New constrained "molecular tongs" designed to dissociate HIV-1 protease dimer

Article abstract of DOI:10.1021/jm040833q

New "molecular tongs" based on naphthalene and quinoline scaffolds linked to two peptidic strands were synthesized. They were designed to prevent dimerization of HIV-1 protease by targeting the antiparallel β-sheet involving N- and C-termini of each monom

Full text of DOI:10.1021/jm040833q

6392
J. Med. Chem. 2004, 47, 6392-6400
New Constrained “Molecular Tongs” Designed To Dissociate HIV-1 Protease
Dimer
Na¨ıma Merabet,† Julien Dumond,‡ Bruno Collinet,‡ Laurence Van Baelinghem,† Nicole Boggetto,‡
Sandrine Ongeri,† Fariza Ressad,‡ Miche`le Reboud-Ravaux,‡ and Sames Sicsic*,†
Biocis, UMR-CNRS 8076, 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,
Institut Jacques Monod, UMR 7592, CNRS-Universite´s de Paris VI et VII, 2 place Jussieu, F-75251 Paris Cedex 05, France
Received April 30, 2004
New “molecular tongs” based on naphthalene and quinoline scaffolds linked to two peptidic
strands were synthesized. They were designed to prevent dimerization of HIV-1 protease by
targeting the antiparallel â-sheet involving N- and C-termini of each monomer. Compared to
“molecular tongs” previously described (Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.;
Sicsic, S.; Reboux-Ravaud, M. J. Med. Chem. 1999, 42, 957-962), two main different structural
features were introduced: positively charged quinoline as a new scaffold and two peptidic
strands displaying different sequences. Seventeen new “molecular tongs” with dipeptidic or
tripeptidic strands were synthesized. These molecules were assayed on HIV-1 protease using
the Zhang kinetic technique. Eleven molecules behaved as pure dimerization inhibitors, mostly
at the submicromolar range. Compared to a naphthalene scaffold, the quinoline one was shown
in several cases to favor dimerization inhibition. The simplified hydrophobic Val-Leu-Val-OMe
strand was confirmed as particularly favorable. The C-terminal analogue strand Thr-Leu-Asn-
OMe was shown to be the best one for inducing dimerization inhibition (Kid of 80 nM for
compound 30). The mechanism of inhibition was ascertained using ANS binding and gel
filtration. Experimental results are in agreement with the dissociation of the HIV-1 protease
dimeric form in the presence of the synthesized molecular tongs.
Introduction
be highly conserved in HIV-1 and most HIV-2 isolates.13
Blocking the formation or disrupting the homodimer
was shown to be a means of inhibiting protease activ-
ity.14 C- and N-terminal mimetics were the first reported
dimerization inhibitors.15-17 Variation of peptide se-
quence and addition of a lipophilic terminal group
favored targeting the antiparallel â sheet and improved
the efficiency.18,19 The cross-linking of interface peptides
with flexible20 or semirigid21 spacers was explored.
Recently, guanidinium derivatives displaying short pep-
tide mimics and a lipophilic group have been designed
as dimerization inhibitors.22 Our contribution to the
antidimer strategy was the synthesis of the first “mo-
lecular tongs” based on a conformationally constrained
scaffold attached to two peptidic strands through two
carboxypropyl links.23 The advantage of this strategy
is that the two peptidic strands could be suitably
oriented by the scaffold allowing the putative formation
of an antiparallel â-sheet with the C-terminal end of
one HIV-1 PR monomer, thus leading to an entropy
benefit (Figure 1). The integrity of inner C-terminal
strands is essential for dimer formation.11 Two scaffolds
based on naphthalenediol and pyridinediol were found
to lead to active molecules as antidimers conversely to
resorcinol (Kid ) 0.560 µM for the best inhibitor).23 The
“â-sheet nucleator” 4-(2-aminoethyl)-6-dibenzopropionic
acid introduced by Kiso to connect two peptide frag-
ments was less efficient with Kid ) 5.4 µM.24 In order
to improve the activity of this type of inhibitor, it was
tempting to introduce a new scaffold and modifications
of the peptidic strands. Our previous studies showed
that the naphthalenediol spacer was sterically more
HIV-1 protease (PR) has been widely studied due to
its importance as a valuable therapeutic target for
developing anti-AIDS drugs since its inhibition results
in the production of noninfectious virus.1,2 Today seven
HIV-1 PR inhibitors including saquinavir, ritonavir,
amprenavir, indinavir, nelfinavir, lopinavir, and ata-
zanavir have been approved and marketed as anti-AIDS
drugs.3-6 Their efficacy was shown to be remarkable in
tritherapy protocols, combined with two reverse tran-
scriptase inhibitors, reducing the viral load and improv-
ing the number of CD4 lymphocytes in AIDS patients.
However, a rapid emergence of mutations that confer
resistance to all protease inhibitors is observed. Multi-
drug resistance is triggered by mutations located at
different regions of the protease molecule (within the
active site, or not) due to rapid amino acid mutations
of the enzyme.7,8 All the protease inhibitors used in
therapy are active site competitive inhibitors which bind
across the active site formed by both subunits of the
active dimeric enzyme. Residues in the dimer interface
are well conserved from the points of view of both
genetic variation and drug resistance.9,10 A network of
interactions around the active site and termini of the
mature protease are critical for dimerization.11 More
particularly, the antiparallel â-sheet formed between
the four monomeric termini contributes over 75% to the
stabilizing force of the dimer12 and has been found to
* To whom correspondence should be addressed. E-mail:
sames.sicsic@cep.u-psud.fr. Tel: 33(0)146835737. Fax: 33(0)146835740.
† Universite´ de Paris-Sud.
‡ CNRS-Universite´s de Paris VI et VII.
10.1021/jm040833q CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/30/2004

“Molecular Tongs” To Dissociate HIV-1 Protease Dimer
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6393
diester and unchanged product 1. Subsequent benzyla-
tion of the other OH group gave compound 3, giving rise
to distinguishable OH functions. Compound 3 was then
hydrolyzed under basic conditions to afford scaffold 4.
Nonsymmetric quinoline-based molecules were syn-
thesized from compound 5 obtained as described in the
literature26 (Figure 4). The 7-OH group of 5 was
selectively protected as the benzyl ether 6, allowing the
O-alkylation of the 2 position with 71% yield. Reaction
of 6 with ethyl 4-bromobutyrate gave ester 7 obtained
in 40% yield along with the N-alkylated derivative 8
obtained in 42% yield. The two isomers were easily
separated and well identified by their NMR and IR
spectra. These results are not surprising as it is well-
known that alkylation of 2(1H)-quinoline derivatives
involves an ambident anion and leads to a mixture of
N- and O-alkylated products.27,28 The yields were com-
parable with those reported in the literature. Compound
7 was subsequently hydrolyzed to afford the desired acid
9.
