- and N-terminal Peptides. BioMed. Chem. Lett. 1993, 3,
765-768.
(12) Arnold, G. J .; Popinga, K.; Schramm, H. J .; Bell, M.; Thumfart,
O.; Wenger, T.; Schramm, W. Inhibition of HIV-1 Protease by
Bifunctional interface Peptides. In Peptides 1994; Maia, H. L.
S., Ed.; ESCOM: Leiden, 1994; pp 345-346.
Ack n ow led gm en t. This work was supported by the
Fondation pour la Recherche Me´dicale (Sidaction) and
by the European Community (concerted action BMH4-
CT960823). Miche`le Ourevitch is acknowledged for her
supporting aid in NMR.
(13) Schramm, H. J .; Boetzel J .; Bu¨ttner, J .; Fritsche, E.; Go¨rhing,
W.; J aeger, E.; Ko¨nig, S.; Thumfart, O.; Wenger, T.; Nagel, N.
E.; Schramm, W. The Inhibition of Human Immunodeficiency
virus proteases by Interface peptides. Antiviral Res. 1996, 30,
155-170.
(14) Zutshi, R.; Franciskovich, J .; Slultz, M.; Schweitzer, B.; Bishop,
P.; Wilson, M.; Chmielewski, J . Targeting the Dimerization
Interface of HIV-1 Protease: Inhibition with Cross-Linked
Interfacial Peptides. J . Am. Chem. Soc. 1997, 119, 4841-4845.
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Peptide Guest Affording a â-Sheet Structure that subsequently
self-assembles. A Simple Receptor Mimic. J . Am. Chem. Soc.
1995, 117, 1655-1656.
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