Figure 1. Model of interaction of a typical “molecular tong”
with one monomer of HIV-1 protease.
After scaffolds 4 and 9 were obtained, nonsymmetric
naphthalene- and quinoline-based tongs were obtained
according to Figure 5. In a first step, the carboxylic acid
scaffold 4 or 9 was coupled via a classical peptide
condensation to H-Val-Leu-Val-OMe to afford compound
10 or 11 respectively in good yields (90% and 93%,
respectively). After a debenzylation step, compounds 12
and 13 were O-alkylated with benzyl 4-bromobutyrate,29
giving compounds 14 and 15, respectively. These benzyl-
protected compounds were used in order to selectively
generate compounds 16 and 17 respectively in the
subsequent step of deprotection without modifying the
terminal ester of the tripeptide strand. In a last step,
condensation of 16 or 17 with a dipeptidic or tripeptidic
strand was done to afford final naphthalene-based tongs
18-20 and 22-24 (X ) C), and quinoline-based tongs
25-27 and 29-32 (X ) N) in moderate yields of 63% to
80% (Supporting Information). Compounds 20 and 27
which are ortho-protected at the side chain level of
glutamate with a tert-butyl group, were hydrolyzed
under acidic conditions to afford respectively compounds
21 and 28 (Supporting Information).
In order to compare activities of these new compounds
with activities of former ones,23 the two symmetric
quinoline-based tongs 36 and 37 having Val-Leu-Val-
OMe or Thr-Ile-Val-OMe respectively as peptidic strands
were also synthesized in three steps from molecule 5
(Figure 6). In a first step dialkylation of 5 with ethyl
bromobutyrate afforded the N,O-dialkylated compound
33 along with O,O′-dialkylated compound 34, which
gave compound 35 after separation and hydrolysis
under basic conditions. This compound was then sub-
mitted to a condensation step using the HBTU coupling
method with H-Val-Leu-Val-OMe giving rise to com-
pound 36, or with H-Ile-Thr-Val-OMe giving rise to
compound 37 in moderate yields (66% and 70%) (Sup-
porting Information).
Figure 2.
favorable than the pyridinediol one, but the latter could
form a favorable ionic interaction with the negatively
charged Phe-O- 99 at the C-terminus strand of the
monomer.23 To get advantage of the structure of these
two previous scaffolds, we have introduced a quino-
linediol spacer which has the steric structure of naph-
thalenediol and an electronic structure close to that of
pyridinediol (Figure 2). We conserved the same carboxy-
propyl linker between the quinolinediol and peptide
strands. In our previous study, identical peptidic strands
were introduced (“symmetric tongs”) leading to the
identification of the best sequence Val-Leu-Val-OMe, a
simplified mimetic of the C-terminal strand of the
monomer. In the new molecular tongs, the peptide Val-
Leu-Val-OMe constitutes one strand and the nature of
the second one has varied: essentially elements of the
N-terminal end of the monomer, in one case of the
C-terminus, and of YEL found in the powerful lipopep-
tides.19,25 Corresponding molecular tongs displaying a
naphthalenediol spacer were synthesized for compari-
son.
Chemistry
Nonsymmetric naphthalene-based molecules were
synthesized after differential protection of the two OH
groups of 2,7-dihydroxynaphthalene. Product 4 was
synthesized from commercially available 2,7-dihydroxy-
naphthalene 1 using classical reactions according to
Figure 3. Using 0.9 equiv of ethyl-4-bromobutyrate,
monoester 2 was obtained in a 36% yield, along with
The peptidic strands Z-Ile-Thr-NH2, Z-(O-tBu)Glu-
Leu-NH2, Z-Gln-Ile-Thr-NH2, Z-Tyr-(O-tBu)Glu-Leu-
NH2, and Z-Ile-Thr-Leu-OMe were synthesized accord-
ing to a linear peptide coupling method which consists
of condensing the acid group of one N-protected amino
acid with the amino group of another O-protected amino

6394 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Merabet et al.
Figure 3. (a) K2CO3, Br(CH2)3COOEt, DMF, 24 h, rt. (b) K2CO3, BrBn, CH3CN, 4 h, reflux. (c) Methanolic KOH 10%, 4 h, reflux.
Figure 4. (a) DBU, BrBn, iPrOH, 4 h, reflux. (b) K2CO3, Br(CH2)3COOEt, DMF, 24 h, rt. (c) Methanolic KOH 10%, 3 h, reflux.
Figure 5. (a) HBTU, triethylamine, H-VLV-OMe, DMF, 24 h, rt. (b) H2 Pd/C, MeOH, 24 h. (c) K2CO3, Br(CH2)3COOBn, DMF,
24 h, rt. (d) H2 Pd/C, DMF, 24 h. (e) HBTU, triethylamine, H-P2, DMF, 24 h, rt. (f) Trifluoroacetic acid, 6 h, rt.
acid or peptide. The coupling agents used were HBTU
or chloroformates. The N-protective group Z was cleaved
by hydrogenolysis with H2 Pd/C 10% in MeOH or DMF
since the N-protective group Boc was hydrolyzed with
a solution of 3 N HCl/MeOH (Table 1 of Supporting
Information). During the deprotection step of the amino
group of dipeptides Z-Ile-Thr-OMe and Z-(O-tBu)Glu-
Leu-OMe, we observed that cyclization of the dipeptide
occurred to afford lactam, when a methyl carboxylic acid
protective group was used. Consequently we opted for
the use of an amide carboxylic acid protective group to
avoid the cyclization since an amide group is less
sensitive to nucleophilic attack than an ester. The other
peptide strands were synthesized as previously re-
ported.23
Results and Discussion
In our previous work, we have shown that symmetric
molecular tongs, displaying two identical peptidic strands
and based on naphthalenediol and pyridinediol as rigid
scaffolds, could lead to efficient inhibitors of HIV-1 PR
dimerization (Kid ) 0.560 µM for compound 38 in Table
1, spacer 2,7-naphthalenediol and peptide Val-Leu-Val-
OMe).23 To improve the potency of this family of HIV-1
PR dimerization inhibitors, we have modified the struc-
ture of the scaffold and also introduced nonsymmetric
tongs, which consist of two different peptidic strands
attached to each scaffold. For the first point, we decided
to study a quinoline-based scaffold, as quinoline con-
tains structural requirements of both naphthalene and
pyridine (Figure 2). For the second point, we decided to

“Molecular Tongs” To Dissociate HIV-1 Protease Dimer
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6395
Figure 6. (a) K2CO3, Br(CH2)3COOEt, DMF, 24 h, rt. (b) KOH, MeOH 10%, 3 h, reflux. (c) HBTU, triethylamine, HN-pep, DMF,
24 h, rt.
Table 1. Biological Results of Molecular Tongsa
scaffold, and with nonsymmetric quinoline-based tongs.
Table 1 summarizes measured inhibitions by molecular
tongs expressed as IC50, calculated inhibition constants
Kic attributed to competitive inhibition, and calculated
inhibition constants Kid attributed to dimerization
inhibition. Inhibitions were obtained with all compounds
except for compound 22, which precipitated at 2 µM.
Weak inhibitory effects were observed with compounds
28 and 31 (20-30% at 5.7 µM), and no inhibitory effect
compd X
P1
P2
IC50 (µM)
Kic (µM) Kid (µM)
was detected for compound 22, which precipitates at 2
µM. Furthermore, except for compounds 18, 19, and 26,
which led to mixed inhibition in which the inhibitors
could bind to the interfacial region but also to the active
site, the other tested compounds gave a series of parallel
lines in a Zhang plot, which is diagnostic of pure
dissociative inhibition.23 The values of the inhibition
constants Kic (competitive inhibition) and Kid (dimer-
ization inhibition) were determined as explained in the
Experimental Section. The observed results may be
discussed as follow: (i) The new symmetric quinoline-
based tongs 36 and 37 behaved as dimerization inhibi-
tors; more particularly, compound 36, which is the
quinoline analogue of the naphthalene-based compound
38, showed a good Kid ) 0.13 µM. This result is a first
indication that the quinoline spacer is slightly more
favorable to interface inhibition than the naphthalene
spacer. The detrimental effect of sequence Thr-Ile-Val-
OMe (compound 37, Kid ) 1.90 µM) confirms the
superiority of the sequence Val-Leu-Val-OMe for dimer-
ization inhibition. (ii) Compounds which contained P2
as a part of the dimerization inhibitor Pam-Tyr-Glu-
Leu (20, 27, 21, 24, and 32) behaved as pure dimeriza-
tion inhibitors. These results confirm the beneficial
effect induced by Tyr-Glu-Leu for the interfacial inhibi-
tion, a good inhibition being also induced by only the
dipeptide Glu-Leu. For both quinoline and naphthalene
spacers, deprotection of the glutamate residue decreased
the inhibitory potency (21 vs 20, 28 vs 27), confirming
a favorable influence of the hydrophobic character of the
tong arms on efficiency.22,23 In the case of compound 28,
self-association of the inhibitor by intramolecular or
intermolecular ion pairing between glutamic acid car-
boxylate and quinolinium ion may be evoked to explain
the noticeable decrease in the inhibitory efficiency
compared to 21. (iii) For other molecular tongs, it could
be emphasized that reproducing in P2 a part of C-
terminal HIV-1 PR monomers Thr-Leu-Asn-Phe se-
quence is more favorable to interface inhibition than
reproducing a part of the N-terminal monomer se-
quence. Thus, in quinoline-based series, compound 25
(P2 ) Thr-Leu-OMe) is a better interface inhibitor than
18
25
19
26
20
27
21
28
C VLVOMe TLOMe
N VLVOMe TLOMe
C VLVOMe ITNH2
N VLVOMe ITNH2
C VLVOMe E*LNH2 1.1
N VLVOMe E*LNH2 1.8
4
10.3
1.40
1.50
1.50
0.25
0.42
0.53
0.90
1.4
4.3
1.7
6.2
4.3
b
C VLVOMe ELNH2
N VLVOMe ELNH2
2.8
20% at 5.7 µM
38c C VLVOMe VLVOMe 3.5
0.56
0.13
1.90
36
37
22
29
30
23
31
24
32
N VLVOMe VLVOMe 1.4
N TIVOMe TIVOMe 45% at 5.7 µM
C VLVOMe ITLOMe ND
N VLVOMe ITLOMe 5.3
N VLVOMe TLNOMe 5.1
C VLVOMe QITNH2 2.8
N VLVOMe QITNH2 30% at 5.7 µM
C VLVOMe YE*LNH2 1.1
N VLVOMe YE*LNH2 1.0
0.40
0.08
2.30
0.23
0.41
a Binding analysis used the Zhang procedure described previ-
ously.23 b E* for tert-butyl ester of the glutamate. c From ref 23.
mimic the four-stranded â-sheet structure of HIV-1 PR.
In order to easily establish a structure-activity rela-
tionship, it was decided to keep unchanged one of the
two peptidic strands of these nonsymmetric tongs.
According to our previous results, the peptidic strand
P1 was fixed to be Val-Leu-Val-OMe for all compouds
except 37. The other peptidic strand P2 varied in size
(di- or tripeptide) and sequence. In one case P2 was a
part of the C-terminal Thr-Leu-Asn-Phe strand of
monomers of HIV-1 PR (30); in other cases it was a part
of the N-terminal Pro-Gln-Ile-Thr-Leu strand (19, 22,
23, 26, 29, and 31) of monomers. For compounds 18 and
25, P2 was fixed to Thr-Leu-OMe which is a part of both
C-terminal Thr-Leu-Asn-Phe and N-terminal Gln-Ile-
Thr-Leu strands of each monomer. The peptides Glu-
Leu and Tyr-Glu-Leu and their O-protected glutamate
analogues introduced in compounds 20, 21, 24, 27, 28,
and 32 are parts of the powerful dimerization inhibitor
lipopeptide Pam-Tyr-Glu-Leu designed by Schramm.19
Symmetric compounds 36 (P1 ) P2 ) Val-Leu-Val-OMe)
and 37 (P1 ) P2 ) Thr-Ile-Val-OMe) based on the
quinoline scaffold were also synthesized for comparison
with symmetric tongs 38 based on the naphthalene

6396 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Merabet et al.
Figure 7. Analytical gel filtration chromatogram of PR in the absence (A) or presence of compound 25 (B). Arrows indicate
peaks corresponding to the dimeric (D) and monomeric (M) forms of PR (Superdex 75; flow rate 0.1 mL/min; 100 mM sodium
acetate; 5% (v/v) DMSO, pH ) 4.7 and 25 °C).
compound 26 (P2 ) Ile-Thr-OMe), and compound 30 (P2
) Thr-Leu-Asn-OMe) is better than compounds 29 (P2
) Ile-Thr-Leu-OMe) and 31 (P2 ) Gln-Ile-Thr-OMe).
Also in naphthalene-based series, compound 38 (P2 )
Val-Leu-Val-OMe) is a better interface inhibitor than
compound 23 (P2 ) Gln-Ile-Thr-OMe). (iv) Finally, we
observed that the quinoline spacer which is protonated
in the assay medium improves the interface inhibition
of molecular tongs in several cases (25 vs 18, 26 vs 19,
and 36 vs 38) where the P2 strand has a hydrophobic
character. This phenomenon was not observed when the
P2 strand possesses a more polar character (27 vs 20,
and 32 vs 24). Due to the fact that the quinoline ring is
weakly basic and that biological assays were done at
pH 4.7, the importance of this interpretation should be
modulated for physiological mediums which are at pH
7.0. Further evaluation under neutral conditions could
address this aspect. Taken together all these results
show that dimerization inhibition may be promoted,
when the two peptidic strands P1 and P2 reproduce a
part of the C-terminal Thr-Leu-Asn-Phe sequence of
HIV-1 PR monomers or when one strand fulfills this
condition and the other one reproduces a part of the
Pam-Tyr-Glu-Leu sequence. Furthermore, as observed
by Bouras,23 tripeptidic sequences are more convenient
than dipeptidic sequences to promote a good dimeriza-
tion inhibition. This is exemplified by tongs 24, 30, 32,
and 36 which are sub-micromolar dimerization inhibi-
tors. Finally, the good potency of the quinoline-based
tong 30 (Kid ) 80 nM) should be pointed out, since this
result represents a new advance in the research of
potent dimerization inhibitors of HIV-1 PR.
To further probe the mechanism of inhibition we
performed two types of experiments. Analytical gel
filtration chromatography was used to test the ability
of compound 25 to disassemble the dimeric protease.30
As seen in Figure 7, gel filtration of HIV-1 protease in
the presence of 25 led to apparition of two peaks, one
at 17.2 min corresponding to dimeric protease and the
other at 19.5 min attributed to dissociated monomers.
Clearly the ratio monomer over dimer increased in the
presence of compound 25, in agreement with a dissocia-
tive effect for this compound.
Figure 8. ANS emission spectra at pH 4.7 and 25 °C (λex
)
376 nm): PR (0.35 µM) was incubated in the absence of
inhibitor (a), or in the presence of 0.1 µM acetylpepstatin (b),
3.75 µM 23 (c), or 1.66 µM 32 (d). The fluorescence intensity
was corrected from the fluorescence due to the same concen-
tration of ANS (30 µM) without enzyme (a) and without
enzyme and inhibitor (b-d).
increase of fluorescence of ANS. As ANS is a hydropho-
bic molecule, it is generally considered that binding of
this molecule occurred at hydrophobic surfaces of pro-
teins.31 As seen in Figure 8 the fluorescence due to ANS
binding at the protease surface is considerably enhanced
in the presence of compounds 23 and 32 if compared to
the negligible increase observed in the presence of the
active site inhibitor acetylpepstatin. This is consistent
with the exposure of hydrophobic areas located at the
interface between the two monomers upon binding of
the putative dimerization inhibitor which dissociates or
destabilizes the dimer.25 Conversely to active site in-
hibitor binding, fixation of dimerization inhibitors un-
masks hydrophobic protein surfaces. This agrees with
dissociation or destabilization of the dimer enzyme
favoring the exposure of hydrophobic areas located at
the interface between monomers.25
Conclusions
The ANS binding technique was also performed. It is
well-known that ANS binding to protein promoted an
Our previous finding of “molecular tongs” as interfa-
cial inhibitors of HIV-1 protease23 prompted us to

“Molecular Tongs” To Dissociate HIV-1 Protease Dimer
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6397
Enzyme and Inhibition Assays. The proteolytic activity
of PR was assessed by using the fluorogenic substrate (DAB-
CYL-γ-Abu-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-EDANS) at pH
4.7 and 30 °C in 100 mM sodium acetate containing 1 mM
EDTA and 1 M NaCl. Excitation and emission wavelengths
were 340 and 490 nm, respectively. Substrate alone (blank)
or substrate and inhibitor previously dissolved in DMSO (final
concentration: 3% v/v) were preincubated for 3 min at 30 °C.
Enzyme was added to start the reaction. The final enzyme
continue this work in order to get new insights in the
structure-activity relationship of this kind of inhibitor.
Thus, we have introduced quinoline as a new rigid
scaffold, along with modifications of the structure of
peptidic strands attached to naphthalene- and quino-
line-based scaffolds. The main results are (i) quinoline
is a valuable scaffold, as most of quinoline-based tongs
were found as pure interfacial inhibitors of HIV-1 PR
leading to efficient molecules (Kid ) 80 nM for 30); (ii)
the attached tripeptidic strands induce generally better
inhibitions than the dipeptidic ones; and (iii) hydropho-
bicity of the attached peptidic strands is a favorable
property to induce inhibition. However two points
remain to be clearly established: the superiority of
inhibitors with two different tripeptides over inhibitors
with two identical ones, and the superiority of the
quinoline scaffold over the naphthalene one. These two
points are under current evaluation by preparation of
a quinoline scaffold endowed with two Thr-Leu-Asn-
OMe chains and of the naphthalene counterpart of
compound 30. Further studies on the HIV dimer inhibi-
tors reported here will include (i) optimization of the
electrostatic interaction with the C-terminus, including
evaluation at pH values closer to neutrality; (ii) estab-
lishment of the antiviral efficacy in cell culture assays;
(iii) evaluation of binding to the proteases from other
strains of the HIV virus to assess the potential, if any,
of differential binding; (iv) creating proteolysis-resistant
inhibitors by replacement of the peptide strands by
pseudopeptides; and (v) assessment of binding specificity
with other â-sheet proteins as a early indicator of
toxicity.
concentration was 7.5 nM. The increase of fluorescence (λex
)
340 nm; λem ) 490 nm) was followed during 3 min. The IC50
values were determined using 0.52 µL of a 3 mM substrate
solution and 8.5 µL of 8-10 different concentrations of
inhibitor in a total volume of 300 µL. The enzymatic reaction
was begun by adding of 1.6 µL of enzyme (prediluted in buffer
with BSA). IC50 values were calculated by fitting experimental
data to the equation % inhibition ) 100[I]0/(IC50 + [I]0). The
Zhang’s kinetic analyses were carried out at constant initial
substrate and using at least four concentrations of enzyme
(4.7-13.6 nM). Inhibition of PR was measured at different
concentrations of inhibitor: 18 (2.5 and 4 µM); 19 (4.33 µM);
20 (0.8 µM); 21 (1.5 µM); 23 (2 and 4.33 µM); 24 (0.66 µM); 25
(1 and 2.33 µM); 26 (2 and 5.66 µM); 27 (1.33 and 2.83 µM);
29 (4 µM); 30 (1 and 3 µM); 32 (0.66 and 1.66 µM); 36 (1.5 and
4 µM) and 37 (4 µM). The Kic and Kid values were determined
as previously described.16,22,23
Fluorescence Experiments. The experiments were per-
formed by diluting the PR from a protein stock solution (stored
in ice) into the appropriate buffer: 100 mM sodium acetate, 1
mM EDTA, 1 M NaCl, 3% DMSO (v/v), pH 4.7 and 25 °C at a
final concentration of 350 nM. Then, PR was preincubated in
the absence or in the presence of the inhibitor (compound 23
at 3.75 µM or compound 32 at 1.66 µM) prior to the addition
of ANS (20 or 30 µM). The excitation wavelength was 376 nm.
Excitation and emission bandwidths were 5 and 20 nm,
respectively. Acetylpepstatin (0.1 µM) was studied under the
same experimental conditions. Each spectrum was collected
three times and averaged. The spectrum for buffer containing
ANS + inhibitor or ANS + DMSO was subtracted from the
corresponding curve obtained in the presence of enzyme. It
has been verified that the intrinsic fluorescence of PR and of
compounds 23 and 32 was negligible under the experimental
conditions used.
Experimental Section
Materials. Usual solvents were purchased from commercial
sources. Dimethylformamide (DMF) was distilled on BaO.
Tetrahydrofuran (THF) was distilled on sodium/benzophenone;
acetonitrile was distilled on CaCl2. Liquid chromatography was
performed on Merck silica gel 60 (70/30 mesh); TLC was done
on silica gel, 60F-250 (0.26 mm thickness) plates. Benzyl
bromide, ethyl 4-bromobutyrate, 2,7-dihydroxynaphthalene 1,
protected amino acids, O-benzotriazol-1-yl-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate (HBTU), and 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) were purchased from com-
mercial sources. Benzyl 4-bromobutyrate,29 H-Val-Leu-Val-
OMe, H-Thr-Leu-Asn-OMe, H-Thr-Ile-Val-OMe,23 and 7-hy-
droxy-1H-quinolin-2-one 526 were prepared according to methods
reported in the literature. Melting points were determined on
a Kofler melting point apparatus. NMR spectra were per-
formed on a Bruker AMX 200 (1H, 200 MHz; 13C, 50 MHz) or
a Bruker AVANCE 400 (1H, 400 MHz; 13C, 100 MHz). Unless
otherwise stated CDCl3 was used as solvent. Chemical shifts
δ are in ppm, and the following abbreviations are used: singlet
(s), doublet (d), doublet of doublets (dd), triplet (t), multiplet
(m), broad triplet (bt), and broad singlet (bs). IR spectra were
performed on a Bruker Vector 22 apparatus. Mass spectra
were obtained using a Bruker Esquire electrospray ionization
apparatus. Elemental analyses were performed at the Mi-
croanalysis Service of the Faculty of Pharmacy at Chaˆtenay-
Malabry (France).
Analytical Gel Filtration Chromatography. Inhibitor
25 (5 mM) was incubated for 30 min at 25 °C in 100 mM
sodium acetate (pH 4.7) containing PR (95 nM). The blank (no
added inhibitor) contained the same final DMSO concentration
(5% v/v). The mixture was loaded onto a column of Superdex
75 (30 cm × 3.2 mm2, Amersham Biosciences). The preequili-
brated column was eluted with the same sodium acetate buffer
(100 µM, pH 4.7), flow rate 0.1 mL‚min-1. Absorbance at 280
nm was followed using the Empower program (Waters). The
column was previously calibrated with cytochrome c, horse
skeletal muscle myoglobin, bovine serum albumin, and human
thrombin. Bichromate potassium and dextran blue were used
for determination of the column total and void volumes.
4-(7-Hydroxy-naphthalen-2-yloxy)butyric Acid Ethyl
Ester (2). To a solution of 1 (5 g, 31 mmol) and K2CO3 (6.5 g,
47 mmol) in anhydrous DMF (50 mL) was added dropwise
ethyl 4-bromobutyrate (4 mL, 28 mmol). The mixture was
stirred for 24 h, and then solvent was evaporated in vacuo.
The residue was dissolved in CH2Cl2, and the solution was
washed with water (5 × 30 mL), dried (Na2SO4), and concen-
trated in vacuo. The resulting crude product was chromato-
graphed on silica gel (petroleum ether/ethyl acetate 4:1) to give
1
2.8 g (36%) of 2 as white needles, mp ) 91-93 °C. H NMR:
HIV-1 PR, from the HIV (BRU) DNA clone (subclone of
λJ19),32 was expressed in Escherichia coli BL21(DE3)pLysS,
isolated and purified as described by Billich et al.33 1-Anilino-
8-naphthalene sulfonate (ANS) was purchased from Sigma-
Aldrich Chimie. Fluorescence experiments were performed on
a Perkin-Elmer LS 50 B luminescence spectrofluorometer
equipped with a thermostated cell holder. A Suprasil quartz
cell with a 1-cm path length from Hellma (Germany) was used
for all experiments.
δ 7.6 (d, 2H), 7.0-6.9 (m, 4H), 5.5 (bs, 1H), 4.2-4.0 (m, 4H),
2.5 (t, 2H), 2.1 (m, 2H), 1.2 (t, 3H). 13C NMR: δ 174.4, 157.2,
136.3, 129.3, 123.4, 116.2, 115.5, 108.4, 105.8, 66.7, 60.2, 30.5,
24.6, 14.4. Anal. (C16H18O4) C, H.
4-(7-Benzyloxy-naphthalen-2-yloxy)butyric Acid Ethyl
Ester (3). To a solution of 2 (6.5 g, 24.4 mmol) and K2CO3
(4.1 g, 29.3 mmol) in anhydrous CH3CN (70 mL) was added
benzyl bromide (3.48 mL, 29.3 mmol). The mixture was

6398 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25
Merabet et al.
refluxed for 3 h and stirred at room temperature for 24 h. After
filtration, the filtrate was concentrated in vacuo, the residue
was dissolved in CH2Cl2 (70 mL), and the solution was washed
with water (3 × 10 mL). The organic layer was dried (Na2SO4),
filtered, and concentrated. The crude product was chromato-
graphed on silica gel (petroleum ether/ethyl acetate 9:1) to give
4.98 g (60%) of 3 as white crystals, mp ) 84-85 °C (petroleum
ether/ethyl acetate 95:5). 1H NMR: δ 7.7-6.9 (m, 11H), 5.1
(s, 2H), 4.2-4.0 (m, 4H), 2.5 (t, 2H), 2.1 (m, 2H), 1.3 (t, 3H).
13C NMR: δ 174.4, 157.2, 136.3, 129.3, 128.0, 123.4, 116.2,
115.5, 108.4, 105.8, 69.5, 66.7, 60.2, 30.5, 24.6, 14.4. Anal.
(C23H24O4) C, H.
148.4, 139.0, 137.0, 129.0, 128.4, 120.4, 117.0, 110.8, 107.8,
70.5, 65.0, 31.3, 24.8. Anal. (C20H19O4N) C, H, N.
4-(7-Benzyloxy-naphthalen-2-yloxy)butyryl-Val-Leu-
Val-OMe (10). To a stirred solution of 4 (1.24 g, 3.68 mmol),
HBTU (1.53 g, 4.04 mmol), and TEA (0.56 mL, 4.04 mmol) in
anhydrous DMF (25 mL) were added H-VLVOMe (1.26 g, 3.68
mmol) and 0.5 mL of TEA. The mixture was stirred for 24 h,
DMF was evaporated in vacuo, the residue was dissolved in
CH2Cl2, and the solution was washed with 1 N HCl (30 mL),
1 N NaOH (30 mL), and water. The organic layer was dried
(MgSO4) and evaporated in vacuo to give 2.7 g (90%) of a crude
product, which was recrystallized (ethyl acetate/methanol) to
afford 10 as a white powder, mp 215-217 °C. 1H NMR: δ 7.6
(t, 2H), 7.4-7.3 (m, 5H), 7.1 (bs, 1H), 7.05 (dd, 1H), 7.0 (bs,
1H), 6.9 (d, 1H), 5.1 (s, 2H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m,
1H), 4.1 (t, 2H), 3.7 (s, 3H), 2.6-2.4 (m, 2H), 2.2-2.1 (m, 3H),
1.9 (m, 1H), 1.8-1.5 (m, 2H), 0.9-0.8 (m, 18H). 13C NMR: δ
173.0, 172.5, 172.0, 157.3, 157.1, 137.0, 128.9, 128.0, 124.3,
116.3, 116.2, 106.4, 106.1, 70.0, 66.5, 58.4, 57.2, 52.1, 51.9, 41.0,
31.2, 30.1, 29.6, 24.6, 24.2, 22.5, 22.2, 19.0, 17.7. Anal.
(C38H51N3O7) C, H, N.
4-(7-Benzyloxy-quinoline-2-yloxy)butyryl-Val-Leu-Val-
OMe (11). The same procedure as described for preparation
of 10 was used: 93%, mp 214-216 °C (methylene chloride/
methanol).1H NMR (DMSO-d6): δ 8.2 (d, 1H), 8.1-7.9 (m, 3H),
7.7 (d, 1H), 7.4-7.3 (m, 5H), 7.1 (bs, 1H), 6.9 (dd, 1H), 6.7 (d,
1H), 5.1 (s, 2H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H), 4.1 (t,
2H), 3.7 (s, 3H), 2.6-2.4 (m, 2H), 2.2-2.1 (m, 3H), 1.9 (m, 1H)
1.8-1.5 (m, 3H), 0.9-0.8 (m, 18H). 13C NMR (DMSO-d6): δ
172.0, 162.0, 160.0, 139.0, 129.0, 128.7, 128.3, 116.2, 106.4,
106.1, 70.0, 65.0, 51.9, 41.0, 32.0, 31.2, 30.1, 25.0, 24.0, 23.0,
17.7. Anal. (C37H50N4O7) C, H, N.
4-(7-Hydroxy-naphthalen-2-yloxy)butyryl-Val-Leu-Val-
OMe (12). A solution of 10 (2.2 g, 3.32 mmol), and 10% Pd/C
(0.3 g) in MeOH (50 mL) was stirred under hydrogen. After
24 h, the suspension was filtered through Celite, and the Celite
bed was washed thoroughly with ethyl acetate (100 mL). The
filtrate was evaporated to dryness to yield 1.88 g of a crude
powder, which was purified by liquid chromatography (ethyl
acetate/petroleum ether, 20/80) (1.72 g, 90%). 1H NMR: δ 7.7-
7.5 (m, 3H), 6.9-6.8 (m, 4H), 4.5-4.2 (m, 3H), 3.9 (t, 2H), 3.6
(s, 3H), 2.6-2.4 (m, 2H), 2.1-1.3 (m, 7H), 0.8-0.7 (m, 18H).
13C NMR (DMSO-d6): δ 173.0, 172.0, 156.2, 136.0, 128.8, 124.6,
116.0, 115.9, 106.0, 66.5, 58.0, 52.5, 52.0, 41.0, 31.2, 30.1, 29.0,
24.6, 24.2, 22.5, 22.2, 19.0, 18.8, 18.4, 17.7. Anal. (C31H45N3O7)
C, H, N.
4-(7-Benzyloxy-naphthalen-2-yloxy)butyric Acid (4). A
suspension of 3 (0.9 g, 2.5 mmol) in a 10% KOH methanolic
solution (25 mL) was stirred at reflux for 4 h. The solid
obtained after evaporation of the solvent was suspended in
water (10 mL) and the mixture acidified with 1 N HCl. Product
was then extracted twice (methylene chloride), and the organic
layers were dried (MgSO4) and concentrated in vacuo. The
crystalline crude product was recrystallized (ethyl acetate/
petroleum ether 85:15) to give 0.76 g (92%) of the desired acid
1
4 as white crystals, mp ) 132-134 °C. H NMR: δ 7.9-7.6
(m, 11H), 5.1 (s, 2H), 4.1 (t, 2H), 2.5 (t, 2H), 2.1 (m, 2H). 13C
NMR: δ 174.4, 157.3, 157.1, 137.3, 136.0, 129.3, 128.0, 124.2,
116.4, 107.1, 106.5, 69.5, 66.9, 30.5, 24.6. Anal. (C21H20O4) C,
H.
7-Benzyloxy-1H-quinolin-2-one (6). To a solution of 526
(1 g, 6.2 mmol) in 2-propanol (15 mL) were added benzyl
bromide (0.9 mL, 7.52 mmol) and DBU (1.4 mL, 9.28 mmol).
The mixture was refluxed for 4 h. After evaporation of the
solvent, the residue was dissolved in CH2Cl2 and the organic
layer was washed with aqueous 10% Na2CO3 (20 mL), 1 N HCl
(20 mL), and water (20 mL). The organic layer was dried
(MgSO4) and concentrated in vacuo, and the solid residue was
recrystallized (methanol) to afford 1.1 g (71%) of 6 as white
1
crystals, mp ) 219-221 °C. H NMR (DMSO-d6): δ 11.6 (s,
1H), 7.8 (d, 1H), 7.6 (d, 1H), 7.3-7.5 (m, 5H), 6.8-6.9 (dd, 2H),
6.3 (d, 1H), 5.1 (s, 2H). 13C NMR (DMSO-d6): δ 162.2, 160.0,
140.5, 139.9, 136.4, 129.2, 128.4, 127.6, 118.7, 113.5, 110.9,
99.2, 69.4. IR: ν ) 1658 cm-1. Anal. (C16H13O2N) C, H, N.
4-(7-Benzyloxy-quinoline-2-yloxy)butyric Acid Ethyl
Ester (7) and 4-(7-Benzyloxy-quinoline-2-oxo-2H-quino-
line-1-yl)butyric Acid Ethyl Ester (8). A solution of 6 (0.5
g, 2.0 mmol) and K2CO3 (0.41 g, 3 mmol) in anhydrous DMF
(10 mL) was stirred for 1 h. Then ethyl 4-bromobutyrate (0,5
mL, 3.95 mmol) was added, the mixture was stirred for 24 h
at room temperature. After evaporation of the solvent in vacuo,
the residue was dissolved in CH2Cl2 and the solution washed
with water. The organic layer was dried (MgSO4) and evapo-
rated in vacuo, and the crude product was chromatographed
on silica gel, giving rise to the two isomers 7 and 8 (petroleum
ether/AcOEt, 8:2). Ester 7: 0.126 g (40%), mp ) 105-107 °C
(petroleum ether/ethyl acetate, 8:2); IR ν 1734 cm-1; 1H NMR
(DMSO-d6) δ 7.9 (d, 1H), 7.5 (d, 1H), 7.2-7.5 (m, 5H), 7.2 (dd,
1H), 7.0 (d, 1 H), 6.7 (d, 1H), 5.1 (s, 2H), 4.4 (t, 2H), 4.1 (m,
2H), 2.5 (t, 2H), 2.1 (q, 2H), 1.2 (t, 3H); 13C NMR (DMSO-d6)
δ 173.1,162.4,160.0,148.2, 138.2, 136.6, 128.5, 127.4, 119.9,
116.3, 110.3, 107.7, 70.0, 64.6, 60.2, 31.0, 24.4, 14.3. Anal.
(C22H23O4N) C, H, N. Ester 8: 0.132 g (42%), mp ) 79-80 °C;
4-(7-Hydroxy-quinoline-2-yloxy)butyryl-Val-Leu-Val-
OMe (13) was synthesized from 11 by the same procedure as
1
described above: 99%, foam. H NMR (DMSO-d6): δ 8,1 (d,
1H), 8.0-7.9 (m, 3H), 7.6 (d, 1H), 7.3 (bs, 1H), 6.9 (dd, 1H),
6.7 (d, 1H), 4.4 (m, 3H), 4.5 (m, 2H), 3.6 (s, 3H), 2.4-2.3 (m,
2H), 2.1-1.9 (m, 4H), 1.6 (m, 1H), 1.4 (m, 2H), 0.9-0.8 (18H,
m). 13C NMR (DMSO-d6): 172.6, 172.4, 172.3, 162.0, 160.0,
149.0, 139.0, 129.0, 119.5, 116.0, 109.5, 65.0, 57.5, 52.0, 50.5,
41.5, 31.5, 31.0, 25.0, 24.5, 22.5, 17.7. Anal. (C30H44N4O7‚3/
2H2O) C, H, N.
4-[7-(3-Benzyloxycarbonyl-propoxy)naphthalen-2-yloxy]-
butyryl-Val-Leu-Val-OMe (14). A solution of 12 (1.9 g, 3.32
mmol), K2CO3 (0.7 g, 5 mmol) and benzyl 4-bromobutyrate (1
g, 4 mmol) in anhydrous DMF (20 mL) was stirred for 24 h at
50 °C. The solvent was evaporated in vacuo, the residue was
dissolved in CH2Cl2 (30 mL), and the solution was washed with
Na2CO3 10% (30 mL), NH4Cl 10% (30 mL), and water (30 mL).
The organic layer was dried (MgSO4), filtered, and concen-
trated. The crude product was chromatographed on silica gel
(methylene chloride/methanol: 95/5) to yield 2 g (80%) of the
benzyl ester 14. 1H NMR: δ 7.8 (d, 1H), 7.6 (d, 1H), 7.5 (d,
1H), 7.3-7.2 (m, 5H), 7.2 (d, 1H), 7.0-6.9 (m, 4H), 5.1 (s, 2H),
4.7 (m, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.1 (t, 4H), 3.7 (s, 3H),
2.6 (t, 2H), 2.5-2.4 (m, 2H), 2.2-2.1 (m, 4H), 2.0-1.9 (m, 2H),
1.7 (m, 1H), 1.6 (m, 1H), 1.5 (m, 1H), 0.9-0.8 (m, 18H). 13C
NMR (DMSO-d6): δ 172.0-171.0, 170.0, 157.0, 135.0, 129.0,
128.0, 124.0, 116.0, 106.0, 67.0, 65.1, 57.0, 52.0, 51.0, 41.0, 32.5,
31.0, 24.0, 22.0, 18.0. Anal. (C42H57N3O9) C, H, N.
1
IR ν 1727 and 1646 cm-1; H NMR δ 7.4 (d, 1H), 7.3-7.1 (m,
5H), 7.2 (d, 1H), 7.1 (bs, 1H), 6.7 (d, 1H), 6.3 (d, 1H) 5.1 (s,
2H), 4.4 (m, 2H), 4.1 (t, 2H), 2.4 (t, 2H), 2.1 (q, 2H), 1.2 (t,
3H); 13C NMR (DMSO-d6) δ 173.1, 162.4, 160.0, 148.2, 138.2,
136.6, 128.5, 127.4, 119.9, 116.3, 110.3, 107.7, 70.0, 64.6, 41.5,
31.0, 21.9, 14.1. Anal. (C22H23O4N) C; H calcd 6.3, found 6.8;
N.
4-(7-Benzyloxy-quinoline-2-yloxy)butyric Acid (9). The
same procedure was used as for the preparation of 4. Com-
pound 9 was obtained, from the ester 7, as white crystals
(84%), mp 159-161 °C (methanol, ethyl acetate). 1H NMR
(DMSO-d6): δ 7.9 (d, 1H), 7.7 (d, 1H), 7.5-7.3 (m, 5H), 7.3 (d,
1H), 7.1 (dd, 1H), 6.8 (dd, 1H), 5.2 (s, 2H), 4.5 (t, 2H), 2.6 (t,
2H), 2.2 (m, 2H). 13C NMR (DMSO-d6): δ 178.8, 163.0, 160.6,

“Molecular Tongs” To Dissociate HIV-1 Protease Dimer
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 25 6399
4-[7-(3-Benzyloxycarbonyl-propoxy)quinolin-2-yloxy]-
butyryl-Val-Leu-Val-OMe (15) was synthesized from 13 by
the same procedure as described above: 65%, mp 139-140 °C.
1H NMR: δ 8.1-7.8 (m, 5H), 7.5 (m, 5H), 7.0-6.9 (dd, 2H),
6.6 (d, 1H), 5.2 (s, 2H), 4.2-4.1 (m, 3H), 4.0-4.1 (m, 4H), 3.6
(s, 3H), 2.2 (m, 2H), 2.1-1.9 (m, 5H), 1.4-1.3 (m, 4H), 0.9 (m,
18H). 13C NMR: δ 138.0, 130.0, 128.0, 117.0, 109.0, 107.0, 72.0,
66.0, 60.0, 58.0, 51.0, 41.0, 33.3, 32.2, 31.0, 25.0, 23.0, 21.5.
Anal. (C41H56N4O9) C, H, N.
Synthesis of Symmetric Tongs 36 and 37. Tongs 36 and
37 were synthesized using HBTU coupling methods from 35,
as described in the general synthesis of nonsymmetric tongs.
Acknowledgment. Sidaction (Ensemble contre le
Sida) is warmly acknowledged for its financial support
(grant of L.V.B.) together with University Paris 7-Denis
Diderot (Appel d’Offres sur le the`me Sida).
4-[7-(3-Carboxy-propoxy)naphthalen-2-yloxy]butyryl-
Val-Leu-Val-OMe (16). A solution of 14 (0.32 g, 0.42 mmol)
and 10% Pd/C (0.03 g) in DMF (10 mL) was stirred under
hydrogen. After 24 h, the suspension was filtered through
Celite, and the Celite bed was washed thoroughly with ethyl
acetate (100 mL). The filtrate was evaporated to one-fourth
volume, the final product precipitated as a white powder after
addition of water (2 mL), and the precipitate was dried to give
0.2 g (80%) of the desired acid. 1H NMR: δ 7.6 (d, 2H), 7.3 (d,
1H), 7.1 (d, 1H), 7.0 (m, 2H), 6.9 (m, 2H), 6.8 (d, 1H), 4.5-4.4
(m, 2H), 4.3 (m, 1H), 4.1 (t, 2H), 4.0 (t, 2H), 3.7 (s, 3H), 2.6 (t,
2H), 2.5-2.4 (m, 2H), 2.2-2.1 (m, 5H), 2.1-1.9 (m, 1H), 1.7-
1.4 (m, 3H), 0.9-0.8 (m, 18H). 13C NMR: δ 173.1, 171.8, 157.8,
128.9, 124.3, 116.3, 116.2, 107.0, 70.0, 67.0, 58.6, 58.1, 51.7,
50.9, 41.0, 32.4, 31.2, 30.1, 29.6, 24.6, 24.8, 24.2, 22.5, 22.2,
19.0, 17.7. Anal. (C35H51N3O9) C, H, N.
Supporting Information Available: NMR data of syn-
thesized peptides not described and analytical data of com-
pounds 18, 20, 22-27, 29-32, 36, and 37. Table of elemental
analysis of compounds 18-32, 36, and 37. This material is
available free of charge via the Internet at http://pubs.acs.org.
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General Synthesis of Nonsymmetric Tongs (18-20,
22-27, 29-32): Example of 19. To a stirred solution of 16
(0.143 g, 0.21 mmol), HBTU (0.09 g, 0.24 mmol), and TEA
(33.24 µL, 0.24 mmol) in DMF (5 mL) was added a solution of
Ile-Thr-NH2 (0.05 g, 0.21 mmol) in DMF (2 mL). The mixture
was stirred for 24 h. The solvent was removed, and the residue
was dissolved in CH2Cl2 (10 mL). A product precipitated, which
was filtered, washed with 1 N NaOH (10 mL), 1 N HCl (10
mL), water (10 mL), methylene chloride (10 mL), and metha-
nol, and dried at 60 °C in vacuo.
4-[7-(3-Ethoxycarbonyl-propoxy)-2-oxo-2H-quinoline-
1-yl]butyric Acid Ethyl Ester (33) and 4-[7-(3-Ethoxy-
carbonyl-propoxy)quinoline-7-yloxy]butyric Acid Ethyl
Ester (34). To a solution of 5 (0.5 g, 3 mmol) and K2CO3 (0.63
g, 9 mmol) in DMF (5 mL) was added ethyl 4-bromobutyrate
(0.43 mL, 6 mmol). The mixture was stirred for 24 h. The
solvent was evaporated, and the crude product was chromato-
graphed on silica gel (petroleum ether/ethyl acetate, 60:40) to
give the N-alkylated product 33 and its O-alkylated isomer
1
34. Product 33: 49%; IR ν ) 1646 cm-1; H NMR (DMSO-d6)
δ 7.7 (d, 1H), 7.5 (d, 1H), 7.0 (d, 1H), 6.9 (d, 1H), 6.7 (d, 1H),
4.5 (t, 2H), 4.4 (m, 6H), 2.6 (m, 4H), 2.3 (m, 4H), 1.2 (m, 6H);
13C NMR (DMSO-d6) δ 173.2, 173.1, 162.5, 160.0, 148.0, 138.0,
128.0, 119.0, 116.0, 110.0, 107.0, 67.0, 64.7, 41.0, 31.1, 30.9,
1
24.0, 14.0. Anal. (C21H27NO6) C, H, N. Product 34: 43%; H
NMR (DMSO-d6) δ 7.9 (d, 1H), 7.6 (d, 1H), 7.2 (d, 1H), 7.0 (d,
1H), 6.7 (d, 1H), 4.5 (t, 2H), 4.2 (m, 6H), 2.6 (m, 4H), 2.2 (m,
4H), 1.3 (m, 6H); 13C NMR (DMSO-d6) δ 173.2, 173.1, 162.5,
160.1, 148.3, 138.2, 128.3, 119.8, 116.2, 110.3, 107.2, 66.8, 64.7,
60.3, 31.1, 30.8, 24.5, 14.1. Anal. (C21H27NO6) C, H, N.
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Dick, A.; Schramm, W. Lipopeptides as Dimerization Inhibitors
of HIV-1 Protease. Biol. Chem. 1999, 380, 593-596.
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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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Bioorg. Med. Chem. Lett. 1998, 8, 3281-3286.
4-[7-(3-Carboxy-propoxy)quinolin-7-yloxy]butyric Acid
(35). The carboxylic acid 35 was obtained from 34 by the same
procedure as described for 4 and 8: 90%. 1H NMR (DMSO-
d6): δ 12.1 (s, 2H), 8.1 (d, 1H), 7.8 (d, 1H), 7.2 (d, 1H), 7.0 (d,
1H), 6.9 (d, 1H), 4.4 (t, 2H), 4.1 (m, 2H), 2.4 (m, 4H), 2.0 (m,
4H).13C NMR (DMSO-d6): δ 173.2, 173.1, 162.5, 160.1, 148.3,
140.0, 129.3, 119.8, 116.2, 110.3, 107.2, 67.0, 65.0, 30.0, 24.5.
Anal. (C17H19NO6) C, H, N.

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